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Regulation of the Drosophila ID protein Extra macrochaetae by proneural dimerization partners

  1. Ke Li
  2. Nicholas E Baker  Is a corresponding author
  1. Albert Einstein College of Medicine, United States
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Cite this article as: eLife 2018;7:e33967 doi: 10.7554/eLife.33967

Abstract

Proneural bHLH proteins are transcriptional regulators of neural fate specification. Extra macrochaetae (Emc) forms inactive heterodimers with both proneural bHLH proteins and their bHLH partners (represented in Drosophila by Daughterless). It is generally thought that varying levels of Emc define a prepattern that determines where proneural bHLH genes can be effective. We report that instead it is the bHLH proteins that determine the pattern of Emc levels. Daughterless level sets Emc protein levels in most cells, apparently by stabilizing Emc in heterodimers. Emc is destabilized in proneural regions by local competition for heterodimer formation by proneural bHLH proteins including Atonal or AS-C proteins. Reflecting this post-translational control through protein stability, uniform emc transcription is sufficient for almost normal patterns of neurogenesis. Protein stability regulated by exchanges between bHLH protein dimers could be a feature of bHLH-mediated developmental events.

https://doi.org/10.7554/eLife.33967.001

Introduction

Proneural bHLH genes play a fundamental role in neurogenesis. Genes from Drosophila such as atonal (ato) and genes of the Achaete-Scute gene complex (AS-C) define the proneural regions that have the potential for neural fate (Baker and Brown, 2018; Bertrand et al., 2002Gómez-Skarmeta et al., 2003). At least two pathways restrain proneural gene activity and neurogenesis. Lateral inhibition mediated by the Notch pathway blocks neural fate determination by extinguishing proneural gene expression from most proneural cells, with only those that maintain proneural gene expression becoming determined as neural precursors (Artavanis-Tsakonas et al., 1999; Bertrand et al., 2002). When the Notch pathway is blocked, entire proneural regions can differentiate as neural cells, whereas ectoderm outside of proneural regions is generally unaffected (Heitzler and Simpson, 1991). The second restriction is the expression of the Inhibitor of DNA binding (ID) proteins, exemplified in Drosophila by Extra macrochaetae (Emc), which contain HLH domains but lack a basic DNA-binding domain (Benezra et al., 1990; Ellis et al., 1990). Emc (or, in mammals, ID1-4) antagonizes functions of proneural bHLH proteins by forming inactive heterodimers with them (Benezra et al., 1990; Ellis et al., 1990; Cabrera et al., 1994; Ellis, 1994; Norton, 2000).

The transcription patterns of proneural genes are highly regulated. Surprisingly, therefore, uniform expression of a proneural gene can be sufficient for a normal pattern of neurogenesis (Rodríguez et al., 1990; Brand et al., 1993; Domínguez and Campuzano, 1993; Usui et al., 2008). It has been suggested that a pre-exisiting spatial distribution of Emc defines a prepattern of competence for proneural gene function that can help define a restricted pattern of neurogenesis even if proneural gene transcription is uniform (Cubas and Modolell, 1992; Usui et al., 2008; Troost et al., 2015). Consistent with this model, Emc protein levels are low in proneural regions and neural precursor cells, potentially sensitizing these cells to respond to proneural proteins (Cubas and Modolell, 1992; Bhattacharya and Baker, 2011; Troost et al., 2015). This suggests that the spatial regulation of Emc expression is important for neural patterning.

An important aspect of Emc expression is the regulatory relationship between Emc and Da. Da is required for Emc protein expression, which has been thought to reflect transcriptional regulation (Bhattacharya and Baker, 2011). On the other hand, Emc limits Da expression, potentially by heterodimerizing with Da to prevent transcriptional autoregulation of da (Bhattacharya and Baker, 2011). Variation in Emc levels may be responsible for all spatial distinctions in Da levels, because in the absence of emc Da levels are both high and uniform in all Drosophila tissues yet examined (Bhattacharya and Baker, 2011). Emc can be considered a negative feedback regulator of Da, and some E- and ID- protein genes regulate one another similarly in mammalian cells (Bhattacharya and Baker, 2011). Because of their low Emc, most proneural regions express higher levels of Da protein, which is expected to further enhance their competence for productive proneural protein function (Cronmiller and Cummings, 1993; Vaessin et al., 1994; Brown et al., 1996; Bhattacharya and Baker, 2011). These reciprocal changes in Emc and Da levels have been seen in proneural cells from all imaginal discs. If Emc expression is restored to proneural regions, changes in Da level do not occur and proneural gene function is affected (Bhattacharya and Baker, 2011).

One reason for regulation of Da expression is that proliferating, non-proneural imaginal disc cells cannot tolerate high levels of Da (Bhattacharya and Baker, 2011). They respond by regulating the Hippo pathway and other genes to repress cell proliferation and survival (Andrade-Zapata and Baonza, 2014; Wang and Baker, 2015a). In mammals also, E-proteins and ID-proteins are critical regulators of cell cycle and of cell senescence, and accordingly are tumor suppressors and oncoproteins, respectively (Perk et al., 2005; Lasorella et al., 2014). In B cells, negative feedback of ID3 on autoregulation of the E-protein TCF3 represents an important barrier to the development of Burkitt’s Lymphoma (Richter et al., 2012; Schmitz et al., 2012). Mammalian E-proteins and ID-proteins are also associated with multiple neurocognitive diseases, including Pitt Hopkins Syndrome, schizophrenia and Rett Syndrome (Wang and Baker, 2015b).

Our initial focus was the cross regulation of Emc and Da. The results did not indicate the homeostatic feedback that had been expected, and instead revealed extensive regulation of Emc expression at the level of protein stability, controlled by its binding partners. In most cells, Emc levels were simply matched to Da levels, apparently as a consequence of stabilization of Emc protein by Da. Once proneural genes were expressed, these alternative dimerization partners affected Da and Emc levels in multiple proneural regions including: the morphogenetic furrow of the eye imaginal disc, corresponding to a stripe of cells expressing the proneural gene ato that sweeps across the imaginal disc progressively defining the onset of retinal differentiation (Treisman, 2013); the wing imaginal disc, where ac and sc expression specifies dorsal and ventral rows of presumptive sensory bristles of the anterior wing margin(Skeath and Carroll, 1991; Skeath et al., 1994); the notum region of the wing disc, which differentiates sensory bristles of the adult thorax(Gómez-Skarmeta et al., 2003). Consistent with the notion that Emc levels were regulated post-translationally, patterning could continue almost normally when the only source of Emc protein was a uniformly-transcribed transgene. Our findings indicate that Emc (and Da) levels don’t define prepattern that precedes regulated proneural gene expression, but are patterned downstream of proneural gene activity. However, proneural proteins are not sufficient to destabilize Emc proteins at all locations. Therefore, other mechanisms must exist that contribute to define the proneural prepattern.

Results

Da and emc protein levels are proportional to da gene dose

Da and Emc show fairly uniform protein levels in most imaginal disc cells but they change dynamically in proneural regions (Cronmiller and Cummings, 1993; Brown et al., 1995; Bhattacharya and Baker, 2011)(Figure 1A–B). We first investigated the non-proneural regions. If Da and Emc levels were kept even by homeostatic negative feedback, they should compensate for modest changes in expression levels. We decided to compare protein levels in cells homozygous for a null allele (‘clones’), heterozygous for a null allele and a wild type allele (non-recombined cells), and homozygous for wild type alleles (‘twin-spots’). Since the da and emc genes map to different chromosomes, parallel mitotic recombination of both chromosomes can generate up to nine different genotypes in the same tissue (1. da-/-; emc-/-, 2.da-/-; emc-/+, 3. da-/-; emc+/+; 4. da-/+; emc-/-, 5. da-/+; emc-/+, 6. da-/+ emc+/+, 7. da+/+; emc-/-, 8. da+/+; emc-/+, 9. da+/+; emc+/+). Comparing Da and Emc expression in those genotypes in parallel should reveal any homeostatic regulation.

Da and Emc are broadly expressed proteins that are modulated in proneural regions.

Panels show immunofluorescence labeling of Drosophila eye imaginal discs (A) and wing imaginal discs (B). Yellow arrowheads indicate the morphogenetic furrow of the eye disc (A) and future anterior wing margin of the wing disc (B). Neural precursor cells arise in those proneural regions and are labeled by Sens (blue). Da (green) and Emc (red) proteins are broadly detected. Da levels are elevated within proneural regions whereas Emc levels are reduced. At the wing margin Da may be elevated in fewer cells than those where Emc is reduced. In addition, higher Emc protein levels are often higher in the equatorial region of the anterior eye disc (white arrowhead, panel A;see text). Genotype: w1118.

https://doi.org/10.7554/eLife.33967.002

Unless otherwise regulated, gene expression is proportional to gene copy number (Ciferri et al., 1969). This was the case for GFP expressed from the [Ubi-GFP] transgene. Mitotic recombination in [Ubi-GFP] transgene heterozygotes led to clones with 0 or two transgene copies in the background of cells with one copy. GFP fluorescence intensity from confocal images was proportional to [Ubi-GFP] copy number (Figure 2A–B). When GFP expression was instead detected using indirect immunohistochemistry with an anti-GFP antibody, this signal was also proportional to [Ubi-GFP] copy number and to GFP fluorescence (Figure 2A–B). Thus, immunostaining and confocal microscopy were consistent with linear detection of protein expression levels in wing imaginal discs.

Figure 2 with 1 supplement see all
Both Da and Emc protein levels depend on da gene dose.

(A) GFP signals in wing imaginal disc mosaic for the ubi-GFP transgene detected simultaneously by native GFP fluorescence (green) and by anti-GFP antibody (magenta). (B) Quantification of native GFP signal and anti-GFP antibody signal in (A), showing their linearity to the gfp gene dose (N = 4). Panels (C–D) show mosaic imaginal disc tissues obtained after mitotic recombination of heterozygous genotypes (see text). Homozygous da and emc mutant clones are negatively marked by GFP (green) or βGal (blue), respectively, within the same wing disc. Reciprocal twin spots are brightly labeled while unrecombined heterozygous cells show intermediate labeling. (C) Anti-Da labeling (red) in cells with different da and emc gene copies. Note that da-/+ emc+/+ (red arrowheads) and da-/+; emc-/+ (yellow arrowheads) cells have indistinguishable levels of Da protein. Cells with higher da gene dose have more Da protein (green arrowheads). (D) Anti-Emc labeling (red) in cells with different da and emc gene copies. Note that da-/+; emc-/+ (red arrowheads) and da-/+ emc+/+ (yellow arrowheads) have similar Emc protein levels, while da+/+; emc-/+ (green arrowheads) cells express higher levels of Emc than da-/+; emc-/+ (red arrowheads) cells do. (E–F) Quantification of Da (E) and Emc (F) antibodies fluorescence intensities. Mean ± SEM is shown (N = 7). X-axes represent the endogenous da gene dose and different colors represent different emc gene dose (E–F). In panel (E), the Da level in da+/-; emc+/+ cells appears greater than half that in to da+/+; emc+/+ cells but this was not reproduced in other studies (see panel G and Figure 2—figure supplement 1C). The Da level in da+/-; emc+/+ cells was not statistically different from that in da+/-; emc+/- cells. Remarkably, da+/+; emc-/+ cells have higher Emc protein levels than da-/+ emc+/+ cells do (p=0.00068, two-tailed t-test). (G) Quantification of Da antibody labeling from mosaic wing discs where clones vary the copy number of the endogenous da locus from 0–2 and independently vary the copy number of an unlinked genomic rescue transgene from 0 to 2 (see text and Figure 2—figure supplement 1E). X-axis represents the endogenous da gene dose and different colors represent da rescue transgene dose. Dashed horizontal lines represent mean Da levels for 1,2,3 and 4 gene copy genotypes. Note that the genomic transgene consistently expresses more Da protein than the endogenous locus. Mean ± SEM is shown,(N = 10). (H–I) Random flip-on clones overexpressing emc using a UAS-emc line are marked by GFP (green). Emc (red) over-expression abolishes Da (blue) upregulation in the morphogenetic furrow of the eye disc (H, yellow arrowheads) and the presumptive wing margin in the wing disc (I, yellow arrowheads), but has no discernible effect elsewhere. Genotypes: (A) hsFLP; Ubi-GFP FRT40/FRT40, (C–D) hsFLP; da3 FRT40/Ubi-GFP FRT40; emcAP6 FRT80/arm-LacZ FRT80, (H–I) hsFLP; UAS-emc 5.3/+; act > CD2>Gal4, UAS-GFP/+.

