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Proliferation-independent regulation of organ size by Fgf/Notch signaling

  1. Agnė Kozlovskaja-Gumbrienė
  2. Ren Yi
  3. Richard Alexander
  4. Andy Aman
  5. Ryan Jiskra
  6. Danielle Nagelberg
  7. Holger Knaut
  8. Melainia McClain
  9. Tatjana Piotrowski Is a corresponding author
  1. Stowers Institute for Medical Research, United States
  2. Skirball Institute of Biomolecular Medicine, New York University Langone Medical Center, United States
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Cite as: eLife 2017;6:e21049 doi: 10.7554/eLife.21049

Abstract

Organ morphogenesis depends on the precise orchestration of cell migration, cell shape changes and cell adhesion. We demonstrate that Notch signaling is an integral part of the Wnt and Fgf signaling feedback loop coordinating cell migration and the self-organization of rosette-shaped sensory organs in the zebrafish lateral line system. We show that Notch signaling acts downstream of Fgf signaling to not only inhibit hair cell differentiation but also to induce and maintain stable epithelial rosettes. Ectopic Notch expression causes a significant increase in organ size independently of proliferation and the Hippo pathway. Transplantation and RNASeq analyses revealed that Notch signaling induces apical junctional complex genes that regulate cell adhesion and apical constriction. Our analysis also demonstrates that in the absence of patterning cues normally provided by a Wnt/Fgf signaling system, rosettes still self-organize in the presence of Notch signaling.

https://doi.org/10.7554/eLife.21049.001

Introduction

Organ morphogenesis relies on the integration of complex processes, such as cell migration, cell shape changes and cell specification to generate the correct three-dimensional geometry necessary for function. Additionally, these cell behaviors must be coupled with mechanisms that regulate the final size of the organ for correct integration into the organism. Understanding the mechanisms that underlie these phenomena is a major focus of modern biology. Even though the intracellular mechanisms leading to cell shape changes are fairly well understood, we are only beginning to elucidate how signaling pathways coordinate cell shape changes with the development of a whole tissue or embryo. Additionally, the importance of multicellular, epithelial rosettes in Drosophila axis elongation and retina development, mouse pre-implantation embryo morphogenesis, pancreas development, brain tumors or the neural stem cell niche has only fairly recently been recognized (Bedzhov and Zernicka-Goetz, 2014; Blankenship et al., 2006; Harding et al., 2014; Martin and Goldstein, 2014; Wippold and Perry, 2006).

The zebrafish lateral line is a powerful model to study sensory organ morphogenesis, as it develops superficially in the skin and is amenable to experimental manipulation and in vivo imaging. The lateral line is a sensory system for the detection of water movements and consists of rosette-shaped sensory organs (neuromasts) that are arranged in lines along the body of the animal. Each neuromast is composed of sensory hair cells surrounded by support cells. Lateral line hair cells are homologous to vertebrate inner ear hair cells and are specified by the same molecules (Nicolson, 2005). The lateral line system on the trunk develops from an ectodermal placode posterior to the ear that migrates to the tail tip. The migrating placode (now called primordium) periodically deposits clusters of cells that mature into neuromasts, thus forming a line of sensory organs. This migrating primordium consists of a mesenchymal leading region and a trailing region in which cells apically-basally polarize and then apically constrict to form garlic bulb/rosette-shaped proneuromasts (reviewed in [Harding et al., 2014]). The two domains are maintained by a feedback mechanism between the Wnt and Fgf pathways (Aman and Piotrowski, 2008). Activation of the Wnt pathway in the leading region induces Fgf ligands that activate the Fgf pathway in the trailing region. Fgf ligand expression is uniform in the leading region but then becomes restricted to one central cell as organs (proneuromasts) begin to form. Fgf ligand expression by a central proneuromast cell that activates Fgf signaling in surrounding cells is crucial for proneuromast formation and maintenance (Durdu et al., 2014; Ernst et al., 2012; Harding and Nechiporuk, 2012; Lecaudey et al., 2008; Nechiporuk and Raible, 2008). Fgfr-Ras-Mapk signaling is thought to directly induce apical constriction and rosette formation via the activation of the actin-binding protein shroom3. shroom3 leads to apical localization of Rock2a kinase that phosphorylates non-muscle myosin II (pNMII) driving actomyosin constriction (Ernst et al., 2012; Harding and Nechiporuk, 2012).

Of all the mutants/manipulations thus far analyzed that affect lateral line development only very few lead to an increase in organ size. The only manipulation described that causes an increase in neuromast size is the upregulation of Wnt signaling, while the size of the primordium increases after inhibition of the Hippo pathway member amotl2a (Agarwala et al., 2015; Head et al., 2013; Wada et al., 2013; Wada and Kawakami, 2015; Jacques et al., 2014). The Hippo pathway controls organ size via a kinase cascade that leads to the phosphorylation and degradation of the transcriptional co-activators Yap/Taz (Sun and Irvine, 2016). In the absence of pathway activation, Yap/Taz are translocated to the nucleus where they activate proliferation and survival genes. The Wnt pathway affects organ size via controlling proliferation, which, at least partially, is regulated by amotl2a (Agarwala et al., 2015). In contrast, loss of the transcriptional co-activator yap1 rescues the amotl2a overproliferation phenotype but does not interact with the Wnt pathway. Thus, the details of how Wnt, amotl2a and yap1 are integrated to affect proliferation are not well understood. Here we describe how the upregulation of Notch signaling by overexpression of the Notch1a intracellular domain (ICD; NICD, [Scheer and Campos-Ortega, 1999]) in the primordium, leads to a significant increase in neuromast size. The increased neuromast size in both Notch and Wnt overexpressing embryos is yap1-independent, and in case of NICD the organ size increase is even proliferation-independent.

Apical constriction is driven by cortical tension-generating actomyosin contractions that are transmitted to neighboring cells via apically localized cadherins (reviewed in [Heisenberg and Bellaïche, 2013; Martin et al., 2009; Pilot and Lecuit, 2005; Schwayer et al., 2016]). We demonstrate that Fgf-dependent Notch signaling cell-autonomously, and downstream of Fgf signaling upregulates adherens and tight junction molecules. These molecules together form an apical junctional complex associated with a circumferential actomyosin belt (Niessen, 2007). Thus, Notch signaling increases organ size independently of proliferation by coordinating actomyosin activation and cell adhesion.

Results

Constitutive activation of Notch or Wnt signaling generates larger sensory organs

When we constitutively activate Notch signaling in the lateral line by driving the NICD in (Tg(cldnB:lynGFP);Tg(cldnB:gal4) x Tg(UAS:nicd)) embryos, we observe that deposited neuromasts are substantially larger than in sibling embryos (Figure 1A–B’, Figure 1—figure supplement 1A–A'; Video 1). The NICD transgene is strongly induced when the lateral line placode is forming at 13 hr post fertilization (hpf) (data not shown). It is also myc-tagged and and shows widespread expression in the lateral line and skin (Figure 1C–D). The neuromast size increase is reminiscent of Wnt overexpressing apc mutant neuromasts (Figure 1F–F’, [Wada et al., 2013]). In contrast, a reduction of Notch signaling in mib1 mutants causes fragmentation of neuromasts (Figure 1E,E’; Video 2; [Matsuda and Chitnis, 2010]).

Figure 1 with 1 supplement see all
Induction of Notch signaling induces larger lateral line organs.

(A and B and E and F) First deposited trunk neuromasts (L1) in Tg(cldnB:lynGFP). (B) NICD and (F) apcmcr neuromasts are larger than (A) WT and (E) mib1ta52b neuromasts. Scale bar is 25 μm. (A’ and B’ and E’ and F’) Posterior lateral line in Tg(cldnB:lynGFP). (A’) WT, (B’) NICD, (E’) mib1ta52b and (F’) apcmcr. Scale bar is 100 μm. (C and D) NICD transgenic embryos are labeled by a Myc-tag. The c-Myc antibody in magenta. Scale bar is 50 μm. (GH’) L1 neuromasts stained with Phalloidin at 32 hpf. In NICD neuromasts (H and H’) the apical F-actin meshwork (arrow) is larger compared to wildtype (WT) (G and G’), suggesting that NICD neuromasts are composed of more apically constricted cells. Scale bar is 25 μm. (IN’’) Expression of Notch pathway genes in the primordium. (I’) notch3, (J’) her4 and dgfp in the Notch reporter Tg(Tp1bglob:EGFP) (K’) are upregulated in NICD primordia compared to WT (I and J and K) and mib1ta52b (I’’ and J’’) primordia. The Notch reporter only seems to be activated by high levels of Notch signaling, as it is not active in wildtype primordia (K), even though the Notch target her4 is expressed (J). Scale bar is 25 μm. (LN’’) Expression of delta ligands in the primordium. (L’) deltaa, (M’) deltab and deltac (N’) are largely downregulated in NICD compared to WT (L and M and N) and mib1ta52b (L’’ and M’’ and N’’) primordia. Scale bar is 25 μm. (O) NICD and sibling primordia sizes decrease overtime. NICD primordia start out (24 hpf) with significantly more cells compared to sibling primordia, but after the second deposition cycle at ~32 hpf NICD and sibling primordia have a similar amount of cells. By 40 hpf NICD primordia are composed of significantly less cells. Error bars represent standard error (p<0.01=** Student’s t test). (P) NICD neuromasts (L1-4) along the trunk of the embryo at 2.5 dpf consist of significantly more cells compared to siblings. NICD primordia deposit big neuromasts, even at 32 hpf when NICD and sibling primordia possess the same cell number (time point (32 hpf) is marked in the (O) green box, also see L3 deposition in the Video 3). Error bars indicate standard error (p<0.001=*** Student’s t test). (QR’) Notch activation in hs:NICD embryos after the primordium and ganglion have separated still significantly increases neuromast sizes (R’) compared to a (Q’) sibling (see Figure 1—figure supplement 1E for quantification). These results indicate that the number of cells in neuromasts is independent of primordium size. Notch overexpression was induced by a 39°C heat-shock for 45 min starting at 25 hpf (L1 was still a part of the primordium). Embryos were fixed after L4, L5 deposition (~40 hpf). Scale bar is 25 μm.

https://doi.org/10.7554/eLife.21049.002
Video 1
3D rendering of a 2 dpf sibling and NICD L1 neuromast using FluoRender image visualization software (University of Utah).

Tg(cldnB:lynGFP) embryo was fixed in 4% PFA and stained with DAPI to visualize nuclei.

https://doi.org/10.7554/eLife.21049.004
Video 2
Time-lapse movie of a mib1ta52b mutant in the Tg(cldnB:lynGFP) background showing the gradual disintegration of neuromasts and the primordium.

Embryo was imaged every 5 min starting at 28 hpf.

https://doi.org/10.7554/eLife.21049.005

Labeling of the first deposited trunk neuromast (L1) with the actin marker phalloidin shows that the NICD apical F-actin meshwork is larger compared to wildtype neuromasts, suggesting that NICD neuromasts are composed of more apically constricted cells (Figure 1G–H’). The robust induction of NICD in (Tg(cldnB:lynGFP);Tg(cldnB:gal4) x Tg(UAS:nicd)) transgenic embryos, is also evidenced by upregulation of the Notch targets notch3, her4, strong activation of the Notch reporter Tp1bglob:EGFP and downregulation of delta ligands (Figure 1I–K’ and Figure 1L–N’). On the other hand, in mib1 mutants notch3 and her4 are reduced but not completely absent and delta genes are upregulated (Figure 1I’’–J’’ and L’’–N’’; Matsuda and Chitnis, 2010). NICD primordia migrate more slowly and deposit fewer neuromasts than wildtype primordia and 80% of the NICD primordia do not reach the tail tip, as they deposit too many cells and run out of cells (Figure 1O and Figure 1—figure supplement 1A–C).

During early posterior lateral line development, Notch determines the proportion of placodal cells that contribute to the lateral line ganglion posterior to the ear and how many cells will become part of the migrating primordium (Mizoguchi et al., 2011). Consequently, ectopic activation of Notch signaling generates an initially larger primordium at the expense of the ganglion, which could explain the increase in neuromast size (Figure 1O and Figure 1—figure supplement 1D). Indeed primordium size limits how large NICD neuromasts can grow. NICD primordia become increasingly smaller during migration and NICD neuromasts accordingly decrease in size the further posterior they are located along the trunk of the embryo (Figure 1O and P). However, by 32 hpf NICD and sibling primordia consist of the same number of cells but NICD primordia still deposit larger L3 neuromasts (Figure 1O and PVideo 3). Also, heat-shock activation of Notch signaling after the primordium and ganglion have separated significantly increases the neuromast cell number (Figure 1Q–R’, Figure 1—figure supplement 1E). These results indicate that, although the primordium size determines the maximum size of an enlarged neuromast, the increased NICD neuromast size is independent of the early role of Notch in allocating cells to the primordium.

Video 3
Time-lapse movie showing the primordium depositing the L3 proneuromast in a sibling- Tg(cldnB:lynGFP) and a Tg(cldnB:lynGFP);NICD embryo.

The NICD primordium deposits a larger proneuromast compared to the wildtype sibling. Embryos were imaged every 5 min.

https://doi.org/10.7554/eLife.21049.006

The increased organ size after Notch and Wnt activation is independent of yap1

The size of the migrating primordium is partially controlled by the Hippo pathway components amotl2a (a tight junction-associated scaffolding protein) and yap1 (a transcriptional co-activator) (Agarwala et al., 2015). amotl2a inhibits proliferation, whereas yap1 is required in the primordium to reach its normal size. To test if the Hippo pathway might modulate neuromast size downstream of Notch or Wnt signaling we tested if (a) Manipulating Wnt or Notch signaling affects the expression of Hippo pathway genes and (b) if the downregulation of yap1 rescues neuromast size in NICD and apc embryos. yap1 is expressed in the leading 2/3 of the wildtype primordium (Figure 2A’) and is upregulated in NICD (Figure 2B’). yap1 is also increased in apc primordia correlating with the increased proliferation observed in these embryos (Figure 2D’; [Aman et al., 2011]). In contrast, yap1 is downregulated in mib1 and Wnt depleted hs:dkk1b primordia (Figure 2C’,E’). However, yap1 is not expressed in deposited wildtype, NICD, mib1, apc or hs:dkk1b neuromasts suggesting that proliferation in deposited neuromasts is yap1-independent (Figure 2A–E). We also analyzed the canonical Hippo pathway inhibitor stk3 (the ortholog of hippo kinase in Drosophila) and amotl2a, which inhibits Wnt-induced proliferation (Figure 2—figure supplement 1A–J'Agarwala et al., 2015). The expression of these genes does not correlate with proliferation in the primordia with manipulated Wnt or Notch signaling, showing that we do not yet fully understand the integration of Hippo signaling with other mitogens and that further studies are needed.

Figure 2 with 1 supplement see all
The Hippo pathway member yap1 regulates primordium size but not neuromast size.

(AE) yap1 is not expressed in WT neuromasts (A) or neuromasts in which Notch (B and C) or Wnt signaling (D and E) are manipulated. (A’E’) yap1 is upregulated in the primordium after (B’) Notch and (D’) Wnt overexpression compared to (A’) wildtype siblings and Wnt downregulation (E’). (C’) Notch loss in mib1ta52b mutants leads to downregulation of yap1. (F) Loss of yap1 by morpholino injections significantly reduces the number of cells in the leading 2/3 of WT, NICD and apcmcr primordia, as well as the overall primordium size (H). (G) Notch and Wnt signaling significantly increases the number of cells in deposited L1 neuromasts, which is not rescued to a wildtype level by yap1 morpholino injections. Even though the size of NICD L1 and L2 pro-neuromasts is significantly reduced in yap1 morphants, they are still significantly larger than the corresponding WT neuromasts (F and G). Error bars represent standard error from one independent experiment (p<0.05=*, p<0.01=**, p<0.001=*** Student’s t test). Scale bar is 25 μm.

https://doi.org/10.7554/eLife.21049.007

However, the upregulation of yap1 in primordia correlates with the increased neuromast cell numbers in apc and NICD. In addition, yap1 morpholino injections inhibit the ectopic proliferation in the primordium that is induced by loss of amotl2a, phenocopying the yap1 mutant (Agarwala et al., 2015). We therefore injected yap1 morpholino into NICD and apc embryos and counted the number of cells in the deposited neuromasts, as well as migrating primordium. yap1 morpholino reduces the number of primordium cells in its expression domain in the leading 2/3 of wildtype, apc and NICD primordia (Figure 2F). As morpholino injections can cause cell death in the lateral line (Aman et al., 2011), we injected the yap1 morpholino into homozygous p53 mutant embryos (Figure 2—figure supplement 1K). The primordium cell number decreases from approximately 100 to 80, which is similar to what we find in yap1 morpholino-injected wildtype embryos (Figure 2H), suggesting that the decrease in cell number is not caused by morpholino-induced p53 activation (Kok et al., 2015). Although yap1 downregulation significantly reduces the size of the primordium, it does not rescue neuromast size in apc or NICD embryos to a wildtype level (Figure 2G). The reduction in NICD L1 and L2 pro-neuromast size in yap1 morphants (Figure 2F and G) can be attributed to the role of yap1 in primordium size regulation and that the primordium size limits the number of cells available for allocation into forming neuromast (Figure 1O and P). The above experiments show that the increase in neuromast size in apc and NICD is yap1 independent.

