1. Developmental Biology

Detailed analysis of chick optic fissure closure reveals Netrin-1 as an essential mediator of epithelial fusion

  1. Holly Hardy
  2. James GD Prendergast
  3. Aara Patel
  4. Sunit Dutta
  5. Violeta Trejo-Reveles
  6. Hannah Kroeger
  7. Andrea R Yung
  8. Lisa V Goodrich
  9. Brian Brooks
  10. Jane C Sowden
  11. Joe Rainger  Is a corresponding author
  1. University of Edinburgh, United Kingdom
  2. UCL Great Ormond Street Institute of Child Health, United Kingdom
  3. National Eye Institute, National Institutes of Health, United States
  4. Harvard Medical School, United States
https://doi.org/10.7554/eLife.43877
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Cite this article
  1. Holly Hardy
  2. James GD Prendergast
  3. Aara Patel
  4. Sunit Dutta
  5. Violeta Trejo-Reveles
  6. Hannah Kroeger
  7. Andrea R Yung
  8. Lisa V Goodrich
  9. Brian Brooks
  10. Jane C Sowden
  11. Joe Rainger
(2019)
Detailed analysis of chick optic fissure closure reveals Netrin-1 as an essential mediator of epithelial fusion
eLife 8:e43877.
https://doi.org/10.7554/eLife.43877
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Abstract

Epithelial fusion underlies many vital organogenic processes during embryogenesis. Disruptions to these cause a significant number of human birth defects, including ocular coloboma. We provide robust spatial-temporal staging and unique anatomical detail of optic fissure closure (OFC) in the embryonic chick, including evidence for roles of apoptosis and epithelial remodelling. We performed complementary transcriptomic profiling and show that Netrin-1 (NTN1) is precisely expressed in the chick fissure margin during fusion but is immediately downregulated after fusion. We further provide a combination of protein localisation and phenotypic evidence in chick, humans, mice and zebrafish that Netrin-1 has an evolutionarily conserved and essential requirement for OFC, and is likely to have an important role in palate fusion. Our data suggest that NTN1 is a strong candidate locus for human coloboma and other multi-system developmental fusion defects, and show that chick OFC is a powerful model for epithelial fusion research.

https://doi.org/10.7554/eLife.43877.001

eLife digest

Our bodies are made of many different groups of cells, which are arranged into tissues that perform specific roles. As tissues form in the embryo they must adopt precise three-dimensional structures, depending on their position in the body. In many cases this involves two edges of tissue fusing together to prevent gaps being present in the final structure.

In individuals with a condition called ocular coloboma some of the tissues in the eyes fail to merge together correctly, leading to wide gaps that can severely affect vision. There are currently no treatments available for ocular coloboma and in over 70% of patients the cause of the defect is not known. Identifying new genes that control how tissues fuse may help researchers to find what causes this condition and multiple other tissue fusion defects, and establish whether these may be preventable in the future.

Much of what is currently known about how tissues fuse has come from studying mice and zebrafish embryos. Although the extensive genetic tools available in these ‘models’ have proved very useful, both offer only a limited time window for observing tissues as they fuse, and the regions involved are very small. Chick embryos, on the other hand, are much larger than mouse or zebrafish embryos and are easier to access from within their eggs. This led Hardy et al. to investigate whether the developing chick eye could be a more useful model for studying the precise details of how tissues merge.

Examining chick embryos revealed that tissues in the base of their eyes fuse between five and eight days after the egg had been fertilised, a comparatively long time compared to existing models. Also, many of the genes that Hardy et al. found switched on in chick eyes as the tissues merged had previously been identified as being essential for tissue fusion in humans. However, several new genes were also shown to be involved in the fusing process. For example, Netrin-1 was important for tissues to fuse in the eyes as well as in other regions of the developing embryo.

These findings demonstrate that the chick eye is an excellent new model system to study how tissues fuse in animals. Furthermore, the genes identified by Hardy et al. may help researchers to identify the genetic causes of ocular coloboma and other tissue fusion defects in humans.

https://doi.org/10.7554/eLife.43877.002

Introduction

Fusion of epithelia is an essential process during normal human development and its dysregulation can result in birth defects affecting the eye, heart, palate, neural tube, and multiple other tissues (Ray and Niswander, 2012). These can be highly disabling and are among the most common human birth defects, with prevalence as high as 1 in 500 (Ray and Niswander, 2012; Morrison et al., 2002; Nikolopoulou et al., 2017). Fusion in multiple embryonic contexts displays both confounding differences and significant common mechanistic overlaps (Ray and Niswander, 2012). Most causative mutations have been identified in genes encoding transcription factors or signalling molecules that regulate the early events that guide initial patterning and outgrowth of epithelial tissues (Ray and Niswander, 2012; Nikolopoulou et al., 2017; Patel and Sowden, 2019; Kohli and Kohli, 2012). However, the true developmental basis of these disorders is more complex and a major challenge remains to fully understand the behaviours of epithelial cells directly involved in the fusion process.

Ocular coloboma (OC) is a structural eye defect that presents as missing tissue or a gap in the iris, ciliary body, choroid, retina and/or optic nerve. It arises from a failure of fusion at the optic fissure (OF; also referred to as the choroid fissure) in the ventral region of the embryonic eye cup early in development (Patel and Sowden, 2019; Onwochei et al., 2000; Gregory-Evans et al., 2004). OC is the most common human congenital eye malformation and is a leading cause of childhood blindness that persists throughout life (Morrison et al., 2002; Williamson and FitzPatrick, 2014). No treatments or preventative measures for coloboma are currently available.

The process of optic fissure closure (OFC) requires the coordinated contributions of various cell types in the fusion environment along the proximal-distal (PD) axis of the ventral eye cup (reviewed in Patel and Sowden, 2019; Onwochei et al., 2000). In all vertebrates studied so far, these include epithelial cells of both the neural retina (NR) and retinal pigmented epithelium (RPE), and periocular mesenchymal (POM) cells of neural crest origin (Patel and Sowden, 2019; O’Rahilly, 1966; Hero, 1990; Hero, 1989; Gestri et al., 2018). As the eye cup grows, the fissure margins come into apposition along the PD axis and POM cells are gradually excluded. Through unknown mechanisms, the basal lamina that surround each opposing margin are either breached or dissolved and epithelial cells from each side intercalate and then subsequently reorganise to form a continuum of NR and RPE, complete with a continuous basal lamina. The function, requirement and behaviour of these epithelial cells in the fusing tissue, and their fates after fusion, are not well understood.

Some limited epidemiological evidence suggests environmental factors may contribute to coloboma incidence (Gregory-Evans et al., 2004; Hornby et al., 2003). However, the disease is largely of genetic origin, with as many 39 monogenic OC-linked loci so far identified in humans and the existence of further candidates is strongly supported by evidence in gene-specific animal models (Patel and Sowden, 2019). Most known mutations cause syndromal coloboma, where the eye defect is associated with multiple systemic defects. A common form of syndromal coloboma is CHARGE syndrome (MIM 214800) for which coloboma, choanal atresia, vestibular (inner-ear) and heart fusion defects are defining phenotypic criteria (Verloes, 2005). Palate fusion defects and orofacial-clefting are common additional features of CHARGE (~20% of cases) and in other monogenic syndromal colobomas (e.g. from deleterious mutations in YAP1, MAB21L1, and TFAP2A [Rainger et al., 2014; Williamson et al., 2014; Lin et al., 1992]), suggestive of common genetic mechanisms and aetiologies, and pleiotropic gene function.

Isolated (i.e. non-syndromal) OC may be associated with microphthalmia (small eye), and the majority of these cases are caused by mutations in a limited number of transcription-factor encoding genes that regulate early eye development (e.g. PAX6, VSX2 and MAF [Patel and Sowden, 2019; Williamson and FitzPatrick, 2014]), implying that abnormal growth of the eye prevents correct OF margin apposition and that fusion defects are a secondary or an indirect phenotype. Indeed, none of these genes have yet been implicated with direct functional roles in epithelial fusion. However, many isolated coloboma cases also exist without microphthalmia, suggesting that in these patients, eye growth occurs normally but the fusion process itself is defective. These OCs are highly genetically heterogeneous and known loci are not recurrent among non-related patients (Rainger et al., 2017). Furthermore, despite large-scale sequencing projects, over 70% of all cases remain without a genetic cause identified (Rainger et al., 2017).

The most effective and informative models for studying OFC so far have been mouse (Mus musculus) and zebrafish (Danio rerio). Both have significant experimental advantages, including powerful genetics and robust genomic data. In particular, live-cell imaging with fluorescent zebrafish embryos has proven to be useful in revealing some intricate cell behaviours at the fissure margin during fusion (Gestri et al., 2018). However, both models are restrictive for in-depth molecular investigations due to their limited temporal windows of fusion progression and the number of cells actively mediating fusion and subsequent epithelial remodelling.

Here, we present accurate staging and anatomical detail of the process of chick OFC. We show the expansive developmental window of fusion, and the sizable fusion seam available for experimentation and analysis. We take advantage of this to perform transcriptional profiling at key discrete stages during fusion and show significant enrichment for known human OFC genes, and reveal multiple genes not previously associated with OFC. Our analyses also identified specific cellular behaviours at the fusion plate and found that apoptosis was a prominent feature during chick OFC. Furthermore, we reveal Netrin-1 as a mediator of OFC that is essential for normal eye development in evolutionarily diverse vertebrates, and that has a specific requirement during fusion in multiple developmental contexts. This study presents the chick as a powerful model system for further OFC research, provides strong evidence for a novel candidate gene for ocular coloboma, and directly links epithelial fusion processes in the eye with those in broader embryonic tissues.

Results

OFC in the chick occurred within a wide spatial and temporal window

The eye is the foremost observable feature in the chick embryo and grows exponentially through development (Figure 1a, Figure 1—figure supplement 1). The optic fissure margin (OFM) was first identifiable as a non-pigmented region at the ventral aspect of the eye that narrowed markedly in a temporal sequence as the eye increased in size (Figure 1a). To gain a clearer overview of gross fissure closure dynamics we first analysed a complete series of resected flat-mounted ventral eye tissue from accurately staged embryos at Hamburger Hamilton stages (HH.St) 25 through to HH.St34 (n > 10 per stage; Figure 1—figure supplement 1). The OFM was positioned along the proximal-distal (P-D) axis of the eye, from the pupillary (or collar) region of the iris to the optic nerve. Progressive narrowing of the OFM was observed between HH.St27 to HH.St31, characterised by the appearance of fused OFM in the midline that separated the non-pigmented iris from the posterior OFM (Figure 1—figure supplement 1). Both these latter regions remained unpigmented throughout development and we found they were associated, respectively, with the development of the optic nerve and the pecten oculi - a homeostasis-mediating structure that extends out into the vitreous from the optic nerve head and is embedded in the proximal OFM (Figure 1—figure supplements 1 and 2) (Wisely et al., 2017). The distal region of the pecten was attached to blood vessels that invade the eye globe through the open region of the iris OFM. This iris region remained open throughout development and well after hatching (Figure 1—figure supplement 2). A recent study reported that the proximal chick OFM closes via the intercalation of incoming astrocytes and the outgoing optic nerve (Bernstein et al., 2018), in a process that does not reflect the epithelial fusion seen during human OFC (e.g. mediated by epithelial cells of the RPE and neural retina) (O’Rahilly, 1966; Bernstein et al., 2018). To assess the utility of the chick as a model for human OFC and epithelial fusion, we therefore focused our study on OFC progression in the distal and medial eye.

Figure 1 with 2 supplements see all
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Analysis of chick optic fissure closure.

(a) Chicken embryos at HH.St25 and HH.St30 illustrated the optic fissure (OF; arrows) as a non-pigmented region in the ventral aspect of the developing eye. (b) Left: Schematic showing orientation of the developing chick optic fissure with respect to the whole embryonic eye. Dorsal-ventral and proximal-distal axes are indicated. This study focused on the medial optic fissure (marked by white hatching) distal to the developing pecten and optic nerve. Right: brightfield and fluorescent confocal microscopy using memGFP cryosections illustrated the open (arrow) and fused seam (arrowhead) regions in chick OFM. The location and planes of the cut sections along the D-P axis are indicated in the accompanying schematic. (c) Brightfield and fluorescent confocal microscopy of memGFP OFM sections unambiguously defined the location of fusion plates (arrowheads, top and middle panels) at all stages throughout OFC, combined with flat-mounted memGFPs. Bottom panel: representative single plane confocal z-stack projection image clearly indicated FP2. (d) Brightfield microscopy of flat-mounted ventral eyes revealed the tissue dynamics during closure and coinciding with location of fusion plates (FPs). At HH.St29 the medial OFM had narrowed markedly along the P-D axis between the iris and the proximal region, with FP1 and FP2 (arrowheads) closely positioned in the distal OF. At HH.St31 the medial OFM had become fully pigmented in the fused seam, and the distance between FP1 and FP2 (arrowheads) had lengthened in the P-D axis. An opening remained in the OFM at the iris region (asterisk). (e) Histogram to illustrate fused seam length at each HH stage (error bars = s.d.). Quantitative data of OFM progression obtained from flat mounts and cryosections are provided in Table 1. (f) Schematic representation of chick OFC progression in the distal and medial retina. 1. Pre-fusion: A fully open OFM is evident in the ventral retina at stages HH.St25-27; 2. Initiation: At HH.St27-28 the first fused region is observed in the distal-medial OFM; 3. Active fusion: fusion extends briefly in the distal direction but then stops in the presumptive iris to leave an open region throughout development. Fusion proceeds markedly proximally with FP2 extending towards the pecten. 4. Complete fusion: Fusion stops proximally when FP2 meets the fused pecten region. The fusion seam is complete with a complete continuum of both NR and RPE layers in the ventral eye. Abbreviations: L, lens; OC, optic cup, OF, optic fissure; ON, optic nerve; FP, fusion plate; HH, Hamburger Hamilton staging; RPE, retinal pigmented epithelia; NR, neural retina; POM, periocular mesenchyme.