https://doi.org/10.7554/eLife.33967.003

Next, mitotic recombination was induced in the da-/+; emc-/+ genotype and Da protein levels were quantified in cell populations with different doses of the wild type da and emc genes. Contrary to the idea that uniform levels of Da protein were subject to homeostatic feedback, Da protein levels were instead proportional to da gene dose (Figure 2C,E and Figure 2—figure supplement 1A,C). In the background wild type for emc (i.e. emc+/+), cells with two copies of the wild type da gene had almost twice as much Da protein as cells with only one copy (Figure 2E and Figure 2—figure supplement 1A,C). This did not support the notion that the level of Da expression was buffered by negative feedback regulation from emc. Accordingly, Da protein levels did not change when one copy of emc was removed, that is Da protein levels were indistinguishable in the emc+/+ and emc-/+ backgrounds, so long as da gene copy number was the same (Figure 2C,E and Figure 2—figure supplement 1B,D). In the total absence of emc (i.e. emc-/-), Da levels were elevated, as reported previously (Bhattacharya and Baker, 2011)(Figure 2E and Figure 2—figure supplement 1B,D). We extended these observations using a genomic rescue transgene to vary da copy number from 0 to 4. Extra da gene dose increased Da protein levels linearly (Figure 2G and Figure 2—figure supplement 1E). In summary, Da expression was proportional to da gene dose and unaffected by emc gene dose unless the emc gene was completely deleted. This suggested that Da autoregulation was not significant at the Emc levels normal for imaginal disc cells outside proneural regions.

We used Emc over-expression to look for Da regulation in another way. Outside proneural regions, Da levels were not affected by Gal4-driven Emc (Figure 2H–I). Ectopic Emc only reduced Da levels in the proneural cells of the morphogenetic furrow and wing margin, where endogenous Emc is very low (Bhattacharya and Baker, 2011)(Figure 2H–I). Since Emc is expected to prevent autoregulatory da expression, this result suggests three things: that da does not autoregulate outside of proneural regions; that Emc is already in excess outside of proneural regions; and that feedback from Emc is not what maintains steady Da levels.

Emc protein levels were measured in the same da and emc genetic combinations resulting from mitotic recombination in the da-/+; emc-/+ genotype (Figure 2D,F). Remarkably, Emc levels were also proportional to da gene dose, and cells with the same da gene dose (and therefore the same Da protein level) had indistinguishable Emc protein levels, regardless of whether one or two copies of the emc genes was present (Figure 2F and Figure 2—figure supplement 1B,D). These observations were exemplified by the finding that da+/+; emc-/+ cells had more Emc protein than da-/+; emc+/+ cells, despite the double emc gene dose in the latter (Figure 2D,F). As reported previously, da-/- cells expressed only low levels of Emc protein (Bhattacharya and Baker, 2011)(Figure 2F and Figure 2—figure supplement 1A,C). In summary, Emc protein levels did not seem to be buffered against fluctuations in Da levels, in fact da gene dose, rather than emc gene dose, was the determinant of Emc protein level.

We focused on the wing disc for quantification of Emc and Da levels since it mostly consists of similar cells, developing synchronously (see Materials and methods for details). Similar results were observed in eye discs, although we did not perform quantitative analysis because of the multiple cell types and developmental stages present in eye discs.

Emc is stabilized by Da in S2 cells

Emc dimerizes with bHLH proteins, including Da, through HLH-mediated interactions (Van Doren et al., 1991; Cabrera et al., 1994). Our observations on Emc levels could be explained if Emc protein was unstable except in a heterodimer with Da. To test this, the half-life of Emc was measured in cultured S2 cells.

A V5-tagged Emc open reading frame cloned under the control of an actin promoter was transiently transfected into S2 cells. The expression of the full-length Emc protein was confirmed by western blot analyses. Emc protein half-life was estimated by following a time course after cycloheximide (CHX) addition to block new protein synthesis. Emc was a short-lived protein with half-life around 30 min (Figure 3A,E). Treatment of the cells with the proteasome inhibitor MG132 significantly extended the half-life of Emc to more than 300 min (Figure 3B,E). Therefore, in S2 cells Emc was an unstable protein degraded via the proteasome-dependent pathway.

Emc is unstable alone but stabilized in the presence of Da.

In panels (A-F) proteins from S2 cells were analyzed by western blot following a time course after cycloheximide (CHX) addition. (-) indicates absence of CHX treatment. αTubulin is used as a loading control. (A) Western blot of Emc-V5 show Emc had a short life in S2 cells. (B) Cells are pretreated with a proteasomal inhibitor MG132 to block ubiquitin-proteasome mediated degradation before CHX addition. Emc-V5 degradation is significantly slower. (C) Western blot of Da-Flag show Da is a very stable protein in S2 cells. (D) Co-transfection of Da-Flag with Emc-V5 make Emc a stable protein, while the half-life of Da seems shorter in the presence of Emc. (E) Quantification of Emc-V5 half-lives in panels A,B and D. Mean ± SEM is shown and calculated from 3 to 5 biological replicates(ie independent transfections). All experiments performed were included for quantification. (F) Quantification of Da-Flag half-lives in panels C and D. Mean ± SEM is shown and calculated from 3 to 5 biological replicates. All experiments performed were included for quantification. (G) Expression of Da in the developing wing disc using nub-Gal4 drives a high level of Da protein (green: image underexposes the normal Da expression in surrounding cells in order to record the Da over-expression). Emc protein (magenta) is stabilized in a precisely corresponding pattern (normal Emc expression in surrounding cells underexposed). Note that Da over-expression is likely also to increase transcription of the endogenous emc gene(Bhattacharya and Baker, 2011).

https://doi.org/10.7554/eLife.33967.005

Cotransfection of Flag-tagged Da rendered Emc very stable (Figure 3D–E), increasing the half-life of Emc at least as much as blocking proteasomal degradation (Figure 3E). Similar studies showed that Da itself was a stable protein (Figure 3C,F) although Da stability might be somewhat shortened by costransfection with Emc (Figure 3D,F). The half-life of Da alone was estimated at > 300 min, but that of Da co-transfected with Emc at 139 min (Kiparaki et al., 2015)(Figure 3D,F). It would be interesting to investigate whether this difference affects Da levels in vivo when emc is mutated.

To verify these findings in vivo, Da was overexpressed in wing imaginal discs using the Gal4 system. Da overexpression led to Emc protein accumulation in exactly the same cells (Figure 3G).

Altogether, these data suggested Emc becomes stabilized in Da/Emc heterodimers. This could explain both why Emc protein levels depend on Da levels and are relatively homogenous outside proneural regions, rather than transcriptional regulation of emc by da, as suggested previously(Bhattacharya and Baker, 2011). Emc might also affect Da stability, to a lesser degree.

Ato is required for altering Da and Emc levels in the morphogenetic furrow

Emc instability could also explain its reduction in proneural regions. Da might become limiting where Da also heterodimerizes with proneural proteins. Significantly, Dpp and Hh, the same signals that induce Ato expression in the morphogenetic furrow, are also required to change Da and Emc levels (Greenwood and Struhl, 1999; Curtiss and Mlodzik, 2000; Bhattacharya and Baker, 2011), consistent with the possibility that destabilization of Emc is linked to Ato expression.

Apparently contradicting this idea, however, Da and Emc levels continue to change in the morphogenetic furrow in clones of cells homozygous for the ato1 mutation (Bhattacharya and Baker, 2011). The ato1 mutation contains three coding substitutions, A25T, K253N and N261I (Jarman et al., 1994)(Figure 4A). K253 and N261 lie in the basic domain that is required for DNA-binding (Figure 4A). The ato1 allele has been considered genetically amorphic, since its effects on neurogenesis resemble that of a deletion of the gene (Jarman et al., 1994), but it still encodes detectable protein that is expected to contain a helix-loop-helix domain and therefore may be able to heterodimerize with Da (Figure 4—figure supplement 1A) (Jarman et al., 1995). To characterize a true protein null allele we determined the sequence of ato3, which has the same loss-of-function phenotype as ato1 with respect to neurogenesis but does not encode detectable protein (Jarman et al., 1995)(Figure 4—figure supplement 1B). Sequencing of ato3 genomic DNA revealed a single base-pair change 8278687C > T that introduced a premature stop codon (Q188X) upstream of the bHLH domain (Figure 4A). Therefore even if the ato3 mutant cells contain a protein not detected by the available antibody, this protein should lack the bHLH domain and thus not be able to form heterodimers.

Figure 4 with 1 supplement see all
Proneural proteins are necessary for modulating Da and Emc levels.

(A) Cartoon of Ato protein showing sequence changes in ato1 and ato3 mutants. ‘B’ indicating the basic domain and ‘HLH’ indicating the helix-loop-helix domain of wild type Ato. (B–E) Homozygous ato1 (B and C) or ato3 (D and E) mutant clones are marked by the absence of βGal (green). (B) Emc (magenta) goes down in ato1 clones in the furrow (arrow). (C) Da (magenta) goes up in ato1 clones in the furrow (arrow), at levels comparable to (if not higher) than the normal high level of Da in the furrow. (D) Emc (magenta) is retained in ato3 clones in the furrow (arrow), at levels comparable to the normal Emc levels ahead of the furrow. (E) Da (magenta) fail to upregulate in ato3 clones in the furrow (arrow). (F–G) ato1 MARCM clones are positively labeled by GFP. (F) cells homozygous for ato1 mutant (arrow) upregulatd Da (magenta). (G) Overexpression of Emc in ato1 mutant clones prevents Da upregulation (arrow). (H–I) Homozygous scB57 mutant clones are marked by the absence of GFP (green). (H) Emc (magenta) is retained in cells lacking all the four AS-C genes in the wing margin of the wing discs (arrow). (I) Da (magenta) is not elevated in wing margin cells homozygous for the AS-C mutant (arrow). Genotypes: (B–C) hsFLP; FRT82 ato1/FRT82 arm-lacZ; (D–E) hsFLP; FRT82 ato3/FRT82 arm-lacZ; (F) hsFLP, UAS-GFP; tub-Gal4/+; FRT82 tub-Gal80/FRT82 ato1; (G) hsFLP, UAS-GFP; tub-Gal4/UAS-emc 5.3; FRT82 tub-Gal80/FRT82 ato1; (H–I) Df(1)scB57FRT101/Ubi GFP FRT101; hsFLP/+..

https://doi.org/10.7554/eLife.33967.006

As reported previously, cells homozygous for ato1 mutant downregulated Emc (Figure 4B) and upregulated Da (Figure 4C) in the morphogenetic furrow, like wild type cells (Bhattacharya and Baker, 2011)(Figure 1A). By contrast, cells homozygous for ato3 retained Emc in the morphogenetic furrow (Figure 4D) and failed to upregulate Da (Figure 4E). Thus, ato function does regulate Da and Emc expression levels in the morphogenetic furrow, but independently of aspects of ato function altered in the ato1 allele, which behaves as a null allele for neurogenesis. We have been unable to express Ato in S2 cells and therefore could not test whether Ato destabilizes Emc directly or by sequestering Da.

To gain further insight into the upregulation of Da that occurs in ato1 clones, we performed MARCM (Lee and Luo, 1999; Lee and Luo, 2001) experiments to overexpress Emc in ato1 mutant cells. Unlike plain ato1 mutant cells (Figure 4C,F), cells homozygous for ato1 and also overexpressing Emc failed to upregulate Da and maintained pre-existing Da levels (Figure 4G). This suggests that Da upregulation is bHLH-mediated, for example by transcriptional autoregulation of the da gene, or by greater stability of Da-Da and Da-Ato1 dimers in the absence of Emc.