Activated Notch leads to proliferation-independent neuromast growth

Activation of Wnt signaling produces larger neuromasts via hyperproliferation and we therefore tested if proliferation is upregulated in a yap1-independent fashion in NICD primordia and neuromasts (Wada et al., 2013). In contrast to apc neuromasts, which show a significant increase in proliferation, NICD neuromasts show a significant decrease (Figure 3A–D). Accordingly, apc neuromasts gain a significant number of cells after deposition between 32–56 hpf, whereas NICD neuromasts do not significantly grow in cell number (Figure 3E). The BrdU index and number of primordium cells in 31 hpf apc primordia is not significantly higher than in the siblings (Figure 3F,F’,H,I), although 33 hpf apc primordia possess a significant increase in proliferating cells (Aman et al., 2011). Similarly, the BrdU index and the number of cells in 35 hpf NICD primordia is unchanged (Figure 3G–I).

Proliferation is not responsible for large neuromasts in NICD embryos.

(A) BrdU treatment strategy. (BC’) Representative images of BrdU-positive nuclei in L1 apcmcr (B’) and NICD neuromasts (C’). (B and B’ and D) The BrdU index is increased in apcmcr L1 neuromasts and significantly reduced in NICD L1 neuromasts (C and C’ and D). (E) Nevertheless, the number of cells is significantly higher in the apcmcr and NICD L1 neuromasts. While the apcmcr neuromast grows overtime, the NICD neuromast size does not significantly change from 32 hpf to 56 hpf. (FG’) Representative images of BrdU positive nuclei in apcmcr (F’) and (G’) NICD primordia. (FH) There is no significant difference in the BrdU index (H) or the number of cells (I) between sibling and apcmcr or sibling and NICD primordia. (J) Hyroxyurea and aphidicolin treatment strategy. (K) Cell cycle inhibition with hydroxyurea and aphidicolin significantly reduces the number of DAPI- positive cells in the last deposited neuromasts in apcmcr embryos. There is no significant reduction in sibling or NICD neuromast sizes after cell cycle inhibition. (L) Cell cycle inhibition significantly reduces the number of cells in sibling, apcmcr and NICD primordia. Scale bars are 25 μm. Treatment of sibling and NICD embryos with the GSK3β inhibitor BIO causes significant enlargement of neuromasts (L1–3). Scale bar is 100 μm. Error bars represent standard error from one independent experiment (p<0.05=*, p<0.01=**, p<0.001=*** Student’s t test).

https://doi.org/10.7554/eLife.21049.009

The striking increase of the BrdU index in deposited apc neuromasts compared to the primordium suggests that apc neuromasts only grow larger once deposited, whereas the increase in the size of NICD neuromasts is controlled by a proliferation-independent process within the primordium, prior to deposition. To further test the hypothesis that aberrant proliferation does not contribute to increased neuromast size in NICD embryos, we inhibited proliferation by treating embryos with the DNA replication inhibitors hydroxyurea and aphidicolin (HUA and Aph, Figure 3J–L). Indeed, the size of the last deposited NICD neuromast is not significantly reduced, even though the primordium cell number is smaller (Figure 3K–L). On the other hand, apc neuromasts were rescued to a wildtype size and apc primordia became smaller (Figure 3K–L). To further test if Wnt and Notch affect neuromast size via independent mechanisms we simultaneously activated Wnt and Notch signaling. The induction of Wnt signaling in NICD embryos causes even bigger neuromasts than in untreated NICD embryos, suggesting an additive effect of the two pathways (Figure 3M–O). These results demonstrate that Wnt signaling affects neuromast size via upregulating proliferation in the deposited neuromasts, whereas activation of Notch causes an increase in neuromast size independently of the proliferation rate.

The increase in organ size is independent of the role of Notch in cell type specification

As proliferation does not contribute to the large proneuromast size in NICD primordia and Notch is crucial for cell fate specification via lateral inhibition, we asked if in NICD primordia a switch in cell fate could be responsible for the increase in organ size. As yet, four cell types have been identified in the primordium: (a) mesenchymal, unpatterned cells in the leading region, (b) hair cell precursors, (c) support cells in the center and (d) future interneuromast cells that are deposited in between neuromasts (Figure 1A’, arrows). Interneuromast cells are lateral line stem cells that postembryonically give rise to additional sensory organs (Grant et al., 2005; López-Schier and Hudspeth, 2006; Lush and Piotrowski, 2014). To test if Notch changes the fate of one cell population into another we counted the number of cells in these four populations in wildtype and NICD primordia.

In the ear and lateral line, Delta/Notch signaling is essential for the specification of sensory hair and support cells (Haddon et al., 1999; Itoh and Chitnis, 2001; Riley et al., 1999; Millimaki et al., 2007). The deltad- (and atoh1a) – expressing cell differentiates into a hair cell, whereas the surrounding Notch-expressing cells are specified as support cells (Matsuda and Chitnis, 2010). As Notch signaling inhibits atoh1a, NICD neuromasts consist only of support cells (Figure 4A–L). However, within the primordium only one atoh1a-labeled hair cell precursor exists per proneuromast (Figure 4K) and their cell fate switch to a support cell cannot account for an increase of an average of 15 or 11 cells in the first proneuromast (orange, Figure 4M–N’) and an increase of 12 or 19 cells in the last proneuromast cells at 26 hpf and 30 hpf (green, Figure 4M–N’). Nevertheless, we tested if the ectopic generation of hair cells in NICD neuromasts would rescue the neuromast size. We induced hair cells in NICD primordia and neuromasts by activating the hair cell specification gene atoh1a using a heat-shock inducible transgenic line (Figure 4—figure supplement 1A–E’’). Hair cell containing neuromasts in heat-shocked Tg(hs:atoh1a;NICD) larvae are not significantly different in size from sibling NICD neuromasts (Figure 4—figure supplement 1F). Thus, the loss of hair cells in NICD neuromasts does not cause the increase in NICD neuromast size. Interestingly, hs:atoh1a alone induces a significant increase in neuromast size (Figure 4—figure supplement 1C'’ and F). The increase of her4 expression in response to ectopic atoh1a activation suggests that Atoh1a possibly induces larger neuromasts through Notch signaling activation in the primordium (Figure 4—figure supplement 1B–E).

Figure 4 with 1 supplement see all
NICD neuromasts are not larger because of a switch in cell fate.

Scanning electron micrograph of a 2 dpf sibling (A) and NICD neuromast (B) shows that no hair cells (white arrow in WT) are present in the NICD neuromast. Scale bar is 10 μm. (CF) Transmission electron sections through sibling (C,E) and NICD neuromasts (D,F). (C,E) Hair cells are false colored in yellow. (D,F) No hair cells are present in NICD neuromasts. (CF) Scale bars are 5 μm. (G and H) Acetylated tubulin antibody stains hair cells in a sibling neuromast (G), but staining is absent in a NICD neuromast (H). (G and H) Scale bar is 25 μm. (I and L) The proneural gene atoh1a is not expressed in NICD neuromasts (J) and the primordium (L). (IL) Scale bar is 25 μm. (MN’) Cell number (DAPI counts) in the different parts of the primordium. Magenta indicates mesenchymal tip cells, orange indicates the first proneuromast and green indicates the about to be deposited proneuromast. The gray cells are calculated by subtracting mesenchymal, first pro-neuromast and last pro-neuromast cell numbers from the total number of cells in the primordium. (M and M’) Scale bar equals 25 μm. (O,P) Tg(cxcr4b:H2A-GFP) labels all lateral line nuclei. (Q) NICD neuromasts consist of significantly more cells compared to sibling neuromasts, but there is no difference between the number of interneuromast cells (INCs) or the primordium cells at 2 dpf. (O and P) Scale bar equals 100 μm. (R) Significantly fewer proneuromasts are formed in a NICD primordium at 30 hpf. Error bars represent standard error from one independent experiment (p<0.05=*, p<0.01=**, p<0.001=*** Student’s t test).

https://doi.org/10.7554/eLife.21049.010

We then asked if a cell fate switch in mesenchymal leading cells contributes to the large neuromast phenotype in NICD. We determined that the number of mesenchymal cells in the leading region is initially normal in 26 hpf embryos and is only reduced from an average of 9 to 5 cells in 30 hpf NICD primordia (Figure 4M and N’). Therefore, the reduction in the number of leading region cells is equally insufficient to explain the increase in organ size. The last potential cell fate switch that could contribute to the organ size increase is the loss of future interneuromast cells from the primordium. Future oblong-shaped interneuromast cells are located at the periphery of the primordium (Figure 4M’, arrow) but their number is not affected in NICD embryos (Figure 4O–P arrows, Figure 4Q). The only significant difference we detected between NICD and sibling primordia is that NICD primordia possess on average fewer, larger proneuromasts at the expense of a normal-sized proneuromast (Figure 4M and M’, grey cells, and Figure 4R). We therefore conclude that cell fate changes are not major contributors to the increase in organ size in NICD primordia but that the allocation of support cells into fewer but larger proneuromasts is the major cause.

Notch is sufficient to induce organ morphogenesis and apical constrictions cell autonomously in the absence of Fgf signaling

The loss of Fgf signaling leads to the loss of proneuromasts/rosettes (Lecaudey et al., 2008; Nechiporuk and Raible, 2008). It has therefore been proposed that the Fgf-expressing central cell in a proneuromast acts as a signaling center that recruits surrounding support cells by inducing Fgf signaling in these cells (Lecaudey et al., 2008; Nechiporuk and Raible, 2008). We therefore tested if in NICD proneuromasts, Fgf ligand expression or Fgf signaling is increased. Surprisingly, even though fgfr1 is normally expressed, fgf3, fgf10a and the Fgf targets pea3 and dkk1b are substantially abrogated in NICD primordia (Figure 5A,A’,B,B’,C,C’,D,D’, Figure 8L,L’). Fgf signaling is likely reduced because: (a) Notch inhibits atoh1a, which is required for fgf10a expression in the most mature rosettes in the primordium (Figures 4J,L and 5C,C’ arrows; [Matsuda and Chitnis, 2010]) and (b) because Notch inhibits Wnt, which normally induces Fgf signaling in the first forming rosette (see below, Figure 8I’–K’). Therefore, increased Notch signaling causes decreased Wnt and Atoh1a dependent Fgf signaling. The loss of Notch signaling in mib1 mutants also leads to loss of Fgf signaling in central cells, however fgfr1a and pea3 are strongly expressed in peripheral cells of the primordium (Figure 5A’’,D’’). In mib1 primordia Fgf signaling is reduced in central cells because of the upregulation of atoh1a, which is normally restricted to a central cell by Notch signaling. Atoh1a, inhibits fgfr1a, thus causing the loss of Fgf signal transduction (Figure 5A’’–D’’; [Matsuda and Chitnis, 2010]).

Figure 5 with 1 supplement see all
Notch induces proneuromast (rosette) formation independent of Fgf-MAPK-Shroom3 signaling.

(AD’’) The expression of Fgf pathway members in the primordium is altered by Notch signaling. Fgf ligand (B’ and C’) and target gene expression (D’) is largely downregulated in NICD primordia compared to siblings (BD). (B’’) fgf3 and (D’’) pea3 expression is downregulated in mib1ta52b primordia, but fgf10 expression (C’’) is largely expanded. fgfr1 expression remains normal in NICD (A’) but is downregulated in the center of the mib1ta52b primordia (A’’). (EH’) Notch induces proneuromast formation independent of Fgf-MAPK signaling. NICD primordia form proneuromasts in the presence of Fgfr1 and MAPK inhibitors (F’ and F’’ and F’’’) as well as after the induction of dominant-negative Fgfr1 (H’), while sibling proneuromast formation fails after these manipulations (E’E’’’ and G’). (GH’) Embryos were heat-shocked 2 times at 39°C for 20 min with 20 min incubation at a room temperature in between, starting at 26 hpf and fixed 4 hr later. (IL’’) Notch regulates apical Rock2a accumulation independent of Fgf signaling. Contrary to sibling primordia (J’), the Rock2a expression is maintained in the apices of the forming proneuromasts in NICD primordia treated with the Fgfr1 inhibitor SU5402 (L’). (J’’ and L’’) Fgf downregulation is confirmed by the loss of di-pERK1/2 expression in the primordium. (MP’) Notch regulates Phosphorylated Myosin Regulatory Light Chain (pMRLC) expression independent of Fgf signaling. (N’) Sibling primordia lose the pMRLC expression after Fgfr1 inhibitor treatment, but pMRLC expression is maintained in the apices of treated (P’) NICD proneuromasts. (QV’) NICD primordia form proneuromasts (black arrows) in the absence of shroom3 expression (V’). Treatment with the Fgfr1 inhibitor SU5402 depletes pea3 (Q’ and R’), her4 (S’) and shroom3 (U’ and V’) expression in control and NICD primordia. (T’) her4 expression is maintained in NICD primordia after Fgfr1 inhibition. (W) shroom3 is upregulated in mib1ta52b primordia, although Fgf signaling is downregulated. (XY’’’) Constitutive activation of Fgf signaling by heat-shock induction of Fgfr1 does not rescue rosette formation in mib1ta52b primordia (Y’’’) suggesting that Fgf signaling is not sufficient for rosette formation in the absence of Notch signaling. (XY’’’) Embryos were heat-shocked at 39°C for 40 min, starting at 33 hpf and fixed 4 hr later. Scale bar in all panels is 25 μm.

https://doi.org/10.7554/eLife.21049.012

As the expression of Fgf pathway genes is not completely abrogated in NICD primordia, we depleted Fgf signaling in NICD embryos with (a) the pharmacological Fgfr1 inhibitors SU5402 and PD173074 (Figure 5E–F’’), (b) treated NICD embryos with an inhibitor of MAPKK/Di-pErk (PD0325901) (Figure 5E’’’–F’’’, Figure 5—figure supplement 1A-B’) and (c) heat-shock induction of dominant-negative fgfr1 in NICD embryos (Figure 5G–H’). Irrespective of by which method we deplete Fgf signaling in NICD embryos, proneuromasts form, even though the primordium still eventually stalls (Figure 5F’,F’’,F’’’,H’). Also, the molecular analysis of Fgf-depleted wildtype and NICD embryos shows that, although the Fgf signal transduction protein di-pErk is absent from all embryos (Figure 5J’’, L’’), only in NICD embryos Rock2a and pMRLC are still properly localized to the apical constrictions of proneuromast cells (Figure 5L',P'). These results demonstrate that in the absence of Fgf signaling, Notch signaling is sufficient to apically localize Rock2a and pMRLC and induce/maintain apical constrictions (Figure 5I-P').

To test if Notch signaling acts downstream of Fgf signaling we performed in situ analyses with the Notch target her4 in Fgf-depleted sibling and NICD embryos (Figure 5Q-T'). Indeed, Notch signaling is highly reduced in the siblings in the absence of Fgf signaling (Figure 5Q–S’; [Nechiporuk and Raible, 2008]) but the loss of Fgf signaling has no effect on her4 expression in NICD primordia (Figure 5R–T’). We determined that Notch acts downstream of the RAS/MAPK pathway, as the MAPKK inhibitor PD0325901 leads not only to the loss of the Fgf targets pea3 and fgfr1a but also of notch3 and her4 (Figure 5—figure supplement 1C–F’).