https://doi.org/10.7554/eLife.43877.003

Using serial sections from memGFP (Rozbicki et al., 2015) and wild-type embryos, we then unambiguously identified open fissure and fused seam regions of the medial-distal OFM (Figure 1b). The fused seams were defined by epithelial continuum in both the developing retinal pigmented epithelia (RPE) and neural retina (NR) layers. We also identified the fusion plates undergoing active fusion using sections and z-stack confocal microscopy (Figure 1c). Serial sectioning at stages HH.St25-34 provided qualitative data for the identification of fusion plates during the progression of chick OFC (Table 1). We then combined these data with fusion seam length measurements taken from flat mounted fissures to provide a robust quantitative framework of fusion progression (Table 2). In all analyses, we observed no evidence for fusion in the medial or distal OFM at stages before HH.St27 (Figure 1; Figure 1—figure supplement 1; Table 1). Fusion was first initiated between HH.St27-28 as confirmed by the definitive appearance of joined epithelial margins at a single fusion point (FP). By HH.St29, the fused area had expanded to generate a fused seam of 0.56 mm (SD: ± 0.12 mm; Figure 1d–e) with two fusion plates, FP1 and FP2 at the distal and proximal limits, respectively. The position of FP1 became fixed at approximately 0.5 mm (SD: ± 0.04 mm) from the developing pupillary region of the iris in all subsequent developmental stages (Table 2, n = 60 fissures analysed), and the region between FP1 and the iris remained fully open throughout ocular development (Figure 1—figure supplement 2 and Table 1). In contrast, the location of FP2 became progressively more proximal until HH.St34 (Table 2), when FP2 was no longer distinguishable from the pecten (by flat mount or cryosections). This total expansion created a fused epithelial seam of ~1.7 mm at its maximum length (SD: ± 0.07 mm, Figure 1e). In summary, we observed four distinct phases of fusion (Figure 1f): (1) pre-fusion when the entire OFM is open (up to HH.St27); (2) fusion initiation at HH.St27-28 in the medial OFM with the appearance of a single medial FP; (3) active fusion as two FPs separate with the expansion of a fused seam along the P-D axis (HH.St29-33); and (4) complete fusion as the entire OFM is fully fused in the medial OFM (by HH.St34). The process is active between HH.St27-HH.St34 and proceeds over ~66 hr.

Table 1
Qualitative analysis of fusion plates observed per developmental stage by cryosections and H and E.
https://doi.org/10.7554/eLife.43877.006
Fusion plates identified
HH stage1x FP onlyBoth FP1 and FP2N per stage
 25004
 26004
 27103
 28314
 29145
 30044
 31033
 32055
 33123
 34303
Table 2
Quantitative measurements of key features during OFC progression using flat mounted WT and mem-GFP fissures.

Total OFM length includes optic nerve and pecten. * Fused fissures observed were too small to measure accurately (<0.1 mm).

https://doi.org/10.7554/eLife.43877.007
HH stageMean total
OFM length (mm)

± SD		Mean length of fused seam (mm)

± SD
272.200.15--
282.920.33**
293.580.280.560.12
304.380.170.930.09
314.500.251.090.13
324.770.161.150.10
335.310.231.390.10
345.670.161.700.07

Chick OFC was characterised by the breakdown of basement membranes, loss of epithelial morphology and localised apoptosis

By defining fusion progression and the location of the fusion plates during chick OFC, we could then accurately assess the cellular environment within these regions. Immunostaining for the basement membrane (BM) (or basal lamina) marker Laminin-B1 on cryo-sectioned fissure margins (Figure 2a) indicated that fusion occured between cells of the RPE and neural retinal, as observed in human OFC (O’Rahilly, 1966). Fusion between opposing margins was defined by a reduction of Laminin-B1 at the edges of the directly apposed fissures, followed the appearance of a continuum of BM overlying the basal aspect of the neural retina. Periocular mesenchymal cells were removed from between the fissure margins as fusion progressed. Using a histological approach, we then provided evidence that both the RPE and NR directly contribute cells to the fusion plate (Figure 2b). We also observed that within the fusion plates there was marked epithelial remodelling of both cell types, beginning after apposition of the OFM edges. In contrast, at the fused seam we observed NR and RPE cells were realigned into apical-basal orientation and were indistinguishable from regions outside of the OFM, indicating that the fusion process was complete.

Figure 2 with 2 supplements see all
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Basement membrane remodelling, loss of epithelial characteristics and apoptosis are defining features of Chick OFC.

(a) Immunostaining for the basement membrane (BM) component Laminin-B1 and nuclear staining (DAPI) using confocal microscopy illustrated that fusion was preceded by the dissolution of BM (compare arrowheads in boxes) as the fissure margins came into contact at the fusion plate, and that fusion was characterised by the generation of a BM continuum at the basal aspect of the neural retina (arrows). Nuclear staining indicated that cells of the retinal pigmented epithelium (RPE) and neural retina (NR) contributed to the fusion plate and that periocular mesenchymal cells were removed from the region between the apposed margins. Images are from a single OFM and are representative of n ≥ 3 samples. (b) H and E staining on paraffin sections at FP2 showed apposed fissure margins with well organised epithelia in NR and RPE (−40 µm from FP2); subsequent sections at the fusion plate showed loss of epithelial organisation in both cell types (within hatching); at the fused seam (+200 µm from FP2) continuous organised layers were observed in both NR and RPE epithelia. Note that fusion occurred from contributions of both NR and RPE. (c) Immunostaining for the apoptosis marker activated Caspase-3 (A-Casp3) on serial cryo-sectioned OFMs (HH.St30) using confocal microscopy (z-stack projections) indicated that A-Casp3 positive foci (arrows) were enriched in epithelia at the OFM and in the nascently fused seam. The midline OFM, including the fusion points, is indicated by yellow arrowheads in all panels. OFMs were counterstained with DAPI. (d) Quantitation of A-Casp3 foci from serially-sectioned OFMs confirmed significant enrichment at FP2, with a graded reduction in apoptotic cells in both directions away from the fusion plate. Data shown are the mean of all measurements (n = 4); error bars = 95% Confidence intervals. Scale bars = 25 µm in a and c, =20 µm in b.

https://doi.org/10.7554/eLife.43877.008

To determine whether the expanding seam between FP1 and FP2 was a result of active directional fusion (e.g. ‘zippering’), or was driven by localised cell-proliferation within the OFM seam (e.g. pushing forward static fusion plates), we used phospho-Histone-H3A (PH3A) as a marker for S-phase nuclei in mitotic cells and revealed there was no significant enrichment within the fusion seam (Figure 2—figure supplement 1). These results suggested that localised cell-proliferation within the seam was not a major mechanism for seam expansion during chick OFC, and further work is required to elucidate the precise mechanisms that drive seam expansion. We then sought to establish whether axonal ingression was a feature of chick OFC in the distal-medial OFM. Using Neurofilament-145 immunofluorescence, we found a complete absence of axonal processes in open, fusing, and fused regions of the distal-medial chick OFM (Figure 2—figure supplement 2). In contrast, at the same stages we found marked enrichment for axons within the proximal OFM and pecten region, providing further evidence that these regions of the chick optic fissure are distinct (Bernstein et al., 2018).

Programmed-cell death has been previously associated with epithelial fusion in multiple developmental contexts but the exact requirements for this process remain controversial (Ray and Niswander, 2012). Even within the same tissues differences arise between species - for example, apoptotic cells are clearly observed at the mouse fusion plate during OFC (Hero, 1990) but are not routinely found in zebrafish (Gestri et al., 2018). We therefore asked whether apoptosis was a major feature of chick OFC. Using HH.St30 eyes undergoing active fusion, we performed immunofluorescence staining for the pro-apoptotic marker activated Caspase-3. We consistently identified apoptotic foci within RPE and NR at both fusion plates, in the adjacent open fissure margin, and at the nascently fused seam with both cryo-section and whole-mount samples (Figure 2c; Figure 2—figure supplement 2). Foci were not found consistently in other regions of the eye or ventral retina (not shown). By quantifying the number of positive A-Casp-3 foci at FP2, we found that apoptosis was specifically enriched in the active fusion environment but was absent from fused seam >120 µm and from open regions > 250 µm beyond FP2 (Figure 2d), indicating that apoptosis is a specific feature of OFC in the chick eye.

Transcriptional profiling revealed genetic conservation between chick and human OFC

We took advantage of the size and accessibility of the embryonic chick eye to perform transcriptomic profiling with the objectives of: (i) assessing the utility of the chick as a genetic model for human OFC by expression of chick orthologues for known disease genes; and (ii) to identify novel genes that are required for OFC. Using HH.st25-26 eyes (pre-fusion; approx. embryonic day E5), segmental micro-dissection of the embryonic chick eye was first performed to obtain separate OFM, ventral eye, dorsal eye and whole eye samples (Figure 3—figure supplement 1). We took care to not extract tissue from the pecten or optic nerve region of the developing OFM to ensure we obtained transcriptional data for the distal and medial OFM only. Cognate tissues were pooled, RNA was extracted, and region-specific transcriptomes were determined using total RNAseq and analysed to compare mean transcripts per million (TPM) values (Figure 3—source data 1). Pseudoalignment to the Ensembl chicken transcriptome identified 30,265 expressed transcripts across all tissue types. To test whether this approach was sensitive enough to reveal domain-specific expression in the developing chick eye, we compared our RNAseq expression data for a panel of genes with clear regional specific expression from a previous study of mRNA in situ analyses in the early developing chick eye cup (Peters and Cepko, 2002). Markers of the early dorsal retina (Efnb1, Efnb2, Vsx2, Tbx5, Aldh1A1) clustered as dorsal-specific in our RNAseq data, whereas known ventral markers (Crx, Maf1, Pax2, Aldh6 [Ald1a3], Vax1, and Rax1) were strongly expressed in our fissure and ventral transcriptomes (Figure 3—figure supplement 1), which validated this approach to reveal OFC candidate genes.

We then repeated the analysis, collecting OFM, ventral tissue and whole eye and included stages HH.st27-28 (~E6; during initiation) and HH.st28-30 (~E7; during active fusion) as discrete time-points (Figure 3—figure supplement 1). Dorsal tissue was not extracted for these stages. Correlation matrices for total transcriptomes of each sample indicated one of the HH.st25-26 fissure samples as an outlier, but otherwise that there was close correlation between all the other samples (Pearson’s correlation coefficient >0.9; Figure 3—figure supplement 1). Quantitative analyses identified 14,262 upregulated genes and 14,125 downregulated genes in the fissure margin at the three time points (Figure 3a; fissure versus whole eye. False discovery rate (FDR) adjusted p-value<0.05). The largest proportion of these differential expressed genes (DEGs) were observed at HH.st25-27, most likely reflecting the periocular tissue between the fissure margins. Remarkably few DEGs were shared between stages. We used fold change (FC) analysis to identify biologically-relevant differential gene expression (Log2FC ≥1.5 or ≤−1) in the fissure compared to whole eye, we found 1613, 2971 and 1491 DEGs at pre-fusion, initiation, and active fusion, respectively (Figure 3—source data 2). Refining our analysis to identify only those DEGs common across all stages revealed 12 genes with increased expression in the fissure and 26 with decreased expression (Figure 3b; Table 3 ). Of these upregulated fissure-specific genes, causative mutations have previously been identified in orthologues of PAX2, SMOC1, ALDH1A3, and VAX1 in human patients with coloboma or structural eye malformations (Patel and Sowden, 2019; Williamson and FitzPatrick, 2014), and some of these genes, such as PAX2 and inhibitors of BMP expression, induce coloboma phenotypes when overexpressed in the developing ventral chick eye (Gregory-Evans et al., 2004; Sehgal et al., 2008). In addition, targeted manipulations of orthologues of both CHRDL1 and CYP1B1 have recently been shown to cause coloboma phenotypes in Xenopus and zebrafish, respectively (Pfirrmann et al., 2015; Williams et al., 2017). The remaining fissure-specific genes (NTN1, RTN4RL1, TFEC, GALNT6, CLYBL and RGMB) had not been previously associated with OFC defects to the best of our knowledge.

Figure 3 with 1 supplement see all
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Transcriptional profiling in chick optic fissure closure.

(a) Transcriptional profiling using microdissected regions of the developing chick eye at E5 (HH.St25-27; pre-fusion), E6 (HH.St27-28; initiation), and E7 (HH.St28-30; during active fusion) revealed multiple DEGs at each stage. (b) NTN1 was the highest expressing gene of 12 fissure-specific DEGs (fissure vs whole eye) throughout all stages of chick OFC (Log2 FC >1.5; FDR < 0.05). These included the known human coloboma associated genes (indicated by #): SMOC1, PAX2, VAX1 and ALDH6, in addition to the coloboma candidates from other animal studies CHRDL1 and CYP1B1 (indicated by •). (c) Clustering for relative expression levels at active fusion stages (HH.St28-30) revealed three independent clusters (2, 3, and 5) where expression levels trended with Fissure >ventral > whole eye. (d) Analysis of normalised mean expression values (TPM, n = 3 technical replicates; error bars = 1 x standard deviation) from clusters 2, 3, five at HH.St28-30 for the Gene Ontology enriched pathways (p<0.0001; Biological fusion [GO:0022610], and Epithelial fusion [GO:0022610]) revealed significant fissure-specific expression for highly expressed (TPM >100) genes - NTN1, FLRT3, CYP1B1 and COL18A1 - in addition to other potential candidate genes for roles in OFC. NTN1 (TPM >200) was the highest expressed fissure-specific DEG identified during active fusion.

https://doi.org/10.7554/eLife.43877.011
Figure 3—source data 1

Kallisto analysis of RNAseq data from segmentally dissected HH.St25-26/E5 chick eyes.

https://doi.org/10.7554/eLife.43877.013
Figure 3—source data 2

Limma analysis of RNAseq data from segmentally dissected chick eyes at all stages.

https://doi.org/10.7554/eLife.43877.014
Table 3
Fissure-Specific Differentially expressed genes (q < 0.05; LogFC:≥1.5 and ≤−1) at all stages analysed.