AS-C is required for altering Da and Emc levels in wing disc proneural regions

Like the morphogenetic furrow, proneural cells of the anterior wing margin also elevate Da and reduce Emc (Bhattacharya and Baker, 2011) (Figure 1B). When all the four AS-C bHLH genes were deleted, Da was no longer elevated at the wing margin (Figure 4I) and Emc was not downregulated (Figure 4H). Thus AS-C gene function regulates Da and Emc levels in the anterior wing margin, as ato does in the eye disc.

It has been reported that Emc regulation is independent of AS-C in the notum primordium of the wing disc (Cubas and Modolell, 1992; Troost et al., 2015). These studies used the viable mutation sc10-1, which deletes ac but not l’sc or ase, and truncates the C-terminus of Sc after the penultimate residue of the bHLH domain (Villares and Cabrera, 1987; Rodríguez et al., 1990). Although sc10-1 behaves genetically as a mutation of both ac and sc, it has the potential to encode a truncated Sc protein that includes much of the HLH domain (Villares and Cabrera, 1987). At the anterior wing margin, sc10-1 appeared to present an intermediate phenotype between wild type (Figure 1B) and scB57 (Figure 4H–I), expressing Da but to a lower degree than wild type, and retaining more Emc expression than wild type (Figure 5A), although a clonal analysis would be useful to confirm this impression.

Proneural genes regulate Emc and Da in the notum.

(A) In sc10-1 wing discs, fewer cells are labeled by Sens (green) in the wing margin (yellow arrowheads). Emc (red) and Da (blue) levels are not affected as strongly as in the wild type (compare Figure 1B). (B) In wild type notum, Sens (green) marks single sensory organ precursor (SOP) cells. Emc (red) and Da (blue) proteins are expressed widely, although the SOP cells generally have lower Emc and higher Da (eg yellow arrowheads)(Bhattacharya and Baker, 2011). In addition, Emc protein levels are higher in particular domains (white arrowheads). High-Emc domains lack SOP cells. (C) Homozygous scB57 clones lack GFP. SOP cells with lower Emc(red) and higher Da(blue) were never observed in scB57 clones, although the regions of high Emc were unchanged (white arrowheads). (D) The whole sc10-1 notum lacks Sens positive SOP cells (green). SOP cells with lower Emc(red) and higher Da(blue) were never observed in sc10-1notum, although the regions of high Emc were unchanged (white arrowheads). (A, D) Df(1)sc10-1; (B) w1118; (C) Df(1)scB57FRT101/Ubi GFP FRT101; hsFLP/+.

https://doi.org/10.7554/eLife.33967.008

The notum differentiates a number of innervated bristles derived from individual sensory organ precursor (SOP) cells which express elevated Da and low Emc (Bhattacharya and Baker, 2011). In addition, other more subtle differences in Emc occur (Figure 5B)(Troost et al., 2015). Emc expression is higher in a large domain along the anterior margin, and two small ventral domains located posteriorly and centrally (Figure 5B). Proneural regions, which can be identified by Sca-LacZ (Mlodzik et al., 1990), lie in between these higher Emc domains (Troost et al., 2015). These Emc domains are not affected by sc10-1 (Troost et al., 2015) (Figure 5D) or by scB57 clones (Figure 5C). By contrast, we did not succeed in locating cells lacking Emc at the locations of the missing SOP cells (Figure 5C–D). Although it might be difficult to locate individual cells lacking Emc expression in the absence of any SOP marker, we also did not see cells with higher Da, which would be expected if regulation of Emc and Da was independent of AS-C (Figure 5C–D), suggesting the AS-C may regulate Emc and Da levels in the precursors of thoracic macrochaetae as well as at the anterior wing margin.

Proneural genes regulate Emc levels post-transcriptionally

To further investigate how proneural proteins regulate Emc expression in proneural regions, the regulation of emc transcription was examined. Three enhancer trap lines, emc-GFPYB0040, emc-GFPYB0067 and emc04322 largely recapitulating the mRNA distribution (Figure 6A and data not shown)(Baonza et al., 2000; Baonza and Freeman, 2001; Bhattacharya and Baker, 2009; Spratford and Kumar, 2015). They exhibited reduced expression in the morphogenetic furrow and the anterior wing margin (Figure 6A).

Figure 6 with 1 supplement see all
Atonal regulates Emc expression post-transcriptionally.

(A) emc enhancer trap expression in the eye disc in the emc-GFPYB0067 line. Downregulation in the morphogenetic furrow (arrow) is broader and less complete than seen fpr the Emc protein (compare Figure 1A). (B) ato3 mutant clones are marked by the absence of βGal (green). emc enhancer trap (magenta) is lower in the furrow both inside and outside ato3 mutant clones. Genotypes: (A) emc-GFPYB0067; (B) eyFLP; emc-GFPYB0067, FRT82 arm-lacZ/FRT82 ato3.

https://doi.org/10.7554/eLife.33967.009

In both ato1 and ato3 mutant clones, emc reporter expression remained low in the morphogenetic furrow region (Figure 6B and Figure 6—figure supplement 1). Mutant cells posterior to the furrow also exhibited lower reporter expression (Figure 6B and Figure 6—figure supplement 1), possibly due to eye differentiation being prevented by ato mutations (Jarman et al., 1994). These data indicated ato was not required to repress emc transcription in the morphogenetic furrow. Therefore, regulation of Emc expression by Ato was post-transcriptional, like regulation of Emc expression by Da.

Proneural genes are not sufficient to regulate Da or Emc protein levels

If proneural proteins destabilize Emc by sequestering Da, then ectopic expression of Ato (or AS-C proteins) should have this effect in other, non-proneural regions of imaginal discs. Gal4-mediated Ato expression was driven in clones of cells to test this. When HA-tagged Ato was induced in eye discs or wing discs clones, Da was slightly upregulated but Emc was not reduced (Figure 7A and Figure 7—figure supplement 1A). Similar results were obtained with weaker expression of untagged Ato from a different transgene (Figure 7—figure supplement 1B). The levels of ectopic HA-tagged Ato were generally similar to the endogenous levels in the morphogenetic furrow of wild type eye discs (Figure 7B) and in many cases were sufficient to express scabrous, a general reporter of proneural gene activity (Mlodzik et al., 1990)(Figure 7E). The ectopic Ato levels were somewhat heterogenous, however, with individual clones containing cells with higher and lower Atonal in a salt-and-pepper fashion. We measured the levels of Emc and Da in individual cells with different Ato levels of ectopic Ato, without observing any correlation (Figure 7C–D and Figure 7—figure supplement 1C–D). Interestingly, the GAL4-induced expression level of ectopic Ato was lower in a region spanning the morphogenetic furrow (Figure 7B).

Figure 7 with 2 supplements see all
Ectopic Ato or Sc does not reduce Emc levels.

Flip-on clones expressing ato or sc using act-Gal4 and UAS-ato or UAS-sc lines are marked by GFP (green). (A) Ectopic Ato expression from UAS-ato.ORF-3HA had little effect on Emc levels (red) but slightly elevated Da (blue). (B) Ectopic Ato (red) levels ahead of the furrow were comparable to normal physiological levels in the furrow but failed to induce ectopic neuronal differentiation (Elav: blue). Notably, ectopic Ato levels declined close to the furrow, both anteriorly and posteriorly. (C) Cells with higher (yellow arrowheads) and lower (green arrowheads) levels of ectopic Ato had similar levels of Emc protein. White arrowhead indicates the morphogenetic furrow. (D) Cells with higher (yellow arrowheads) and lower (green arrowheads) levels of ectopic Ato had similar levels of Da protein. White arrowhead indicates the morphogenetic furrow. (E) Ectopic Ato expression activates its downstream target Sca (red) in the eye disc (arrows) but only affected neuronal differentiation (blue) posterior to the furrow (green arrow). (F) Ectopic Sc expression from UAS-sc in wing discs sightly elevated Da (red) in clones and perhaps also Emc (blue) expression. Genotypes: (A–E) hsFLP; act > CD2>Gal4, UAS-GFP/UAS-ato.ORF-3HA; (F) hsFLP; UAS-sc.39/+; act > CD2>Gal4, UAS-GFP/+.

https://doi.org/10.7554/eLife.33967.011

Consistent with previous conclusions (Bhattacharya and Baker, 2011), Ato expression by itself was insufficient to induce premature neuronal differentiation anterior to the morphogenetic furrow (Figure 7B,E). In similar experiments, ectopic Sc expression in wing or eye discs only slightly upregulated Da and mildly increased Emc expression (Figure 7F and Figure 7—figure supplement 2). These results indicated that although proneural genes like Ato and AS-C genes may be required to downregulate Emc, they were not sufficient.

Uniform emc transcription supports neural patterning

To confirm the primacy of post-translational control of Emc protein, we used the Gal4-UAS system to replace endogenous emc expression with ubiquitous transcription under the control of Actin-Gal4 in the background of the embryonic lethal, amorphic genotype emcAP6/emc∆1. High levels of ubiquitous Emc in the absence of the endogenous locus abolished sensory neurons to various degrees in many tissues (Figure 8—figure supplement 1A–F), just like ectopic Emc in the presence of the endogenous locus (Bhattacharya and Baker, 2011). At lower temperatures, lower levels of uniform transcription led to different results. Despite uniform transcription, Emc protein patterns resembled wild type (Figure 8C,F and I). Emc protein was reduced in the morphogenetic furrow, and higher in regions of the notum primordium, and Da protein levels also resembled the wild type (Figure 8C,F and I). One difference, however, was that whereas in wild type higher Emc protein levels were often noticed around the equator near the anterior of the eye disc, a region where emc transcription is positively regulated by Notch signaling (Bhattacharya and Baker, 2009)(Figure 1A), Emc protein levels were uniform here in the flies rescued by uniform emc transcription (Figure 8C). The rescued emc mutants survived to pharate adults, and a small proportion emerged as adults. Both adults and pharate adults exhibited significant rescue of neural patterning. This included almost normal eye development, including the interommatidial bristles (Figure 8B), an essentially normal pattern of thoracic macrochaetae, a spaced pattern of some microchaetae (Figure 8E), and essentially normal pattern of sensory bristles along the anterior wing margin (Figure 8H). Therefore, uniform emc transcription was sufficient for most neural patterning, which did not depend critically on patterns of emc transcription.

Figure 8 with 1 supplement see all
Ubiquitous emc transcription confers normal expression pattern and neurogenesis.

(A) Wild type adult eye showing ommatidia and interommatidial bristles. (B) Actin-Gal4-mediated ubiquitous transcription of emc at 18C in the absence of the endogenous locus gives rise to normal adult eye. (C) Eye imaginal discs from the rescued larvae show almost normal protein patterns, including the downregulation of Emc and upregulation of Da in the morphogenetic furrow, and normal neurogenesis shown by Sens staining. Unlike wild type discs, however, Emc protein levels are not discernibly elevated near the equatorial anterior margin (contrast with Figure 1A). Scattered cells show higher Emc levels. (D) Wild type thorax displays 11 pairs macrochaetae (at least seven pairs are shown here) and evenly-spaced microchaetae. (E) Ubiquitous emc expression gives rise to nearly all macrochaetae. Spaced microchatae are present over some regions. (F) Wing imaginal discs with ubiquitous emc expression elevated Emc in many cells. The number and position of Sens positive SOP cells resemble the wild type notum, and they also also showed higher Da expression (compare Figure 5B). (G) Anterior wing margin from wild type adult flies display mechanosensory and chemosensory bristles. (H). Uniform emc expression gives rise to normal bristles on the anterior and posterior wing margin (wings from this genotype do not inflate properly). (I) Wing imaginal discs from (H) show broad Emc expression with higher levels in scattered cells, more frequently in central portions of the wing pouch. Sens and Da shows normal upregulation in the presumptive anterior wing margin. Genotypes: (A, D, G) w1118; (B–C, E–F and H–I) act > Gal4/UAS-emc 5.3; emcΔ1 FRT80/emcAP6 FRT80.

https://doi.org/10.7554/eLife.33967.014

Discussion

Although proneural gene transcription is highly regulated, uniform proneural transcription still results in a pattern of neurogenesis (Rodríguez et al., 1990; Brand et al., 1993; Domínguez and Campuzano, 1993). A candidate prepattern gene is Emc, a widely-expressed negative regulator of proneural protein function that is down-regulated in proneural regions and neuronal precursor cells (Cubas and Modolell, 1992; Brown et al., 1995; Usui et al., 2008). We, and others, have focused previously on transcriptional regulation of emc (Bhattacharya and Baker, 2009; Bhattacharya and Baker, 2011). Here we report, however, that Emc protein levels were largely determined post-transcriptionally and by its dimerization partners, and also that neuronal patterning was almost normal in the presence of uniform emc transcription. Although other proneural prepatterns may exist, Emc is regulated downstream of proneural genes, not upstream. Our findings also suggest that dynamics of HLH protein heterodimer formation and exchange, and ensuing changes in protein stability, may play important roles in neurogenesis and perhaps other processes regulated by bHLH transcription factors (Figure 9).