To functionally test if Fgf signaling affects organ morphogenesis via the induction of Notch signaling, we crossed Notch-depleted mib1 mutants with a heat-shock line that allows us to constitutively upregulate Fgf signaling (Tg(hs:cafgfr1), Figure 5X–Y’’’). The analysis of the resulting embryos demonstrates that in the absence of Notch signaling, the activation of Fgf signaling is not sufficient to rescue proneuromast formation (Figure 5Y–Y’’). Also, the expression of ZO1, a component of the apically localized tight junctions is also still reduced in mib1;hs:cafgfr1 embryos (Figure 5Y',Y''') confirming that Notch acts downstream of Fgf signaling in rosette formation.

shroom3 is an Fgf-dependent scaffolding protein thought to be required for preneuromast cell apical constriction (Figure 5U, [Ernst et al., 2012]). shroom3 is lost in Fgf-depleted primordia, reduced in NICD primordia, completely lost in Fgf-depleted NICD primordia and upregulated in mib1 primordia (Figure 5U’, V, V’, W). As NICD primordia are forming proneuromasts in the absence of shroom3 (Figure 5V’, arrows), shroom3 is not required for rosette formation in the presence of Notch. We also expressed dominant negative shroom3 in wildtype primordia and did not observe any rosette or lateral line defects (Figure 5—figure supplement 1G–I). Likewise, in contrast to published data (Durdu et al., 2014; Ernst et al., 2012), shroom3 morpholino injections did not delay rosette formation. To investigate if other shroom genes might act redundantly with shroom3 we performed in situ hybridization with shroom1, shroom2a, shroom2b and shroom4 (Figure 5—figure supplement 1J–K''). shroom2b and shroom4 are not expressed in the lateral line. shroom1 and 2a are not expressed in NICD primordia and we therefore conclude that shroom3 does not act redundantly with other shrooms in NICD rosette formation.

Our results show that in wildtype primordia Fgfr activation induces Ras-MAPK signaling, which in turn activates Notch signaling. Notch leads to the apical localization of Rock2a and pMRLC in a shroom3-independent fashion, leading to apical constriction of the cell.

A reduction in Notch leads to loss of cell-cell adhesion and apical constrictions

As Notch is sufficient to induce apical constrictions in the absence of Fgf signaling, we wondered if apical constriction is affected in mib1 mutant primordia compared to wildtype and NICD primordia (Figure 6). Like in wildtype proneuromasts, cells in NICD proneuromasts constrict apically (Figure 6A,B,E). In 36 hpf mib1 mutant primordia cells in younger proneuromast constrict normally (R2), however cells in the most trailing, about to be deposited rosette (R3) lose apical constrictions and no longer constrict apically (Figure 6C–E; Video 4). We measured cell constrictions in the R3 proneuromast by calculating the ratios between the narrowest cell surface area value by the widest area value. Results confirmed that mib1 R3 cells are much less constricted than wildtype or NICD R3 cells (Figure 6E). We also visualized and quantified apical constriction volumes by coloring the constriction volume in magenta using Imaris (Bitplane) (Figure 6F). Subtracting the constriction volumes of R2 from R3 of WT and mib1 proneuromasts reveals that the difference is positive for wildtype proneuromasts, whereas it is negative for mib proneuromasts. Therefore, the constriction volumes decrease in mib1 as they mature and before they are deposited. These observations support the findings by Matsuda et al., that mib1 proneuromasts fall apart (Matsuda and Chitnis, 2010). The disintegration of apical constrictions becomes more apparent between 32–36 hpf, as the loss of Notch signaling in mib1 mutants becomes progressively more severe, likely because of depleted maternal stores (Matsuda and Chitnis, 2010).

Loss of Notch signaling disrupts apical constrictions.

(AE) Analysis of cell shapes using Imaris software. R1–R3 indicate proneuromast numbers. (A) In wildtype and (B) NICD primordia all analyzed proneuromast cells in all rosettes constrict apically. Cells in a mib1ta52b primordium (C) constrict in the more immature rosette (R2) but are lacking constricted apices in the last proneuromast (R3) at 36 hpf. The scale bars equal 10 μm. While the individual cells within one graph are scaled the same with respect to each other, no comparison can be made between different samples. This is due to the limitations of the Imaris scale bar function, rooted in the complexity of displaying 3D data. (D) Still image of an animation of the same primrodium as in (C) demonstrating the shape changes in R3. (E) Quantification of apical constrictions (area). Cells from the most mature proneuromasts (R3) were selected for analysis. Boxplot defines standard error and the ends of the whiskers show standard deviation between different cells. (F) The bar graph shows the apical constriction volume value when the R2 volumes are subtracted from R3 volumes. Constriction volumes gradually diminish in maturing proneuromasts in mib1ta52b primordia, therefore, the last proneuromast (R3) in mib mutants has a significantly smaller constriction volume in comparison to R2, whereas, in the siblings R3 is larger than R2, which results in a positive value.

https://doi.org/10.7554/eLife.21049.014
Video 4
Animation of the cells in the mib1ta52b mutant primordium at 36 hpf (see Figure 6C–D).

Cells constrict less in the trailing proneuromast (R3 in Figure 6C–E). Cell shapes are outlined and colored using Imaris (Bitplane) software.

https://doi.org/10.7554/eLife.21049.015

Notch-positive cells self-organize into rosettes

To test if NICD expressing cells might act as signaling centers that recruit surrounding wildtype cells into a proneuromast, we tested if the phenotype is non cell-autonomous. We transplanted cells (magenta color) from NICD transgenic embryos or wildtype embryos (as a control) into wildtype Tg(cldnb:lynGFP) embryos and compared neuromast size (green color) and cell composition between these two conditions (Figure 7A–B’).

Figure 7 with 2 supplements see all
Notch cell-autonomously induces cell clustering.

(A,B and B’) Wildtype cells transplanted into a wildtype embryo do not cause larger neuromasts. (A’B’) Mosaic, NICD-positive cells that are transplanted into a wildtype embryo are clumping together and form bigger neuromasts. (A’’) Mosaic L3 neuromast from panel (A’) where NICD positive cells cluster together to form an enlarged neuromast. (B) Images at the top of the graph show 2 examples of L1-L4 neuromasts with clone cell numbers measured below. Left: WT cells transplanted into WT lateral line and right: NICD cells transplanted into WT lateral line. Colored dots below indicate three values measured for each neuromast: green- total number of cells in the neuromast, magenta- number of transplanted cells, black- number of host cells (a calculated value of magenta cells subtracted from the number of green cells). The grey line indicates the average neuromast size of all WT neuromasts containing WT clones. (B’) Quantification of cell transplantation experiments. The significant increase in the neuromast size (green bars; 38 versus 45 cells) in NICD>WT experiments compared to WT>WT situation is only due to addition of transplanted NICD cells, since the number of host cells (black bar) is not significantly changed between two types of cell transplantation strategies (26 versus 30 cells). (CCC’’) e-cadherin expression in the primordium is upregulated by Notch (C’) and by Wnt signaling (CC) compared to a wildtype primordium (C). (C’’) e-cadherin is downregulated in mib1ta52b primordia and in mib1ta52b in which Wnt is activated with BIO (CC’) suggesting that Wnt requires Notch signaling to induce e-cadherin. (CC’’) e-cadherin is upregulated in hs:atoh1a primordia in which her4 (Notch target) is also expanded (see Figure 4—figure supplement 1C). (DI’’’) Notch upregulates E-cadherin protein expression in neuromasts (D’), proneuromasts in the primordium (E’’) and in interneuromast (INCs) cells (yellow arrow) (I’’’) compared to a wildtype sibling (D, E’ and I,I’’). In mib1ta52b embryos e-cadherin is downregulated (E’’’). Strongest E-cadherin expression is marked by the yellow arrows in the x,z plane, white arrows in the x,y plane and green arrows in the y,z plane. (FH) NICD causes a significant increase in apical adherens junction (AJ) lengths. (F’ and G’) Magnification of the areas in yellow boxes in F and G. AJs are marked by the white arrows. Error bar indicates standard error (p<0.05=* Student’s t test). (II’’’) E-cadherin expression in the NICD interneuromast cells, which tend to form clusters (arrow), quantified in (K). (J) Transplanted NICD cells contribute in similar proportions to neuromasts and interneuromast cells as transplanted wildtype cells. (K) Transplanted NICD cells form significantly more clusters, as defined by groups of cells that contain two or more cells. (LZ’) Apical junction genes, such as adherens junction genes and tight junction components are upregulated by NICD. (LN’) cd9b is upregulated in NICD neuromasts and the primordium (M and M’) compared to siblings (L and L’), and is downregulated in mib1ta52b embryos (N and N’). (OQ’) cldnB is upregulated in NICD neuromasts and the primordium (P and P) compared to a sibling (O and O’) and slightly reduced in mib1ta52b embryos (Q and Q’). The cldnB signal is especially low in the center of mib1ta52b neuromasts (Q). (RT’) cldnE is upregulated in NICD neuromasts (S) compared to a sibling (R). cldnE is unchanged in mib1ta52b neuromasts (T). (R’, S’ and T’) No change in cldnE expression is seen in wildtype, NICD and mib1ta52b primordia. (UW’) epcam is overexpressed in NICD neuromasts and the primordium (V and V’) compared to siblings (U and U’) but epcam is downregulated in mib1ta52b embryos (W and W’). (XZ’) celsr2 is overexpressed in NICD neuromasts and the primordium (Y and Y’) compared to siblings (X and X’) and mib1ta52b embryos (Z and Z’). All scale bars are 25 μm, unless stated otherwise. Error bars represent standard error (p<0.05=*, p<0.01=**, p<0.001=*** Student’s t test).

https://doi.org/10.7554/eLife.21049.016

We observed that neuromasts only get bigger (green dots) than the average wildtype neuromast (grey line) if they contain NICD clones but not if they contain wildtype clones (Figure 7B). If NICD cells acted as a signaling center enlarged neuromasts should not only contain NICD-positive cells but also possess an above average increase in wildtype cells (black dots). The quantification of all cell transplantation experiments confirmed that the significant increase in neuromast size in NICD>WT experiments (Figure 7B’, green bars) is only due to the excessive incorporation of transplanted NICD cells into proneuromasts, as the number of wildtype host cells (black bars) is not significantly changed between two types of cell transplantation strategies. Therefore, we conclude that wildtype cells are not recruited to contribute to larger neuromasts (Figure 7A–B’; Video 5). Thus, the increase in organ size after Notch activation is cell-autonomous and Notch transducing cells do not act as a signaling center.

Video 5
Time-lapse movie of a migrating Tg(cldnB:lynGFP) wildtype primordium into which magenta-colored NICD cells were transplanted.

The NICD-positive cells contribute to a larger neuromast. The embryo was imaged every 5 min starting at 25 hpf.

https://doi.org/10.7554/eLife.21049.021

Notch signaling upregulates e-cadherin expression

It was suggested that self-organization into rosettes depends on cell adhesion molecules, such as e-cadherin (cdh1) and n-cadherin (cdh2), and a reduction in e-cadherin expression correlates with lateral line adhesion defects in mib1 mutants (Matsuda and Chitnis, 2010). The loss of e-cadherin expression was attributed to the inhibitory effect of the expanded atoh1a domain in mib1 primordia. Correspondingly, we found that in NICD primordia e-cadherin mRNA and E-cadherin protein are highly upregulated, accompanied by an increase in the length of adherens junctions in deposited neuromasts (Figure 7C–C’,D–E’’,F–H).

To determine if e-cadherin is regulated by Atoh1a, Wnt, Fgf or Notch signaling we analyzed e-cadherin expression after different signaling pathway manipulations (Figure 7C–CC’’). Our data does not support the hypothesis that atoh1a negatively regulates e-cadherin and that therefore the upregulation of e-cadherin could be caused by the loss of atoh1a in NICD primordia. Ectopic expression of atoh1a in wildtype primordia causes increased e-cadherin levels and neuromast size (Figure 7CC’’ and Figure 4—figure supplement 1F). Simultaneously, her4 is also induced (Figure 4—figure supplement 1C and F). These data suggest that heat-shock induction of atoh1a leads to the upregulation Notch signaling which induces e-cadherin.

Wnt overexpressing apc mutant primordia also show an increase in e-cadherin mRNA and protein expression suggesting that e-cadherin is regulated by Wnt signaling as in many other cell types (Figure 7CC and Figure 7—figure supplement 1A,B; Heuberger and Birchmeier, 2010). Our data show that Wnt signaling regulates e-cadherin via the induction of Notch signaling, as e-cadherin is reduced in mib1 primordia and the upregulation of Wnt with the GSK3β inhibitor BIO is not sufficient to induce e-cadherin in Notch-deficient primordia (Figure 7C’’ and 7CC’). Likewise, Fgf signaling indirectly upregulates e-cadherin expression via the induction of Notch signaling. Loss of Fgf signaling leads to the loss of e-cadherin expression but if Notch is simultaneously activated, e-cadherin is expressed (Figure 7—figure supplement 1C–F'). n-cadherin, on the other hand, is regulated by Wnt in a Notch-independent fashion, as it is not affected in mib1 or NICD primordia (Matsuda and Chitnis, 2010) but it is upregulated in apc mutants (Figure 7—figure supplement 1G–J). N-cadherin also localizes to apical constrictions (Revenu et al., 2014), however, we did not observe a rosette defect in n-cadherin mutants (pactm101b, cdh2hi3644, data not shown). Together these experiments show that e-cadherin is induced by Notch signaling downstream of Wnt, Fgf and Atoh1a.

To test the function of e-cadherin in rosette formation we transplanted cells from e-cadherin morpholino-injected embryos into wildtype embryos, as e-cadherin mutants die during epiboly (Figure 7—figure supplement 1K–M’’; [Kane et al., 2005]). E-cadherin-deficient cells behave normally in wildtype primordia and neuromasts, even if almost the entire neuromast consists of mutant cells, suggesting that other adhesion molecules act redundantly (Figure 7—figure supplement 1M–M’’; Video 6).

Video 6
Time-lapse movie of a migrating Tg(cldnB:lynGFP) wildtype primordium into which magenta-colored E-cadherin morphant cells were transplanted.

E-cadherin deficient cells integrate into the proneuromast normally and cause no aberrant rosette formation or maintenance phenotype. Embryo was imaged every 5 min starting at 25 hpf.

https://doi.org/10.7554/eLife.21049.022

To determine if cell adhesion is affected at all in the NICD lateral line, we analyzed if transplanted NICD cells sort out from wildtype cells. First we analyzed if transplanted NICD cells preferentially contribute to neuromast or interneuromast cells and observed that NICD and wildtype cells contributed equally to either lateral line cell type, again demonstrating that cell fate is not affected (Figure 7J). However, NICD cells that contribute to interneuromast cells cluster significantly more often than wildtype cells, which are usually aligned in a string (Figure 7I–I’’’, arrow and Figure 7K; Figure 7—figure supplement 1N,O). Almost 76% of NICD cells formed clusters of 2 or more cells, compared to 13% of wildtype cells. Combined with the finding that transplanted NICD cells contribute to wildtype neuromasts in larger clusters we conclude that Notch overexpression changes cell adhesion properties.

Notch signaling upregulates cell-cell adhesion molecules and tight junction genes

To identify genes that are transcriptionally controlled by Notch signaling during proneuromast formation, we isolated primordium cells from dissected tails of 36 hpf Tg(cldnB:lynGFP);Tg(cldnB:gal4) x Tg(UAS:nicd) and sibling Tg(cldnB:lynGFP);Tg(cldnB:gal4) embryos by FACS and performed RNASeq analysis. We identified 187 genes that are upregulated in the lateral line of NICD embryos (Figure 7—source data 1). GO term analysis revealed that enriched cellular components are: ‘apical junction complex’, containing the tight junction components cldnb, cldna, cldne and cingulinb and ‘actin cytoskeleton’ (Figure 7O–P’ and R–S’). Upregulated actin cytoskeleton genes are formin1 (fmn1), myo5c, baiap2b and cingulinb (cgnb) (Figure 7—figure supplement 2, Figure 7—source data 1 and 2). cgnb is a tight-junction associated protein that links tight junctions to the actomyosin cytoskeleton and regulates RhoA signaling (Aijaz et al., 2005; Terry et al., 2011; Van Itallie and Anderson, 2014). Other molecules that interact with this complex are also upregulated, such as rab25a, a Rab11 GTPase family member, as is celsr2, an atypical cadherin (Figure 7X–Y’). Celsr1, if reduced, causes otic defects because of loss of apical constrictions due to disturbed actomyosin recruitment to the apical junctional complex (Sai et al., 2014). RNASeq analysis also revealed the induction of cd9b by Notch, a Tetraspanin family member implicated in cell-matrix adhesion and Sdf1 (Cxcl12a) mediated migration (Figure 7L–M’, [Arnaud et al., 2015; Leung et al., 2011]).