Genes with increased expression are depicted in grey.

https://doi.org/10.7554/eLife.43877.015
ENSEMBL IDHGNC IDLogFC: Fissure vs whole
(HH.St25-27)~E5		FDR adjusted P valueLogFC: Fissure vs whole
(HH.St27-28)~E6		FDR adjusted P valueLogFC: Fissure vs whole
(HH.St28-30)~E7		FDR adjusted P value
ENSGALG00000023626NTN13.985.11E-054.348.16E-055.413.06E-07
ENSGALG00000005689PAX23.489.36E-063.412.11E-053.184.14E-06
ENSGALG00000033365ALDH62.971.00E-053.941.75E-043.004.91E-05
ENSGALG00000016875novel gene2.214.57E-053.568.72E-073.552.62E-08
ENSGALG00000009415SMOC13.491.46E-052.363.92E-032.608.93E-05
ENSGALG00000025822CYP1B12.951.11E-052.031.34E-023.021.55E-05
ENSGALG00000021589RTN4RL12.415.79E-032.438.00E-032.694.22E-04
ENSGALG00000040557TFEC1.938.70E-032.355.01E-032.909.86E-04
ENSGALG00000009261VAX11.996.95E-041.963.15E-032.491.55E-05
ENSGALG00000041101GALNT61.783.45E-042.076.97E-042.556.85E-06
ENSGALG00000008072CHRDL11.793.85E-051.861.03E-031.942.49E-05
ENSGALG00000015284RGMB1.891.37E-021.732.43E-021.767.63E-03
ENSGALG00000011413novel gene−1.531.13E-02−1.343.34E-02−1.691.60E-02
ENSGALG00000004270ALDH1A2−1.204.06E-02−1.809.46E-03−1.762.74E-03
ENSGALG00000010801TMEM61−2.078.63E-03−1.493.77E-02−1.935.90E-03
ENSGALG00000003842GHRH−1.334.58E-02−2.601.48E-02−2.625.28E-03
ENSGALG00000012712RBM24−2.579.39E-04−2.001.69E-02−2.352.80E-03
ENSGALG00000012644novel gene−1.854.91E-03−2.581.38E-02−3.189.33E-04
ENSGALG00000003324PRRX1−1.524.65E-02−2.772.21E-02−3.421.32E-03
ENSGALG00000007706FGF8−2.202.94E-03−3.108.72E-04−2.649.86E-04
ENSGALG00000010929SPARCL1−3.163.03E-03−1.774.19E-02−3.176.48E-04
ENSGALG00000034585CP49−3.656.32E-06−1.931.55E-02−2.593.60E-04
ENSGALG00000038848MSX2−2.194.15E-03−3.351.15E-02−2.925.01E-03
ENSGALG00000004279GRIFIN−3.977.94E-04−2.712.55E-02−1.924.89E-02
ENSGALG00000004569UNC5B−1.414.81E-03−4.144.56E-08−3.212.62E-08
ENSGALG00000019802novel gene−2.241.56E-02−3.434.36E-02−3.599.52E-03
ENSGALG00000043175novel gene−3.597.36E-03−2.993.27E-02−2.912.59E-02
ENSGALG00000005613novel gene−2.966.50E-04−2.211.99E-02−4.402.06E-04
ENSGALG00000015015CYTL1−2.433.39E-02−3.134.74E-02−5.145.01E-03
ENSGALG00000004035CRYBA1−5.041.21E-04−2.561.95E-02−3.332.00E-03
ENSGALG00000006189CRYGN−4.666.22E-04−4.251.82E-02−4.979.33E-04
ENSGALG00000012470LYPD6−2.491.20E-02−4.646.97E-04−7.135.09E-06
ENSGALG00000008253TBX5−3.503.48E-04−6.735.98E-04−4.396.02E-05
ENSGALG00000015147ALDH1A1−5.061.46E-05−4.961.22E-04−4.791.55E-05
ENSGALG00000042119MIP−4.472.10E-03−5.433.97E-02−6.153.54E-03
ENSGALG00000005634CRYBA4−5.472.65E-04−4.941.61E-02−7.176.56E-04
ENSGALG00000005630CRYBB1−5.361.72E-04−6.974.56E-03−6.241.37E-04
ENSGALG00000008735BFSP1−6.485.53E-04−6.231.76E-02−8.631.97E-03

Clustering analysis revealed NTN1 as a fusion-specific OFC gene

Clustering for relative expression levels of the RNAseq data at active fusion stages (HH.St28-30) revealed three independent clusters (2, 3, and 5) where expression profiles matched Fissure >ventral > whole eye (Figure 3c). We hypothesised that analysis of these clusters would reveal genes with fusion-specific functions during OFC. Of the three clusters with this profile, ontology analyses showed significant enrichment for sensory organ development and eye development processes (FDR q < 0.001, 10 genes) and for adhesion processes (Figure 3—figure supplement 1; FDR q < 0.05, 25 genes; Biological adhesion [GO:0022610] and cell adhesion [GO:0022610]), of which 17 genes had mean TPM values > 10. Within this group, multiple candidates for roles during OFC fusion were revealed, such as several transmembrane proteins, Integrin-A2, Cadherin-4, Collagen 18A1 and FLRT3 (Figure 3d). However, of these NTN1 was the highest expressed and most fissure-specific (mean TPM values: Fissure = 204; ventral = 35; and whole eye = 4).

Netrin-1 was specifically and dynamically expressed in the fusing OFM

We used RNAscope, colorimetric in situ hybridisation, and immunostaining with NTN1-specific antibodies to determine the precise location of Netrin-1 in the chick eye (Figure 4 and Figure 4—figure supplement 1). We observed highly specific expression in both neuroepithelial retina and RPE cells at the fissure margins during active fusion at HH.St29-30 (Figure 4a). This was consistent at both fusion plates (FP1 and FP2), and in both locations NTN1 expression was markedly reduced in the fused seam compared to expression in the adjacent open margins. Immunofluorescence revealed that, consistent with NTN1 mRNA, NTN1 protein was specifically localised to the basal lamina at the opposing edges of the OFM, and to both RPE and neuroepithelial retina cells in this region (Figure 4b–c, Figure 4—figure supplement 1). To test the significance of our findings to other vertebrates, we first asked whether this localisation was conserved to the human OFM. Immunofluorescence analysis for NTN1 (hNTN1) in human embryonic fissures during fusion stages (Carnegie Stage CS17) displayed remarkable overlap with our observations in chick, with protein signal localised specifically to open and fusion plate regions of OFM at the NR and RPE (Figure 4d), and an absence of hNTN1 in fused seam. Consistent with the protein localisation, RNAseq analysis on laser-captured human fissure tissue showed a 32x fold increase in hNTN1 expression compared to dorsal eye (Patel and Sowden; manuscript in preparation). Microarray analyses had previously observed enrichment for Ntn1 in the mouse fissure during closure stages (Brown et al., 2009), so we then analysed Ntn1 protein localisation in equivalent tissues in the mouse optic fissure (fusion occurs around embryonic day E11.5 and is mostly complete by E12.5 (Hero, 1990). We observed consistency in both cell-type and positional localisation of Ntn1 protein (Figure 4e), and that Ntn1 protein was not detected in the fused seam at E12.5 (immunoreactivity for NTN1 was observed in the proximal optic nerve region at this stage; Figure 4—figure supplement 2).

Figure 4 with 4 supplements see all
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A conserved fusion-specific requirement for NTN1 in OFC and palate development.

(a) RNAscope analysis of NTN1 mRNA (green, and grey in insets) in HH.St29 OFMs revealed fissure-specific NTN1 expression (arrows) with strongest signal observed at open regions and in the fusion plate, and reduced expression in the adjacently fused seam. NTN1 expression was localised to cells of both the NR and RPE. Fusion progression was indicated using anti-laminin co-immunofluorescence (magenta). Images shown are maximum intensity projections of confocal Z-stacks. (b) Single-plane confocal images of immunofluorescence analysis for NTN1 on flat-mounted distal (FP1) and proximal (FP2) OFM revealed enriched protein localisation at the edges of the open fissure margins and reduced in the fused seam. (c) Immunostaining on cryosectioned OFM at the open and fusion plate at HH.St29 revealed NTN1 was specifically localised to the basal lamina (arrowheads) and to the epithelia of the neural retina and RPE (arrows) at the OFM. (d) Immunostaining on CS17 human foetal eye sections revealed human Netrin-1 (hNTN1) was localised to NR epithelia (arrows) and at the overlying basal lamina (dented arrowheads) at the fissure margins. hNTN1 was absent from the fused seam epithelia. (e) Immunostaining for mouse Netrin-1 (mNtn1) in during active fusion stages (E11.5) showed mNtn1 was localised at the open fissure margins (arrow) in the basal lamina and to cells at the NR-RPE junction. mNtn1 was absent from this region in fused OFM seam at E12.5. (f) Ntn1-/- mice exhibited highly penetrant (~90%) bilateral coloboma (arrows; n = 10/11 homozygous E15.5-E16.5 animals analysed). (g) Cleft secondary palate (arrows) was observed in ~36% of Ntn1-/- embryos at E15.5-E16.5 (4/11 homozygous animals).

https://doi.org/10.7554/eLife.43877.016

Complete loss of netrin caused coloboma and multisystem fusion defects in vertebrates

Our results suggested that Netrin-1 has an evolutionarily conserved role in OFC and prompted us to test if NTN1 is essential for this process. We therefore analysed mouse embryos of WT and Netrin-null (Ntn1-/-; Yung et al., 2015) littermates at embryonic stages after OFC completion (E15.5-E16.5) (Hero, 1990) and observed highly penetrant ocular coloboma in Ntn1-/- mutants (>90%; n = 10/11; Figure 4f). Mutant eyes analysed at earlier stages of eye development (E11.5) when fusion is first initiated (Hero, 1990) were normal (n = 4 Ntn1-/- embryos; 8x eyes analysed in total), with fissure margins positioned directly in appositional contact each other (Figure 4—figure supplement 2). We also observed variably penetrant orofacial and palate fusion defects in mutant mice (Figure 4g;~36%; n = 4/11 Ntn1-/- embryos), indicating that NTN1 may also have an important role in fusion during palatogenesis and craniofacial development.

Finally, we then tested whether Netrin deficiency would cause similar ocular defects in other vertebrates and generated germline netrin-1 mutant zebrafish by creating a nonsense mutation in the first exon of ntn1a using CRISPR/Cas9 gene editing (Figure 4—figure supplement 3). We inter-crossed heterozygote G0 fish (ntn1a+/-) and observed several G1 embryos displaying bilateral ocular defects including coloboma and microphthalmia (Figure 4—figure supplement 3). DNA sequencing of the targeted ntn1a locus confirmed 100% (n = 3) of the phenotypic embryos were homozygous, whereas ocular defects or colobomas were not observed in any heterozygous (n = 6) or wild-type (n = 12) embryos. A recent study applied morpholino (MO) translation-blocking knockdown approaches to target ntn1a in zebrafish embryos and observed bilateral ocular colobomas in all fish injectected (Richardson et al., 2019), with normal early eye development and appropriately apposed fissure margins obvious prior to fusion. We were also able induce colobomas using MOs designed to target the translational start site of ntn1a (Figure 4—figure supplement 3). Bilateral colobomas were observed in 31/71 (43.7%) of MO injected embryos with no ocular phenotypes observed in control injections (n = 40). In combination, these results are in agreement with our data presented in chicken and mouse OFMs and that Netrin-1 is also essential for zebrafish eye development and is likely to have a specific role in tissue fusion. It also confirms an evolutionarily essential requirement for Netrin in ocular development, including OFC, in diverse vertebrate species.

Discussion

NTN1 is a strong candidate gene for coloboma and multisystem fusion defects

Our study provides strong evidence that Netrin-1 is essential for OFC in the developing vertebrate eye and is required for normal orofacial development and palate fusion. The transient and specific NTN1 expression at the fusion plate, and the subsequent reduction/loss in fused OFM, suggests NTN1 has a direct role in the fusion process. Indeed, Netrin1-deficient mouse eyes displayed highly penetrant colobomas but their fissure margins were normally apposed during fusion initiation, arguing against a broad failure of early eye development. In further support for a direct role in epithelial fusion was previously published work showing fusion failure during development of the vestibular system of both chick and mice where NTN1-expression was manipulated (Yung et al., 2015; Salminen et al., 2000; Nishitani et al., 2017). In this developmental context, otic epithelia must fuse normally for the correct formation of the semicircular canal structures. Although we and others (Richardson et al., 2019) found coloboma in zebrafish knockdown experiments of ntn1a, we observed coloboma with microphthalmia in the context of complete knockout of ntn1a. This more severe phenotype in the complete absence of ntn1a implies there could be a more general requirement for Netrin-1 during early eye development, or could reflect teleost-specific eye developmental processes not shared among higher vertebrates (Martinez-Morales et al., 2017). Further work is required to elucidate the precise role of Netrin-1 during OFC and broader eye development among different species.

Taken in combination, these findings strongly implicate NTN1 as a multipotent factor required for tissue fusion in multiple distinct developmental contexts. In humans, variants near NTN1 have been associated with cleft lip in human genome wide association studies (Leslie et al., 2016; Leslie et al., 2015). While these are not monogenic disease mutations, this observation adds additional further relevance for future genetic studies of patients with coloboma. It is also consistent with our observations in Netrin-1 knock-out animals having a high penetrance of both coloboma and cleft palate phenotypes. Therefore, we propose that NTN1 should be included as a candidate gene in diagnostic sequencing of patients with human ocular coloboma, and should also be carefully considered for those with other congenital malformations involving defective fusion.