Model for HLH protein regulation inside and outside proneural regions.

The top part compares gene transcription in most imaginal disc cells (non-proneural) with that in cells in proneural regions. The bottom part compares the protein species active in these cells. In non-proneural regions (left), only da and emc are transcribed. Emc is short-lived unless dimerized with Da, so that Da levels determine Emc levels. In proneural regions, one or more proneural bHLH genes are activated, and in the morphogenetic furrow emc transcription is repressed. Proneural Ato or AS-C proteins bind to Da and are responsible for Emc degradation. Emc may be degraded after displacement from heterodimers. It is possible that Ato (or AS-C) heterodimerizes with Emc, or Da homodimers are also present, and that these species also have distinct stabilities. Our findings show how the changes in Emc and Da levels that are a feature of in all proneural regions depend on post-translational regulation of HLH protein stability.

https://doi.org/10.7554/eLife.33967.016

Our study first addressed non-proneural regions, where Da levels are uniform and Emc levels are also quite steady. Because da is required for Emc expression, while Emc restrains Da expression, their levels might be maintained by homeostatic feedback (Bhattacharya and Baker, 2011). The predicted feedback mechanism was not born out by experiment, however. The level of Da expression in fact was not buffered against variation in da gene copy number (Figure 2E,G and Figure 2—figure supplement 1C), and not affected by emc gene dose or even Emc over-expression (Figure 2E,H–I and Figure 2—figure supplement 1D). It seems that uniform Da levels outside proneural regions reflect uniform transcription from the simple da proximal promoter region, with no contribution from the Da-dependent da transcription.

The matching of Emc protein levels with levels of Da led us to discover that Emc protein was short-lived in S2 cells unless Da was co-expressed (Figure 3E). We hypothesize that the Emc protein level in vivo is significantly influenced by protein stability and depends on the amount of Da protein available to form heterodimers. Emc and Da proteins may approach 1:1 stoichiometry in imaginal disc cells, with most (if not all) Da and Emc molecules existing as heterodimers. This simple model could explain the lack of evidence for Da-dependent da transcription, and the failure of da mutations alone to enhance growth, even though da can inhibit growth in the absence of emc (Bhattacharya and Baker, 2011), because Da is heterodimerized with Emc outside of proneural regions (Figure 9). Dependence of Emc on Da for stability probably breaks down above a certain threshold, since it is possible to achieve higher Emc levels in over-expression experiments, and for strong Emc over-expression to affect development (Figure 8—figure supplement 1C–E)(Baonza et al., 2000; Adam and Montell, 2004).

The finding that Emc stability depended on Da prompted us to re-evaluate the effects of other potential heterodimer partners. We showed that proneural bHLH proteins were required for reducing Emc levels in the morphogenetic furrow of the eye disc, anterior wing margin, and in SOP primordial of the notum (Figure 4D,H and Figure 5C–D). Previous studies of the eye had drawn the opposite conclusion, because of studies of an ato mutation that encodes a protein expected to lack a functional DNA binding domain but that could contain an intact HLH domain (Figure 4A)(Bhattacharya and Baker, 2011). Because changes in Emc and Da levels correlated with expression of an Ato HLH domain, which mediates dimerization, it is likely to be changes in heterodimer partners that altered the levels of Da and Emc proteins in the morphogenetic furrow. We also saw that changes in Emc and Da levels that occur at the anterior wing margin (Figure 4H–I) depended on the AS-C, and the same may be true for the SOP cells of the thorax (Figure 5C). Another group reported that some aspects of the Emc pattern in the notum primordium were independent of AS-C(Troost et al., 2015). Although we confirmed this finding using clones of an AS-C deletion (the sc10.1 genotype used before might encode a mutant Sc protein that perhaps could heterodimerize), these patterns reflect mainly regions where Emc is higher than elsewhere. We would describe three levels of Emc in the notum: regions of highest Emc, which do not include proneural regions and where Da expression is not modified; SOP cells, where Emc is reduced and Da elevated; the rest of the notum, where Emc and Da levels are relatively uniform and resemble those of the wing pouch or eye disc. Proneural regions (as defined by Sca-LacZ expression) lie within the latter regions. In our view, the notum region of the wing disc differs from other imaginal discs in that Emc and Da levels are not altered in proneural regions, but only in neural precursor cells themselves, where these changes depend on AS-C. It is worth mentioning that regions of higher Emc are not unique to the notum: the eye disc often has higher Emc along the equatorial region of the anterior eye disc, where it is known that higher emc transcription is induced by Notch signaling (Figure 1A)(Bhattacharya and Baker, 2009).

Despite the importance we demonstrate for proneural proteins in regulating Emc, ectopic Ato or Sc proteins were not sufficient to lower Emc levels prematurely or outside of neurogenic regions (Figure 7A,F, Figure 7—figure supplement 1 and Figure 7—figure supplement 2). In proneural regions, proneural proteins may have different properties from the proteins expressed ectopically in other cells. Interestingly, ectopic expression of either Emc or Ato achieved lower protein levels in the vicinity of the morphogenetic furrow (Figure 2H and Figure 7B).

Our findings challenge the view that Emc levels define a prepattern for neurogenesis sufficient to impose a normal pattern of neurogenesis on a uniform proneural expression pattern, because we found that most variation in Emc levels was downstream of proneural genes. Consistent with this, uniform emc transcription was sufficient for most patterns of Emc and Da protein expression and most neural patterning, showing that if Emc contributed to a proneural prepattern, it was not essential for it. This result parallels the earlier discovery that almost normal thoracic neurogenesis can occur in the presence of only uniform AS-C transcription (Rodríguez et al., 1990; Usui et al., 2008), and suggests that the basis for the neural prepattern could lie elsewhere, for example in post-translational modification of HLH proteins(Baker and Brown, 2018). When Emc is expressed ectopically, we do see that Emc protein levels accumulate differently in some locations (Figure 8), and this is seen for proneural proteins also, consistent with undescribed factors that determine expression level of these proteins. It can’t yet be ruled out, however, that transcriptional regulation of proneural genes and of emc each provide redundant patterning information, because we have not investigated whether neural patterning would be normal if both emc and proneural genes were transcribed uniformly. In addition, even though the emc expression pattern may not be the source of prepattern, Emc may still be a component of the mechanism, since it clearly does suppress neural differentiation at inappropriate locations, which in many cases are locations where ectopic proneural genes seem unable to destabilize Emc.

Like Emc, mammalian ID1, ID2 and ID3 proteins are also short-lived proteins degraded through the ubiquitin proteasome pathway and there is evidence they can be stabilized by heterodimerization with E-proteins (Deed et al., 1996; Bounpheng et al., 1999; Lingbeck et al., 2005). Our studies of the simpler Drosophila system indicate that in most cells the E-protein Da is the major single determinant of ID protein stability. It is possible this will be found to be the case in mammalian cells also, which typically express multiple E-proteins and ID-proteins, although it might be necessary to examine cells that lack all mammalian E-proteins. A recent study suggested that Da is made less stable by heterodimerization with Sc, and that Da-Sc heterodimers also affect Enhancer-of-split protein stability and vice versa (Kiparaki et al., 2015). Thus, the equilibrium and dynamics of HLH protein dimerization and stability must change when transcription of proneural bHLH genes begins and ends when proneural regions are established and decay, and may also be affected by the Notch signaling that induces expression of bHLH proteins from the E(spl)-C. It would be of interest, in the future, to investigate the binding properties HLH dimer species and the dynamics of their mixtures more quantitatively than has been done in the past.

Materials and methods

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
gene (Drosophila melanogaster)emcFlyBase: FBgn0000575
gene (D. melanogaster)daFlyBase: FBgn0267821
gene (D. melanogaster)atoFlyBase: FBgn0010433
gene (D. melanogaster)scFlyBase: FBgn0004170
genetic reagent (D. melanogaster)da[3]PMID: 3802198
genetic reagent (D. melanogaster)emc[AP6]PMID: 7947322
genetic reagent (D. melanogaster)emc[Δ1]this study
genetic reagent (D. melanogaster)act > CD2>Gal4, UAS-GFPPMID: 9053304
genetic reagent (D. melanogaster)ato[1]PMID: 8196767
genetic reagent (D. melanogaster)ato[3]PMID: 7635049
genetic reagent (D. melanogaster)emc-GFP[YB0040]PMID: 17179094
genetic reagent (D. melanogaster)emc-GFP[YB0067]PMID: 17179094
genetic reagent (D. melanogaster)P{PZ}emc[04322]PMID: 9529525
genetic reagent (D. melanogaster)Df(1)sc[B57]PMID: 2510998
genetic reagent (D. melanogaster)Df(1)sc[10-1]PMID: 3111716
genetic reagent (D. melanogaster)UAS-HA-daPMID: 25579975
genetic reagent (D. melanogaster)UAS-ato.ORF-3HAPMID: 23637332
genetic reagent (D. melanogaster)UAS-scPMID: 8978666
genetic reagent (D. melanogaster)UAS-ato-4PMID: 8324823
genetic reagent (D. melanogaster)UAS-emc5.3PMID: 10804180
cell line (D. melanogaster)S2DGRCStock Number: 6
antibodyanti-βGal (mouse)DSHB40-1a(1:100)
antibodyanti-ElaV (rabbit)DSHB7E8A10(1:50)
antibodyanti-Da (mouse)PMID: 3802198(1:200)
antibodyanti-Emc (rabbit)Y.N. Jan(1:8000)
antibodyanti-Ato (rabbit)PMID: 8196767(1: 50000)
antibodyanti-Sca (mouse)PMID: 8622662(1:200)
antibodyanti-GFP (rat)Nacalai TesqueGF090R(1:1000)
antibodyanti-Sens (guinea pig)PMID: 10975525(1:50)
antibodyanti-V5 (mouse)Invitrogen46–0706(1:5000)
antibodyanti-Flag (mouse)SigmaF3165(1:8000)
antibodyanti-Tubulin (mouse)Abcamab18251(1:5000)
antibodyanti-Tubulin (rabbit)Abcamab7291(1:5000)
antibodyanti-HA (rabbit)Cell Signaling TechC29F4(1:1000)
antibodyanti-HA (mouse)Roche12CA5(1:1000)
recombinant DNA reagentEmc-V5 (plasmid)this study
recombinant DNA reagentDa-Flag (plasmid)PMID: 25694512
recombinant DNA reagentGFP (plasmid)PMID: 25694512

Mosaic analysis

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Mosaic clones were obtained using FLP/FRT mediated mitotic recombination(Xu and Rubin, 1993). Larvae were subjected to heat shock for 1 hr at 37°C at 60 ± 12 hr after egg laying, and dissected 72 hr after heat shock. To make ‘flip-on’ clones, larvae were heat shocked for 30 min instead. All flies were maintained at 25°C unless otherwise stated.

Drosophila Strains

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w1118, da3 (Cronmiller and Cline, 1987); emcAP6(Ellis, 1994); emcΔ1(an apparent null allele corresponding to a 1 bp deletion that frameshifts the open reading frame in the 5th codon whose characterization will be described elsewhere); act > CD2>Gal4, UAS-GFP (Pignoni and Zipursky, 1997), Neufeld, Neufeld et al., 1998); UAS-emc5.3 (Baonza et al., 2000); ato1(Jarman et al., 1994); ato3(Jarman et al., 1995); UAS-HA-da (Wang and Baker, 2015a); UAS-ato.ORF-3HA (Bischof et al., 2013); UAS-sc(Parras et al., 1996); emc-GFPYB0040 and emc-GFPYB0067 (Quiñones-Coello et al., 2007); P{PZ}emc04322 (Röttgen et al., 1998); Df(1)scB57(González et al., 1989); UAS-ato-4(Jarman et al., 1993); Df(1)sc10-1 (Villares and Cabrera, 1987).