By performing in situ hybridization experiments with a number of candidate adhesion molecules, we identified that epithelial cell adhesion molecule (epcam), which regulates claudin expression and tight junctions is also strongly upregulated in NICD embryos (Wu et al., 2013Figure 7U–V’). Conversely, in situ expression analyses demonstrated that molecules induced by NICD are downregulated in mib1 mutants supporting the finding that Notch is regulating the expression of tight junction and adhesion molecules (Figure 7N–Z’).

To determine if tight junction associated genes are responsible for the large organ phenotype we injected a cldnb morpholino into wildtype embryos, and also tested celsr2rw71 mutants for lateral line phenotypes (Kwong and Perry, 2013; Wada et al., 2006). But like with e-cadherin mutant cells, we did not observe apical junction disassembly phenotypes, likely due to the presence of other claudins and celsr1a/1b in the primordium (data not shown). Likewise, a mutation in the adhesion molecule epcam does not cause a lateral line phenotype (Slanchev et al., 2009). cd9b has been previously knocked down by morpholino injections. Primordium migration is normal but neuromast formation is affected (Gallardo et al., 2010). As morpholino injections can be toxic (Aman et al., 2011), the function of cd9b is currently unclear and has to be reinvestigated.

Our data show that (a) cell adhesion is upregulated in NICD cells, as transplanted cells cluster and (b) cell adhesion molecules act redundantly as their individual downregulation does not cause a phenotype. Given that adhesion and tight junction genes belong to the most highly enriched GO term in NICD primordia, and NICD-positive cells form clusters, we conclude that Notch signaling induces an increase in organ size by activating a combination of adherence and tight junction molecules.

Notch signaling is a component of the Wnt/Fgf signaling network that coordinates lateral line morphogenesis

Collective cell migration and organ morphogenesis are coordinated in the primordium via signaling interactions between Wnt and Fgf signaling (Aman and Piotrowski, 2008; Chitnis et al., 2012). It is therefore important to determine how Notch signaling fits into this gene interaction network. Wnt signaling in the leading primordium region activates Fgf signaling in the trailing region. Both pathways repress the other pathway via the activation of inhibitors in their respective expression domain (dkk1b and sef/dusp6).

Notch signaling is activated by Fgf signaling in the primordium (Figure 5S’ and Figure 9A,B; [Matsuda and Chitnis, 2010; Nechiporuk and Raible, 2008]). Fgf signaling activates the transcription of notch3 in hair cell progenitors and support cells and of atoh1a and delta ligands just in central hair cell progenitors (Figure 9B). Notch signaling, in turn, inhibits atoh1 expression (Baker et al., 1996; Baker and Yu, 1997; Matsuda and Chitnis, 2010; Millimaki et al., 2007). However, if Notch signaling also acts downstream of Wnt signaling has not been investigated.

In apc mutants or in embryos treated with the GSK3β inhibitor BIO, the Fgf pathway genes pea3 and atoh1a and the Notch target her4 are upregulated (Figure 8A–A’, B-B', C-C' and Figure 8—figure supplement 1). As Notch is activated by Fgf signaling we asked if Wnt activates Notch via the activation of Fgf signaling. We treated sibling and apc mutants with the Fgfr1 inhibitor PD173074. The loss of Fgf signaling attenuates pea3, atoh1a and her4 in sibling and apc mutants, especially in the youngest proneuromast (Figure 8D–F’), which demonstrates that Wnt signaling is not sufficient to activate either atoh1a or her4 to wildtype levels in the absence of Fgf (Figure 8E,E' and F,F'). Also, rosette formation still occurs in NICD primordia with depleted Wnt signaling by heat-shock activation of dkk1b demonstrating that Notch signaling acts downstream of Wnt signaling (Figure 8G–H’). In conclusion, Wnt activates Fgf signaling, which in turn activates atoh1a, delta ligands and Notch signaling (Figure 9A,B).

Figure 8 with 1 supplement see all
Wnt induces Notch signaling in the primordium via Fgf.

(A and A’) The expression of the Fgf target pea3 is strongly increased in apcmcr primordia at 36.5 hpf. (B and B’) In apcmcr primordia (B’) atoh1a expression is increased compared to a sibling (B), but it still is restricted to individual cells. (C and C’) her4 expression is slightly expanded in the primordium after Wnt upregulation by apcmcr (C’). (DF’) The Fgf inhibitor PD173074 inhibits the expression of pea3 (D’) and prevents the upregulation of atoh1a (E’). The expression of atoh1a is lost in the first proneuromast, as atoh1a is Fgf dependent. The more trailing central cells express atoh1a because it becomes self-regulatory. The Fgf inhibitor downregulates her4 in sibling (F) and apcmcr mutant primordia (F’). (GH’) Loss of Wnt signaling in the primordium by heat-shock induction of dkk1b does not disrupt proneuromast formation in NICD (H’) compared to heat-shocked sibling primordia (G’) suggesting that Notch acts downstream of Wnt and Fgf in proneuromast formation. (IL’’) Notch inhibits Wnt signaling in the primordium. The expression of Wnt targets lef1 (I’), sef (J’), wnt10a (K’) and dkk1b (L’) expression is downregulated in NICD primordia. Conversely, in mib1ta52b primordia lef1 (I’’), sef (J’’) and wnt10a (K’’) are upregulated. (L’’) dkk1b is downregulated in mib1ta52b primordia, because Fgf signaling is secondarily lost (see text). (MR’) wnt10a expression expands only in the absence of Notch signaling in the primordium. (MN’) In apcmcr primordia wnt10a expands towards the trailing region but is restricted to more central cells (arrows) (N,N’). (O,O’) Downregulation of Notch with the γ-secretase inhibitor DAPT, causes a much more complete expansion of wnt10a in the trailing region of apcmcr primordia, demonstrating that Notch signaling inhibits wnt10a in the primordium. (QR’) wnt10a expression is expanded in the absence of Notch signaling when primordia are treated with Fgf inhibitor (Q,Q’) but is once again restricted to the leading region of the primordium if NICD is activated in the absence of Fgf (R,R’). (S) Treatment of sibling embryos with the GSK3β inhibitor and Wnt activator BIO causes the upregulation of pea3. (S’) BIO treatment of NICD embryos rescues the loss of pea3 in NICD primordia demonstrating that Wnt activates Fgf signaling upstream of Notch and that the loss of Fgf signaling in NICD is secondary to the loss of Wnt signaling. Wnt signaling is lost because Notch negatively feeds back to Wnt downstream of Fgf. All scale bars are 25 μm.

https://doi.org/10.7554/eLife.21049.023
Model of the signaling interactions between Wnt, Fgf and Notch.

(A) Signaling in the leading primordium region. Arrows in red indicate interactions described in this study. Other interactions are described in (Agarwala et al., 2015; Chitnis et al., 2012; Matsuda and Chitnis, 2010). Notch acts downstream of Wnt and Fgf signaling to form proneuromasts in the posterior lateral line via the upregulation of cell adhesion or/and cell apical constriction. Notch signaling also inhibits amotl2a and promotes yap1 transcription, which induces some of the proliferation observed in the primordium. However, yap1 does not affect neuromast size. Notch also negatively feeds back to restrict Wnt signaling, thus being an essential component of the signaling interactions that coordinate migration with organ formation. (B) Signaling in the most nascent proneuromast. Fgf signaling (fgf3/10 and fgfr1) is induced by Wnt ligands in all proneuromast cells where it then induces atoh1a and Notch signaling (Chitnis et al., 2012) . As a result, Notch regulates apical constriction and cell adhesion in all proneuromast cells. (C) Signaling in the trailing proneuromasts. Fgf and Delta ligand expression now depends on Atoh1a and not Wnt anymore in central hair cell precursors (green). Fgf signaling is activated through Fgfr1 in the peripheral cells where it also induces Notch signaling (Matsuda and Chitnis, 2010). Notch signaling activates apical constriction and cell adhesion machinery in the support cells.

https://doi.org/10.7554/eLife.21049.025

To test if Notch signaling negatively feeds back on Wnt signaling as in mature neuromasts (Romero-Carvajal et al., 2015), we assessed the expression of Wnt target genes in NICD and mib1 mutant primordia. In NICD primordia, lef1, wnt10a and sef (an Fgf inhibitor that is regulated by Wnt; (Aman and Piotrowski, 2008)) are reduced, whereas in mib1 the expression domains of these genes expand to the trailing region of the primordium (Figure 8I-K'',P). This finding was surprising as the Fgf target and Wnt inhibitor dkk1b is strongly reduced in NICD primordia and wnt10a should expand. dkk1b can therefore not be responsible for the repression of Wnt signaling in NICD primordia (Figure 8L–L’). A clue was provided by our observation that in apc primordia the Wnt target wnt10a is not expanded uniformly in the trailing region but is mostly upregulated in a group of central cells suggesting that Wnt signaling is inhibited in more peripheral cells (Figure 8N,N', arrows). To test if Notch signaling restricts wnt10a expression in peripheral cells, we treated apc mutant embryos with the γ-secretase and Notch inhibitor DAPT. Indeed, wnt10a is now expressed in the most trailing cells demonstrating that Notch inhibits wnt10a, a feedback interaction not previously described in the primordium (Figure 8O,O'). A Fgf-independent inhibition of Wnt signaling by Notch is also supported by our finding that wnt10a expression is still inhibited in NICD primordia treated with Fgf inhibitor, even though loss of Fgf signaling in wildtype embryos leads to expanded wnt10a expression (Figure 8Q–R'). The inhibition of wnt10a by Notch also explains why wnt10a is restricted to a single central cell in proneuromast of a wildtype primordium (Figure 8M,M'). wnt10a seems to be fluctuating in central cells, as it is not obvious in all primordia imaged (Figure 8K).

In conclusion, as illustrated by the schematic representation of gene expression in forming neuromasts, changes in the wnt10a domain only correlate with the presence or absence of Notch (notch3, Figure 8M’–R’). Thus, the analysis of NICD primordia revealed that Wnt signaling is normally not only inhibited by the Fgf target dkk1b but also independently by Notch signaling.

The fact that Notch signaling negatively feeds back on Wnt signaling also explains why Fgf signaling is reduced in NICD primordia. Fgf signaling in the first forming proneuromast depends on Wnt signaling, whereas the expression of fgf10a in the central cell of formed proneuromasts depends on atoh1a (Aman and Piotrowski, 2008; Matsuda and Chitnis, 2010). Therefore, the loss of Fgf signaling in NICD primordia can be attributed to both the inhibition of Wnt signaling and the inhibition of atoh1a expression by Notch in the trailing region. The conclusion that the loss of Fgf signaling in NICD primordia is mostly caused by Notch-mediated repression of Wnt signaling is confirmed by our finding that Fgf signaling (pea3) can be activated in NICD primordia by activating Wnt signaling with the GSK3β inhibitor BIO (Figure 8S,S'). Combined our pathway analyses revealed that Notch is a component of the Wnt/Fgf signaling interaction network that controls migration with organ morphogenesis.

Discussion

Notch induces larger sensory organs independently of the Hippo pathway or proliferation

Even though overexpression of both Wnt and Notch signaling induce larger neuromasts, they produce this effect by different mechanisms. Wnt signaling upregulates proliferation in deposited neuromasts, whereas Notch signaling induces an increase in organ size as they form within the primordium in a proliferation-independent manner. Accordingly, cell cycle inhibition in NICD embryos does not rescue the neuromast size, in line with our previous study that demonstrated that proliferation does not influence wildtype neuromast size (Aman et al., 2011). Likewise, although inhibition of the Hippo pathway component amotl2a causes an increase in proliferation in the primordium (Agarwala et al., 2015) and yap1 morpholino injections reduce the primordium size, neither manipulation affects the size of the forming sensory organs in wildtype, apc or NICD embryos (Figure 2G–H; [Agarwala et al., 2015]). Thus, primordium size and lateral line organ size are regulated by independent mechanisms. However, in amotl2a mutant embryos on average one more neuromast develops (Agarwala et al., 2015). Thus, primordium size does not affect neuromast size in a wildtype embryo but rather affects neuromast number. yap1 is upregulated and amotl2a is reduced by Notch signaling without affecting the primordium cell number suggesting that yap1 also possesses proliferation-independent functions that need to be further explored (Figures 3H–I, 9A).

Notch signaling upregulates components of the epithelial apical junctional complex

RNASeq and GO term analyses of FACS isolated primordium cells and the analyses of candidate genes revealed that members of the epithelial junctional complex are upregulated in the lateral line of NICD embryos. Epithelial junctional complexes consist of cadherin-based adherens junctions and tight junctions that encompass a large number of transmembrane proteins connected to the actomyosin belt via cytoplasmic scaffolding proteins (González-Mariscal et al., 2003). Because of the enrichment of cell adhesion molecules, such as E-cadherin and because transplanted NICD cells coalesce (Figure 7K, Figure 7—figure supplement 1O), we hypothesize that an increase in cell adhesion is contributing to the increase in organ size. However, transplanted E-cadherin mutant cells do not show a phenotype likely due to redundancy, as the primordium expresses several other adhesion molecules, such as epcam and claudins. Epcam is a calcium-independent, epithelial adhesion molecule and its reduction in mutant zebrafish and cultured cells changes their adhesiveness and induces ectopic, localization of apical junctional complexes (Slanchev et al., 2009; Wu et al., 2013). epcam (tacstd) is required for zebrafish gastrulation movements, and its loss also does not cause a lateral line phenotype suggesting that it acts redundantly with other adhesion molecules in the primordium. A previously reported morpholino-induced lateral line defect was likely caused by morpholino toxicity (Villablanca et al., 2006).

The upregulated Claudin proteins could act redundantly with E-cadherin and Epcam adhesion molecules. Although, Claudins are tight junction molecules and play an important role in controlling permeability of epithelia, they also affect cell adhesion as demonstrated in Xenopus development and cell culture (Brizuela et al., 2001; Kubota et al., 1999). In addition, their loss leads to epithelial to mesenchymal transition and the induction of epithelial characteristics if overexpressed (Bhat et al., 2015). Interestingly, loss of claudin-6 leads to the loss of apical actin accumulation in the developing Xenopus pronephros demonstrating that Claudins regulate a variety of morphogenetic processes (Sun et al., 2015). Thus, it is possible that Claudins are also involved in apical constriction, as has been demonstrated for the tight junction protein ZO-1 (Bhat et al., 2015; Tornavaca et al., 2015). The finding that the knockdown of individual adhesion and tight junction molecules does not cause a phenotype is reminiscent of the collectively migrating Drosophila border cells, which have only been shown to fall apart when JNK signaling is inhibited, which has many downstream targets (Llense and Martín-Blanco, 2008).

Other molecules previously implicated in proneuromast formation also act redundantly. For example, the combined downregulation of lgl1 and lgl2 leads to loss of the leading-most rosettes in the primordium (Hava et al., 2009). Lgl proteins act upstream of Notch signaling, as in the zebrafish retina loss of lgl1 leads to an increase in the apical domain of neuroepithelia, which induces the upregulation of Notch signaling (Clark et al., 2012). Likewise, in the mouse brain lgl1 regulates Notch via the localization of the Notch inhibitor Numb (Klezovitch et al., 2004). In NICD lateral line primordia lgl2 expression is normal (data not shown) suggesting that lgl2 acts upstream of Notch signaling in the lateral line as well. If lgl1/2 are regulated by Fgf signaling has not been determined but lgl1/2 could be a possible link between Fgf and Notch signaling, which needs further exploration.

Shroom3 is not required for apical constriction of lateral line cells in the presence of Notch signaling

Apical constrictions are induced by myosin regulatory light chain activity that is phosphorylated by Rho associated coiled-coil protein kinase (ROCK; [Aguilar-Cuenca et al., 2014; Bresnick, 1999; Sawyer et al., 2010]). In the neural tube of mice and frogs the scaffolding and actin-binding protein shroom3 is important for the apical localization of ROCK and neurulation (Haigo et al., 2003; Hildebrand, 2005; Hildebrand and Soriano, 1999; Nishimura and Takeichi, 2008). In contrast to previously published studies, our experiments did not reveal a function for shroom3 in lateral line rosette formation (Durdu et al., 2014; Ernst et al., 2012). Most importantly, neuromast cells apically constrict normally in the absence of shroom3 in NICD embryos (Figure 5V–V’, arrows). In chicken otocyst invagination also occurs independently of shroom3 providing further evidence that apical constriction is achieved via different mechanisms in different contexts (Martin and Goldstein, 2014; Sai et al., 2014).

