INTRODUCTION
Signaling by c-Jun N-terminal kinase (JNK) mediates one of the major stress response pathways (Chen, 2012; Stronach and Perrimon, 1999) . Indeed, activation of JNK signaling often boosts or triggers apoptosis (Dhanasekaran and Reddy, 2008; Igaki, 2009; Leppä and Bohmann, 1999) . JNK can exert its proapoptotic effect through phosphorylation of Jun, a component of the AP-1 transcriptional activator, or of other cellular proteins (Bogoyevitch and Kobe, 2006) . It is important to note, however, that JNK signaling does not always trigger apoptosis (Weston and Davis, 2007) and has been shown to control nonapoptotic processes such as cytoskeletal rearrangements (Homsy et al., 2006) , cell migration (Ríos-Barrera and Riesgo-Escovar, 2013), and cell proliferation (Shaulian and Karin, 2002) during development and regeneration. It is generally thought that the cellular context or the activity of other signaling pathways determines whether JNK signaling leads to apoptosis or not. In well-documented instances, this involves downregulation or blunting of JNK signaling itself, e.g., through the activity of Gadd45b, an NF-kB-induced factor (De Smaele et al., 2001; Papa et al., 2004) , or by Puckered, a feedback inhibitor of JNK signaling (McEwen and Peifer, 2005) . Mechanisms that dampen JNK's proapoptotic influence without affecting core pathway activity have also been documented. For example, in the developing Drosophila eye, mitogen-activated protein kinase phosphorylates and destabilizes Hid, a proapoptotic protein transcriptionally activated by JNK signaling in this tissue (Bergmann et al., 1998) . Another documented process involves transcriptional repression of hid, which would otherwise be overactivated by JNK in response to irradiation damage (Luo et al., 2007) . Noncell-autonomous protective mechanisms could also jacobharper@example.com. For example, the transcriptional modulator Schnurri limits radiation-induced tissue damage by recruiting macrophages through activation of the PDGF-related growth factor Pvf1 (Kelsey et al., 2012) . All the aforementioned mechanisms have been shown to operate in response to cellular stress. However, so far, little is known about the regulatory processes that prevent JNK from causing apoptosis during normal development. One well-characterized nonapoptotic JNK-dependent developmental process is the morphogenetic movement of dorsal closure (Glise and Noselli, 1997; Hou et al., 1997; Riesgo-Escovar and Hafen, 1997) . Here, we set out to investigate the molecular mechanisms that prevent JNK from activating apoptosis during this process.
INTRODUCTION
Dorsal closure is a morphogenetic movement that closes a large gap left on the embryo's dorsal side after germ band retraction. It involves the concerted movement of the dorsal epidermis toward the midline and requires both JNK and Dpp signaling. One current view is that JNK signaling at the leading edge promotes expression of Decapentaplegic (Dpp, a transforming growth factor b [TGF-b] homolog), which, in turn, orchestrates the cell shape changes required for dorsal closure (Ferná ndez et al., 2007; Ríos-Barrera and Riesgo-Escovar, 2013) . It is likely that a protective mechanism is at work at the leading edge since JNK does not trigger apoptosis there. Additional evidence that the dorsal epidermis is protected from apoptosis came from the analysis of crumbs (crb) (abbreviated as crb in genotypic descriptions) mutants, where JNK target genes are upregulated in response to loss of apicobasal polarity . In such embryos, most epidermal cells undergo apoptosis, except in an approximately ten-cellwide band of dorsal cells, despite strong activation of JNK signaling there. It appears, therefore, that the protective mechanism acts over a broader domain than just within the leading edge (see Figures 1A and 1B for a diagram of the relevant region of the embryonic epidermis). Most apoptosis in Drosophila requires the H99 locus (White et al., 1994) , which comprises the three main proapoptotic genes: reaper, hid, and grim. Among these, reaper is the most likely mediator of the response to epithelial disruption since it is upregulated in crb mutant embryos in a pattern prefiguring that of caspase immunoreactivity . Moreover, overexpression of Puckered, a phosphatase that inhibits JNK signaling prevents reaper upregulation, as well as apoptosis, in crb mutants ).