NTN1 may have a role in CHARGE syndrome

Coloboma in association with additional fusion defects of the inner ear are two of the key clinical classifications for a diagnosis of CHARGE syndrome (Verloes, 2005). Further phenotypes commonly associated with the syndrome are septal heart defects and orofacial clefting, both with aetiologies likely to involve fusion defects (Ray and Niswander, 2012). CHARGE syndrome cases are predominantly caused by heterozygous loss-of-function pathogenic variants in the chromodomain helicase DNA-binding protein 7 (CHD7) gene (Vissers et al., 2004). Mice lacking Chd7 display CHARGE syndrome-like phenotypes and exhibit abnormal expression of Ntn1 (Hurd et al., 2007; Hurd et al., 2012). In addition, ChIP-seq analyses have shown direct binding of Chd7 to the promoter region of Ntn1 in mouse neural stem cells (Engelen et al., 2011). Given the amount of tissue available in the chick model, it would be possible and intriguing to confirm whether CHD7 directly regulates NTN1 expression in ovo in the chick optic fissure. There is also emerging evidence that CHD7 and the vitamin A derivative retinoic acid (RA) indirectly interact at the genetic level during inner ear development (Yao et al., 2018). Defective RA signalling also leads to significant reduction of Ntn1 expression in the zebrafish OFM (Lupo et al., 2011), implicating a possible genetic network involving RA and CHD7, where NTN1 could directly mediate developmental fusion mechanisms from these hierarchical influences.

How does Netrin-1 mediate fusion?

Netrin-1 is well-studied for its canonical roles in guidance of commissural and peripheral motor axons and growth-cone dynamics, with attraction or repulsion mediated depending on the co-expression of specific receptors (reviewed in Lai Wing Sun et al., 2011; Larrieu-Lahargue et al., 2012). We found that axonal processes were absent from the chick fissure margin during fusion stages, suggesting that the normal function of NTN1 may be to prevent axon ingression into the OFM to permit fusion. However, the phenotypic evidence from both the palate and vestibular system strongly support the argument that NTN1 has a non-guidance mechanistic role during OFC. Netrin orthologues have been recently associated with the regulation of cell migration and epithelial plasticity in the apparent absence of co-localised canonical Netrin-1 receptors (Manhire-Heath et al., 2013; Lee et al., 2014; Yan et al., 2014). In contrast, netrin acting together with its receptor neogenin combined to mediate close adhesion of cell layers in the developing terminal end buds during lung branching morphogenesis (Srinivasan et al., 2003). Although we observed strong NTN1 expression in cells lining the chick OFM, and similar localisation of Netrin-1 protein in chick, human and mouse, we did not observe reciprocal expression of any canonical NTN1 receptors in our RNAseq datasets (e.g. UNC5, DCC or Neogenin; Figure 4—figure supplement 4). Indeed, the Netrin repulsive cue UNC5B was the most significantly downregulated DEG in fissure versus whole eye in our data and was also downregulated in human OFM (Sowden and Patel; manuscript in preparation). Therefore, it will be vitally important for future studies to elucidate interaction partners of Netrin in fusing tissues, or to reveal if Nerin-1 can act autonomously in these contexts and to provide deeper insight into its mechanistic function during fusion.

The chick is a powerful model for OFC

The chick is one of the earliest established models for developmental biology and has provided many key insights into human developmental processes (Stern, 2018). Despite this, and extensive historical study of eye development in chicken embryos, the process of chick OFC has not been well analysed until now. Indeed, the first study appeared only recently and specifically defined aspects of tissue fusion at the proximal (optic nerve and pecten) region of the OF (Bernstein et al., 2018), and did not observe complete fusion of epithelia in these regions. Indeed, closure of the proximal OF was characterised by intercalation of pecten and the lack of true epithelial continuum of neuroepithelial retina and RPE. By focusing on the epithelial fusion events in the distal and medial eye, our study complements the Bernstein et al study (Bernstein et al., 2018) to provide a comprehensive framework of OFC progression in the chick. Indeed, taken together, our analyses clearly define three distinct and separate anatomical regions in the developing chick OFM: the iris, the medial OFM, and the pecten. In addition, we present the spatial and temporal sequence of chick OFC at the anatomical and molecular level, and provide strict criteria for staging the process - based on a combination of broad embryonic anatomy, ocular, and fissure-specific features. Fusion initiated at the medial OFM at HH.St27/28 and continued until HH.St34, with predominantly distal to proximal directionality. In addition, we found that closure of the medial OFM is a true epithelial fusion process that occurs over a large time window of approximately 60 hr, involving two fusion plates, and that closes over 1.5 mm of complete fusion seam. This temporal window, the number of directly contributing cells, and the accurate staging of its progression allows unique opportunities for further experimentation. Importantly, one whole chick optic fissure (from HH.St29 onwards) can simultaneously provide data for unfused, fusing, and post-fused contexts.

In addition, our transcriptional profiling, including the identification of OFM-specific genes in the chick that include multiple human coloboma orthologues, builds on previous work that illustrate the chick as an excellent model for human eye development and the basis of embryonic malformations (Wisely et al., 2017; Vergara and Canto-Soler, 2012; Trejo-Reveles et al., 2018). These features, in combination with recent advances in chick transgenics and genetic manipulations (Davey et al., 2018), project the chick as a powerful to analyse cell behaviours during OFC and epithelial fusion. For example, the stable multi-fluorescent Cre-inducible lineage tracing line (the Chameleon chicken [Davey et al., 2018]) will be valuable to determine how the fissure-lining cells contribute to the fusing epithelia, while the very-recent development of introducing gene-targeted or gene-edited primordial germ cells into sterile hosts for germ-line transmission (Taylor et al., 2017) provides a rapid and cost-effective way to develop stable genetic lines to interrogate specific gene function (Davey et al., 2018; Woodcock et al., 2017). Thus, our study illustrates the powerful utility of the chick as a model for investigating OFC and for the discovery of novel candidate genes for coloboma, and is perfectly timed to coincide with major new developmental biology techniques in avian systems to place the chick model as a powerful addition to OFC and fusion research.

Summary

This study provides the first detailed report of epithelial fusion during chick OFC and illustrates the power of the embryonic chick eye to investigate the mechanisms guiding this important developmental process further and to provide insights into human eye development and broader fusion contexts. We clearly define the temporal framework for OFC progression and reveal that fusion is characterised by loss of epithelial cell types and a coincidental increase in apoptosis. We reveal the specific expression of orthologues of known coloboma-associated genes during chick OFC, and provide a broad transcriptomic dataset that can be used to improve the identification of candidate genes from human patient exome and whole-genome DNA sequencing datasets. Finally, we identify that NTN1 is specifically and dynamically expressed in the fusing vertebrate fissure - consistent with having a direct role in epithelial fusion, and is essential for OFC and palate development. We propose that NTN1 should therefore now be considered as a new candidate for ocular coloboma and congenital malformations that feature defective epithelial tissue fusion.

Materials and methods

Key resources table
Reagent type
(species) or
resource		DesignationSource or referenceIdentifiersAdditional
information
Genetic reagent (M. musculus)Ntn1-/-PMID 26395479MGI:5888900Lisa Goodrich (Harvard Medical School, Boston MA).
Biological sample (G. gallus)memGFPPMID 25812521Rozbicki et al., 2015Maintained at The Greenwood Building, Roslin Institute, UK.
Biological sample (G. gallus)Chicken eye and OFM dissectionsThis paperHy-Line BrownMaintained at The Greenwood Building, Roslin Institute, UK.
AntibodyNTN1 (Mouse monoclonal)R and D SystemsMAB128one in 100 dilution for whole mount IF
AntibodyNTN1 (Rabbit polyclonal)Abcamab126729one in 300 dilution for human and mouse IF; 1 in 500 dilution for chick cryosection IF
AntibodyLaminin-B1 (Mouse monoclonal)DSHB3H11one in 20 dilution for all IF
AntibodyNF145 (Rabbit polyclonal)MerkAB1987one in 100 dilution for all IF
AntibodyPhospho-Histone H3A (Rabbit monoclonal)Cell Signalling Technologies#3377one in 200 for cryosections, 1 in 1000 for flat-mount
AntibodyActivated Caspase-3 (Rabbit polyclonal)BD Pharminagen#559565one in 400 dilution for all IF
Commercial assay or kitAlexa Fluor Phalloidin (488 nm)Thermo-Fisher#A12379one in 40 dilution for all IF
Software, algorithmKallistoPMID 27043002NANA
Software, algorithmLimmaPMID 25605792NANA

Embryo processing

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Hy-Line Eggs were incubated at 37°C at day 0 (E0), with embryo collection as stated throughout the text. Whole embryos were staged according to Hamburger Hamilton (Hamburger and Hamilton, 1992; Hamburger and Hamilton, 1951). Heads were removed and either ventral eye tissue was resected and flat-mounted and imaged immediately, or whole heads were placed in ice cold 4% paraformaldehyde (PFA) in pH 7.0 phosphate buffered saline (PBS), overnight and then rinsed twice in PBS. OFMs used for fusion progression measurements (flat mounts) were mounted in glycerol between a coverslip and glass slide, without fixation. Whole embryo, flat mounted OFMs, and dissected eye images for were captured on a Leica MZ8 light microscope and measurements were processed using FIJI (NCBI/NIH open source software [Schindelin et al., 2012]).

Immunofluorescence

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For cryosections, resected ventral chick eyes were equilibrated in 15% Sucrose-PBS then placed at 37°C in 7% gelatin:15% Sucrose, embedded and flash-frozen in isopentane at −80°C. Sections were cut at 20 µm. Immunofluorescence was performed on chick fissure sections as follows: 2 × 30 min rinse in PBS, followed by 2 hr blocking in 1% BSA (Sigma) in PBS with 0.1% Triton-X-100 [IF Buffer 1]. Sections were incubated overnight at 4°C with primary antibodies diluted in 0.1% BSA in PBS with 0.1% Triton-X-100 [IF Buffer 2]. Slides were then washed in 3 × 20 min PBS, followed by incubation for 1 hr with secondary antibodies (Alexa Fluor conjugated with 488 nm or 594 nm fluorophores; 1:800–1000 dilution, Thermo Fisher), and mounted with ProLong Antifade Gold (Thermo Fisher) with DAPI. Alexa Fluor Phalloidin (488 nm; Thermo-Fisher #A12379) was added at the secondary antibody incubation stages (1:50 dilution). Human foetal eyes were obtained from the Joint Medical Research Council UK (grant # G0700089)/Wellcome Trust (grant # GR082557) Human Developmental Biology Resource (http://www.hdbr.org/). For Netrin-1 immunostaining in human and mouse tissues, cryosections were antigen retrieved using 10 mM Sodium Citrate Buffer, pH 6.0 and blocked in 10% Goat serum +0.2% Triton-X100 in PBS, then incubated overnight at 4°C with primary antibody (Abcam #ab126729; 1: 300) in block. Secondary antibody staining and subsequent processing were the same as for chick (above). For anti-NTN1 immunostaining in chick tissues, cryosections were hydrated in phosphate buffer (PB) pH7.2, antigen retrieved using 1% SDS in PB and blocked 2% bovine serum albumen +0.2% Tween-20 in PB (blocking buffer). Primary antibody was diluted in blocking buffer and incubated at room temperature for 4 days. Secondary antibody staining and subsequent processing were as stated above, but PB was used instead of PBS. For whole-mount immunofluorescence we followed the protocol from Ahnfelt-Rønne et al (Ahnfelt-Rønne et al., 2007), with the exception that we omitted the TNB stages and incubated instead with IF Buffer 1 (see above) overnight and then in IF Buffer two for subsequent antibody incubation stages, each for 24 hr at 4°C. No signal amplification was used. Antibodies were used against Phospho-Histone H3A and Netrin-1. Imaging was performed using a Leica DM-LB epifluorescence microscope, or a Nikon C1 inverted confocal microscope and Nikon EZ-C1 Elements (version 3.90 Gold) software. All downstream analysis was performed using FIJI. Image analysis for proliferation in the OFM on flat-mounts was performed by counting Phospho-Histone H3A positive foci using a region of interest grid with fixed dimensions of 200 µm2 and throughout the entire confocal Z-stack. To quantitate apoptotic foci at the OFM, we used Activated-Casp3 immunofluorescence on serial cryosections of HH.St29-30 OFMs and collected confocal images for each section along the P-D axis. Image analysis was performed by counting A-Casp3 positive foci at the OFM in sequential sections using a region of interest with fixed dimensions of 100 µm2. For histology and subsequent haematoxylin and eosin staining, resected eyes processed and image captured according to Trejo-Reveles et al (Trejo-Reveles et al., 2018).

In situ hybridization

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RNAscope was performed on HH.St29 cryosections using a probe designed specific to chicken NTN1 according to Nishitani et al (Nishitani et al., 2017). For colourimetric in situ hybridisation, a ribprobe was for NTN1 was designed using PCR primers to amplify a 500 bp product from cDNA prepared from chick whole embryos at HH.St28-32 (Oligonucleotide primers: Fwd 5’-ATTAACCCTCACTAAAGGCTGCAAGGAGGGCTTCTACC-3’ and Rev 5’-TAATACGACTCACTATAGGCACCAGGCTGCTCTTGTCC-3’). The PCR products were purified and transcribed into DIG-labelled RNA using T7 polymerase (Sigma-Aldrich) and used for In Situ hybridization on cryosectioned chick fissure margin tissue (prepared as described above for immunofluorescence) or whole embryos using standard protocols (described in J. Rainger's doctoral thesis - available on request).