Large organ size is not due to cell fate changes in NICD embryos

Notch signaling specifies support cells by inhibiting the proneural gene atoh1a, which is required for the acquisition of a neurogenic cell fate ([Bermingham et al., 1999], Figure 4J,L and Figure 9C). Accordingly, constitutive activation of Notch by overexpression of NICD leads to the loss of atoh1a-positive hair cell progenitors, whereas the loss of Notch signaling in mib1 primordia causes an increase in atoh1a and delta-positive hair cell progenitors (Figure 1L’’–N’’, [Itoh and Chitnis, 2001]). Downregulation of atoh1a in mib1 mutants modestly rescued e-cadherin expression and improved proneuromast cohesion, suggesting that atoh1a inhibits e-cadherin (Matsuda and Chitnis, 2010). A role for neurogenic genes in epithelial morphology was also described in Drosophila, where the loss of these genes causes delamination of cells from epithelia (Hartenstein et al., 1992). Nevertheless, it is unlikely that the NICD phenotype is caused by the loss of the central hair cell precursor and loss of atoh1a expression because: (a) re-expression of Atoh1a in NICD primordia does not rescue neuromast size (Figure 4—figure supplement 1E’’,F) (b) apc and hs:atoh1a primordia show an increase in e-cadherin, even though atoh1a is robustly expressed, arguing that atoh1a does not inhibit e-cadherin expression (Figures 7CC, 8CC’’; Figure 7—figure supplement 1A,B), (c) the loss of atoh1a/b by morpholino injection into wildtype embryos does not affect e-cadherin expression and does not lead to larger neuromasts (Matsuda and Chitnis, 2010; Nechiporuk and Raible, 2008). On the contrary, our data show that ectopic atoh1a causes a significant increase in neuromast size. Interestingly, Atoh1a also upregulates Notch target her4 expression (Figure 4—figure supplement 1B,C,F). This suggests that neuromast size in hs:atoh1a embryos is possibly regulated by Notch signaling acting downstream of Atoh1a dependent Delta ligand expression. During zebrafish inner ear development Atoh1b is required for the initial activation of her4 (Radosevic et al., 2014). Whether Atoh1a regulates Notch in the lateral line still needs to be further investigated.

The above experiments show that, even though NICD primordia loose hair cells, which could lead to signaling changes in neighboring cells, the loss of hair cells is not the cause of organ size increase of NICD proneuromasts.

Signaling in forming and mature proneuromasts

Interestingly, during proneuromast formation and maturation signaling between central hair cell precursors and surrounding support cells changes slightly between the first forming proneuromast and the trailing, more mature proneuromasts (Figure 9B and C; [Matsuda and Chitnis, 2010]). In the first forming proneuromast fgf3/10a is expressed in all cells, whereas in trailing, more mature rosettes these ligands are restricted to a central cell. This restriction is controlled by Notch signaling that inhibits the proneural gene atoh1a in support cells (Figure 9C). In contrast, even though Fgf signaling is active in all cells in the first forming proneuromast, the downstream targets atoh1a and deltaa/b are immediately restricted to a central hair cell precursor. Likewise, wnt10a is only expressed in one central cell. The restriction of atoh1a to a central cell contrasts findings in flies where atoh1 is first broadly expressed, marking a prosensory field and only subsequently becomes restricted during cell specification (Jarman et al., 1995; Millimaki et al., 2007). The mechanisms by which Notch signaling immediately inhibits atoh1a, delta and wnt10a expression in support cells and confines them to a central cell are still unknown. Another difference between signaling in the first forming and trailing proneuromasts has previously been described. Fgf signaling is induced in all cells of the first forming proneuromast by Wnt ligands, possibly wnt10a (Figures 8K, 9A,B). In more trailing proneuromasts, Fgf signaling depends on atoh1a, rather than Wnt signaling (Figure 9C, [Matsuda and Chitnis, 2010]). Likewise, atoh1a expression becomes Fgf-independent and self-regulatory (Matsuda and Chitnis, 2010; Millimaki et al., 2007). Even though, Fgf and Notch signaling are particularly high in support cells because of lateral inhibition, some Fgf and Notch signaling occurs even in central cells, as evidenced by her4 expression (Figures 1J and 5S, not shown in Figure 9C), as well as notch3, albeit at lower levels (Figure 1I and Figure 5—figure supplement 1E). Possibly, this low level of Notch signaling ensures that also central cells maintain their apical constrictions.

Notch cell-autonomously induces apical constriction and rosette formation downstream of Fgf signaling

The expression of Fgf ligands in a central cell in a forming neuromast suggests that this cell acts as a signaling center that organizes neighboring cells into a rosette (Harding and Nechiporuk, 2012; Lecaudey et al., 2008; Nechiporuk and Raible, 2008). Indeed, loss of Fgf signaling causes existing neuromasts to fall apart and a failure of new neuromasts to form (Lecaudey et al., 2008; Lush and Piotrowski, 2014; Nechiporuk and Raible, 2008). Durdu et al., reported that Fgf signaling in trailing proneuromasts depends on Fgf ligands being secreted into a dorsal lumen and that only central cells that are apically in contact with the lumen activate the Fgf target pea3 (Durdu et al., 2014). They concluded that Fgf signaling and/or Fgf-dependent rosette formation is important for cell migration and the periodicity of organ deposition (Durdu et al., 2014; Lecaudey et al., 2008). However, lumina only form in the trailing region of the primordium and are not required for Fgf-mediated formation of the first proneuromast (Figure 9B). Our results demonstrate that Fgf signaling is dispensable for rosette formation in the presence of ubiquitous Notch signaling. This finding begs the question why fgf10a production is restricted to a central cell that limits the number of Fgf-responding and Notch-activating cells. A likely explanation is that restricting Fgf signaling and thereby Notch signaling is important to control organ size.

An interesting question is why in NICD primordia two larger rosettes form, rather than three smaller ones as in their siblings. We hypothesize that the Notch-driven increases in cell adhesion and cortical tension change the morphology of the organ to achieve minimal surface tension (Heisenberg and Bellaïche, 2013). Measurements and manipulations of physical forces are needed to test if physical forces determine organ size control and the number of organs that can self-organize in a defined tissue such as the lateral line primordium.

Summary

Together, our experiments demonstrate that Notch signaling is an essential part of the feedback loop between Wnt and Fgf signaling that coordinates cell migration with sensory cell specification and organ morphogenesis. Importantly, we show that Notch signaling acts cell-autonomously and downstream of Fgf signaling in sensory organ rosette formation via the regulation of cell adhesion and tight junction molecules. Therefore, Notch is involved in neuromast rosette self-organization that is analogous to rosettes and that precedes lumen formation in pre-implantation mouse embryos (Bedzhov and Zernicka-Goetz, 2014). Similarly, rosettes self-organize during C. elegans gastrulation but are also characteristic for some brain tumors (Pohl et al., 2012; Wippold and Perry, 2006). We speculate that Notch signaling might be involved in these other developmental events as well. Therefore, our results do not only inform lateral line biology but also contribute to our understanding of morphogenesis of other organs and tissues and how rosettes form in certain brain tumors.

Materials and methods

Fish maintenance and fish strains

The following fish strains were used

Tg(cldnb:lynGFP)zf106 (Haas and Gilmour, 2006), Tg(Tp1bglob:eGFP)um13 (Parsons et al., 2009), Tg(UAS:myc-Notch1a-intra)kca3 (Scheer and Campos-Ortega, 1999), Tg(hsp70l:Gal4-VP16)VU22 (Shin et al., 2007), apcmcr (Hurlstone et al., 2003), mindbomb, mib1ta52b (Itoh and Chitnis, 2001), Tg(hsp70l:dkk1b-GFP)w32 (Stoick-Cooper et al., 2007), Tg(hsp70l:dnfgfr1-EGFP)pd1 (Lee et al., 2005), Tg(hsp70:ca-fgfr1)pd3 (Marques et al., 2008), Tg(hsp70:atoh1a)x20 (Millimaki et al., 2010), Tg(UASDNshroom3) (created by injecting plasmid: pT2dest(bidirectional UAS)-dsred-shrm3DN(520-874)) received from B. Link (Clark et al., 2012; Kwan et al., 2007), Tg(ubi:Zebrabow) (Pan et al., 2013), p53zdf1, pactm101b, cdh2hi3644 kind gift from Anand Chandrasekhar. To generate the Tg(−8.0cldnB:gal4vp16)psi8, we used the zebrafish Tol2 kit (Kwan et al., 2007). The 8 kb claudinB promoter fragment (Haas and Gilmour, 2006) was cloned into the 5’ entry vector and combined via a Gateway reaction with the Gal4-VP16 middle entry vector of the Tol2 kit. The DNA was injected into one-cell stage zebrafish embryos to generate transgenic fish. To generate the TgBAC(cxcr4b:H2A-GFP)p2 transgenic line, the BAC clone DKEY-169F10 was modified in two ways by recombineering. First, the Tol2 sites and the cryaa:dsRed transgenesis marker were inserted into the BAC backbone (Fuentes et al., 2016). Second, a cassette consisting of H2A-GFP flanked by 791 bp and 1042 bp of homology upstream of cxcr4b exon 2, and downstream of the cxcr4b stop codon, respectively, was inserted to replace the cxcr4b coding sequence in cxcr4b exon 2 (amino acid 6–358, the last amino acid before the stop codon) using seamless galK-mediated recombineering (Warming et al., 2005). This transgene expresses the first five amino acids from cxcr4b exon 1 fused to H2A-GFP from the cxcr4b promoter. The final BAC transgene was characterized by SpeI and EcoRI restriction digestion, sequencing of PCR amplicons of the modified locus, and BAC-end sequencing. The DKEY-169F10 BAC library were obtained from ImaGenes GmbH, Germany. BAC transgenes were purified with the nucleobond BAC 100 kit (Clontech). We co-injected 1 nl of 50–250 ng/ml BAC transgene DNA, and 40 ng/ml Tol2 mRNA into the lifting cell of the zygote of 0- to 20-minute-old embryos. The Tol2 mRNA was transcribed from pCS2FA-transposase (Kwan et al., 2007) using the mMessage Machine SP6 Transcription Kit (Thermo Fisher). At 4 days post fertilization (dpf), the rate of mosaic expression of the fluorescent protein in the lens was scored. Clutches with 50% or more embryos showing mosaic fluorescent protein expression in the lens were raised to adulthood. Stable transgenic larvae were identified by out-crossing adults injected with the cxcr4b:H2A-GFP BAC transgene, and by raising larvae positive for the fluorescent transgenesis marker in the lens at 4 dpf. We screened 100 or more embryos from eight adults injected with the cxcr4b:H2A-GFP BAC transgene for fluorescent transgenesis marker expression in the lens and identified three founder fish. The full names of the transgenic lines identified are TgBAC(cxcr4b:H2A-GFP; cryaa:dsRed) p1 through p3. All experiments were performed according to guidelines established by the Stowers Institute IACUC review board.

In situ hybridization

In situ hybridization procedures were performed as described (Kopinke et al., 2006). The following probes were used: lef1, pea3, sef, axin2, fgf3, fgf10, fgfr1, dkk1b (Aman and Piotrowski, 2008), wnt10a (Lush and Piotrowski, 2014), deltaa, deltab, deltac, deltad (Itoh and Chitnis, 2001), dGFP, notch3, her4.1, atoh1a (Jiang et al., 2014), s100t (Venero Galanternik et al., 2015), shroom3 (Ernst et al., 2012), epcam (probe cb6 from ZIRC), celsr2 (Wada et al., 2006; Siddiqui et al., 2010), cldne kind gift from Ashley Bruce. The following primers were used to clone other probes: yap1 (Fw 5’-CGACTTTCCTTGAAAACGGT-3’ and Rv 5’-AAGGTGTAGTGCTGGGTTCG-3’), stk3 (Fw 5’-GCAGTGCTTCCTTAAACTCCAAAC-3’ and Rv 5’-GCAGGAATCTAGAGTAAGATGCAG-3’), amotl2a (Fw 5’-TGGAGAAGGTGGAAAGGATG-3’ and Rv 5’-GCTGGGCTCTTCTGAATCAC-3’), shroom1 (Fw 5’-CGTCTATGATGGGCAAACCT-3’ and Rv 5’-GGCAGGTCGTATGAGATGGT-3’), shroom2a (Fw 5’-AACAAGCAAACCCAATGGAG-3’ and Rv 5’-CTTTGGAGGCGAGTTGTAGC-3’), cdh1 (Fw 5’-TGGAAGAACAAGGACGCTCT-3’ and Rv 5’-TCTCAGGGACAGATGCAGTG-3’). The following probes were cloned into pPR-T4P vector using AA18 and PR244 primers: cldnb (Fw 5’-CATTACCATCCCGAAACGAAAAAGCATGGCATC-3’ and Rv 5’- CCAATTCTACCCGAGAGGCTGTTTCAAACGTGG-3’), cd9b (Fw 5’-CATTACCATCCCGTTGTGTTCACACACTCGCTG-3’ and Rv 5’- CCAATTCTACCCGACAACAGGACAACCACTCGC-3’).

Genotyping

The following primers were used to identify Tg(hsp70:ca-fgfr1)pd3 embryos by PCR: Fw 5’- GCAGCCTGACAGGACTTTTC-3’ and Rv 5’-GATCCGACAGGTCCTTTTCA-3’.

Transplantation assay

Transplantation assays were performed as previously described (Aman and Piotrowski, 2008). Donor embryos were injected with 3% lysine-fixable biotinylated-dextran AF568 (D22912) (Invitrogen, USA) at the one cell stage. Alternatively, Tg(ubi:Zebrabow) embryos were used as donors in Figure 7.

Morpholinos

All embryos were injected at the one cell stage. The splice-blocking yap1 morpholino (5’-AGCAACATTAACAACTCACTTTAGG-3’) was injected at a concentration of 10 ng/2 nL (Skouloudaki et al., 2009). Translation blocking E-cadherin morpholino (5’-ATCCCACAGTTGTTACACAAGCCAT-3’) was injected at a concentration of 8.4 ng/2 nL (Babb and Marrs, 2004).

Immunohistochemistry and Phalloidin staining

Embryos for Phalloidin staining were fixed for 30 min in 4% paraformaldehyde (PFA), washed 3 times for 5 min with 0.8% TritonX in PBS (PBT) and incubated for 2 hr at room temperature with Phalloidin (Invitrogen, 1:40 in 2% PBT). For immunohistochemistry embryos were fixed in 4%PFA at 4°C for the following antibodies: c-Myc (9E10) Santa Cruz Biotechnology (sc-40) (1:500), Anti-Acetylated Tubulin (T679) Sigma-Aldrich (1:500), mouse anti-ZO1 (Zymed; 61–7300) (1:200). Glyo-Fixx (Thermo Scientific, UK) at 4°C was used as a fixative for these antibodies: Di-pERK1/2 (Diphosphorylated ERK-1 and 2 antibody-mouse (M8159) Sigma-Aldrich) (1:500), Anti – ROCK – 2a (CT), Z – FISH- rabbit (AS-55431) (1:50), Mouse Anti-E-Cadherin (610182) BD-Biosciences (1:500), and Bouin’s fixative (Polysciences) was used for Phospho-Myosin Light Chain 2 (Ser19) Antibody (#3671 Cell Signaling (1:20)).

Electron microscopy

Transmission electron microscopy (TEM)

Samples were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in PBS for 1 hr at room temperature (then stored in 4°C until processing), followed by a secondary fixation in 1% aqueous osmium tetroxide with potassium ferricyanide overnight at 4°C. Samples were then dehydrated in a graded series of ethanol with propylene oxide as a transitional solvent, and infiltrated in Epon. Sections were cut on a Leica UC6 ultramicrotome in the range of 50 nm to 70 nm thickness and post stained with 5% uranyl acetate in 70% methanol and Sato’s lead stain. Sections were viewed in a FEI Technai Spirit BioTWIN TEM and imaged with a Gatan Ultrascan digital camera.

Scanning electron microscopy (SEM)

Samples were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in PBS for 1 hr at room temperature (then stored at 4°C until processing) and treated with tannic acid, osmium, thiocarbohydrazide, and osmium (TOTO) as described in Jongebloed et al. (1999). Samples were then dehydrated in an ethanol dilution series, critical point dried in a Tousimis Samdri 795 CPD, mounted on stubs and coated with gold palladium. Samples were imaged in a Hitachi TM-1000 tabletop SEM.