INTRODUCTION
These observations suggested that loss of apicobasal polarity triggers JNK signaling (through an unknown mechanism), which, in turn, causes reaper expression and, hence, apoptosis. However, in the dorsal epidermis, JNK signaling does not activate reaper expression. Here, we show the molecular mechanism underpinning such protection and thus explain how JNK can control cell migration without triggering apoptosis.
RESULTS AND DISCUSSION

Activation of reaper by Canonical JNK Signaling Mediates Apoptosis in Response to Epithelial Disruption
In crb mutant embryos, reaper is strongly upregulated ( Figures  1C-1F ) in a pattern similar to that of apoptosis (Figures S1A and S1B available online), while the other two main proapoptotic genes, hid and grim, remain largely silent . No activated caspase immunoreactivity was detectable in rpr 87 crb double mutant embryos ( Figure S1C ), confirming the essential role of reaper and highlighting the need to uncover the mechanisms that activate reaper expression following loss of apicobasal polarity. As suggested previously , JNK signaling is likely involved. Indeed, JNK signaling, as measured with a transcriptional reporter (AP-1 > GFP) was strongly activated in crumbs mutant embryos (Figures 1G-1J ; Movie S1). Moreover, little reaper transcription was detectable in crumbs embryos that also lack jra or kayak, which encode the two components of AP-1, Jun, and Fos (Figures 1K-1N ; see Figures S1F-S1I for reaper expression in the single mutants), and this was associated with a near-absence of apoptosis, as reported by activated caspase-3 immunoreactivity (Figures S1D and S1E). Notably, JNK signaling does not seem to necessarily cause apoptosis. In the ventral epidermis, the patterns of reaper expression and apoptosis (highlighted with anti-activated caspase) appeared to mirror the early segmental activation of JNK signaling (compare Figure 1D with Figure S1B ), suggesting a relatively straightforward, likely causal, relationship there. However, neither reaper expression nor apoptosis were significantly activated in the dorsal epidermis, even at the dorsal edge, where JNK signaling is particularly active both in wild-type and crumbs mutants. What is the mechanism that protects the dorsal epidermis from the proapoptotic effect of JNK?
Activation of reaper by Canonical JNK Signaling Mediates Apoptosis in Response to Epithelial Disruption
Dpp Signaling Prevents JNK from Activating reaper Expression in the Dorsal Epidermis One feature of the dorsal epidermis is that it is under the influence of Dpp, a member of the BMP family of secreted growth factors (Hamaratoglu et al., 2014) . Indeed, phospho-Smad (p-Smad) immunoreactivity (a mark of Dpp signaling) (Tanimoto et al., 2000) was detectable in this region before and during the time when JNK is active (Figure 2A ). Therefore, Dpp signaling could prevent JNK signaling from activating reaper expression both in the dorsal epidermis of crumbs mutant embryos and, physiologically, at the dorsal edge of wild-type embryos. This was tested in embryos expressing a Dpp RNA interference (RNAi)-encoding transgene (Supplemental Information) under the control of the ubiquitous tubulin-gal4 driver. This led to reduced signaling as indicated by the loss of p-Smad immunoreactivity from stage 11 onward ( Figure 2B ). In these embryos, a band of reaper transcription was observed at the dorsal edge ( Figure 2C ), where JNK is known to be activated in the wildtype (see Figure 1I) . A similar result was seen in embryos lacking zygotic (but not maternal) activity of thickveins (tkv), which encodes an essential Dpp receptor (Nellen et al., 1994) (Figure 2D) . Expression of reaper was also seen in the approximately ten-cell-wide dorsal region in tkv crumbs double mutants (as well as in the rest of the epidermis; Figure 2F ), suggesting that Dpp signaling prevents reaper expression throughout the dorsal epidermis. Caspase immunoreactivity became detectable throughout the epidermis of tkv crumbs mutants ( Figure S1J ), consistent with the notion that repression of reaper expression by Dpp signaling translates into antiapoptotic activity. 