Immunohistochemistry and image processing

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Antibody staining was performed as previously described(Baker et al., 2014). The following primary antibodies were used: mouse anti-βGal (1:100, DSHB 40-1a), rabbit anti-βGal, rat anti-ElaV(1:50, DSHB 7E8A10), mouse anti-Da(1:200)(Cronmiller and Cummings, 1993), rabbit anti-Emc (1:8000, a gift from Y. N. Jan)(Brown et al., 1995), rabbit anti-Ato(1:50000)(Jarman et al., 1994), mouse anti-Sca (1:200)(Lee et al., 1996), rat anti-GFP(1:1000, Nacalai Tesque GF090R), guinea pig anti-Sens (Nolo et al., 2000), mouse anti-HA (1:1000, Roche 12CA5), rabbit anti-HA (1:1000, Cell Signaling Tech C29F4). Seondary antibodies conjugated with Cy2, Cy3 and Cy5 dyes (1:200) were from Jackson ImmunoResearch Laboratories. Multi-labeled samples were sequentially scanned with Leica SP2 or SP5 confocal microscopes. Z-stacks were projected using Max Intensity and processed with ImageJ. Genotypes were identified according to GFP and βGal staining. For quantification of GFP, βGal, Da and Emc levels in mosaic discs, mean fluorescence intensities were measured for all areas of each genotype and averaged for each wing disc. Fluorescence intensities in gfp-/-, lacZ-/-, da-/- and emc-/- genotypes were measured as an estimate of background to be substracted from anti-GFP, anti- anti-βGal, anti-Da and anti-Emc fluorescence intensities. The wing margin and notum regions were excluded from this analysis of the main wing disc.

DNA constructs

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ORFs of each gene were cloned from cDNA of 0–6 hours w1118 embryos to make constructs used in transfection. Emc open reading frame with Kozak sequences were cloned in-frame into pAc5.1/V5-His vector (Invitrogen) to make pAc-Emc-V5 construct. pAc-Da-Flag and pAc-GFP constructs were obtained from was obtained from Dr. Marianthi Kiparaki (Kiparaki et al., 2015).

Cell culture, transient transfection and western blotting

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Drosophila S2 cells obtained from Drosophila Genomics resource Center were cultured at 25°C in Schneider’s Medium supplemented with 10% heat inactivated fetal bovine serum and Penicillin-Streptomycin. Cells were transiently transfected with Effectene Transfection Reagent (Qiagen, Valencia, CA) or TransIT-2020 Transfection Reagent (Mirus, Madison, WI) according to manufacturer’s instructions. Cells were treated with 50 µM MG132 or 50 µg/ml cycloheximide where noted (Kiparaki et al., 2015). Whole cell lysates were collected 48–72 hr after transfection using RIPA buffer (150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM Tris, pH 8.0) with addition of protease inhibitors cocktail (Roche) and phosphatase inhibitors cocktails (Sigma). Total protein concentration was determined using Pierce BCA Protein Assay Kit (ThermoFisher Scientific). Protein lysates were separated on 10–12% homemade SDS–polyacrylamide gels and electrotransferred onto PVDF membranes (Bio-Rad) for following detection by western blotting. The following primary antibodies were used for western blotting: mouse anti-V5 (1:5000, Invitrogen 46–0706), mouse anti-Flag (1:8000, Sigma F3165), mouse anti-Tubulin (1:5000, Abcam ab18251), rabbit anti-Tubulin (1:5000, Abcam ab7291). Secondary antibodies conjugated with IRDye 680RD and IRDye 800CW were used (LI-COR, Lincoln, NE). Membranes were imaged on LI-COR Odyssey scanner and images were quantified in ImageJ.

Sequencing of ato mutant alleles

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Both ato1 and ato3 flies were outcrossed to w1118 flies to obtain ato1/+ and ato3/+ flies. Genomic DNA was isolated from w1118, ato1/+ and ato3/+ flies and PCR products were obtained using primers flanking the endogenous ato locus. Amplified products were gel purified and subjected to Sanger sequencing. Re-sequencing of ato1 confirmed three point mutations (8278198G > A, 8278884G > T and 8278907A > T, numbers represented genomic coordinates on chromosome 3L).

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Decision letter

  1. Hugo J Bellen
    Reviewing Editor; Baylor College of Medicine, United States

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.

[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for submitting your work entitled "Regulation of the Drosophila ID protein Extra macrochaetae by proneural dimerization partners" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife as the findings at this stage.

Specifically, two of the three reviewers, who are all experts in your field, request a significant number of additional experiments to bolster your conclusions. The data and hypothesis have potential, but it is most likely that it will take more than two months to address the concerns and we can therefore not accept the manuscript. Furthermore, from some of the current inconsistencies between data and text pointed out in all three reviews, the consultations suggest that the suggested work may well show far more complexity to the regulatory network that will require a significantly more fine-tuned model. We suggest that you carefully read the reviews and determine if you are willing and able to address these concerns. If you can detail that this is indeed feasible and decide to submit a manuscript to eLife rather than elsewhere, we will be pleased to afresh consider an extensively edited and revised manuscript in which you address all key concerns and decide if it should be reviewed.

Reviewer #1:

Li and Baker report a very interesting set of observations suggesting complex post-transcriptional, and very likely post-translational cross-regulation between Proneural Proteins (Ato, Ac-Sc), E-Proteins (Da) and ID proteins (Emc) in Drosophila. Building upon previous work by the same group, the authors uncover evidence consistent with the idea that heterodimer formation between Emc and Da may compete with heterodimer formation between Da and Ato/Ac-Sc and that this competition to a significant degree determines Emc levels and thus potentially neurogenic regions in the fly PNS.

While the idea of a critical role for post-translational regulation of key proteins in neurogenesis, in this case that of ID proteins, is very interesting and consistent with a growing body of evidence supporting this notion, this study does not provide sufficient evidence for the mechanisms of such regulation and its impact on neurogenesis. Much more work is needed to consolidate the findings, extend them to a level that allows the extraction of a model and understand their consequences for cell fate transitions in the nervous system.

1) The genetic evidence presented is largely consistent with the model, but not entirely. In subsection “Da and Emc protein levels are proportional to da gene dose” it is stated that the evidence in Figure 2 argues against homeostatic feedback regulation because "In the background wild type for emc (i.e. emc+/+), cells with two copies of the wild type da gene had almost twice as much Da protein as cells with only one copy (Figure 2M)". Looking at Figure 2M, this is clearly not the case. The levels of Da protein are far less than double. In contrast in emc-/- cells the levels of Da are more than double the gene dosage. This, in fact, is precisely consistent with homeostatic feedback. On the same page, it is stated, correctly, that Da levels are indistinguishable between emc-/+ and emc+/+. Again, this seems precisely what one would predict from homeostasis: that it buffers against minor changes in the dosage of the regulator. Thus, the very notion the study start with discounting seems to be supported by, rather than contradicted by, this particular set of data.

2) Aside from measuring steady state protein levels in S2 cells, the paper lacks biochemical evidence for the model, particularly for competition between Emc and Proneural Proteins for forming heterodimers with Da. More biochemical and and genetic evidence is needed to demonstrate this model. For example, there are no in vitro competition experiments performed. Another example: the difference in the effect of the two Ato alleles (Ato1 vs Ato3) is intriguing and an important piece of data. It is consistent with the idea that the bHLH domain in needed as opposed to the transcriptional activity, but Ato3 lacks much more than the bHLH domain. A more careful dissection is needed.

3) There is clear evidence in the paper for a complex set of regulatory interactions, because over expression of Ato has no effect on Emc levels and because there is clearly some level of transcriptional control. The paper however provides no data to explain these effects, and how both transcriptional and post-transcriptional mechanisms interact to ensure the precision of the Emc pattern.

4) What are the implications for neurogenesis for interfering with the post-translational regulation of Emc?

Reviewer #2:

This paper addresses the role of Emc (Id in mammals) in patterning proneural domains in Drosophila. Earlier studies have indicated that Emc provides a pre-pattern that is decoded by Da to direct the pattern of proneural gene expression. The proposed decoding mechanism involved positive regulation of proneural factors by Da, titration (inhibition) of Da by Emc and Da auto-regulation. The data presented here challenges this view.

First, the authors nicely show that the physiological levels of Emc proteins are set by the level of Da proteins (Figure 2; this effect does not seem to be mediated by a transcriptional regulation of emc by Da, Figure 7) and that Emc is stabilized by Da in S2 cells (Figure 3). They further show that the presence of a mutant/inactive Ato protein is sufficient to up-regulate Da, possibly via a stabilization mechanism (not further investigated here), and to down-regulate Emc (Figure 4). How Ato down-regulates Emc remains, however, unclear. Based on the findings reported in Figure 2 and Figure 3, a possible mechanism would be a competition for the binding of Da, resulting in the destabilization of free Emc. However, since ectopic Ato (or Sc) is not sufficient to down-regulate Emc (Figure 5), this mechanism is not favored. Another possibility would be that Ato (or Sc) represses the transcription of the emc gene. However, Ato is not required to repress emc transcription in the morphogenetic furrow. Thus, how Emc is down-regulated in proneural domains at the mRNA and protein levels remain unclear. Also, the functional significance of this down-regulation for proneural patterning is not clear (more generally, whether the patterned expression of emc is relevant for its function remains to be addressed; see below). Finally, the authors suggest that Da is required to activate (or at least maintain) emc gene expression, but only in a region close to/ahead of the furrow in the eye disc (Figure 7; emc is then repressed in the furrow; and Da does not seem to be required in wings).

In summary, this manuscript reports one interesting finding i.e. Da stabilizes Emc and sets Emc protein levels. However, the extent to which this mechanism contributes to the endogenous Emc protein pattern is not entirely clear to me. This manuscript also reports a new model (Figure 8) suggesting that the pattern of Ato/Sc is established in response to positional information independently of the Emc pattern and that Emc protein levels are merely a read-out of proneural activity. In this model, Emc appears to merely keep Da inactive in the absence of proneural factors. This model is supported by the data but remains largely untested.

Essential revisions:

1) To further test the relevance (or lack thereof) of the expression pattern of the emc gene, the authors should test whether ubiquitous expression of emc (at the gene level; i.e. ubi-emc in emc mutants) results in a normal Emc protein pattern and provides proper emc activity. Also, the model predicts that changes in the level of expression of the emc gene should have no significant effect on proneural patterning. This should be tested.

2) A potential limitation of this study is that it challenges a view proposed by several groups (Modolell, Simpson, Klein) who worked on a slightly different context (proneural clusters of the dorsal thorax). It could be appropriate to revisit this view also in this context (or at least justify why conclusions obtained in the eye necessarily applies to another developmental context).

Reviewer #3:

Li and Baker present a thorough analysis of the interdependence of the expression levels of two interacting HLH proteins, Da (a DNA-binding activator) and Emc (a non-DNA-binding inhibitor of Da). Earlier work from the same group had shown that Da activates emc expression, which then feeds back to repress da transcription by preventing Da from activating a da autoregulatory enhancer (Bhattacharya et al., 2011). The complex Emc expression pattern was thought to provide a prepattern for neural differentiation, in the sense that Da/proneural activators would be more active in regions of lowest emc expression. The authors claim that emc does not set the prepattern for neurogenesis, but instead its complex expression pattern depends to a great extent on the activity of Da and its proneural patterns.

The crucial findings of Li and Baker are:

1) Da protein levels are proportional to the da gene dosage in the range of 0-2. However, Emc protein levels do not similarly reflect the emc gene dosage (range of 0-2), instead they also reflect the da gene dosage! Da levels are not affected by emc gene dosage in the range of 1-2, but are greatly upregulated in emc null clones, due to the above-mentioned release of inhibition.

2) The unexpected dependence of Emc protein levels on Da protein levels probably comes from the dramatic stabilization of Emc by Da, as determined by half-life measurements.