Heat-Shock treatments

Heat-shock induction was done at various developmental time points, different temperature combinations and time intervals as indicated in the text. After the heat-shock, embryos were allowed to develop at 28.5°C.

BrdU and Pharmacological inhibitors

BrdU incorporation, Hydroxyurea and Aphidicolin treatment (inhibition of proliferation) was performed as described in (Aman et al., 2011). Rat anti-BrdU (Accurate; OBT0030G) was used at a dilution of 1:500. To visualize nuclei embryos were placed into 0.1 ng/mL DAPI (Invitrogen). All chemical inhibitors were diluted in DMSO (final concentration of 1%) to concentrations as indicated in the text. Used drugs were purchased from Tocris, USA: Fgfr1a inhibitors PD173074 and SU5402, MAPK/ERK inhibitor PD0325901, GSK-3β inhibitor BIO and γ-secretase inhibitor DAPT.

Time-lapse imaging

Time-lapse imaging was performed as described (Lush and Piotrowski, 2014).

Image analysis

Cell shape/volume analysis

Individual cells were reconstructed in 3D using Bitplane’s Imaris software. Cells were reconstructed manually by drawing the cell boundary in every single z-slice using Imaris’ surface function. After the cell boundary was drawn in every z-slice, a surface was created, and the cell volume was exported for analysis.

For illustrative purposes, the cell surfaces were exported, oriented with the cell constriction upward, and placed side by side for comparison. Every attempt was made to export a view of the cells at the same scale, and at an angle, which shows the most representative perspective.

Apical constriction analysis (area)

Rosette constrictions for each cell were analyzed using the ratio of two area measurements in 2D. First we measured the area of the bounding box of the smallest discernable constriction. Second we measured the area of the bounding box of the largest part of the cell. We then used the ratio of those two measurements to determine how much the cell is constricting on the apical side with respect to the bulk of its structure.

Apical constriction analysis (volume)

Rosette constrictions were analyzed using the Imaris surface function. Because so much membrane from multiple cells comes together at a single point, constrictions are the brightest feature in the image. A global threshold was set, selecting the brightest portion of the image. Smaller insignificant features were eliminated using a size threshold, leaving only the largest and brightest rosette constrictions.

Cell counts

Cell counting was performed in Fiji software where every DAPI positive nuclei was counted by hand using a Wand tool. An average number of cells between different samples was calculated and plotted with the standard error bars. Additionally, Student’s t tests were performed in Microsoft Excel software to assess the significance between the samples.

Sequencing data analysis

For the RNASeq experiment we cut tails from 36 hpf Tg(cldnb:lynGFP) embryos and isolated primordium and neuromast cells by FACS. Reads were mapped to the Danio rerio genome version danRer10 from the University of California, Santa Cruz (UCSC) using TopHat version 2.0.13, with gene annotations from Ensembl 84. RNASeq analysis and Gene Ontology (GO) Enrichment analysis was performed in the R environment using the bioconductor packages edgeR, topGO, and biomaRt. Reads counted on Ensembl transcripts from UCSC using HTSeq version 0.6.1 were analyzed with edgeR to generate P values to form comparisons between wildtype and NICD embryos. Of the 32,105 genes detected, only 19,643 genes with a sum across all samples of at least three reads per million were considered for the analysis. P values were adjusted for multiple hypothesis testing using the Benjamini–Hochberg method. The fragments per kilobase of transcript per million mapped reads (FPKM) values were generated using Cufflinks version 2.2.1. To define differentially expressed genes in the NICD embryos, genes were selected with the following criteria: log2 ratio >0.5 with p value <0.01 and log2 ratio <−0.5 with p value <0.01, resulting in 187 genes up- and 502 genes downregulated, respectively. The necessary GO IDs were obtained from Ensembl using biomaRt. Of the 19,643 expressed genes, 11,757 were associated with GO terms. Therefore, only 117 of the 187 genes and 323 of the 502 genes were used in the GO analysis.

Data access

The whole-genome sequence data from this study have been submitted to the NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/), accession number GSE86571.

References

  1. 1
  2. 2
  3. 3
  4. 4
  5. 5
  6. 6
  7. 7
  8. 8
  9. 9
  10. 10
  11. 11
  12. 12
  13. 13
  14. 14
  15. 15
  16. 16
  17. 17
  18. 18
  19. 19
  20. 20
  21. 21
  22. 22
  23. 23
  24. 24
  25. 25
  26. 26
  27. 27
  28. 28
  29. 29
    The function of the neurogenic genes during epithelial development in the Drosophila embryo
    1. AY Hartenstein
    2. A Rugendorff
    3. U Tepass
    4. V Hartenstein
    (1992)
    Development 116:1203–1220.
  30. 30
  31. 31
  32. 32
  33. 33
  34. 34
  35. 35
  36. 36
  37. 37
  38. 38
  39. 39
    Role of the proneural gene, atonal, in formation of Drosophila chordotonal organs and photoreceptors
    1. AP Jarman
    2. Y Sun
    3. LY Jan
    4. YN Jan
    (1995)
    Development 121:2019–2030.
  40. 40
  41. 41
  42. 42
  43. 43
  44. 44
  45. 45
  46. 46
  47. 47
  48. 48
  49. 49
  50. 50
  51. 51
  52. 52
  53. 53
  54. 54
  55. 55
  56. 56
  57. 57
  58. 58
  59. 59
  60. 60
  61. 61
  62. 62
  63. 63
  64. 64
  65. 65
  66. 66
  67. 67
  68. 68
  69. 69
  70. 70
  71. 71
  72. 72
    The deltaA gene of zebrafish mediates lateral inhibition of hair cells in the inner ear and is regulated by pax2.1
    1. BB Riley
    2. M Chiang
    3. L Farmer
    4. R Heck
    (1999)
    Development 126:5669–5678.
  73. 73
  74. 74
  75. 75
  76. 76
  77. 77
  78. 78
  79. 79
  80. 80
  81. 81
  82. 82
  83. 83
  84. 84
  85. 85
  86. 86
  87. 87
  88. 88
  89. 89
  90. 90
  91. 91
  92. 92
  93. 93
  94. 94
    Neuropathology for the neuroradiologist: rosettes and pseudorosettes
    1. FJ Wippold
    2. A Perry
    (2006)
    AJNR. American Journal of Neuroradiology 27:488–492.
  95. 95

Decision letter

  1. Tanya T Whitfield
    Reviewing Editor; University of Sheffield, United Kingdom

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Proliferation-independent regulation of organ size by Notch signaling" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Tanya Whitfield as the Reviewing Editor and Marianne Bronner as the Senior Editor. The following individual involved in review of your submission have agreed to reveal their identity: Ajay B. Chitnis (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

This manuscript provides a detailed assessment of the role of Notch signaling in establishing neuromast size in the zebrafish lateral line. Previous studies from the Chitnis lab (Matsuda et al. 2010) described how loss of Notch signaling in mib1 mutants triggers problems in the morphogenesis of the lateral line primordium, including instability of deposited neuromasts and eventual disintegration of the primordium. In contrast to what was shown in this previous study, Kozlovskaja-Gumbriene et al. from the Piotrowski Lab have now shown, using a transgenic line expressing an active form of Notch (NICD), that broad activation of Notch in the primordium formation results in larger and fewer proneuromasts in the migrating primordium. This key observation now reveals a previously unappreciated, more direct role for Notch signaling in determining morphogenesis of epithelial rosettes. While this study confirms many observations made previously by Matsuda et al., the direct comparison of both gain and loss of function effects of Notch signaling allows them to go further by demonstrating the role of Notch signaling in inhibiting Wnt signaling and in acting downstream of FGF signaling to determine morphogenesis of epithelial rosettes.

The study also demonstrates that Notch has a direct role in determining formation and stability of epithelial rosettes, and now provides a clearer explanation for the early phenotype in Mib1 mutants associated with deposition of smaller unstable neuromasts. The study also reveals an important additional role played by Notch ligands expressed in the central Atoh1a expressing cell in both nascent and maturing proneuromasts. In addition to preventing neighboring cells from expressing atoh1a, activation of Notch contributes to formation of stable apical constrictions in neighboring cells. Contrary to previous studies that suggested FGF signaling-dependent shroom3 expression is required for morphogenesis of epithelial rosettes, this study shows that Shroom's role is dispensable.

For the most part, the reviewers found that the authors' conclusions are well supported by the data, and that the study provides an important mechanistic advance for the field. However, the reviewers had a number of comments where additional detail or discussion could help to clarify the findings. Most of these suggestions require relatively minor changes to the text or schematic diagrams. In a few places, additional quantitative analysis would help to support the conclusions. In addition, there were some places where it was felt that the conclusions were over-stated, and should be toned down.

Essential revisions:

1) Ensure descriptions of cell shape are accurate, and distinguish between quantitative and qualitative data (rev. 1).

2) State expected outcomes for the hypotheses that are being tested (rev. 1).

3) Include quantitative data to address the comment about mosaicism from reviewer 1, or tone down the conclusions from this section.

4) Consider changing the title (rev. 2).

5) Add a caveat about the control of expression of Ecad by Atoh1a (rev. 2).

6) Discuss or rebut the suggestion that control of wnt10a expression may be indirect (rev. 2).

7) Clarify the schematic of the 1st proneuromast (rev. 2).

8) Address and clarify the relationship between primordium and neuromast size in the NICD overexpression experiments (rev. 3).

9) State exactly how the cell counts were done, and provide additional data to clarify the relationship between NICD, Yap1 and primordium cell number, if possible (rev. 3).

10) Ensure that the schematics are close representations of the data shown (rev. 3).

11) Make the manuscript shorter and more concise where possible (rev. 3).

Detailed versions of the reviews are appended below for further information.

Reviewer #1:

1) In the subsection “A reduction in Notch leads to loss of cell-cell adhesion and apical constrictions”, Figure 6: The data presented in Figure 6 are difficult to interpret. First, the rows of isolated cells in panels A-C are difficult to relate to their counterparts in the intact images. Cell shapes in L1 appear similar in NICD and mib embryos but are quite distinct from wt, yet are described as normal. Cell constrictions in L2 are said to be lost in mib mutants. This is evident in some cells (e.g. blue and pink) but less so in others. And the pink cell in L1 in NICD also appears to lack an obvious apical constriction. Somehow interpretations in this part of the paper seem highly subjective. Note, Figure 6D is labeled to direct attention to Video 5, whereas the text mentions Video 4. Figure 6E-H marks "constriction volume", but it is not clear what this means – and is this quantitative as the term suggests, or strictly qualitative as the images suggest? In the last sentence it is stated that the phenotypic defects in mib mutants become progressively more severe "because of maternal rescue". It would make more sense to say the phenotype worsens as maternal stores become depleted.

2) In the subsection “Notch-positive cells self-organize into rosettes”, Figure 7: Mosaic analysis is often a powerful means of distinguishing between competing hypotheses, but it is important to clearly articulate expected outcomes that might support one hypothesis over the other. In this case expectations are not discussed, nor is it clear whether data support the "signaling center" hypothesis or the "altered cell adhesion" hypothesis. With either model, one might expect that the number of wt cells recruited into a neuromast might vary depending on the number of transgenic cells present. Yet there was no attempt to correlate neuromast size with the percentage of wt vs. transgenic cells. Moreover, it is concluded that wt cells were "never recruited to make neuromasts larger than they would normally be". The basis for this is unclear. The image in Figure 7B" appears to show a larger than normal neuromast that includes a mix of both wt and NICD cells, in apparent contradiction of the conclusion. Size measurements and cell counts (wt & NICD) are needed to make a convincing argument. Other data in Figure 7 are more convincing. On a technical note, the high mag image in 7G' is inverted horizontally relative to the low mag image in 7G.

Reviewer #2:

While overall the paper is excellent and reveals another example of a context in which Notch has a more general role in promoting an epithelial morphology, there are a few details related to interpretation or emphasis with which I did not agree. These issues are enumerated below.

1) I thought the title of the paper, which emphasizes a role for Notch signaling in organ size formation is misleading. While in the gain of function experiments many more cells can be recruited to become part of an epithelial rosette, as noted above, this gain of function phenotype does not represent that pattern of Notch activation or epithelial rosette formation seen under normal circumstances, where formation of rosettes is beautifully coordinated by a central cell that becomes the source of FGFs and Notch ligands. Instead, I believe the paper provides excellent evidence for a role for Notch signaling in not only preventing neighboring cells from expressing atoh1a but also in determining formation of stable epithelial rosettes by activating Notch in the neighbors. The size of these rosettes may, under physiological conditions, still be determined by FGF signaling-though, now as we have learnt from this study, not only by shroom or MAPK dependent mechanisms as previously described but in addition by Notch signaling. As the authors themselves state, by determining Notch3 expression, FGF signaling may still play a critical role in determining which how many cells are recruited to become part of the rosette by Notch signaling. The gain of function experiments with NICD do, however, show how in the absence of all patterning determined by interactions between Wnt and FGF signaling systems, Notch signaling can independently determine a particular configuration of epithelial rosettes in the primordium.

2) In the subsection “Notch signaling upregulates cell-cell adhesion molecules and tight junction genes”, second paragraph: the authors show that e-cadherin expression is promoted by Notch signaling. While data in this paper supports this conclusion, it remains possible that in addition to being promoted by Notch signaling, its expression is inhibited by Atoh1a as suggested by Matsuda et al., since knock-down of atoh1a leads to some recovery of e-cadherin expression in mib1 mutants.

3) "To test if Notch signaling restricts wnt10a expression we treated apc mutant embryos with the γ-secretase inhibitor DAPT (Figure 9M', N'). Indeed, wnt10a is now expressed in many more trailing cells demonstrating that Notch inhibits wnt10a, a feedback interaction not previously described in the primordium (Figure 8A). This negative interaction also explains why wnt10a is restricted to a single central cell in the first forming proneuromast of a wildtype primordium (Figure 9M)".

While this observation superficially supports the assertion of the authors that Notch signaling restricts Wnt10a expression, I feel what is happening here might be more complicated and only indirectly related to Notch signaling. One problem is that the broad spread of wnt10a expression seen in apc mutants is only seen following 20 hours of exposure to DAPT. Does it spread with a shorter treatment? If not, I suspect that the expansion of wnt10a expression is the indirect effect of something that takes a while to develop. One possibility is that it is related to progressive slow expansion in the number of atoh1a expressing cells. As Atoh1a inhibits expression of the FGF receptor, it would therefore permit Wnt signaling in this cell, which is so close to the source of Wnt signals. This may explain why transiently or in an oscillatory pattern Wnt10a is expressed in a spot just adjacent to the leading cloud of wnt10 expression, where it is expressed under normal circumstances. Before the authors can conclude that the restricted pattern is determined by lateral inhibition they should determine if knock-down of atoh1a prevents the expansion. Alternatively, they can skip the experiment and suggest this alternate hypothesis.

4) The schematic for signaling in the 1st proneuromast (most recently formed?) has a number of problems. Notch3 is not being activated by DeltaA/D but by Ras-Mapk which confuses transcriptional regulation of Notch3 and actual activation by its ligand. If there are details the author feels are not well known at this stage they should consider dropping the schematic of early proneuromast or revise it to make it accurate. I feel what’s most clear from their paper is what is presented for the maturing proneuromast.

Reviewer #3:

This paper studies the role of increasing Notch signalling on lateral line development. How organ size is regulated by balancing cell proliferation and cell differentiation is an important question and the lateral line has been a good experimental model to address these questions. The authors deepen into them, and here they propose that in contrast to Wnt signaling that increases neuromast cell numbers by increasing cell proliferation, Notch regulates organ size independently of cell proliferation and by regulating cell adhesion and rosette number in the primordium. The authors combine several approaches and provide an extensive analysis of Notch effects. The work provides interesting, but not fully novel data and some of the conclusions are precipitated and not supported by their data. The link between the effects on cell adhesion and NMs size is not clear. On a side, there are several functional experiments addressing NICD effects on NMs size. On another set of experiments, the authors address the effects of NICD on rosette formation analyzing the primordium without showing the effect on deposited NMs size. In overall, the paper is very long, difficult to follow and without a clear-cut idea of the Notch role on NMs size.