Transgenic animal work

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To obtain Ntn1-/- mouse embryos (Ntn1tm1.1Good, RRID:MGI:5888900), we performed timed matings with male and female heterozygotes and took the appearance of a vaginal plug in the morning to indicate embryonic day (E)0.5. Embryos were collected at E11.5 and E16.6 and genotyped according to Yung et al (Yung et al., 2015). As with this previous report we observed ratios within the expected range for all three expected genotypes (28 total embryos: 13x Ntn1+/-; 10x Ntn1-/-; 5x WT – 46%; 35%; 18%, respectively). Embryos were fixed in 4% paraformaldehyde overnight and then rinsed in PBS and imaged using a Leica MZ8 light microscope. Ntn1-/- and C57Bl/6J animals were maintained on a standard 12 hr light-dark cycle. Mice received food and water ad lib and were provided with fresh bedding and nesting daily. For zebrafish work, we designed gene-editing sgRNA oligos alleles to target ntn1a: 5´-GGTCTGACGCGTCGCACGTG-3´. We then generated founder (G0) animals by zygotic microinjection of CRISPR/Cas9 components according to previous work (Dutta et al., 2015; Varshney et al., 2015; Jao et al., 2013). G0 animals were genotyped and used for crosses to generate G1 embryos which were scored for coloboma phenotypes and genotyped individually (Figure 4—figure supplement 3). All experiments were conducted in agreement with the Animals (Scientific Procedures) Act 1986 and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research (USA). Morpholinos were designed and generated by Gene Tools LLC (Oregon) to target the translation initiating site of ntn1a: 5′-CATCAGAGACTCTCAACATCCTCGC-3′, and a Universal control MO sequence was used as a control: 5′-ATCCAGGAGGCAGTTCGCTCATCTG-3′. One cell stage embryos were injected with 2.5 ng or 5.0 ng of ntn1a or control morpholino and allowed to develop to OFC stages (≥48 hpf). Oligos used for ntn1a genotyping by sanger sequencing were: 5′-TTACGACGAGAACGGACACC-3′ and 5′-GGAGGTAATTGTCCGACTGC-3′.

Transcriptional profiling

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For RNA seq analysis, we carefully dissected regions of (i) fissure-margin, (ii) ventral eye, and (iii) dorsal eye, and (iv) whole eye tissue from ≥10 individual embryos for each HH stage range (Figure 3—figure supplement 1). Samples were collected and pooled for each tissue type and stage to obtain n = 3 technical replicate RNA pools per tissue type per stage. Total RNA was extracted using Trizol (Thermo Scientific). Whole-transcriptome cDNA libraries were then prepared for each pool following initial mRNA enrichment using the Ion RNA-Seq Core Kit v2, Ion Xpress RNA-Seq Barcodes, and the Ion RNA-Seq Primer Set v2 (Thermo Scientific). cDNA quality was confirmed using an Agilent 2100 Bioanalyzer. Libraries were pooled, diluted, and templates were prepared for sequencing on the Ion Proton System using Ion PI chips (Thermo Scientific). Quantitative transcriptomics was performed using Kallisto psuedoalignment (Bray et al., 2016) to the Ensembl (release 89) chicken transcriptome. Kallisto transcript counts were imported into R using tximport (Soneson et al., 2015) and differentially expressed transcripts identified using Limma (Ritchie et al., 2015). Genes not expressed in at least three samples were excluded. To identify the relationships between samples, Log2 transformed counts per million were then calculated using edgeR (Robinson et al., 2010) and Spearman’s rank correlation was used to identify the similarities in genome-wide expression levels between samples. All RNAseq data files are submitted to the NCBI Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo) with the accession number GSE84916.

Statistical analysis

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Bar graphs display means ± SD or 95% confidence intervals as indicated. Sample sizes were n ≥ 3, unless stated otherwise. Statistical analyses were performed using Prism 8 (GraphPad Software Inc). Data were assessed for normal distribution by Shapiro-Wilk test where appropriate. Significance was evaluated by unpaired Student’s t-test, where p≤0.05 was deemed significant. Asterisk indicate significance in Figure 1 as *p≤0.05. **p≤0.01, ***p≤0.001.

Data availability

All RNAseq data files are submitted to the NCBI Gene Expression Ominibus database (http://www.ncbi.nlm.nih.gov/geo) with the accession number GSE84916.

The following data sets were generated
    1. Rainger J
    (2019) NCBI Gene Expression Omnibus
    ID GSE84916. Segmental chick eye transcriptome analysis.
    http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE84916

References

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    (1990)
    Optic fissure closure in the normal Cinnamon mouse: an ultrastructural study
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    2. Ward SJ
    3. Gilbert CE
    (2003)
    Eye birth defects in humans may be caused by a recessively-inherited genetic predisposition to the effects of maternal vitamin A deficiency during pregnancy
    Medical Science Monitor : International Medical Journal of Experimental and Clinical Research 9:23–26.
    1. O’Rahilly R
    (1966)
    The early development of the eye in staged human embryos
    Carnegie Institution of Washington Publication 625:1–42.
    1. Salminen M
    2. Meyer BI
    3. Bober E
    4. Gruss P
    (2000)
    Netrin 1 is required for semicircular canal formation in the mouse inner ear
    Development 127:13–22.

Decision letter

  1. Marianne E Bronner
    Senior Editor; California Institute of Technology, United States
  2. Jeremy Nathans
    Reviewing Editor; Johns Hopkins University School of Medicine, United States
  3. Stephan Heermann
    Reviewer; Freiburg University, Germany
  4. Teri Belecky-Adams
    Reviewer

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 "Detailed analysis of chick optic fissure closure reveals Netrin-1 as a conserved mediator of epithelial fusion" for consideration by eLife. Your article has been reviewed by Marianne Bronner as the Senior Editor, a Reviewing Editor, and three reviewers. The following individuals involved in review of your submission have agreed to reveal their identity: Stephan Heermann (Reviewer #2); Teri Belecky-Adams (Reviewer #3).

I am including the three reviews at the end of this letter, as there are a variety of specific and useful suggestions in them. Some of the comments have to do with clarity of presentation, critical interpretation of the data, and comparison with previously published data – these comments should be relatively straightforward to address. The more substantive ones relate to the experimental evidence that Netrin plays a causal role in fissure closure.

We appreciate that the reviewers' comments cover a broad range of suggestions for improving the manuscript. Please use your best judgment in deciding which of these can be accommodated in a reasonable period of time. We look forward to receiving your revised manuscript.

Reviewer #1:

This manuscript addresses the question of how epithelial fusion occurs within a specific structure in the eye, the optic/choroid fissure. Epithelial fusion is not well understood, and is a feature in the development of a number of different organ systems. The authors describe events that occur during optic fissure fusion in chick embryos, then carry out transcriptome analysis on microdissected tissues in an effort to identify factors that might be directly involved in the fusion process. They identify NTN1 as a factor enriched in the fissure, and then move to mouse and zebrafish for loss-of-function phenotypes.

The work to describe optic fissure fusion in chick is fundamentally sound, although more schematics are necessary to orient the reader (especially a broader audience), and editing should be done to more explicitly state how this work is different (more focused, finer time window; more quantitative analysis) from a recent publication of describing many aspects of chick optic fissure development (Bernstein et al., 2018). The transcriptome analyses are excellent, and the temporal comparisons are useful.

On the other hand, mechanistic conclusions are weaker and often correlative. For example, based on antibody staining at a single timepoint, roles for proliferation and apoptosis are inferred or suggested; conclusions would be much stronger if proliferation and apoptosis had been tested via inhibition (for example, with pharmacological reagents). The idea of netrin as an epithelial fusion factor is exciting and intriguing, but the results are difficult to interpret in this form. The netrin mouse mutant exhibits coloboma, but given that the authors review many direct and indirect causes of coloboma in the introduction, it is not clear that netrin acts in mouse in the way they hypothesize from chick data. Characterization of the mutant phenotype (along with controls) would be necessary to interpret the gross morphological phenotype. The zebrafish data are also difficult to interpret, and no molecular characterization of these novel mutant alleles is shown. Similarly, characterization and controls in zebrafish would be necessary to interpret these data.

Abstract: "Our data reveal that NTN1 is a new locus for human coloboma…"

Unless there are human genetic data, this statement does not seem to be supported by the manuscript in this form.

More information is needed to understand the images (especially the fluorescent confocal images) in the manuscript. For example, in Figure 1C, is this a single confocal optical section? Or a projection? This is important especially for interpreting data such as in Figure 4 (netrin localization; see below).

Table 1 and Table 2: The authors have carried out quantitative analysis of optic fissure fusion in chick. Yet these tables are difficult to interpret, which makes this section of the results confusing. For example, subsection “OFC in the chick occurred within a wide spatial and temporal window”: "FP2 displayed active movement in a posterior direction to create a fused seam between FP1-FP2 that extended until HH.St34 (Table 2)". FP1 and FP2 are not noted in the table, and in the table, the term "fused collar" is confusing. Inclusion of a schematic or a graphical representation of these data (including where these terms fit with respect to the measurements being taken) might help. In addition, the authors note four distinct phases of fusion, with #3 being "active fusion as two FPs separate to generate a fused seam along the A-P axis" (subsection “OFC in the chick occurred within a wide spatial and temporal window”). Can the authors comment on the variability of this in Table 1? The text suggests that there should be two FPs at these stages, but at all of these stages, 50% of the embryos have 1 FP.

Figure 1 and Figure 2 (and associated supplemental figures): regarding the proliferation and apoptosis analysis, the antibody staining is clear at the stage shown. But this is correlative and would not seem to be sufficient to exclude or support a role for either process in optic fissure fusion: can the authors use pharmacological reagents to inhibit proliferation or apoptosis to functionally test their roles?

Figure 2—figure supplement 1: regarding RGC axons, in this orientation, it is difficult to interpret the localization of NF-145 staining. RGC axons, and their positions relative to the fusing fissure, might be easier to see in a fissure flatmount. This may be important, since the authors use these data to exclude a role for RGC axons (and possibly a role for netrin acting through RGC axon guidance) in optic fissure fusion.

Figure 3, also subsection “Transcriptional profiling reveals genetic conservation between chick and human OFC”: the figure suggests that St28-32 was used to represent "Active-fusion", but the results state Hh.st28-30. Please clarify.

Figure 4B’’: the authors state that netrin protein is "specifically enriched in open fissures but not in the fused seam". Although netrin protein may be missing from the specific site where fusion took place, it does still appear to be present in the basal lamina (although it seems absent in Figure 4B – was this a single confocal optical section, and is the remaining netrin "inside" after fusion?). Do the authors think that netrin may be carrying out a different function around the optic nerve after fusion? How does this fit with their model?

Figure 4, regarding netrin expression: more expression data is required, earlier in development. At this stage, netrin RNA is absent from the fused seam, but was it actually present earlier in this region's pre-fusion margins? Might it mark the first site of fusion? A fissure flatmount could be helpful for assessing expression.

Figure 4, regarding mouse mutant data: these data are intriguing, but not enough is shown to demonstrate that netrin is mediating fusion. What is the more detailed phenotype of the mutant mouse? Does loss of netrin affect localization and movement of periocular mesenchyme or RGC axons? Coloboma could be caused via indirect effects through many other factors.

Figure 4—figure supplement 1: the zebrafish data are difficult to interpret, as no molecular characterization of these novel mutant alleles is included. Can the authors demonstrate that these are loss-of-function alleles (for example, via other phenotypes)? Is mRNA still present? In addition, the phenotypic penetrance seems unclear. In subsection “Loss of Netrin causes coloboma and multisystem fusion defects in vertebrates”: "We observed bilateral ocular coloboma in all homozygous mutant animal(s) analysed…"; whereas in subsection “Immunofluorescence”: "In these crosses, we observed coloboma phenotypes in 4/28 offspring, giving approximately 50% penetrance…" Were zebrafish mutant embryos genotyped? This is not clear, and embryos should be genotyped. Other control experiments and further characterization of the mutant phenotype should be carried out (e.g. potential effects periocular mesenchyme migration or RGC axons) in order for the role of netrin as a fusion mediator to be more convincing.

Regarding both mouse and zebrafish mutant data: the move to the other organisms leads to basic questions about optic fissure fusion and netrin expression in those other organisms – are the same fusion mechanisms at play at a descriptive level? Is netrin really expressed along the entire optic fissure margins in mouse and zebrafish at the appropriate timepoints? More characterization of these other systems would be necessary to interpret these phenotypes.

Reviewer #2:

In the manuscript "Detailed analysis of chick optic fissure closure reveals Netrin-1 as a conserved mediator of epithelial fusion", Hardy and colleagues carefully addressed the fusion process of the medial and distal optic fissure domain in chicken embryos and by analysis of fixed samples at different developmental stages identified the onset and the progression of fusion. They also show that cell proliferation was not increased in the fissure margins compared to other regions but that apoptosis could be found in and next to the progressing fusion site. Furthermore, they applied transcriptomic analysis in order to identify genes, which are important during fissure fusion. Among others they identified Netrin-1 (NTN1), which was found most specifically upregulated during fusion in chicken. NTN1 expression and protein localization was found in the fissure region mainly before and during fusion. Protein localization was then also checked in developing human- and mouse eyes. And the loss of NTN1 was addressed in mouse and zebrafish.

Per se this is an important contribution to the field. It is beneficial for the field to include also the chicken model for studying optic fissure fusion. In itself the data seem sound. However, the fusion of the fissure in chicken was addressed also recently (Bernstein et al., 2018). Notably, the data (e.g. onset of apposition, BM modulation and fusion) presented there was different to the presented work here. Could there be strain dependency? Hardy et al. state in the discussion that Bernstein et al. did not report fusion in anterior or midline region and mentioned that in the proximal regions fusion is happening via intercalation. Nevertheless, Bernstein et al. reported fusion. It should be pointed out which of the current findings is new, which is backing up previous data and which is potentially at odds. Along the lines of chicken as a model for fissure fusion, it would be helpful to address the role of the pecten. But beyond that, Hardy et al. provide transcriptional data from well-designed experiments and further addressed the necessity of NTN1.

A lot of effort was put into the analysis of loss of NTN1 in other species, mouse and zebrafish. Technically it would be possible to address the loss of NTN1 also in chicken. What was the rational to use the other species only?

It was proposed that the role of NTN1 would be the same in the different species. However, in zebrafish the remaining fissure seems a bit wider. What is the age of the embryo? It would be helpful to see also histological images to see that the margins were in "apposition" properly.

Is the work with human samples covered by the HDBR? In the ethics statement it is mentioned that in the UK most studies will not need specific approval, but is this study also covered?

In some cases, I was a bit lost in the images presented e.g. Figure 1C. How was the staining achieved? What exactly do we see? Are the cells in FP1 head to head and in FP2 side by side? What is the scale and orientation of the individual images? I will give more examples in the minor point section.

It would be good if the data mentioned in subsection “Chick OFC was characterised by the breakdown of basement membranes, loss of epithelial morphology and localised apoptosis” and subsection “Netrin-1 is specifically and dynamically expressed in the fusing OFM” could be shown.