Developmental Cell

Schnurri Prevents JNK-Induced Apoptosis

Schnurri Prevents reaper Transcription and Apoptosis
Dpp signaling is mediated by the Mad complex, which can activate or repress target genes depending on the sequence context and other cofactors (Affolter and Basler, 2007) . The best characterized instance of repression by Dpp signaling is that of the brinker gene, which occurs via silencer elements where the Mad complex recruits the corepressor Schnurri (Affolter and Basler, 2007; . We therefore asked if Schnurri could mediate the repression of reaper by Dpp. Indeed, reaper transcription was upregulated at the dorsal edge of schnurri mutant embryos ( Figure 2E ; Figures S1O-S1R). In these embryos, reaper expression was also seen to extend segmentally in the lateral region, a feature not seen in Dpp-RNAi-expressing embryos (compare Figure 2E with Figure 2C ), perhaps because Schnurri also has Dpp-independent activity, as suggested by (Kelsey et al., 2012) . In any case, the reaper-repressive role of Schnurri in the dorsal epidermis was confirmed in schnurri crumbs double mutants, where reaper transcription was strongly upregulated in both the dorsal and ventral regions ( Figure 2H ). No reaper upregulation was seen in schnurri kayak (fos) double mutants ( Figure 2G ), showing that JNK signaling is an essential positive input to reaper transcription at the dorsal edge, as well as in segmentally repeated lateral domains, where the JNK sensor appears not to be sufficiently sensitive to detect activity. It is worth noting that staining of schnurri and schnurri crumbs embryos with anti-activated caspase confirmed that the repression of reaper transcription by Schnurri is needed to suppress apoptosis (Figures S1K and S1L). As AP-1 and Schnurri are both transcriptional regulators, we next sought to address whether the opposing influences of Schnurri and JNK signaling converge directly on the reaper promoter.
Binding Sites Upstream of the reaper Promoter Integrate the Effects of Dpp and JNK
The promoter region of brinker that mediates Schnurri-dependent repression has been extensively characterized, and mutation analysis identified an essential 16-base-pair (bp) repressor sequence . Similar elements are found at 350 positions in the Drosophila genome, defining a consensus sequence: GRCGNCNNNNNGTCTG . Two related sites were identified upstream of reaper, in a region that is conserved in the 12 sequenced Drosophila species. In all these species, the proximal site (SE p ) is flanked on either side by a predicted AP-1-binding site, making it a potential regulatory element. Because SE p is not an exact match to the consensus Schnurri binding site, we used an electrophoretic mobility shift assay (EMSA) to test whether it is recognized by Schnurri, using the previously characterized site from the brinker gene (cSE) as a positive control. Recombinant Schnurri protein induced a supershift of the Mad-Medea-DNA complex in both cases, although to a lesser extent with SE p than with cSE ( Figure S2A ). Thus, we conclude that SE p is recognized by Schnurri/Mad/Medea and could therefore mediate the repressive influence of Dpp on reaper expression. By extension, the module comprising SE p and the two putative AP-1 sites could integrate the influence of Dpp and JNK signaling on reaper expression. To test this hypothesis in vivo, we made a reporter construct comprising 5.5 kb of sequence including this module and the basal reaper promoter, upstream of a GFP complementary DNA ( Figure 3A ). This reporter (rpr-GFP) and the variants described later were introduced by PhiC31-mediated integration at the same genomic location to allow comparison without confounding influence from position effects. The wild-type reporter was essentially silent in wild-type embryos ( Figure 3B ), as expected, since reaper expression is barely detectable during normal embryogenesis. By contrast, in crumbs mutants, rpr-GFP became segmentally upregulated in the ventrolateral-but not dorsalepidermis (double-headed arrow in Figure 3C ), thus mirroring the activity of the endogenous reaper gene in this background (compare to Figure 1F ). Critically, like the endogenous reaper gene, this reporter became active in the dorsal epidermis of schnurri mutants ( Figure 3D ). Such dorsal expression was segmentally modulated (see Figure S2B for GFP staining alone), resembling the pattern of endogenous reaper transcription in schnurri mutants ( Figure 2E ). Also, like endogenous reaper, the reporter was widely and strongly activated in the epidermis of schnurri crumbs double mutants ( Figure 3E ). These observations suggest that Schnurri mediates repression of the reporter. To test the contribution of the predicted Schnurri binding site, a mutation was introduced in the reporter (ATCGTCTCGCC GTCTG / ATCGTCTCGCTTTCTG), thus creating rpr[DShn]-GFP ( Figure 3A ). This mutation was found to abrogate formation of the Mad-Medea-DNA complex in vitro (see SEm in Figure S2A) , suggesting that rpr[DShn]-GFP would no longer be subject to repression by Schnurri. Indeed, this transgene became activated in wild-type embryos in the dorsal epidermis ( Figure 3F ). One must point out that this dorsal activity of rpr [DShn]-GFP did not appear before stage 13, 1-2 hr later than the appearance of rpr-GFP in schnurri mutants (compare Figure 3F to Figure 3D ). Also, unlike rpr-GFP in schnurri mutants, rpr [DShn] -GFP was active in segmentally repeated ventral domains at stage 13. Despite these differences (see further discussion in the legend of Figure S2 ), the upregulation of rpr-GFP in schnurri mutants and the expression of rpr [DShn] -GFP in the dorsal epidermis of otherwise wild-type embryos (where rpr-GFP is silent) are consistent with the notion that Schnurri represses reaper expression in the dorsal epidermis, thus allowing JNK signaling to control epithelial migration without triggering apoptosis.
Binding Sites Upstream of the reaper Promoter Integrate the Effects of Dpp and JNK
We next assessed the function of the two predicted AP-1 binding sites by mutating them (TGACTCATA / TGACATTTA) (Chatterjee and Bohmann, 2012) individually or as a pair in the GFP reporter ( Figure 3A ) and assessing their activity in the ventral epidermis of crumbs embryos, where the wild-type reporter is strongly activated. Mutating one or the other site reduced activation, while the double mutant reporter (rpr[DAP1
Binding Sites Upstream of the reaper Promoter Integrate the Effects of Dpp and JNK
had no detectable activity in crumbs mutants (Figures 3H-3J ; wild-type background in Figure S2E ). This reporter is nevertheless functional. It was activated by irradiation ( Figure 3K ), consistent with the presence of a p53-response element previously shown to mediate the response to irradiation (Brodsky et al., 2000) . It is worth pointing out here that reaper expression was still activated in crb p53 5A-1-4 , 2006; Riesgo-Escovar et al., 1996) . Our results suggest an additional role for Dpp signaling during dorsal closure, namely, to ensure the survival of leading edge cells. We propose that such a protective mechanism is needed because of the proapoptotic influence of JNK signaling. Accordingly, the ''dorsal open phenotype'' of schnurri mutants (and possibly other Dpp pathway mutants) would not only be caused by the failure of dorsal edge cells to migrate but also by their reduced survival. To evaluate the contribution of the latter, we assessed the extent of tissue loss in fixed and live schnurri mutant embryos, which, as previously reported (Arora et al., 1995; Grieder et al., 1995; Staehling-Hampton et al., 1995) , do not undergo dorsal closure. Cursory observation suggests that, in schnurri mutant embryos, the dorsal hole that appears at the end of germband retraction gapes open over time (Figures 4A and 4B; Figures S1Q and S1R) . This was confirmed by confocal live imaging of schnurri mutants carrying spider-GFP as a marker of cell outlines ( Figure S3D ; Movie S2). To test if gaping of the