1) Role of NICD in proliferation

The authors make strong emphasis in demonstrating that Notch overactivation increases neuromast size independent on effects on cell proliferation. The authors claim that the size of NICD NM is independent of primordium size (figure legend, Figure 1—figure supplement 1) and this is not fully supported by their data. When overexpressing NICD early by combining cdnB:gal4 with UAS:NICD, the size of the primordium is bigger (while ganglion smaller) and in this condition, the L1 instead of containing approx 35 cells contains 110. Through development, as pLLP decreases its size, the NM size also decreases, indicating that the size of the primordium has an influence on NM size. If NICD is overexpressed after 25 hpf, the L1 or L2 NM only increases its size from 35 cells to 45-55. The authors suggest that as the size of the primordium in this condition is not bigger (due to the effect of ganglion-primordium fate switch) but the NM size is still bigger, the size of the primordium is not the cause of NM increase. However, in this later condition, the effect over NM size is much milder, indicating that if NICD is overactivated early, this has a very strong effect of first neuromasts deposited. How the authors explain that, if the size of the primordium has no effect on NM size, the early effects are much stronger than in the hs:NICD. It is not clear why all the data is shown in embryos in which NICD has been manipulated early instead of hsp:NICD to overcome possible effects of initial primordium size. The authors should also provide data on the size of the primordium in hs:NICD experiments.

Again, the authors state "whereas the increase in the size of NICD neuromasts is controlled by a proliferation-independent process within the primordium".

The difference in the proliferation status in NICD NMs and apc NMs is clear but the authors also show that the BrdU index at the NICD primordium is higher than controls and similar to apc mutants (Figure 3H, although stated that does not change). Could the increased proliferation at the primordium affect deposited NMs size?

The authors also show that the neuromast size in NICD is independent of yap1, while there is a strong reduction in primordium cell numbers in NICD-yap MO embryos (Figure 2G), the size of the NMs is not significantly reduced in this condition. In this particular experiment the deviation bars are higher, thus this mild reduction might become significant if increasing the number of embryos analysed. This is important because is one of the major conclusions of the paper. I suggest to repeat this experiment and add more embryos. Throughout the paper is not clear, which NMs are counted for cell numbers and at which stage the counting is done.

Figure 1P shows that the relative difference of size of wt and NICD NM is reduced overtime, however in Figure 3E this difference is kept. Please explain incongruence. As part of this, it is stated in the text that NICD neuromasts do not significantly grow in cell number (Figure 3E) but instead the NICD NM size increases over time as shown in this figure. How this happens if cell proliferation decreases so drastically in NM (Figure 3D)?

In the schematic representation of the primordium shown in Figure 4, the NICD primordium is not bigger. However, Figure 6B (primordia shown are at different stages!) shows a much bigger primordium in NICD. In particular, the size of the leading part is bigger (not depicted in the drawing). Again, the changes in size of the primordium and in particular the ratio of leading and trailing cells might be relevant for the neuromast size of NICD embryos. This is very little discussed or addressed.

2) Role of NICD in cell fate specification

This section is confusing. If by cell fate the authors refer to the switch between the 4 cell types defined in the primordium, it is clear that there is not a major cell fate switch. The authors conclude that NICD causes the allocation of support cells into fewer but larger proneuromasts and this is clear. But, as the authors mention later, within the primordium, the first forming proneuromast and more trailing proneuromasts are in different stages of cell fate specification due to different activities of FGF signaling. Since Notch affects FGF and Wnt signaling, it is possible that the proneuromast lost is converted to the fate of the other proneuromasts. It is possible that the disappearance of a proneuromast is due to the cell signaling effects and changes of proneuromast commitment stage and not due to increased recruitment of Rock2a that would be secondary. Please discuss this.

3) Cell adhesion:

The authors claim that the main cause of increased NICD neuromast size is their increased adhesivity, more cells sticking together, but not effects on rosette formation are observed when blocking some cell adhesion molecules. In addition, the conclusion is also taken that interneuromasts cells cluster more together in NICD, but these are cells not related to the focus of the study. The conclusion is strong for the little evidences on the cell adhesion and I would suggest to tune-down the conclusions. As in NICD more cells compose a proneuromast, it is obvious that the E-cadherin staining is going to be larger (Figure 7E). Moreover, in the NICD primordium one of the rosette disappears making the proneuromast larger, but if cell adhesivity is increased, why not more rosettes are formed instead? The authors suggest that increased cell adhesion, affects the morphology of the organ to achieve minimal surface tension. If data from microsurgery experiments to measure of surface tensions in wt and NICD cells are available, this would be an interesting addition, but is not essential.

4) Notch and FGF/wnt signaling

The epistatic data showing that Notch is downstream of Wnt and FGF signaling is clear. In parallel Notch negatively feeds-back in FGF signalling by reducing fgf10a, fgf3 expression (not clear about pea3). On the other hand, NICD reduces the levels of Lef1, sef1, dkk1b and wnt10a at the leading region. The effects on Wnt signalling are quite strong and previous reports have analysed the influence of the disruption of Wnt in proliferation and neuromast deposition. The authors make strong emphasis showing that the effects on NMs size is different that the phenotype caused by apc mutants. However, since NICD modifies the balance of FGF/Wnt signalling, it is not clear that Notch and Wnt are not linked in the regulation of primordium size and differentiation. In NICD embryos, what happens if Wnt signaling is raised by crossing with apc mutants? If this is known, please discuss.

https://doi.org/10.7554/eLife.21049.028

Author response

[…] Reviewer #1:

1) In the subsection “A reduction in Notch leads to loss of cell-cell adhesion and apical constrictions”, Figure 6: The data presented in Figure 6 are difficult to interpret. First, the rows of isolated cells in panels A-C are difficult to relate to their counterparts in the intact images.

We now assigned individual colors for every cell to make it easier to find their positions in the intact primordium. Also, we added an additional panel with colored cells in the proneuromasts and without the cldnB:GFP signal in the background to again make it easier to locate each cell (Figure 6A-C).

Cell shapes in L1 appear similar in NICD and mib embryos but are quite distinct from wt, yet are described as normal. Cell constrictions in L2 are said to be lost in mib mutants. This is evident in some cells (e.g. blue and pink) but less so in others. And the pink cell in L1 in NICD also appears to lack an obvious apical constriction. Somehow interpretations in this part of the paper seem highly subjective.

We agree with the reviewer and have added quantifications of the apical constriction ratios (new Figure 6E). These data show that cells in the last proneuromast in the mib primordium constrict less than WT or NICD cells. We defined constriction as a ratio between the area of the portion of the cell that has the smallest discernable constriction, and the area in the slice that has the largest area. While the individual cells within one graph are scaled equally with respect to each other, no scaling comparison can be made between different samples. This is due to the limitations of the Imaris scale bar function, rooted in the complexity of displaying 3D data.

Note, Figure 6D is labeled to direct attention to Video 5, whereas the text mentions Video 4.

We now have changed the labels to indicate the correct Video 4.

Figure 6E-H marks "constriction volume", but it is not clear what this means – and is this quantitative as the term suggests, or strictly qualitative as the images suggest?

We have now included the quantification for the constriction volume analysis (new Figure 6F). The bar graph shows that the subtraction value of the constriction volumes of R3 (more mature proneuromasts) and R2 (less mature proneuromasts) is positive for WT primordia, whereas it is negative for mib primordia. Therefore, the constriction volumes decrease in mib as proneuromasts mature and before they are deposited.

In the last sentence it is stated that the phenotypic defects in mib mutants become progressively more severe "because of maternal rescue". It would make more sense to say the phenotype worsens as maternal stores become depleted.

We have rephrased the last sentence of this paragraph accordingly.

2) In the subsection “Notch-positive cells self-organize into rosettes”, Figure 7: Mosaic analysis is often a powerful means of distinguishing between competing hypotheses, but it is important to clearly articulate expected outcomes that might support one hypothesis over the other. In this case expectations are not discussed, nor is it clear whether data support the "signaling center" hypothesis or the "altered cell adhesion" hypothesis. With either model, one might expect that the number of wt cells recruited into a neuromast might vary depending on the number of transgenic cells present. Yet there was no attempt to correlate neuromast size with the percentage of wt vs. transgenic cells.

Our new quantification strongly favors the “altered cell adhesion” hypothesis over the “signaling center” hypothesis. Specifically, if NICD cells acted as a signaling center we would find enlarged mosaic neuromasts with just one or few NICD cells in it and with more than a normal number of wildtype cells recruited. However, our new quantification shows that larger mosaic neuromasts are observed only when NICD cells contribute to the mosaic neuromast. Moreover, the number of wildtype host cells remains similar between two transplantation conditions (Figure 7B’). Together these findings strongly support the hypothesis that larger neuromasts are formed due to altered cell adhesion between NICD cells and no wildtype cells are recruited.

Moreover, it is concluded that wt cells were "never recruited to make neuromasts larger than they would normally be". The basis for this is unclear. The image in Figure 7B" appears to show a larger than normal neuromast that includes a mix of both wt and NICD cells, in apparent contradiction of the conclusion. Size measurements and cell counts (wt & NICD) are needed to make a convincing argument. Other data in Figure 7 are more convincing.

See above. The new data verifies that wildtype neuromast size does not exceed the normal number of cells when wildtype cells are transplanted into it (cell number in Figure 7B’ is similar to wildtype neuromast size in Figure 1P, which is around 38 cells). Indeed, an increase in the neuromast size from on average 38 cells to 45 cells is due to the number of NICD cells present in it (Figure 7B’).

On a technical note, the high mag image in 7G' is inverted horizontally relative to the low mag image in 7G.

We would like to thank the reviewer for pointing this out. We now have corrected the inversion in Figure 7G’.

Reviewer #2:

While overall the paper is excellent and reveals another example of a context in which Notch has a more general role in promoting an epithelial morphology, there are a few details related to interpretation or emphasis with which I did not agree. These issues are enumerated below.

1) I thought the title of the paper, which emphasizes a role for Notch signaling in organ size formation is misleading. While in the gain of function experiments many more cells can be recruited to become part of an epithelial rosette, as noted above, this gain of function phenotype does not represent that pattern of Notch activation or epithelial rosette formation seen under normal circumstances, where formation of rosettes is beautifully coordinated by a central cell that becomes the source of FGFs and Notch ligands. Instead, I believe the paper provides excellent evidence for a role for Notch signaling in not only preventing neighboring cells from expressing atoh1a but also in determining formation of stable epithelial rosettes by activating Notch in the neighbors. The size of these rosettes may, under physiological conditions, still be determined by FGF signaling-though, now as we have learnt from this study, not only by shroom or MAPK dependent mechanisms as previously described but in addition by Notch signaling. As the authors themselves state, by determining Notch3 expression, FGF signaling may still play a critical role in determining which how many cells are recruited to become part of the rosette by Notch signaling. The gain of function experiments with NICD do, however, show how in the absence of all patterning determined by interactions between Wnt and FGF signaling systems, Notch signaling can independently determine a particular configuration of epithelial rosettes in the primordium.

The reviewer made a convincing argument that in wildtype embryos FGF signaling is likely still crucial for determining rosette size by regulating the number of cells that turn on Notch signaling. We have therefore changed the title to: ‘Proliferation-independent regulation of organ size by FGF/Notch signaling’.

2) In the subsection “Notch signaling upregulates cell-cell adhesion molecules and tight junction genes”, second paragraph: the authors show that e-cadherin expression is promoted by Notch signaling. While data in this paper supports this conclusion, it remains possible that in addition to being promoted by Notch signaling, its expression is inhibited by Atoh1a as suggested by Matsuda et al., since knock-down of atoh1a leads to some recovery of e-cadherin expression in mib1 mutants.

To test Atoh1a function in E-cadherin regulation we overexpressed Atoh1a in the lateral line by heat-shock, which resulted in elevated e-cadherin expression in the primordium (Figure 7CC’’). Thus, e-cadherin is not inhibited by atoh1a. In addition, atoh1a overexpression leads to increased her4 expression in the primordium, which suggests that Atoh1a positively regulates Notch signaling in the lateral line which then leads to the induction of e-cadherin. We added these findings to the second paragraph of the subsection “Notch signaling upregulates e-cadherin expression”.

3) "To test if Notch signaling restricts wnt10a expression we treated apc mutant embryos with the γ-secretase inhibitor DAPT (Figure 8M',N'). Indeed, wnt10a is now expressed in many more trailing cells demonstrating that Notch inhibits wnt10a, a feedback interaction not previously described in the primordium (Figure 9A). This negative interaction also explains why wnt10a is restricted to a single central cell in the first forming proneuromast of a wildtype primordium (Figure 8M).

While this observation superficially supports the assertion of the authors that Notch signaling restricts Wnt10a expression, I feel what is happening here might be more complicated and only indirectly related to Notch signaling. One problem is that the broad spread of wnt10a expression seen in apc mutants is only seen following 20 hours of exposure to DAPT. Does it spread with a shorter treatment? If not, I suspect that the expansion of wnt10a expression is the indirect effect of something that takes a while to develop. One possibility is that it is related to progressive slow expansion in the number of atoh1a expressing cells. As Atoh1a inhibits expression of the FGF receptor, it would therefore permit Wnt signaling in this cell, which is so close to the source of Wnt signals. This may explain why transiently or in an oscillatory pattern Wnt10a is expressed in a spot just adjacent to the leading cloud of wnt10 expression, where it is expressed under normal circumstances. Before the authors can conclude that the restricted pattern is determined by lateral inhibition they should determine if knock-down of atoh1a prevents the expansion. Alternatively, they can skip the experiment and suggest this alternate hypothesis.

To address the reviewer’s concern that wnt10a might be expanding in DAPT-treated primordia due to a gradual FGF loss and consequent expansion of Wnt (Figure 8M-O’), rather than de-repression by the loss of Notch, we tested if Notch is sufficient to restrict wnt10a in the presence of FGF inhibitor. Our experiment shows that wnt10a expression is restricted to the leading domain in the NICD primordium, even in the presence of FGF inhibitor (Figure 8Q-R’), strongly suggesting that Notch inhibits wnt10a independently ofFGF/Atoh1a. We have now added a schematic representation of the status of wnt10a, pea3, atoh1a and notch3 in primordia in which Wnt, Notch and FGF are manipulated individually or in combination (Figure 8M’-R’). wnt10a only expands in primordia in which Notch is depleted, irrespective of the activation status of FGF (pea3) or atoh1a. We therefore conclude that Notch inhibits Wnt signaling in parallel to these factors. Changes were made in the fourth and fifth paragraphs of the subsection “Notch signaling is a component of the Wnt/Fgf signaling network that coordinates lateral line morphogenesis”.

4) The schematic for signaling in the 1st proneuromast (most recently formed?) has a number of problems. Notch3 is not being activated by DeltaA/D but by Ras-Mapk which confuses transcriptional regulation of Notch3 and actual activation by its ligand. If there are details the author feels are not well known at this stage they should consider dropping the schematic of early proneuromast or revise it to make it accurate. I feel what’s most clear from their paper is what is presented for the maturing proneuromast.

To address reviewer’s comment, we changed several aspects of Figure 9. First, in the Figure 9A we isolated hair cell progenitor cell in green to better illustrate a valid point that Notch is not transcriptionally activated by deltaD/A but rather by lateral-inhibition. Also, to convey the same message we used a dashed line from deltaD/A instead of a solid line towards Notch in the dark blue cell in Figure 9B.Reviewer #3:

This paper studies the role of increasing Notch signalling on lateral line development. How organ size is regulated by balancing cell proliferation and cell differentiation is an important question and the lateral line has been a good experimental model to address these questions. The authors deepen into them, and here they propose that in contrast to Wnt signaling that increases neuromast cell numbers by increasing cell proliferation, Notch regulates organ size independently of cell proliferation and by regulating cell adhesion and rosette number in the primordium. The authors combine several approaches and provide an extensive analysis of Notch effects. The work provides interesting, but not fully novel data and some of the conclusions are precipitated and not supported by their data. The link between the effects on cell adhesion and NMs size is not clear. On a side, there are several functional experiments addressing NICD effects on NMs size. On another set of experiments, the authors address the effects of NICD on rosette formation analyzing the primordium without showing the effect on deposited NMs size. In overall, the paper is very long, difficult to follow and without a clear-cut idea of the Notch role on NMs size.

We have shortened and rephrased some of the sections to make the paper easier to read and to emphasize that our data shows that Notch signaling cell-autonomously upregulates apical junction complex genes which induce apical constriction and cell adhesion. These changes in cell adhesion and apical constriction lead to a coalescence of support cells into larger neuromasts in the absence of proliferation.