3) The upregulation of Da and concomitant downregulation of Emc seen in proneural regions of the eye and wing margin need the expression of proneural bHLH proteins with intact HLH domains, but not necessarily DNA binding activity. This suggests that a proneural partner displaces Emc from Da, which leads to degradation of Emc and auto-activation of Da. Some unknown factor must also contribute to this process, since ectopic expression of proneural genes in non-proneural regions cannot recapitulate this effect on Emc and Da levels.

4) emc transcription is not affected by ato or da mutations (exception: there is a moderate effect of da mutations only in the posterior part of the eye disk).

This three-way post-transcriptional interplay among Da, Emc and proneurals represents an important in vivo demonstration of properties of HLH proteins that had only been shown in vitro. It also paves the way for deciphering the biological consequences of these important HLH heterodimerization properties, adding the interesting twist of stability regulation. That said, I have a few concerns that I will list below:

a) The authors focus their "narrative" on how Emc does not represent a proneural prepattern, but rather reflects a proneural "response". I would say that it is both. They themselves show that the complex RNA pattern of emc does not depend (much) on Da or proneural factors, so this is patterned by something else. Granted, this complex pattern is greatly "evened out" at the protein level, thanks to the mutual interactions described by the authors. Still some proneural cluster "lows" in Emc protein (e.g. the morphogenetic furrow) have a clear proneural-independent transcriptional component, so they are expected to predispose these areas to become neurogenic, namely act as a prepattern. I would change the narrative in such a way to clarify that emc contributes to the prepattern (it cannot be the entire prepattern, since there is still some patterning of bristles in the absence of emc), but at the same time it is itself modulated by proneural activity (at the protein level). After all, whether Emc is or is not a prepatterning factor seems of lesser importance than the dissection of its molecular mode of action.

b) They should point out that the previously reported activation of emc transcription by da was a misinterpretation of the destabilization of Emc in da null clones and that instead Da only affects transcription of emc behind the morphogenetic furrow (and that only modestly, as shown in Figure 7—figure supplement 1).

c) They should point out that the dependence of Emc on Da levels does break down after a certain threshold. Overexpression of emc in Figure 2O-R causes an enormous increase of Emc protein without concomitantly increasing Da. Probably the degradation machinery of Emc is saturated. Intriguingly Da may also play some role even at these unnaturally high Emc levels, since in the furrow, where Da is engaged by Ato, this excess Emc seems to accumulate less. Therefore, its high accumulation in non-proneural regions is not solely because of saturation of the degradation machinery, since the latter seems to work better in the furrow.

d) The language needs to be improved. There are many places where the text is hard to follow and the meaning difficult to extract. Probably, with a thorough rewriting, the scientific points (a-c) above will also become clearer.

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Thank you for submitting your article "Regulation of the Drosophila ID protein Extra macrochaetae by proneural dimerization partners" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor, Hugo Bellen, and K VijayRaghavan as the Senior Editor. The reviewers have opted to remain anonymous.

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

Reviewer #1:

The revised manuscript is much improved, especially as it is more focused on the main message. The new experiment showing that uniform moderate levels of EMC driven by an exogenous promoter rescue neurogenesis certainly support the conclusion that post-transcriptional modulation of EMC is key to patterning neurogenesis. The new data showing that Da protein levels increase linearly with gene dosage are more convincing. Finally, the in vivo data showing that availability of Da regulates EMC protein levels are very nice.

What remains rather circumstantially substantiated in my view is their mechanistic model of the regulation of EMC protein levels by Proneural proteins by competition for Da. I agree with the authors that the few data they have in this regard do not contradict this notion, and I in fact like the idea mostly because it fits the strong response of EMC to Da levels within the proneural domain under wild type conditions. I am surprised that they were unable to express Atonal in S2 cells to test their model more directly because expression of Ato in vitro has been reported previously.

The idea that the proneural pre-pattern depends on Da and Proneural proteins, not ID proteins is novel and interesting. Furthermore, it fits nicely with the emerging paradigm that post-transcriptional/translational modification of bHLH proteins is a key determinant of neurogenesis. The authors could do a better job referencing that literature in their Discussion section.

In summary, the authors' model is the simplest and most parsimonious explanation of their data, but their advance of a specific mechanism for their observations remains relatively weak.

Reviewer #2:

This paper challenges the commonly held view that the ID factor Emc acts as a prepattern regulator of proneural activity during neurogenesis in Drosophila. Emc is known to heterodimerize with the E protein Da, thereby blocking its DNA-binding activity, hence its ability to regulate proneural gene expression. This paper reports on an alternative model whereby Da would stabilize Emc in the absence of proneural factor and proneural factors, patterned independently of Emc activity, would negatively regulate this stabilizing activity of Da towards Emc to generate the observed pattern of Emc protein accumulation.

Several interesting observations support this model. First, in contexts of low proneural activity (S2 cells, wing pouch), the accumulation levels of Emc proteins appear to depend on Da levels. The analysis of Emc and Da levels in da, emc clones is really nice (Figure 2) and the stabilization of Emc by Da in S2 cells is clear (Figure 3).

Second, in the proneural region of the eye, Ato is required for the accumulation of Da and the down-regulation of Emc in neural cells (Figure 4). The analysis of the ato1 and ato3 mutations is convincing. The observation that Emc is stabilized in the absence of Ato (Figure 4D and Figure 6) is interesting. However, how Ato destabilizes Emc is not understood: Ato does not appear to be sufficient to destabilize Emc (Figure 7). It is also not clear whether Ato acts indirectly via Da.

Third, non-patterned expression of the emc gene largely rescues the emc mutant phenotype (Figure 8), indicating that Emc transcription does not provide key patterning cues.

In summary, the strength of this paper is that it convincingly shows that the current model needs to be revised. Further experiments are, however, needed to strengthen the alternative model proposed here.

Essential revisions:

1) It would be nice to confirm the changes seen in Da levels by IF using Western blots, for instance by comparing Da levels in emc+/+ vs emc+/- discs (and Emc in da+/+ vs da+/- tissues). Please test.

2) Figure 2E: it is not clear whether the increased Da levels seen in emc mutant cells result from protein stabilization (indicative of Emc targeting Da for degradation, as suggested by Figure 3F) or from increased transcription (due for instance to ectopic/premature expression of proneural factors). Please clarify.

3) Figure 3G: please control that nub>da does not affect emc gene expression (in situ, enhancer-trap…)

4) Subsection “Ato is required for altering Da and Emc levels in the morphogenetic furrow”. The conclusion that Da up-regulation is transcriptional and bHLH-mediated needs to be supported by experimental evidence. Could the authors exclude the possibility that Emc also targets Da for degradation (see Figure 3F)?

5) Subsection “AS-C is required for altering Da and Emc levels in wing disc proneural regions”: This conclusion is not convincing. I find it hard to compare Da levels based on different IF experiments. Clonal analysis is required.

6) Subsection “AS-C is required for altering Da and Emc levels in wing disc proneural regions”: Whether AS-C regulates Emc/Da levels in sensory cells is difficult to study in the absence of these cells. The data shown in Figure 5B-D are not convincing.

7) Figure 7B: heterogenous levels of Ato appear to be detected in the non-proneural region (Figure 7B). Therefore, a per-cell quantification of the relative Emc/Da levels is needed (Figure 7A). It would be nice to test whether Ato levels correlate with Emc/Da levels. Also, what is causing the observed heterogeneity in Ato levels?

8) Figure 7—figure supplement 1A: the clone of act>ato cells (right) exhibits high Emc and high Da levels. This seems to contradict the model whereby Ato competes Emc for binding Da. Please comment.

9) Figure 8: what is the phenotype of emcD1/emcAP6 flies? are these mRNA null? what is the emc mRNA pattern in the mutant and rescued eye discs? Is the latter similar to the protein pattern shown in 8C? does the UAS-ems construct contain the 3'UTR of the emc gene? Does it matter? Also, is the emc mutant combination used in Figure 8 protein null?

https://doi.org/10.7554/eLife.33967.019

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

Specifically, two of the three reviewers, who are all experts in your field, request a significant number of additional experiments to bolster your conclusions. The data and hypothesis have potential, but it is most likely that it will take more than two months to address the concerns and we can therefore not accept the manuscript. Furthermore, from some of the current inconsistencies between data and text pointed out in all three reviews, the consultations suggest that the suggested work may well show far more complexity to the regulatory network that will require a significantly more fine-tuned model. We suggest that you carefully read the reviews and determine if you are willing and able to address these concerns. If you can detail that this is indeed feasible and decide to submit a manuscript to eLife rather than elsewhere, we will be pleased to afresh consider an extensively edited and revised manuscript in which you address all key concerns and decide if it should be reviewed.

In addition to a complete rewrite of the manuscript, we have added three additional experiments requested by reviewers and which strengthen the paper: (1) We extended the analysis of Da protein levels in da gene doses from 0-2 to 0-4; (2) We showed that uniform transcription of emc is sufficient to rescue nearly all gene expression patterns and most of the emc mutant phenotype; (3) We explored the regulation of Emc and Da in the notum region of the thorax and explained how our findings are related to previously-published models.

Reviewer #1:

Li and Baker report a very interesting set of observations suggesting complex post-transcriptional, and very likely post-translational cross-regulation between Proneural Proteins (Ato, Ac-Sc), E-Proteins (Da) and ID proteins (Emc) in Drosophila. Building upon previous work by the same group, the authors uncover evidence consistent with the idea that heterodimer formation between Emc and Da may compete with heterodimer formation between Da and Ato/Ac-Sc and that this competition to a significant degree determines Emc levels and thus potentially neurogenic regions in the fly PNS.

While the idea of a critical role for post-translational regulation of key proteins in neurogenesis, in this case that of ID proteins, is very interesting and consistent with a growing body of evidence supporting this notion, this study does not provide sufficient evidence for the mechanisms of such regulation and its impact on neurogenesis. Much more work is needed to consolidate the findings, extend them to a level that allows the extraction of a model and understand their consequences for cell fate transitions in the nervous system.

1) The genetic evidence presented is largely consistent with the model, but not entirely. On page 8 it is stated that the evidence in Figure 2 argues against homeostatic feedback regulation because "In the background wild type for emc (i.e. emc+/+), cells with two copies of the wild type da gene had almost twice as much Da protein as cells with only one copy (Figure 2M)". Looking at Figure 2M, this is clearly not the case. The levels of Da protein are far less than double. In contrast in emc-/- cells the levels of Da are more than double the gene dosage. This, in fact, is precisely consistent with homeostatic feedback. On the same page, it is stated, correctly, that Da levels are indistinguishable between emc-/+ and emc+/+. Again, this seems precisely what one would predict from homeostasis: that it buffers against minor changes in the dosage of the regulator.

Thus, the very notion the study start with discounting seems to be supported by, rather than contradicted by, this particular set of data.

Reviewer 1 complained that Figure 2M (now Figure 2E in the revised submission) shows that the da+/+ genotype encodes less than twice the amount of Da protein as the da(+/-) genotype, supporting the idea of feedback regulation of Da levels rather than discrediting it.

There are multiple responses to this criticism.

The First is that although the reviewer is correct about the relative values of these particular two datapoints, their conclusion is not supported by the rest of the quantification in this and related Figures. We have now measured the relative levels of Da protein in da[+/+] versus da[+/-] cells in 4 independent experiments in the emc[+/+] genotype (Figure 2E, Figure 2—figure supplement 1C, and twice in Figure 2G) as well as once in the emc[+/-] genotype (in Figure 2E). In all the other cases the Da protein level was clearly doubled by doubled Da gene dose. In addition, 3x and 4x Da gene dose has now been added (Figure 2G) and also look linear. In addition, it is not the level for da[+/+] in Figure 2E that was unexpectedly low, it is that the level for da[+/-] was anomalously high. Although the da[+/-] emc[+/+] datapoint in Figure 2E is a little higher than in the other da[+/-] examples, it is not statistically different from them. The same graph shows that the Da level in da[+/ +] is identical to the Da level in da[+/+] emc[+/-]. In fact, we suspect the reviewer might have missed the latter datapoint because it is partially covered by the former and have changed the format of the graphs to help make this clearer. These da[+/+] genotypes also have indistinguishable Emc protein levels (Figure 1F) so how can the identical Da levels be evidence for feedback? It is unfortunate that the First graph of Da protein levels encountered is this one that contains an outlier, and we experimented with different orders of presentation, or moving Supplemental figures into the main Figure, but it is just more logical for the argument to present Figure 2E First, so we have kept the order of presentation and added a comment to the Figure legend.