dorsal hole could be due to tissue shrinkage, stage 13 schnurri mutant embryos were stained with anti-Cut, which marks dorsal and ventral clusters of peripheral neurons along the D-V axis (Figures 4A-4D ; Figures S3A-S3C) , and the number of Cut-positive cells in each cluster was counted. While cell number in the dorsal cluster remained constant in wild-type embryos, it decreased significantly in schnurri mutants ( Figure 4E ). The number of ventral cells was relatively unaffected in both genotypes. It appears, therefore, that the dorsal epidermis is preferentially eliminated prior to this stage. Next, we used multiview light-sheet microscopy (MuVi-SPIM; Krzic et al., 2012) to achieve in toto imaging of schnurri mutants and control embryos carrying histone-red fluorescent protein (histone-RFP) ( Figures 4H-4M) . In schnurri mutants, cell debris could be seen around the dorsal edge, and many macrophages scurried around (Movies S3 and S4). Moreover, the number of epidermal nuclei became reduced, compared to that in control embryos ( Figure S4 ). These observations confirm that, during stages 12-14, the dorsal epidermis of schnurri mutants progressively shrinks ( Figures S4A-S4I ), likely by apoptosis (see also Figures S1K, S1Q, and S1R). Consistent with the involvement of apoptosis, the dorsal hole did not appear to gape open in schnurri mutants that also lacked reaper (Figure 4G) , even though these embryos failed to complete dorsal closure. As shown in Figure 4H , the JNK reporter remains active throughout tissue shrinkage ( Figures 4F-4G ), suggesting that, as 
Developmental Cell
Schnurri Prevents JNK-Induced Apoptosis dorsal edge cells are eliminated, adjacent epidermal cells activate JNK. Taken together, our results suggest that the dorsal open phenotype of schnurri mutants is a combined consequence of tissue loss by apoptosis and lack of cell migration over the amnioserosa.
Developmental Cell
Most functions of Dpp signaling are achieved through Schnurri-dependent repression of the transcriptional repressor Brinker. However, as we have shown, inhibition of reaper by Schnurri is direct and, hence, likely Brinker independent. By contrast, the migration of dorsal edge cells is mediated by Brinker repression, since schnurri brinker double mutant larvae have a sealed dorsal midline (Marty et al., 2000; Torres-Vazquez et al., 2001) . It is interesting that the dorsal epidermis of these double mutants is much reduced in surface area compared to that of wild-type embryos or brinker single mutant embryos, which have expanded dorsal fates (in Torres-Vazquez et al., 2001, compare Figure 2D to Figures 2A and 2B ). We suggest that schnurri brinker double mutant embryos complete dorsal closure despite tissue loss, perhaps because a sufficient number of cells are able to migrate before undergoing apoptosis. To assess directly whether Brinker has any impact on reaper expression, we performed gain-and loss-of-function experiments ( Figures S3E-S3I) . No ectopic reaper expression was seen in embryos lacking or overexpressing brinker ( Figures  S3G and S3H) . Moreover, Brinker overexpression did not prevent reaper expression in crumbs mutant embryos ( Figures  S3E and S3F ), while overexpressing Schnurri did ( Figures S3E  and S3I ). We conclude that Brinker does not affect reaper expression and that Schnurri contributes to dorsal closure through two parallel routes: first, by repressing Brinker and, hence, allowing the derepression of genes regulating the cytoskeletal functions required for cell migration (Ferná ndez et al., 2007; Homsy et al., 2006) ; and second, by repressing reaper, thus ensuring survival of the dorsal edge cells in the face of JNK's proapoptotic pressure. Our results highlight the central role of Dpp and Schnurri in cell survival, extending observations on Dpp in imaginal discs (Adachi-Yamada et al., 1999; Gibson and Perrimon, 2005; Moreno et al., 2002; Shen and Dahmann, 2005) and TGFb in vertebrates (Sabapathy et al., 1999; Taya et al., 1999) .