In some cases, the citations seemed incorrect, e.g. subsection “Transcriptional profiling reveals genetic conservation between chick and human OFC”, Cho and Cepko (2006).

Subsection “Transcriptional profiling reveals genetic conservation between chick and human OFC”: what would be the definition of "biologically significant" in terms of differential gene expression? Would this not be dependent on the gene and gene product? Can this be generalized?

Subsection “Transcriptional profiling reveals genetic conservation between chick and human OFC”: Table 2 is not showing this. Figure 3B shows the 12 increased only.

Figure 1A: Is the OFM really the unpigmented region? Could it be the fissure itself?

Figure 1B: To what extent is the iris developed at the stage of fissure fusion? The pecten could be explained more. On the images, it appears as a gap. Could the axes be renamed to proximal and distal?

Figure 1C: What is the orientation, magnification and how was the labelling achieved?

Figure 1E: Were this and Figure 1—figure supplement 1G independent datasets?

Figure 1F: Is the proliferation increased outside or reduced inside?

Figure 1—figure supplement 1B: What is seen here?

Figure 1—figure supplement 1C: Is this taken from flat mounted tissue? What is the orientation?

Figure 1—figure supplement 1D: It is stated that at HH26 there is no fusion plate. Could higher magnifications be provided? In Figure 1—figure supplement 1Di it is not totally clear, especially comparing it to Figure 2B.

Figure 1—figure supplement 1G: The label "G" is missing in the figure. I do not understand the note that at HH29 the seam length was too small to quantify. The length itself was quantified in Figure 1—figure supplement 1F.

Table 1: Can an animal have FP1 and FP2? At stage HH29 n 5 were analyzed. 4 showed 2FP 5 showed 1FP. Could this be explained?

Figure 2:

Without time-lapse analysis, the term dynamics is potentially misleading.

Figure 2A: Arrowheads in fusion plate image point to a domain in which the basal lamina is still visible.

Figure 2B: Can the authors be sure that the future NR and RPE are both contributing and are both remodeled? Could there be dynamic in the fissure margins?

Figure 2C: The image presented to show the apposition is not corresponding to Figure 2B.

Figure 2D: Which domain was used for quantification? What was considered seam and outside seam and what could be considered OFM and outside OFM (towards nasal and temporal directions respectively)?

Figure 2—figure supplement 1: What is meant by medium? What is the orientation? What is considered central retina?

Figure 3B: Could the boxes be separated for the different stages? The annotation over the boxed could be misunderstood for statistical data. The annotation (.) should be included in the legend. The legends mention CHDL1 and not CHRDL1.

Figure 3—figure supplement 1A: The images are appreciated. Were the individual regions dissected manually and in independent eyes also for fissure and ventral eye, meaning, is ventral eye including the fissure?

Figure 3—figure supplement 1B: How was the dorsal data acquired? Is the transcriptional data available?

Figure 4A-C: What is the orientation?

Figure 4B: What is meant by whole mount? At the FP2, the NTN1 signal seems less intense. Is the arrow positioned correctly? A co-staining with a basal lamina marker would be helpful to conclude the localization.

Figure 4B’’: What is NT? Is the overall annotation correct? Is NTN1 expressed also in the POM?

Figure 4C, D: Higher magnifications are needed and potentially a co-labelling with a basal lamina marker.

Figure 4F: What is shown in the second image?

Figure 4—figure supplement 1B: The anti-laminin staining looks odd.

Figure 4—figure supplement 1C: What was the age of the embryos? Arrows were used twice for different annotations. In the no antibody control, green signal can be seen. Was there a problem with color channel separation? It was stated that there was no staining.

Figure 4—figure supplement 1D: Please add axes for orientation and landmarks for orientation.

Figure 4—figure supplement 1F: If the penetrance of a phenotype is given, could it be also supported by genotyping besides by calculation? Was the phenotype consistent with the image in f? The margins seem farther apart? Where the margins in "apposition" correctly? Histology should be performed, or confocal analysis, to address this issue. What is the age of the embryo presented?

At the end of the legend there is a redundant text section.

It would be good if the data mentioned in subsection “Chick OFC was characterised by the breakdown of basement membranes, loss of epithelial morphology and localised apoptosis” and subsection “Netrin-1 is specifically and dynamically expressed in the fusing OFM” could be also shown.

The RNAseq data was shared. It would be best if it was accessible also without too much bioinformatics background.

Reviewer #3

Summary: The authors have (1) characterized in detail the fusion of the coloboma in chick embryos, (2) identified sets of genes that change across several stages and in relation to other areas of the developing chick optic cup (dorsal, ventral, and whole optic cup), and (3) tested the role of netrin in chick, mouse, and zebrafish optic fissure closure. The reviewer congratulates the authors on a beautifully done study.

https://doi.org/10.7554/eLife.43877.025

Author response

Reviewer #1:

[…]

Abstract: "Our data reveal that NTN1 is a new locus for human coloboma…"

Unless there are human genetic data, this statement does not seem to be supported by the manuscript in this form.

We have made this statement more accurately reflect the findings of the study. This section of the Abstract now reads: “Our data suggest that NTN1 is a strong candidate locus for human coloboma and other multi-system developmental fusion defects, and show that chick OFC is a powerful model for epithelial fusion research.”

More information is needed to understand the images (especially the fluorescent confocal images) in the manuscript. For example, in Figure 1C, is this a single confocal optical section? Or a projection? This is important especially for interpreting data such as in Figure 4 (netrin localization; see below).

We apologise for not making this more clear in the figures or legends and have improved the information available in the manuscript to help with interpretation of the data and figure legends have been amended to clearly describe the data shown.

Table 1 and Table 2: The authors have carried out quantitative analysis of optic fissure fusion in chick. Yet these tables are difficult to interpret, which makes this section of the results confusing. For example, subsection “OFC in the chick occurred within a wide spatial and temporal window”: "FP2 displayed active movement in a posterior direction to create a fused seam between FP1-FP2 that extended until HH.St34 (Table 2)". FP1 and FP2 are not noted in the table, and in the table, the term "fused collar" is confusing. Inclusion of a schematic or a graphical representation of these data (including where these terms fit with respect to the measurements being taken) might help.

We appreciate the reviewer’s efforts to interpret our data as presented and agree that the way we had presented this was confusing. We have made the following amendments to improve the interpretation and clarity of the measurement data: (i) we have included a schematic in Figure 1 to orientate the reader, which includes labelled anatomical features and axes for orientation; (ii) we have added more samples to our data and stratified the data in Table 1 to more clearly indicate the presence of either a single fusion point or two fusion points; (iii) we have removed the data for the “fused collar” measurements in Table 2 as this does not add any useful additional information to the table or study; (iv) we have also significantly edited the text in the results paragraph relating to this data to improve clarity and interpretation. This now reads:

“Fusion was first initiated between HH.St27-28 as confirmed by the definitive appearance of joined epithelial margins at a single fusion point (FP). […] The process is active between HH.St27-HH.St34 and proceeds over ~66 hours.”

In addition, the authors note four distinct phases of fusion, with #3 being "active fusion as two FPs separate to generate a fused seam along the A-P axis" (subsection “OFC in the chick occurred within a wide spatial and temporal window”). Can the authors comment on the variability of this in Table 1? The text suggests that there should be two FPs at these stages, but at all of these stages, 50% of the embryos have 1 FP.

The reviewer raises an important point as we observed subtle variability in the progression of fusion within defined HH stages of development, despite using meticulous adherence to established staging criteria using developmental landmarks. Indeed, this variation should be taken into account when analysing chick OFC processes, as is the case with OFC in other model organisms. However, our model attempts to be inclusive of these variabilities and remains a useful staging framework for studies of chick OFC. For example, in our model “initiation” occurs between HH.St27-28 where 5/7 OFMs (>70%) analysed had observable fusion plates. However, only one OFM (at HH.St28) had an expanded seam with two clear FPs, whereas the majority had only one observable FP. Two had no FPs and no FPs were identified prior to these stages. Subsequently, “active fusion” occurs from HH.St28-33. Our data now shows that in this range 19/24 OFMs (79%) analysed have 2x FPs (i.e. both FP1 and FP2 present). In contrast, by HHSt.34 (“complete fusion”) we could not definitively identify any proximally open fissures in all samples analysed, indicating that the seam had met the pecten and active fusion had been completed (in 100% of samples).

Figure 1 and Figure 2 (and associated supplemental figures): regarding the proliferation and apoptosis analysis, the antibody staining is clear at the stage shown. But this is correlative and would not seem to be sufficient to exclude or support a role for either process in optic fissure fusion: can the authors use pharmacological reagents to inhibit proliferation or apoptosis to functionally test their roles?

We were careful not to assign a functional role for either of these processes but believe both observations are important to accompany the fusion-progression data to establish the chick model of OFC. We specifically included the apoptosis data because the role and requirement for apoptosis during epithelial fusion has been controversial in other animal models of OFC and fusion. Whether apoptosis is indeed necessary or has a direct role in mediating fusion is still undetermined and may vary across divergent species, but our data shows strong spatial and temporal correlation between apoptosis and fusion in the chick OFC. We are currently developing tools to inhibit apoptosis at both the chemical and genetic level in chick, and to coincidentally manipulate Netrin-1 expression and function in this context. We feel these studies are beyond the scope of the current study but will provide valuable information of the specific pathways and processes that lead to cell-death in the chick fissure.

On reflection of the reviewer’s comment, we have moved the proliferation data into supporting data as although the analysis clearly showed fewer dividing cells in the fused seam, we feel that this observation, while interesting, requires additional analyses to reveal the exact mechanisms of seam expansion. Inhibition of cell-proliferation, as suggested by the reviewer, would need to specifically and reproducibly target cells within the OFM to formally rule out a requirement for proliferation in seam expansion. However, these tools are not yet available.

Figure 2—figure supplement 1: regarding RGC axons, in this orientation, it is difficult to interpret the localization of NF-145 staining. RGC axons, and their positions relative to the fusing fissure, might be easier to see in a fissure flatmount. This may be important, since the authors use these data to exclude a role for RGC axons (and possibly a role for netrin acting through RGC axon guidance) in optic fissure fusion.

We agree with the reviewer and have now included flat mounted brightfield OFM image data to help with orientation in this figure. We have also provided additional flat-mount immunofluorescence analyses, which confirm the absence of NF145 in the fusing and nascently fused OFM, supporting our previous conclusion that RGC axons do not have a direct role in mediating chick OFC.

Figure 3, also subsection “Transcriptional profiling reveals genetic conservation between chick and human OFC”: the figure suggests that St28-32 was used to represent "Active-fusion", but the results state Hh.st28-30. Please clarify.

We are grateful for this observation. The segmental dissections for RNAseq were performed at HHSt.28-30, during the active fusion period. We did not dissect at any later stages during active fusion. We have amended these inconsistencies throughout the manuscript and figure.

Figure 4B’’: the authors state that netrin protein is "specifically enriched in open fissures but not in the fused seam". Although netrin protein may be missing from the specific site where fusion took place, it does still appear to be present in the basal lamina (although it seems absent in Figure 4B – was this a single confocal optical section, and is the remaining netrin "inside" after fusion?). Do the authors think that netrin may be carrying out a different function around the optic nerve after fusion? How does this fit with their model?

The dynamic nature of NTN1 is intriguing. The images the reviewer refers to are indeed single-plane confocal sections and this has been made clearer in the figure legend. The data shows the absence of NTN1 within the fused seam and is consistent with NTN1 mRNA localisation. In our mouse data (Figure 4—figure supplement 2), we observed continual Ntn1 localisation in the optic nerve after fusion was completed. Although we have not analysed NTN1 localisation in the chick optic nerve at equivalent stages, we believe that in both species Netrin-1 has a role in the continued development of the optic nerve that progresses after fusion has completed in the medial OFM, consistent with a role in axonal guidance or neuronal migration. The exact relevance of our localisation data in the OFM is unclear but suggests there may be further multiple roles for NTN1 during the progression of fusion that are mediated by its differential localisation in the basal lamina and ECM. We are actively working on elucidating these roles using in vitro and our chick OFC model.

Figure 4, regarding netrin expression: more expression data is required, earlier in development. At this stage, netrin RNA is absent from the fused seam, but was it actually present earlier in this region's pre-fusion margins? Might it mark the first

site of fusion? A fissure flatmount could be helpful for assessing expression.

We have added whole mount in situ hybridisation data showing NTN1 expression in the ventral optic cup before the OFM is established (HH.St22-24; Figure 4—figure supplement 1). Our transcriptomic analysis had revealed NTN1 as the most highly-expressed fissure-specific gene in our dataset, including at pre-fusion stages (HH.St25-27). We also showed in situ data of NTN1 expression in cryosections at the open medial OFM immediately prior to fusion (Figure 4—figure supplement1A), confirming its localisation in the OFM leading up to fusion. We have now added immunofluorescence data on cryosections confirming that NTN1 protein is also localised to this region immediately preceding fusion. In combination, these data show that NTN1 is expressed throughout the OFM long before fusion is initiated, and therefore is not specific to the fusion initiation point. This suggests that NTN1 has a role in mediating fusion at all points along the A-P axis and is important for establishing or maintaining the OFM in the developing ventral eye.

Figure 4, regarding mouse mutant data: these data are intriguing, but not enough is shown to demonstrate that netrin is mediating fusion. What is the more detailed phenotype of the mutant mouse? Does loss of netrin affect localization and movement of periocular mesenchyme or RGC axons? Coloboma could be caused via indirect effects through many other factors.