The second response is to emphasize that while these gene dose experiments certainly generated our hypothesis, this was then tested in a very stringent assay when we over-expressed Emc without seeing any diminution in Da levels in nonproneural cells (Figures 2H,I). This lack of effect of even very high Emc levels is inconsistent with the notion of continuous transcriptional feedback that maintains constant Da levels (please see subsection “Da and Emc protein levels are proportional to da gene dose”).

2) Aside from measuring steady state protein levels in S2 cells, the paper lacks biochemical evidence for the model, particularly for competition between Emc and Proneural Proteins for forming heterodimers with Da. More biochemical and and genetic evidence is needed to demonstrate this model. For example, there are no in vitro competition experiments performed. Another example: the difference in the effect of the two Ato alleles (Ato1 vs Ato3) is intriguing and an important piece of data. It is consistent with the idea that the bHLH domain in needed as opposed to the transcriptional activity, but Ato3 lacks much more than the bHLH domain. A more careful dissection is needed.

We acknowledge that we have not directly shown that Emc and proneural proteins compete for Da in vitro. Such an experiment is not trivial to do with EMSA assays. Because neither Emc/Da nor Emc/Ato heterodimers would bind DNA, only Ato/Da heterodimers could be quantified directly. Because Ato does not bind DNA except as a Ato/Da heterodimer, whether Emc disrupts DNA binding through interaction with Ato or with Da would have to be inferred indirectly. Our lab is considering measuring the interactions directly by biophysical means e.g. analytical ultracentrifugation, but these experiments will be more time-consuming. We also have to take account of what projects can gain funding. Because the significance of directly measuring the affinities of various HLH proteins is not yet generally apparent (perhaps because our work is still unpublished), at present it is a common opinion among grant reviewers that such experiments represent an incremental increase in knowledge of little practical significance. In this regard we state that in vitro data like that suggested by the reviewer would provide further support to our model (Discussion section). We suggest that our paper should stimulate more interest in these questions.

3) There is clear evidence in the paper for a complex set of regulatory interactions, because over expression of Ato has no effect on Emc levels and because there is clearly some level of transcriptional control. The paper however provides no data to explain these effects, and how both transcriptional and post-transcriptional mechanisms interact to ensure the precision of the Emc pattern.

It is clear from several reviewers’ comments that data on the transcriptional regulation of Emc was complicating while adding little to the manuscript and we have removed most of this data. We think the revised manuscript is more focused as a result.

4) What are the implications for neurogenesis for interfering with the post-translational regulation of Emc?

The consequences of preventing Emc degradation for both Da expression and for neural patterning were described in detail previously (Bhattacharya and Baker, 2011).

The best experiment was one in which enGal4 at 23°C drove a level of Emc expression that was not detectable outside of proneural regions but allowed Emc protein to persist in neural cells. This led to loss of ~60% of macrochaetae (Figures 6E-J and S5C-H of Bhattacharya and Baker, 2011.

Reviewer #2:

This paper addresses the role of Emc (Id in mammals) in patterning proneural domains in Drosophila. Earlier studies have indicated that Emc provides a pre-pattern that is decoded by Da to direct the pattern of proneural gene expression. The proposed decoding mechanism involved positive regulation of proneural factors by Da, titration (inhibition) of Da by Emc and Da auto-regulation. The data presented here challenges this view.

First, the authors nicely show that the physiological levels of Emc proteins are set by the level of Da proteins (Figure 2; this effect does not seem to be mediated by a transcriptional regulation of emc by Da, Figure 7) and that Emc is stabilized by Da in S2 cells (Figure 3). They further show that the presence of a mutant/inactive Ato protein is sufficient to up-regulate Da, possibly via a stabilization mechanism (not further investigated here), and to down-regulate Emc (Figure 4). How Ato down-regulates Emc remains, however, unclear. Based on the findings reported in Figure 2 and Figure 3, a possible mechanism would be a competition for the binding of Da, resulting in the destabilization of free Emc. However, since ectopic Ato (or Sc) is not sufficient to down-regulate Emc (Figure 5), this mechanism is not favored. Another possibility would be that Ato (or Sc) represses the transcription of the emc gene. However, Ato is not required to repress emc transcription in the morphogenetic furrow. Thus, how Emc is down-regulated in proneural domains at the mRNA and protein levels remain unclear. Also, the functional significance of this down-regulation for proneural patterning is not clear (more generally, whether the patterned expression of emc is relevant for its function remains to be addressed; see below). Finally, the authors suggest that Da is required to activate (or at least maintain) emc gene expression, but only in a region close to/ahead of the furrow in the eye disc (Figure 7; emc is then repressed in the furrow; and Da does not seem to be required in wings).

In summary, this manuscript reports one interesting finding, i.e. Da stabilizes Emc and sets Emc protein levels. However, the extent to which this mechanism contributes to the endogenous Emc protein pattern is not entirely clear to me. This manuscript also reports a new model (Figure 8) suggesting that the pattern of Ato/Sc is established in response to positional information independently of the Emc pattern and that Emc protein levels are merely a read-out of proneural activity. In this model, Emc appears to merely keep Da inactive in the absence of proneural factors. This model is supported by the data but remains largely untested.

Reviewer 2 states the manuscript contains one interesting finding, that Da stabilizes Emc levels. The revised manuscript also shows that; (a) proneural genes are required to destabilized Emc; (b) uniform Emc transcription is sufFicient for largely normal patterning. Therefore, we hope the reviewer finds the revised manuscript more substantial.

Essential revisions:

1) To further test the relevance (or lack thereof) of the expression pattern of the emc gene, the authors should test whether ubiquitous expression of emc (at the gene level; i.e. ubi-emc in emc mutants) results in a normal Emc protein pattern and provides proper emc activity. Also, the model predicts that changes in the level of expression of the emc gene should have no significant effect on proneural patterning. This should be tested.

As suggested, we have now tested whether ubiquitous Emc transcription can rescue the patterning of Da and Emc proteins and the pattern of neurogenesis and confirmed that it does. This contributes significantly to the significance of the study by showing that the Emc transcription pattern is not essential for the neural prepattern as previously assumed (subsection “Proneural genes are not sufficient to regulate Da or Emc protein levels.”; subsection “Uniform Emc transcription supports neural patterning”; Figure 8).

2) A potential limitation of this study is that it challenges a view proposed by several groups (Modolell, Simpson, Klein) who worked on a slightly different context (proneural clusters of the dorsal thorax). It could be appropriate to revisit this view also in this context (or at least justify why conclusions obtained in the eye necessarily applies to another developmental context).

As suggested we have examined the proneural clusters of the dorsal thorax and explain how our Findings might apply there (subsection “Ato is required for altering Da and Emc levels in the morphogenetic furrow”, Figure 5BD), as well as to the proneural region of the anterior wing margin (subsection “Ato is required for altering Da and Emc levels in the morphogenetic furrow” and Figure 4I-H). We think our Findings apply in all three developmental contexts, but macrochaetae differ in that Emc and Da levels are modulated only in SOP cells, not in the proneural groups, at least at the stages we have examined. Troost et al. have described lower Emc in notum proneural regions but we describe this differently (subsection “AS-C is required for altering Da and Emc levels in wing disc proneural regions”, Figure 5B-D). Unlike the morphogenetic furrow or wing margin, where Emc protein virtually disappears, Emc levels in notum proneural regions seem the same as those in non-neural regions of the wing disc or eye disc. When Troost et al. describe them as ‘lower’ they appear to mean lower than certain other regions of the notum where Emc levels are in fact unusually high. Also, the ‘lower’ Emc (ie normal Emc) regions of the notum do not correspond to the proneural regions but are larger: we think they correspond to the general ground state of the notum which includes proneural regions as well as nonproneural regions.

Regarding the prepattern view of Emc proposed by others (Modolell, Simpson, Klein), what is the evidence for it exactly? It seems to be that Emc loss of function mutants have a phenotype and that Emc expression has a pattern. In our view the new Findings regarding ubiquitous Emc expression require re-evaluation of these views.

Reviewer #3:

Li and Baker present a thorough analysis of the interdependence of the expression levels of two interacting HLH proteins, Da (a DNA-binding activator) and Emc (a non-DNA-binding inhibitor of Da). Earlier work from the same group had shown that Da activates emc expression, which then feeds back to repress da transcription by preventing Da from activating a da autoregulatory enhancer (Bhattacharya et al., 2011). The complex Emc expression pattern was thought to provide a prepattern for neural differentiation, in the sense that Da/proneural activators would be more active in regions of lowest emc expression. The authors claim that emc does not set the prepattern for neurogenesis, but instead its complex expression pattern depends to a great extent on the activity of Da and its proneural patterns.

The crucial findings of Li and Baker are:

1) Da protein levels are proportional to the da gene dosage in the range of 0-2. However, Emc protein levels do not similarly reflect the emc gene dosage (range of 0-2), instead they also reflect the da gene dosage! Da levels are not affected by emc gene dosage in the range of 1-2, but are greatly upregulated in emc null clones, due to the above-mentioned release of inhibition.

2) The unexpected dependence of Emc protein levels on Da protein levels probably comes from the dramatic stabilization of Emc by Da, as determined by half-life measurements.

3) The upregulation of Da and concomitant downregulation of Emc seen in proneural regions of the eye and wing margin need the expression of proneural bHLH proteins with intact HLH domains, but not necessarily DNA binding activity. This suggests that a proneural partner displaces Emc from Da, which leads to degradation of Emc and auto-activation of Da. Some unknown factor must also contribute to this process, since ectopic expression of proneural genes in non-proneural regions cannot recapitulate this effect on Emc and Da levels.

4) emc transcription is not affected by ato or da mutations (exception: there is a moderate effect of da mutations only in the posterior part of the eye disk).

This three-way post-transcriptional interplay among Da, Emc and proneurals represents an important in vivo demonstration of properties of HLH proteins that had only been shown in vitro. It also paves the way for deciphering the biological consequences of these important HLH heterodimerization properties, adding the interesting twist of stability regulation. That said, I have a few concerns that I will list below:

a) The authors focus their "narrative" on how Emc does not represent a proneural prepattern, but rather reflects a proneural "response". I would say that it is both. They themselves show that the complex RNA pattern of emc does not depend (much) on Da or proneural factors, so this is patterned by something else. Granted, this complex pattern is greatly "evened out" at the protein level, thanks to the mutual interactions described by the authors. Still some proneural cluster "lows" in Emc protein (e.g. the morphogenetic furrow) have a clear proneural-independent transcriptional component, so they are expected to predispose these areas to become neurogenic, namely act as a prepattern. I would change the narrative in such a way to clarify that emc contributes to the prepattern (it cannot be the entire prepattern, since there is still some patterning of bristles in the absence of emc), but at the same time it is itself modulated by proneural activity (at the protein level). After all, whether Emc is or is not a prepatterning factor seems of lesser importance than the dissection of its molecular mode of action.

We do agree that Emc may still contribute to the prepattern mechanism and discuss this in the Discussion section. Surely, however, the origin of the spatial pattern of the prepattern is the key question? Our Findings undermine the idea that the Emc expression pattern is the origin of the prepattern.

b) They should point out that the previously reported activation of emc transcription by da was a misinterpretation of the destabilization of Emc in da null clones and that instead Da only affects transcription of emc behind the morphogenetic furrow (and that only modestly, as shown in Figure 7—figure supplement 1).

Since we have removed direct experiments on emc transcription from the paper (they will be submitted elsewhere), it is now less important to comment on the previous transcriptional model, although we do so briefly (subsection “Emc is stabilized by Da in S2 cells”).

c) They should point out that the dependence of Emc on Da levels does break down after a certain threshold. Overexpression of emc in Figure 2O-R causes an enormous increase of Emc protein without concomitantly increasing Da. Probably the degradation machinery of Emc is saturated. Intriguingly Da may also play some role even at these unnaturally high Emc levels, since in the furrow, where Da is engaged by Ato, this excess Emc seems to accumulate less. Therefore, its high accumulation in non-proneural regions is not solely because of saturation of the degradation machinery, since the latter seems to work better in the furrow.