1) Role of NICD in proliferation

The authors make strong emphasis in demonstrating that Notch overactivation increases neuromast size independent on effects on cell proliferation. The authors claim that the size of NICD NM is independent of primordium size (figure legend, Figure 1—figure supplement 1) and this is not fully supported by their data. When overexpressing NICD early by combining cdnB:gal4 with UAS:NICD, the size of the primordium is bigger (while ganglion smaller) and in this condition, the L1 instead of containing approx 35 cells contains 110. Through development, as pLLP decreases its size, the NM size also decreases, indicating that the size of the primordium has an influence on NM size. If NICD is overexpressed after 25 hpf, the L1 or L2 NM only increases its size from 35 cells to 45-55. The authors suggest that as the size of the primordium in this condition is not bigger (due to the effect of ganglion-primordium fate switch) but the NM size is still bigger, the size of the primordium is not the cause of NM increase. However, in this later condition, the effect over NM size is much milder, indicating that if NICD is overactivated early, this has a very strong effect of first neuromasts deposited. How the authors explain that, if the size of the primordium has no effect on NM size, the early effects are much stronger than in the hs:NICD.

We would like to thank the reviewer for this thoughtful comment. We agree that the primordium size has an influence on how big neuromasts can get and we are discussing this point now in the text (subsection “Constitutive activation of Notch or Wnt signaling generates larger sensory organs”, last paragraph). However, we show that the primordium size only limits the maximum size that NICD neuromasts can reach but it does not cause the larger neuromast size in NICD embryos. In support of this conclusion, amotl2a mutant embryos possess much larger primordia but the neuromasts do not increase in size (Agarwala et al., 2015). However, the primordium size limits the maximum size of the NICD neuromasts, cldnB:gal4xUAS:NICD neuromasts get smaller as the primordium gets smaller. Importantly, even though NICD primordia eventually reach the same size as wildtype primordia (Figures 1O,10h [32 hpf time point]), they keep on generating larger neuromasts (Figure 1P, L3 and L4).

It is not clear why all the data is shown in embryos in which NICD has been manipulated early instead of hsp:NICD to overcome possible effects of initial primordium size. The authors should also provide data on the size of the primordium in hs:NICD experiments.

The hs:NICD line only became available to us after the majority of experiments had already been performed. As the experiments with the hs:NICD line supported our findings of experiments that we performed with the UAS:NICD line, we did not redo all experiments in the hs:NICD line.

Again, the authors state "whereas the increase in the size of NICD neuromasts is controlled by a proliferation-independent process within the primordium".

The difference in the proliferation status in NICD NMs and apc NMs is clear but the authors also show that the BrdU index at the NICD primordium is higher than controls and similar to apc mutants (Figure 3H, although stated that does not change). Could the increased proliferation at the primordium affect deposited NMs size?

We agree that there is a slight increase in proliferation in NICD primordia in Figure 3H. However, this change is not statistically significant and has no effect on the primordium cell number in Figure 3I. The results of the experiments in which we reduced proliferation using HUA and Aph (Figure 3K, L) demonstrate that, while the inhibition of proliferation reduces the number of primordium cells significantly in NICD and apc primordia (Figure 3L) it does not significantly rescue the NICD neuromast size, which is still significantly higher than wildtype neuromast size (Figure 3K). apc neuromasts, on the other hand, are reduced back to wildtype size in the absence of proliferation. Together these data show that proliferation does not cause the significant increase in neuromast size in NICD neuromasts.

The authors also show that the neuromast size in NICD is independent of yap1, while there is a strong reduction in primordium cell numbers in NICD-yap MO embryos (Figure 2G), the size of the NMs is not significantly reduced in this condition. In this particular experiment the deviation bars are higher, thus this mild reduction might become significant if increasing the number of embryos analysed. This is important because is one of the major conclusions of the paper. I suggest to repeat this experiment and add more embryos.

As it was suggested by the reviewer we repeated NICD+yap1MO experiment and plotted results from a new experiment in the Figure 2F-H (old results were not added to this quantification). Indeed, by analyzing more embryos we see a significant reduction in NICD+yap1MO L1 and about to be deposited L2 sizes as compared to NICD control. Nevertheless, NICD+yap1MO neuromasts are still significantly larger than WT control neuromasts or WT embryos injected with yap1MO (Figure 2F and G). Therefore, even though the NICD+yap1MO primordium size is rescued back to a wildtype size (Figure 2H), NICD neuromasts remain too large and this increase in size is yap1-independent. We believe that the reduction in neuromast size in yap1 morpholino injected embryos is caused by the effect of yap1 loss in the primordium. Changes to the text were made in the last paragraph of the subsection “The increased organ size after Notch and Wnt activation is independent of yap1”.

Throughout the paper is not clear, which NMs are counted for cell numbers and at which stage the counting is done.

We apologize for the oversight and have now added this information to the following figures:

Figure 2F: L2 proneuromast (still inside the primordium) at 30hpf

Figure 2G: L1 neuromast at 30hpf

Figure 3B-E: L1 neuromast (different time-points are indicated in the figure)

Figure 3K: L3 neuromast at 35hpf

Figure 4A-J: L1 neuromast (different time-points are indicated in the figure)

Figure 5—figure supplement 1I: L3 neuromast at 3dpf

Figure 7D, D’, F, G, L-Z: L1 neuromast (different time-points are indicated in the figure).

Figure 1P shows that the relative difference of size of wt and NICD NM is reduced overtime, however in Figure 3E this difference is kept. Please explain incongruence.

Figure 1P shows the sizes of all posterior lateral line neuromasts (L1-5) along the trunk of the embryo at a single time point (2.5dpf), whereas, Figure 3E describes the growth of the L1 neuromast between 32-56hpf. We have clarified the text, subsection “Constitutive activation of Notch or Wnt signaling generates larger sensory organs”, last paragraph and figure legend.

As part of this, it is stated in the text that NICD neuromasts do not significantly grow in cell number (Figure 3E) but instead the NICD NM size increases over time as shown in this figure. How this happens if cell proliferation decreases so drastically in NM (Figure 3D)?

We agree with the reviewer that NICD L1 neuromast showed on average more cells at 51hpf compared to 26hpf in the old graph, but, importantly, the increase was not statistically significant. We repeated the experiment evaluating L1 neuromast growth over-time in WT, NICD and apc embryos. This time we added more embryos to the quantification. Consistent with our previous result NICD L1 neuromasts do not grow significantly between 32hpf and 56hpf. Therefore, this result is consistent with a significant proliferation decrease in NICD L1 at 35hpf in Figure 3D.

In the schematic representation of the primordium shown in Figure 4, the NICD primordium is not bigger. However, Figure 6B (primordia shown are at different stages!) shows a much bigger primordium in NICD. In particular, the size of the leading part is bigger (not depicted in the drawing). Again, the changes in size of the primordium and in particular the ratio of leading and trailing cells might be relevant for the neuromast size of NICD embryos. This is very little discussed or addressed.

The reviewer is correct. At 27 hpf (Figure 6B) NICD primordia are still significantly larger than wildtype primordia (also see Figure 1O). We now generated new data shown in Figure 4 where we describe the NICD primordium cell composition at 26hpf (Figure 4M, N). The mesenchymal domains are similar between WT and NICD primordia at 26hpf, which indeed suggests that the ratio between the amount of leading and trailing cells is changing in the primordium during migration. However, we do not believe that the size of the leading region significantly affects neuromast size, as 26 hpf primordia have a normal cell number in the leading region but deposit larger neuromasts (subsection “The increase in organ size is independent of the role of Notch in cell type specification”, last paragraph).

2) Role of NICD in cell fate specification

This section is confusing. If by cell fate the authors refer to the switch between the 4 cell types defined in the primordium, it is clear that there is not a major cell fate switch.

We added a sentence to clarify this point: “To test if Notch changes the fate of one cell population into another we counted the number of cells in these four populations in wildtype and NICD primordia.”

The authors conclude that NICD causes the allocation of support cells into fewer but larger proneuromasts and this is clear. But, as the authors mention later, within the primordium, the first forming proneuromast and more trailing proneuromasts are in different stages of cell fate specification due to different activities of FGF signaling. Since Notch affects FGF and Wnt signaling, it is possible that the proneuromast lost is converted to the fate of the other proneuromasts. It is possible that the disappearance of a proneuromast is due to the cell signaling effects and changes of proneuromast commitment stage and not due to increased recruitment of Rock2a that would be secondary. Please discuss this.

As mentioned by the reviewer, Notch does not only balance cell fates, but it also inhibits Wnt and thus FGF signaling, which are critically important for primordium patterning. Therefore, the idea that the disruption of primordium patterning by Notch could be transforming the fates of individual proneuromasts into more or less proneuromasts that then aberrantly coalesce is attractive. However, our data shows that irrespective of the activation status of Wnt and FGF in NICD primordia, always larger neuromasts form in the presence of Notch. Specifically, neuromasts are equally well formed when FGF is completely depleted in NICD primordia (FGF inhibitor treatments in Figure 5E-H’) or upregulated (FGF is elevated upon Wnt overexpression in Figure 3M-O). Similarly, the suppression or upregulation of Wnt in NICD embryos does not rescue NICD neuromast size (Figure 9G-H’ and S, S’). In conclusion, in the presence of Notch signaling, the activation status of Wnt or FGF does not influence NICD neuromast size, as Notch acts downstream of these pathways.

3) Cell adhesion:

The authors claim that the main cause of increased NICD neuromast size is their increased adhesivity, more cells sticking together, but not effects on rosette formation are observed when blocking some cell adhesion molecules. In addition, the conclusion is also taken that interneuromasts cells cluster more together in NICD, but these are cells not related to the focus of the study. The conclusion is strong for the little evidences on the cell adhesion and I would suggest to tune-down the conclusions. As in NICD more cells compose a proneuromast, it is obvious that the E-cadherin staining is going to be larger (Figure 7E).

The reviewer makes a valid point, however the fact that the e-cadherin domain in the apical wall of single cells is increased (Figure 7E’’) and adherens junctions are significantly longer in TEM images (Figure 7G), argues that E-cadherin is upregulated by NICD at the single cell level. Also, our transplantation experiments demonstrate that only transplanted NICD cells cause enlarged neuromasts, arguing that they adhere to each other (Figure 7B’). The reviewer is correct that lateral line interneuromast cells are a different cell population. However, cell adhesion properties are often tested in different cell types of the same genotype (see hanging drop adhesion assays or transplantation of wildtype and mutant cells into gastrulating zebrafish embryos to test their migratory and clonal behaviors). We therefore believe that interneuromast cell behavior is a good proxy for how neuromast cells behave.

Moreover, in the NICD primordium one of the rosette disappears making the proneuromast larger, but if cell adhesivity is increased, why not more rosettes are formed instead? The authors suggest that increased cell adhesion, affects the morphology of the organ to achieve minimal surface tension. If data from microsurgery experiments to measure of surface tensions in wt and NICD cells are available, this would be an interesting addition, but is not essential.

We agree but such experiments are unfortunately beyond the scope of this manuscript.

4) Notch and FGF/wnt signaling

The epistatic data showing that Notch is downstream of Wnt and FGF signaling is clear. In parallel Notch negatively feeds-back in FGF signalling by reducing fgf10a, fgf3 expression (not clear about pea3). On the other hand, NICD reduces the levels of Lef1, sef1, dkk1b and wnt10a at the leading region. The effects on Wnt signalling are quite strong and previous reports have analysed the influence of the disruption of Wnt in proliferation and neuromast deposition. The authors make strong emphasis showing that the effects on NMs size is different that the phenotype caused by apc mutants. However, since NICD modifies the balance of FGF/Wnt signalling, it is not clear that Notch and Wnt are not linked in the regulation of primordium size and differentiation. In NICD embryos, what happens if Wnt signaling is raised by crossing with apc mutants? If this is known, please discuss.

This point is related to the question raised in 2) with regard to how the effect of Notch on the other signaling pathways might contribute to the NICD phenotype. However, in NICD Wnt signaling is reduced and the neuromasts get bigger, whereas in apc mutants Wnt signaling is increased and the neuromasts also get bigger. This finding suggests that the effect of Notch on neuromast size is not caused by its inhibitory effect on Wnt signaling. We also added the experiment suggested by the reviewer where we activated Wnt signaling in NICD and WT embryos by soaking the embryos in BIO. In BIO-treated NICD embryos neuromasts become even larger than in untreated NICD embryos (Figure 3M-O). This shows that Wnt and Notch have an additive effect and therefore affect neuromast size by different mechanisms, namely increasing proliferation and adhesion (subsection “Activated Notch leads to proliferation-independent neuromast growth”, last paragraph.

The reviewer is correct that one would expect to affect proliferation in NICD primordia if Wnt signaling is reduced. However, we have previously shown that Wnt and FGF together (Aman et al., 2011) regulate proliferation. Possibly, proliferation is still triggered in NICD primordia, as in NICD primordia both Wnt and FGF are reduced

https://doi.org/10.7554/eLife.21049.029

Article and author information

Author details

  1. Agnė Kozlovskaja-Gumbrienė

    1. Stowers Institute for Medical Research, Kansas City, United States
    Contribution
    AK-G, Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—review and editing
    Competing interests
    The authors declare that no competing interests exist.
  2. Ren Yi

    1. Stowers Institute for Medical Research, Kansas City, United States
    Present address
    1. Center for Genomics and Systems Biology, Department of Biology, New York University, New York, United States
    Contribution
    RY, Investigation
    Competing interests
    The authors declare that no competing interests exist.
  3. Richard Alexander

    1. Stowers Institute for Medical Research, Kansas City, United States
    Contribution
    RA, Software, Investigation, Visualization
    Competing interests
    The authors declare that no competing interests exist.
  4. Andy Aman

    1. Stowers Institute for Medical Research, Kansas City, United States
    Present address
    1. Department of Biology, University of Washington, Seattle, United States
    Contribution
    AA, Investigation
    Competing interests
    The authors declare that no competing interests exist.
  5. Ryan Jiskra

    1. Stowers Institute for Medical Research, Kansas City, United States
    Contribution
    RJ, Resources, Data curation, Formal analysis
    Competing interests
    The authors declare that no competing interests exist.
  6. Danielle Nagelberg

    1. Developmental Genetics Program and Kimmel Center for Stem Cell Biology, Skirball Institute of Biomolecular Medicine, New York University Langone Medical Center, New York, United States
    Contribution
    DN, Resources
    Competing interests
    The authors declare that no competing interests exist.
  7. Holger Knaut

    1. Developmental Genetics Program and Kimmel Center for Stem Cell Biology, Skirball Institute of Biomolecular Medicine, New York University Langone Medical Center, New York, United States
    Contribution
    HK, Resources
    Competing interests
    The authors declare that no competing interests exist.
  8. Melainia McClain

    1. Stowers Institute for Medical Research, Kansas City, United States
    Contribution
    MM, Resources, Investigation
    Competing interests
    The authors declare that no competing interests exist.
  9. Tatjana Piotrowski

    1. Stowers Institute for Medical Research, Kansas City, United States
    Contribution
    TP, Conceptualization, Supervision, Funding acquisition, Investigation, Writing—original draft, Project administration
    For correspondence
    1. pio@stowers.org
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon 0000-0001-8098-2574

Funding

Stowers Institute for Medical Research (Institutional support)

  • Tatjana Piotrowski

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We would like to thank the Piotrowski lab members and Drs. Krumlauf, Gibson, Li and Trainor for stimulating discussions and Dr. Mark Lush and Joaquin Navajas Acedo for critically reading the manuscript. We are thankful to Boris Rubinstein for a help with mathematical analysis. We are grateful to Dr. Ajay Chitnis for mib1ta52b mutant, Dr. Bruce Riley for Tg(hsp70:atoh1a)x20, Dr. Virginie Lecaudey for the shroom3 morpholino and in situ probe, Dr. Brian Link for the UASDNshroom3 plasmid, Dr. Ashley Bruce for the claudinE in situ probe, Dr. Anand Chandrasekhar for the cdh2hi3644 fish line and Xin Gao for help with the RNASeq analysis. We would also like to thank Helena Boldt for excellent technical support. We are also grateful to Mark Miller for help with illustrations and the Stowers Aquatics facility for excellent help with fish husbandry. We are particularly thankful to the Stowers Institute for Medical Research for Funding.

Ethics

Animal experimentation: All experiments were performed according to guidelines established by the Stowers Institute IACUC review board (IACUC protocol 2014-0129)

Reviewing Editor

  1. Tanya T Whitfield, Reviewing Editor, University of Sheffield, United Kingdom

Publication history

  1. Received: September 2, 2016
  2. Accepted: December 23, 2016
  3. Version of Record published: January 13, 2017 (version 1)

Copyright

© 2017, Kozlovskaja-Gumbrienė et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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