We were careful not to assign a specific mechanism for NTN1-mediated fusion in our manuscript as we are firmly aware that this will require significant additional work to elucidate. However, a requirement for Netrin-1 in the optic fissure was shown in zebrafish in a report published while our manuscript was under review (Richardson et al., 2019). In their study, transient morpholino gene knockdown experiments of the zebrafish orthologue ntn1a resulted in a coloboma phenotype. The authors provided some additional phenotypic characterisation that showed correct apposition of the fissure margins in ntn1 deficient embryos. Consistent with this, and in further support of a direct role for Netrin-1 in the fusion process, we have added additional phenotypic data from sections of Ntn1-/- eyes at stages when fusion initiates in the mouse eye (E11.5; Figure 4—figure supplement 2). This data showed that early growth of the eye in Ntn1-/- embryos was sufficiently normal to bring the opposing optic fissure margins in direct apposition. Furthermore, the tissue morphology in the OFM appeared indistinguishable from the wild type controls and there was no evidence of ectopic cells or axonal processes between the apposed OFM edges. We believe that these new data, in combination with (i) the highly-specific expression of Netrin-1 in multiple vertebrates species during fusion in our study, (ii) prior observations of fusion-specific defects in the vestibular system resulting from Netrin-1 mis-regulation, and (iii) our additional findings of palate fusion defects in the Ntn1-/- mice, all support a fusion-specific role for Netrin-1.

Figure 4—figure supplement 1: the zebrafish data are difficult to interpret, as no molecular characterization of these novel mutant alleles is included. Can the authors demonstrate that these are loss-of-function alleles (for example, via other phenotypes)? Is mRNA still present? In addition, the phenotypic penetrance seems unclear. In subsection “Loss of Netrin causes coloboma and multisystem fusion defects in vertebrates”: "We observed bilateral ocular coloboma in all homozygous mutant animal(s) analysed…"; whereas in subsection “Immunofluorescence”: "In these crosses, we observed coloboma phenotypes in 4/28 offspring, giving approximately 50% penetrance…" Were zebrafish mutant embryos genotyped? This is not clear, and embryos should be genotyped. Other control experiments and further characterization of the mutant phenotype should be carried out (e.g. potential effects periocular mesenchyme migration or RGC axons) in order for the role of netrin as a fusion mediator to be more convincing.

The work presented in our first submission was performed on a gene edited (GE) line produced as part of a wider study, and both the ntn1a and ntn1b loci were targeted. This line therefore required a complex crossing and genotyping strategy to establish the causation of the colobomas observed (4/28) of all offspring. We had observed no colobomas in F1 crosses on the ntn1b mutant background and the colobomas were only present when the ntn1a allele was introduced to the cross. However, we were unable to maintain this line for subsequent analyses as the long-term viability and fertility of ntn1a+/-:ntn1b-/- animals was very poor.

Therefore, we have now derived an additional line with only the ntn1a locus edited. Using the same sgRNA GE design we generated a frame-shift nonsense mutation (p.Cys90Ala.fs15; see Figure 4—figure supplement 3), and the data presented in the revised manuscript is solely for this line, with all G0, F0 and F1 animals individually genotyped (either ntn1a+/-, ntn1a-/-, or Wt). We also have applied morpholino gene-knockdown of ntn1a in zebrafish embryos. These experiments support the ntn1a GE data, that loss of ntn1a causes highly penetrant colobomas.

While our work was under review, a paper from the group of Mariya Moosajee (Richardson et al., 2019) described transcriptional profiling analyses in the zebrafish ventral optic cup during OFC. Remarkably, they revealed specific upregulation of ntn1a coincident with the progression of fusion, with subsequent down-regulation as fusion progressed, similar to our observations in chick. They also provided morpholino knockdown experiments of zebrafish ntn1a with an ocular coloboma phenotype. Consistent with our data from mouse and zebrafish, the mutant phenotype was associated with normal early growth of the ventral optic cup and the tips of the optic fissure margins coming into close contact with each-other. No intervening POM or axonal processes were observed. Therefore, the Richardson et al. study adds further evidence of a fusion-specific role for Netrin-1 in OFC.

Regarding both mouse and zebrafish mutant data: the move to the other organisms leads to basic questions about optic fissure fusion and netrin expression in those other organisms – are the same fusion mechanisms at play at a descriptive level? Is netrin really expressed along the entire optic fissure margins in mouse and zebrafish at the appropriate timepoints? More characterization of these other systems would be necessary to interpret these phenotypes.

The reviewer raises an important question of the evolutionary conservation of fusion mechanisms and we agree with the questions that are raised. Our paper exists as a unique study of fusion at the tissue and genetic level and supports the chick as a solid model for ongoing fusion-specific research. Many additional studies are now emerging in other model organisms applying genetic and imaging data to elucidate fusion mechanisms that reveal both commonality and differences between divergent species. The recent study by Richardson et al. (2019) performed transcriptional profiling in the zebrafish OFM and showed ntn1a is expressed specifically during fusion stages. Our data in mouse also show this at the protein level, and both are consistent with the specific NTN1 expression we observed in the chick OFM. In combination with our phenotypic data, we believe these strongly support the hypothesis of a fusion-specific role for Netrin-1 that is common to all vertebrates. Further work is required to elucidate these mechanisms and address the points the reviewer raises – indeed these form the basis for the long-term research focus of our lab.

Reviewer #2

[…]

A lot of effort was put into the analysis of loss of NTN1 in other species, mouse and zebrafish. Technically it would be possible to address the loss of NTN1 also in chicken. What was the rational to use the other species only?

While overexpression and morpholino analyses are indeed possible in the chick embryo, loss of function studies are more difficult and not well established in the literature. In particular, studies of genes that are secreted (such as Netrin-1) or are predicted to induce cell non-autonomous phenotypes will require highly consistent and widespread cellular transduction of plasmids or RNAi payloads. Current efforts are specifically focused on optimising such approaches and the development of germline knock-out chicken lines, however, these approaches require significant optimisation and costs, respectively and are not as yet “online”. For the purpose of this paper, we chose the mouse and zebrafish knock-out models as they were readily available. However, the data from these models strongly suggests that the requirement for Netrin-1 is highly conserved among evolutionarily diverse vertebrates and our transcriptomic data is consistent with many other studies recently becoming available in these model organisms. Whether the functional mechanisms are consistent among these species remains to be proven but the localisation and then removal from netrin specifically in epithelial cells at the fissure margin in humans, mice and chick are intriguing and support a common mechanism.

It was proposed that the role of NTN1 would be the same in the different species. However, in zebrafish the remaining fissure seems a bit wider. What is the age of the embryo? It would be helpful to see also histological images to see that the margins were in "apposition" properly.

We have added new data to show coloboma phenotypes in both morpholino knockdown and gene edited knockout embryos, at 48hfp, when apposition occurs and fusion initiates. This new data is intriguing in the apparent phenotypic difference between zebrafish and mouse in the absence of Netrin-1. We are keen to understand the mechanisms that lead to coloboma in both zebrafish and mice lacking Netrin-1 but such work is out with the scope of this current study. We hope to provide robust phenotypic analyses in such future studies.

Is the work with human samples covered by the HDBR? In the ethics statement it is mentioned that in the UK most studies will not need specific approval, but is this study also covered?

The HBDR has ethical approval to collect and distribute embryonic and fetal material to all UK based research projects, without the need for individual researchers to obtain their own project specific ethics, providing their project fits within the remit of the HDBR approval. This study is covered for the use of human samples and is in accordance with the HBDR (Newcastle Ethics Form Approval Terms and Conditions – Section 4).

In some cases, I was a bit lost in the images presented e.g. Figure 1c. How was the staining achieved? What exactly do we see? Are the cells in FP1 head to head and in FP2 side by side? What is the scale and orientation of the individual images? I will give more examples in the minor point section.

We are very grateful for this comment and we have made the presented images easier to interpret throughout the manuscript, including the inclusion of a schematic cartoon to help orientate the readers. We have also added scale bars to all images where they were absent, and included new data from cryosections of the memGFP OFM at regions of fusion and fused seam to provide greater clarity for tissue and cellular orientation.

It would be good if the data mentioned in subsection “Chick OFC was characterised by the breakdown of basement membranes, loss of epithelial morphology and localised apoptosis” and subsection “Netrin-1 is specifically and dynamically expressed in the fusing OFM” could be shown.

In subsection “Chick OFC was characterised by the breakdown of basement membranes, loss of epithelial morphology and localised apoptosis”, we have added brightfield microscopy data using sections cut at the nascently fused seam to the updated Figure 1. This data shows the absence of RPE pigmentation in the in the post-fused region. In subsection “Chick OFC was characterised by the breakdown of basement membranes, loss of epithelial morphology and localised apoptosis”, the human NTN1 expression data in the OFM is part of a larger study using RNAseq in the fissure margin to identify fissure specific genes. This manuscript is currently in preparation and should be available as a preprint soon.

In some cases, the citations seemed incorrect, e.g. subsection “Transcriptional profiling reveals genetic conservation between chick and human OFC”, Cho and Cepko (2006).

This reference should be to Peters and Cepko (2002) and not Cho and Cepko (2006). We apologise for this error and have corrected the reference. We have also checked the accuracy of other references throughout the manuscript.

Subsection “Transcriptional profiling reveals genetic conservation between chick and human OFC”: what would be the definition of "biologically significant" in terms of differential gene expression? Would this not be dependent on the gene and gene product? Can this be generalized?

We used fold change (FC) analysis to identify biologically-relevant differential gene expression (Log2FC ≥1.5 or ≤-1) in the fissure compared to whole eye. These are established levels to determine biologically relevant gene expression changes between treatments or groups. The “significance” we used applied a false discovery rate (FDR) adjusted p-value of < 0.05 to determine differential expression. However, we also made use of TPM values as a direct read of gene expression levels, choosing to pay more attention to those genes with observed of >12 transcripts observed per million reads. We included all exons for each gene and therefore were not able to determine alternative splicing events.

Subsection “Transcriptional profiling reveals genetic conservation between chick and human OFC”: Table 2 is not showing this. Figure 3B shows the 12 increased only.

Table 3 has been edited to more clearly highlight genes with increased or decreased expression values.

Figure 1A: Is the OFM really the unpigmented region? Could it be the fissure itself?

The region depicted as OFM in Figure 1A (HH.St25) is actually a combination of non-pigmented OFM and the intervening peri-ocular mesenchyme. This is more clearly illustrated in Figure 1—figure supplement 1.

Figure 1B: To what extent is the iris developed at the stage of fissure fusion?

On the request of the reviewer we have added a supplemental figure (Figure 1—figure supplement 2) showing the superficial development of both the iris and pecten with relevance to OFC progression. The chick iris is apparently unique in among vertebrate models of OFC in that the pupillary region (the anterior-most part of the OFM) fuses early during development (~HH.St26) but remains distinct from the main epithelial fusion events described in our work. Indeed, the region posterior to this fused area remains open throughout development and is abutted by the static FP1. This open region in the iris allows a blood vessel to enter the eye to nourish the pecten, as described in our initial Results section. Other than this, the iris does not play a direct role in OFC in the chick but future studies could investigate the mechanisms by which the development of FP1 is arrested at the iris region.

The pecten could be explained more. On the images, it appears as a gap.

We have added data showing the structural development of the pecten in Figure 1—figure supplement 2, and clearly indicate the pecten region in the schematic we have added to Figure 1. However, the recent report by Bernstein et al. illustrated pecten development in the proximal chick optic fissure and highlighted its distinct intercalation fusion mechanism. We did not observe any pecten involvement at the epithelial fusion in the medial and distal OFM. Very little is known about pecten function, and as humans and mammals do not have pecten we did not study its development further.

Could the axes be renamed to proximal and distal?

Yes. We have changed to use of proximal-distal axes throughout

Figure 1C: What is the orientation, magnification and how was the labelling achieved?

These are flat mount confocal microscopy single plane images of an OFM from the stable memGFP chick line and are now more clearly described in the legend. To aid interpretation, we have now added clearer P-D axis orientation labels and scale bars, and a schematic of the developing OFM in the context of the whole eye.

Figure 1E: Were this and Figure 1—figure supplement 1G independent datasets?

Yes. The section data was not used to quantitate PH3A in the developing OFM, rather it was used to orientate the dividing cells within the fissure. This data is now placed in Figure 2—figure supplement 1.

Figure 1F: Is the proliferation increased outside or reduced inside?

This is an excellent question. We were careful not to over-interpret this data but used it to confirm there are fewer proliferating cells within the seam than in the surrounding tissue, and that therefore cell-division is not a major mechanism for seam expansion.

Figure 1—figure supplement 1B: What is seen here? This image showed the pecten and blood vessel at the iris.

We have added to and clarified this data in a new stand-alone Figure 1—figure supplement 2, and improved the descriptions in the figure legends.

Figure 1—figure supplement 1C: Is this taken from flat mounted tissue? What is the orientation?

Yes, this is flat mounted tissue with P-D axis orientation. We have improved the annotation of this figure.

Figure 1—figure supplement 1D: It is stated that at HH26 there is no fusion plate. Could higher magnifications be provided? In Figure 1—figure supplement 1Di it is not totally clear, especially comparing it to Figure 2B.

We have provided increased magnification images of the iris region in Figure 1—figure supplement 1Di. This panel corresponds to the distal-most region of the iris and is adjacent to the iris OFM that remains open throughout chick eye development (see comments above).

Figure 1—figure supplement 1G: The label "G" is missing in the figure. I do not understand the note that at HH29 the seam length was too small to quantify. The length itself was quantified in Figure 1—figure supplement 1F.

The relevant label has been added. The grids used to quantitate PH3A positive-cells in the flat-mounted confocal analyses were too large (200 µm2) to include the small amount of fused seam in this region (100 µm). Nevertheless, the data at FP1 and FP2 included fused seam tissue and show fewer PH3A positive cells than in regions outwith the seam at this stage. Therefore, our data at both time points does include fused seam and shows a reduced amount of proliferating cells than tissue outwith the fused seams.

Table 1: Can an animal have FP1 and FP2? At stage HH29 n 5 were analyzed. 4 showed 2FP 5 showed 1FP. Could this be explained?

We observed subtle variability in the progression of fusion – see detailed response to similar comments by reviewer 1. We defined a single fusion plate as the presence of RPE and neuroepithelial continuum, and two fusion plates were reported where these were separated by visibly fused seam > 0.1mm. Two FPs, one proximal and one distal, were consistently visible during expansion of the fused seam during active fusion stages.