We agree that the dependence of Emc on Da breaks down above a certain threshold and discuss this in the Discussion section.

d) The language needs to be improved. There are many places where the text is hard to follow and the meaning difficult to extract. Probably, with a thorough rewriting, the scientific points (a-c) above will also become clearer.

We agree that the previous manuscript was not as clear as desirable and believe

the revised manuscript is improved significantly.

[Editors' note: the author responses to the re-review follow.]

Reviewer #1:

The revised manuscript is much improved, especially as it is more focused on the main message. The new experiment showing that uniform moderate levels of EMC driven by an exogenous promoter rescue neurogenesis certainly support the conclusion that post-transcriptional modulation of EMC is key to patterning neurogenesis. The new data showing that Da protein levels increase linearly with gene dosage are more convincing. Finally, the in vivo data showing that availability of Da regulates EMC protein levels are very nice.

What remains rather circumstantially substantiated in my view is their mechanistic model of the regulation of EMC protein levels by Proneural proteins by competition for Da. I agree with the authors that the few data they have in this regard do not contradict this notion, and I in fact like the idea mostly because it fits the strong response of EMC to Da levels within the proneural domain under wild type conditions. I am surprised that they were unable to express Atonal in S2 cells to test their model more directly because expression of Ato in vitro has been reported previously.

A citation to the prior expression of Atonal in Drosophila cells would be most helpful, as we have yet to find such a report

The idea that the proneural pre-pattern depends on Da and Proneural proteins, not ID proteins is novel and interesting. Furthermore, it fits nicely with the emerging paradigm that post-transcriptional/translational modification of bHLH proteins is a key determinant of neurogenesis. The authors could do a better job referencing that literature in their Discussion section.

This is a good point. We added a sentence about post-translational modification of proneural proteins and a reference to an upcoming review (Discussion section).

In summary, the authors' model is the simplest and most parsimonious explanation of their data, but their advance of a specific mechanism for their observations remains relatively weak.

Reviewer #2:

This paper challenges the commonly held view that the ID factor Emc acts as a prepattern regulator of proneural activity during neurogenesis in Drosophila. Emc is known to heterodimerize with the E protein Da, thereby blocking its DNA-binding activity, hence its ability to regulate proneural gene expression. This paper reports on an alternative model whereby Da would stabilize Emc in the absence of proneural factor and proneural factors, patterned independently of Emc activity, would negatively regulate this stabilizing activity of Da towards Emc to generate the observed pattern of Emc protein accumulation.

Several interesting observations support this model. First, in contexts of low proneural activity (S2 cells, wing pouch), the accumulation levels of Emc proteins appear to depend on Da levels. The analysis of Emc and Da levels in da, emc clones is really nice (Figure 2) and the stabilization of Emc by Da in S2 cells is clear (Figure 3).

Second, in the proneural region of the eye, Ato is required for the accumulation of Da and the down-regulation of Emc in neural cells (Figure 4). The analysis of the ato1 and ato3 mutations is convincing. The observation that Emc is stabilized in the absence of Ato (Figure 4D and Figure 6) is interesting. However, how Ato destabilizes Emc is not understood: Ato does not appear to be sufficient to destabilize Emc (Figure 7). It is also not clear whether Ato acts indirectly via Da.

Third, non-patterned expression of the emc gene largely rescues the emc mutant phenotype (Figure 8), indicating that Emc transcription does not provide key patterning cues.

In summary, the strength of this paper is that it convincingly shows that the current model needs to be revised. Further experiments are, however, needed to strengthen the alternative model proposed here.

Essential revisions:

1) It would be nice to confirm the changes seen in Da levels by IF using Western blots, for instance by comparing Da levels in emc+/+ vs emc+/- discs (and Emc in da+/+ vs da+/- tissues). Please test.

We attempted to measure Da levels by western blot using a monoclonal antibody and to measure Emc levels using anti-GFP labeling of the Emc-YFP strain. Unfortunately, we were unable to detect endogenous levels of either protein by western blotting and so could not perform these experiments.

2) Figure 2E: it is not clear whether the increased Da levels seen in emc mutant cells result from protein stabilization (indicative of Emc targeting Da for degradation, as suggested by Figure 3F) or from increased transcription (due for instance to ectopic/premature expression of proneural factors). Please clarify.

We also think this is an interesting question and have been trying to assess experimentally whether Da is primarily restrained at the transcriptional level, as suggested previously (Bhattacharya and Baker, 2011), or at the level of protein stability, as Figure 3F suggests might be possible. So far, we do not have a uniform conclusion. We added a sentence to the paper raising the possibility that Da stability might be relevant to levels in vivo (subsection “Emc is stabilized by Da in S2 cells”).

3) Figure 3G: please control that nub>da does not affect emc gene expression (in situ, enhancer-trap…)

We have shown previously that ectopic Da expression in fact does elevate emc transcription (Bhattacharya et al., 2011, Figure 2C). Another reviewer previously asked us now to show that ectopic Da also leads to more Emc protein, consistent with our current model, and that is why we added Figure 3G to the manuscript. We could also remove Figure 3G again if preferred, but since it was requested by a previous reviewer we added to the figure legend that higher emc transcription is expected in this experiment.

4) Subsection “Ato is required for altering Da and Emc levels in the morphogenetic furrow”. The conclusion that Da up-regulation is transcriptional and bHLH-mediated needs to be supported by experimental evidence. Could the authors exclude the possibility that Emc also targets Da for degradation (see Figure 3F)?

The experimental data do not lead to a uniform conclusion at present (see point 2 above). Therefore, we changed the text in subsection “Ato is required for altering Da and Emc levels in the morphogenetic furrow” to include both possibilities.

5) Subsection “AS-C is required for altering Da and Emc levels in wing disc proneural regions”: This conclusion is not convincing. I find it hard to compare Da levels based on different IF experiments. Clonal analysis is required.

We agree with the reviewer, and this is why we based our definitive conclusions on clonal analysis with a complete deletion of AS-C in Figures 4H,I. We only included sc[10-1] because a reviewer of the previous manuscript asked that we explore the conclusions published by other groups regarding notum patterning in light of our findings. Since those other studies have used the viable sc[10-1] allele, we also included it here for comparison. We could not find any sc[10-1] FRT strain available, and although one should be easy to generate we could not have done so and performed the clonal analysis within two months. We could remove this sc[10-1] data, but in our opinion it might as well be included. We modified the Results section to indicate that a clonal analysis with sc[10-1] could provide stronger results (subsection “AS-C is required for altering Da and Emc levels in wing disc proneural regions”).

6) Subsection “AS-C is required for altering Da and Emc levels in wing disc proneural regions”: Whether AS-C regulates Emc/Da levels in sensory cells is difficult to study in the absence of these cells. The data shown in Figure 5B-D are not convincing.

The cells are present, but not specified as sensory cells. As such there are no markers that could identify them for double labeling, so this cannot be further clarified experimentally. We agree that the failure to find single cells with lower Emc could be equivocal, but we do think it is more significant that we cannot find the single cells with higher Da. Figure 5D shows that we can find these high Da/low Emc SOP cells in the wild type. We changed the text of the Results and Discussion sections to make the uncertainties clear (subsection “AS-C is required for altering Da and Emc levels in wing disc proneural”, Discussion section). This is another example where we only present data on the notum in response to the previous reviewer that requested this.

7) Figure 7B: heterogenous levels of Ato appear to be detected in the non-proneural region (Figure 7B). Therefore, a per-cell quantification of the relative Emc/Da levels is needed (Figure 7A). It would be nice to test whether Ato levels correlate with Emc/Da levels. Also, what is causing the observed heterogeneity in Ato levels?

We performed a per-cell analysis as requested. The data are shown in Figure 7 panels C and D and Figure 7—figure supplement 1C and 1D and described in subsection “Proneural genes are not sufficient to regulate Da or Emc protein levels.”). We saw no evidence that Emc levels were lower in the scattered cells with higher Ato levels. We did see a weak trend towards more Da in high Ato-cells, which is consistent with the slightly higher Da levels already noted in cells expressing ectopic Ato (Figure 7A and Figure 7—figure supplement 1A).

The cause of the heterogenous Ato levels is a mystery at present.

8) Figure 7—figure supplement 1A: the clone of act>ato cells (right) exhibits high Emc and high Da levels. This seems to contradict the model whereby Ato competes Emc for binding Da. Please comment.

Yes, this is part of the data for suggesting that Ato is not sufficient to degrade Emc at ectopic locations (subsection “Proneural genes are not sufficient to regulate Da or Emc protein levels.”). Since ectopic Ato expression in wing discs significantly alters wing disc development, perhaps some effects on Emc and Da levels could be indirect. Overall, however, the data do not clearly support the notion that Ato leads to Emc degradation at ectopic locations.

9) Figure 8: what is the phenotype of emcD1/emcAP6 flies? are these mRNA null? what is the emc mRNA pattern in the mutant and rescued eye discs? Is the latter similar to the protein pattern shown in 8C? does the UAS-ems construct contain the 3'UTR of the emc gene? Does it matter? Also, is the emc mutant combination used in Figure 8 protein null?

We added brief description of the emc∆1 allele and the emcAP6/emc∆1 genotype (subsection “Uniform Emc transcription supports neural patterning”; subsection “Drosophila Strains”). emc∆1 is an early frame-shift allele and protein null, one of two such alleles we generated by Crispr. We will describe these alleles in detail elsewhere, we do not believe it is of sufficient interest to do so here. We used the transheterozygous emcAP6/emc∆1 genotype for the rescue experiments to exclude the possibility of linked mutations on the extant emcAP6 chromosome. We did not sequence the UAS-emc5.3 construct. This was described by Baonza et al., (2000) as “a full-length emc cDNA” so it might include endogenous 3’ UTR that could encode post-transcriptional regulation eg by miRNAs. Therefore we are careful to refer only to uniform transcription in this experiment, although please note that the antibody stainings included in this paper show uniform protein expression in the relevant areas after uniform transcription.

https://doi.org/10.7554/eLife.33967.020

Article and author information

Author details

  1. Ke Li

    Department of Genetics, Albert Einstein College of Medicine, Bronx, United States
    Present address
    Howard Hughes Medical Institute, Department of Physiology, University of California, San Francisco, San Francisco, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0737-1045
  2. Nicholas E Baker

    1. Department of Genetics, Albert Einstein College of Medicine, Bronx, United States
    2. Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, United States
    3. Department of Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, United States
    Contribution
    Conceptualization, Supervision, Funding acquisition, Writing—review and editing
    For correspondence
    nicholas.baker@einstein.yu.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4250-3488

Funding

National Institute of General Medical Sciences (GM047892)

  • Nicholas E Baker

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

Acknowledgements

We thank Drs. Abhishek Bhattacharya, Jorge Blanco, Jean Hebert, Andreas Jenny, Marianthi Kiparaki, Ertugrul Ozbudak, Francesca Pignoni and Lan-Hsin Wang for comments on the manuscript, Dr. Abhishek Bhattacharya for initial contributions to the project, and Dr. Marianthi Kiparaki for DNA constructs. Drosophila stocks were obtained from the Flytrap Project, the Zurich ORFeome Project (FlyORF) and the Bloomington Drosophila Stock Center (supported by NIH P40OD018537). S2 cells were obtained from the Drosophila Genomics Resource Center (supported by NIH 2P40OD010949-10A1). Confocal microscopy was performed in the Analytical Imaging Facility of the Albert Einstein College of Medicine (supported by the NCI P30CA013330). DNA sequencing was performed by the Genomics Core of Albert Einstein College of Medicine. This work was supported by the NIH grant GM047892. Data in this paper are from a thesis submitted in partial fulfillment of the requirement for the degree of Doctor of Philosophy in the Graduate Division of Biomedical Sciences, Albert Einstein College of Medicine, Yeshiva University, USA.

Reviewing Editor

  1. Hugo J Bellen, Baylor College of Medicine, United States

Publication history

  1. Received: November 30, 2017
  2. Accepted: March 25, 2018
  3. Version of Record published: April 24, 2018 (version 1)

Copyright

© 2018, Li 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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