Figure 2:

Without time-lapse analysis, the term dynamics is potentially misleading.

To reflect this comment, we have changed the title of Figure 2 to “Basement membrane remodelling, loss of epithelial characteristics and apoptosis are defining features of Chick OFC.”

Figure 2A: Arrowheads in fusion plate image point to a domain in which the basal lamina is still visible.

The arrowheads have been moved slightly to more accurately indicate the regions of BM dissolution.

Figure 2B: Can the authors be sure that the future NR and RPE are both contributing and are both remodeled? Could there be dynamic in the fissure margins?

We are confident cells from both origins are present in the fusion plate as we have observed both RPE and NR cells contributing to fusion in various experiments (e.g. Figure 2A, Figure 4A and Figure 4 [in human OFM], and in Figure 4—figure supplement 1). Intriguingly, the RPE cells in this region are unpigmented and both cell types lose their normal epithelial organisation. Both of the cell types also express NTN1 (See Figure 4A), suggesting that NTN1 has a role is specifying these cells for fusion, or temporarily prevents them from differentiating into NR or RPE cells until fusion has occurred. We are currently investigating the mechanisms of this process, and believe that it may be this specific role of NTN1 that is essential for normal fusion.

Figure 2C: The image presented to show the apposition is not corresponding to Figure 2B.

We consistently observed A-Casp-3 foci at FP2 in the pre-fusing tissue, FP and adjacently fused seam. We have repeated the cryosection A-Casp-3 immunofluorescence to provide updated and clearer panels for this figure.

Figure 2D: Which domain was used for quantification? What was considered seam and outside seam and what could be considered OFM and outside OFM (towards nasal and temporal directions respectively)?

We apologise this was not made clear in the original submission. The data shown were generated using a fixed-parameter region of interest approach to count apoptotic foci in serial cryosections at HH.St30. The serial sections were cut through the P-D axis and included >200 µm of unfused open fissure and fused seams in full. We have added the following passage to the Materials and methods section for the manuscript to make this clearer: “To quantitate apoptotic foci at the OFM, we used Activated-Casp3 immunofluorescence on serial cryosections of HH.St29-30 OFMs and collected confocal images for each section along the P-D axis. Image analysis was performed by counting A-Casp3 positive foci at the OFM in sequential sections using a region of interest of fixed dimensions of 100 µm2.” The ROI included NR and RPE, but not periocular mesenchyme. We also quantitated A-Casp3 foci in the dorsal, nasal and temporal retina away from the OFM, however we were unable to identify more than >1 foci per eye in many cases and therefore we did not present this data in the study.

Figure 2—figure supplement 1: What is meant by medium?

NF145 protein is also referred to as “neurofilament medium”.

What is the orientation? What is considered central retina?

We are grateful for the opportunity to improve this figure – we have amended this substantially to provide stronger data and to help interpret and orientate the reader, including a brightfield flat mounted OFM. Central retina is now clearly indicated in the schema and refers to a temporal region of optic cup.

Figure 3B: Could the boxes be separated for the different stages? The annotation over the boxed could be misunderstood for statistical data. The annotation (.) should be included in the legend. The legends mention CHDL1 and not CHRDL1.

We have amended the legend to correct these errors. The boxes in b refer to the data presented in Table 3, where the individual stage Log2FC data is shown. We feel the current representation sufficiently shows these genes are consistently enriched during all stages of OFC in the chick.

Figure 3—figure supplement 1A: The images are appreciated. Were the individual regions dissected manually and in independent eyes also for fissure and ventral eye, meaning, is ventral eye including the fissure?

Yes, all were dissected manually and were independent for all sample types. The ventral regions included the fissures to obtain graded expression data.

Figure 3—figure supplement 1B: How was the dorsal data acquired? Is the transcriptional data available?

The dorsal data was similarly segmentally dissected, and the data is available in the excel files supplied as Figure 3—source data 1 and Figure 3—source data 2.

Figure 4A-C: What is the orientation?

The new schematic we have included in the revised Figure 1 will help orientate all of the data in this manuscript and we have added P-D axes in this figure to provide additional clarity.

Figure 4B: What is meant by whole mount? At the FP2, the NTN1 signal seems less intense. Is the arrow positioned correctly? A co-staining with a basal lamina marker would be helpful to conclude the localization.

This is an error in the figure legend, and it should have read as “flat mount”. The tissue was processed as whole OFM for immunofluorescence as described in the methods section. The annotations were incorrectly positioned and have been amended. We agree with the reviewer however we have been unable to find a basal lamina marker that is compatible with the anti-NTN1 antibody in our immunofluorescence method.

Figure 4B’’: What is NT? Is the overall annotation correct? Is NTN1 expressed also in the POM?

This was an error in the figure legend. We have added additional data showing similar NTN1 localisation at FP1. We have also removed the section data as we have optimised the use of the NTN1 antibody for cryosections and have added this new data, which more clearly shows NTN1 localisation in the same regions as the original data. NTN1 was not identified in POM in any of our RNAscope, in situ hybridisation or immunofluorescence analyses.

Figure 4C, D: Higher magnifications are needed and potentially a co-labelling with a basal lamina marker.

In Figure 4C, these tissues were difficult to come by and unfortunately BM markers were not used. However, we have provided higher magnifications to help interpret the data and observe tissue architecture.

In Figure 4D, we have increased the magnification of the images to provide better interpretation of the data.

Figure 4F: What is shown in the second image?

The second image shows a Wild type mouse secondary palate with normal fusion. We have added the label “Palate” to the panels to make this clearer.

Figure 4—figure supplement 1B: The anti-laminin staining looks odd.

These images were taken from the anterior optic fissure margin in the iris region. We appreciate that the Laminin looks to be thicker than is typical and suggest that this is a result of generating a maximum image projection of a confocal Z-stack using sections cut at a slightly oblique angle. Nevertheless, the data in this figure clearly shows a reducing gradient of NTN1 mRNA in post-fused tissue and is representative of 3x biological replicates.

Figure 4—figure supplement 1C: What was the age of the embryos?

HHSt.30 – this information has now been added to the figure legend.

Arrows were used twice for different annotations.

We have updated the figure and now use yellow arrows to define the midline of the OFM in the P-D axis.

In the no antibody control, green signal can be seen. Was there a problem with color channel separation? It was stated that there was no staining.

When merging these raw images to create the composite image in FIJI, some green signal became visible in the NTN1 channel. However, it should be noted that in the single channel fluorescence image for the no-antibody control, there was clearly no signal except for some RBCs. In contrast, the NTN1 positive signal is strong and specifically localised in the samples where antibody was used. This data has been removed from the manuscript and has been replaced by immunofluorescence on cryosections (Figure 4C).

Figure 4—figure supplement 1D: Please add axes for orientation and landmarks for orientation.

We have now added the dorsal and ventral axes to this figure. This data is now in a new stand-alone figure: Figure 4—figure supplement 2.

Figure 4—figure supplement 1F: If the penetrance of a phenotype is given, could it be also supported by genotyping besides by calculation? Was the phenotype consistent with the image in f? The margins seem farther apart? Where the margins in "apposition" correctly? Histology should be performed, or confocal analysis, to address this issue. What is the age of the embryo presented?

At the end of the legend there is a redundant text section.

This has been removed.

It would be good if the data mentioned in subsection “Chick OFC was characterised by the breakdown of basement membranes, loss of epithelial morphology and localised apoptosis” and subsection “Netrin-1 is specifically and dynamically expressed in the fusing OFM” could be also shown.

See previous comments to reviewer 1.

The RNAseq data was shared. It would be best if it was accessible also without too much bioinformatics background.

We are pleased to be able to share the RNAseq data as we believe this is an important resource to ocular and fusion biologists and avian geneticists. The analyses of RNAseq data for all stages is supplied as supplementary Excel files with simple ranked lists that can be downloaded and analysed easily. In addition, the raw RNAseq data will be available form publicly accessible database on publication: NCBI Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo) – accession number GSE84916.

https://doi.org/10.7554/eLife.43877.026

Article and author information

Author details

  1. Holly Hardy

    The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian, United Kingdom
    Contribution
    Formal analysis, Validation, Investigation, Methodology, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4603-7784

  2. James GD Prendergast

    The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian, United Kingdom
    Contribution
    Formal analysis, Methodology
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8916-018X

  3. Aara Patel

    Birth Defects Research Centre, UCL Great Ormond Street Institute of Child Health, London, United Kingdom
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared

  4. Sunit Dutta

    Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Bethesda, United States
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared

  5. Violeta Trejo-Reveles

    The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian, United Kingdom
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared

  6. Hannah Kroeger

    The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian, United Kingdom
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared

  7. Andrea R Yung

    Department of Neurobiology, Harvard Medical School, Boston, United States
    Contribution
    Resources, Visualization
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4053-378X

  8. Lisa V Goodrich

    Department of Neurobiology, Harvard Medical School, Boston, United States
    Contribution
    Resources, Supervision
    Competing interests
    No competing interests declared

  9. Brian Brooks

    Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Bethesda, United States
    Contribution
    Resources, Supervision, Validation, Writing—review and editing
    Competing interests
    No competing interests declared

  10. Jane C Sowden

    Birth Defects Research Centre, UCL Great Ormond Street Institute of Child Health, London, United Kingdom
    Contribution
    Resources, Formal analysis, Supervision, Validation, Writing—review and editing
    Competing interests
    No competing interests declared

  11. Joe Rainger

    The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian, United Kingdom
    Contribution
    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    joe.rainger@roslin.ed.ac.uk
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1091-5100

Funding

Fight for Sight UK (1590/1591)

  • Joe Rainger

Company of Biologists (DMMTF-180520)

  • Joe Rainger

Biotechnology and Biological Sciences Research Council (BB/P013732/1)

  • Joe Rainger

Wellcome (ISSF3)

  • Joe Rainger

Fight for Sight UK (Early Career Investigator Fellowship (1590/1591))

  • Joe Rainger

University of Edinburgh (Institutional Strategic Support Fund)

  • Holly Hardy

Wellcome (Institutional Strategic Support Fund)

  • Holly Hardy

Rosetrees Trust

  • Aara Patel
  • Jane C Sowden

National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children and UCL

  • Aara Patel
  • Jane C Sowden

Great Ormond Street Hospital Children’s Charity

  • Aara Patel
  • Jane C Sowden

Stuart HQ and Victoria Quan Fellow

  • Andrea R Yung

Goldenson Faculty Research Grant

  • Lisa V Goodrich

National Eye Institute (Intramural program)

  • Sunit Dutta
  • Brian Brooks

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

Acknowledgements

We wish to thank Megan Davey at Roslin Institute for academic discussions and support, David FitzPatrick at The MRC IGMM for supporting the RNAseq pilot experiments, Jenny Chen at NIE for technical assistance with zebrafish transgenics, Sadie Schlabach at HMS for help with embryo genotyping and sample collection, Richard Clark at The WTCRF in Edinburgh, and Agnes Gallagher at MRC IGMM for RNA-sequencing.

Ethics

Human subjects: Human foetal eyes were obtained from the Joint Medical Research Council UK (grant # G0700089)/Wellcome Trust (grant # GR082557) Human Developmental Biology Resource (http://www.hdbr.org/). The consent, use and disposal of HDBR samples is regulated by the UK Human Tissue Authority (HTA). The HDBR is a Research Ethics Committee (REC) approved and HTA licenced tissue bank. This means that most research projects based within the UK do not need to obtain their own REC approval.

Animal experimentation: All animal work was carried out in strict accordance with the United Kingdom Home Office Animal (Scientific Procedures) Act 1986. All chicken experiments, breeding and care procedures were approved and carried out under license from the UK Home Office (PPL 7008940 - Prof Helen Sang) and subject to local ethical review by the Roslin Institute AWERB. No regulated procedures were used in this study. Generation and maintenance of memGFP flock were in accordance with annex III of Directive 2010/63 EU and Home Office Codes of Practice. All mouse and zebrafish work was conducted in compliance with protocols approved by the Institutional Animal Care and Use Committee at Harvard Medical School, and at The NIH National Eye Institute. Mice were used from an existing study (Yung et al., Development. 2015). Ntn -/- (Ntn1tm1.1Good, MGI:5888900) and C57Bl/6J animals were maintained on a standard 12hr light-dark cycle. Mice received food and water ad lib and were provided with fresh bedding and nesting daily. All experiments were conducted in agreement with the Animals (Scientific Procedures) Act 1986 and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Pregnant dams were anaesthetised by CO2 asphyxiation and euthanised by cervical dislocation. Embryos were collected at E11.5, E15.5 and E16.5. All embryos were immediately culled on ice by decapitation. All zebrafish embryos/larvae are taken at between 30 hpf-56 hpf and immediately anaesthetised with tricaine methane sulfonate (MS222, 168 mg/l) on ice. Embryos are then euthanised in bleach solution (sodium hypochlorite 6.15%) in water at 1 part bleach to 5 parts water. The larvae remain in this solution at least five minutes prior to disposal to ensure death.

Senior Editor

  1. Marianne E Bronner, California Institute of Technology, United States

Reviewing Editor

  1. Jeremy Nathans, Johns Hopkins University School of Medicine, United States

Reviewers

  1. Stephan Heermann, Freiburg University, Germany
  2. Teri Belecky-Adams

Version history

  1. Received: November 23, 2018
  2. Accepted: June 3, 2019
  3. Accepted Manuscript published: June 4, 2019 (version 1)
  4. Version of Record published: July 2, 2019 (version 2)

Copyright

© 2019, Hardy 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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  1. Holly Hardy
  2. James GD Prendergast
  3. Aara Patel
  4. Sunit Dutta
  5. Violeta Trejo-Reveles
  6. Hannah Kroeger
  7. Andrea R Yung
  8. Lisa V Goodrich
  9. Brian Brooks
  10. Jane C Sowden
  11. Joe Rainger
(2019)
Detailed analysis of chick optic fissure closure reveals Netrin-1 as an essential mediator of epithelial fusion
eLife 8:e43877.
https://doi.org/10.7554/eLife.43877

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