Conclusions
In many contexts, JNK signaling leads to apoptosis, perhaps a remnant of this pathway's ancestral function in stress response (Ríos-Barrera and Riesgo-Escovar, 2013). JNK signaling may have started to regulate cytoskeletal functions in multicellular organisms to facilitate delamination or extrusion of defective cells. It is conceivable that such a regulatory relation might have subsequently been coopted to control other migratory behaviors such as those required for dorsal closure. Evidently, this would have necessitated coevolution of a protective, antiapoptotic mechanism. At the dorsal edge of the epidermis, this is mediated by Dpp, which, intriguingly, is itself under the control of JNK signaling. Therefore, under the right circumstances, JNK contributes to the mechanism that counteracts its own AP-1-dependent proapoptotic pressure. The outcome of the regulatory network linking JNK and Dpp to reaper must be finely balanced, since a mild increase in JNK signaling (e.g., in puckered heterozygous embryos) triggers reaper expression . Unlike the ventral epidermis, the dorsal epidermis seems prone to expressing Dpp in response to JNK, perhaps because of earlier expression in this region or through the action of additional regulators. This is likely to ensure the survival of dorsal edge cells during their migration. As we have shown, the opposing influences of Dpp and JNK are played out within the regulatory region of reaper. The anti-apoptotic activity of Dpp is mediated by Schnurri, a protein that could have more general anti-apoptotic activity since mammalian Schnurri has been shown to dampen cell death during T cell development (Staton et al., 2011) . The function of this or similar regulatory modules in other tissues, contexts, and models can thus yield a broader understanding of the balance between apoptosis and survival at the intersection of signaling pathways.
EXPERIMENTAL PROCEDURES
Details on materials and methods can be found in the Supplemental Information. These include a full list of the Drosophila strains and antibodies as well as step-by-step staining protocols for immunofluorescence and in situ hybridization. For gamma irradiation, embryos were collected for 4 hr, aged a further 4 hr, and introduced in a gamma-cell irradiator for a 4,000 rad exposure (Nordstrom et al., 1996) . The embryos were allowed to recover for 2 hr at 25 C before further analysis. The reaper reporter constructs were created by standard molecular biology with primers listed in the Supplemental Information. They were introduced into the Drosophila genome by PhiC31-mediated integration into PBac[yellow[+]-attP-9A] VK00027 (Bloomington Drosophila Stock Center # 9744, on chromosome III). EMSAs were largely performed as described elsewhere . Live embryo imaging was performed either by classical confocal or MuVi-SPIM. For classical confocal microscopy, we used a Leica SP5 microscope equipped with a resonant scanner and a 203 (NA, 0.8) water immersion objective. For MuVi-SPIM, we used a custom-built set-up (two Nikon 103 0.3-NA illumination objective lenses and two Nikon 253 1.1-NA detection lenses) and protocols as described elsewhere . The custom-modified Hamamatsu Flash 4 cameras were operated in the Lightsheet Readout Mode to reject scattered photons. Details on image processing and data visualization are provided in the Supplemental Information.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures, four figures, and four movies and can be found with this article online at http://dx.doi.org/10.1016/j.devcel.2014.08.015.
ACKNOWLEDGMENTS
We thank the Bloomington Drosophila Stock Center for fly stocks, as well as Dirk Bohmann, Henri Jasper, Markus Affolter, and Kristin White for reagents and Alex Gould and Ingrid Poernbacher for discussions. We also thank Pedro Gaspar for help generating the reporters. Marvin Albert provided an early version of the Mercator projection routine. to the expected dorsal expression). This may not be directly attributable to loss of Schnurri since, at this late stage, similar though weaker expression was also detectable from the rpr-GFP transgene as well as from the endogenous reaper gene in wild type embryos (Fig. S2C,D) . 
SUPPLEMENTAL MOVIE LEGENDS
Movie S1 (related to Figure 1) . Activity of TRE-GFP, a reporter of JNK activity, in an otherwise wild type embryo (left) or a crumbs mutant embryo (right) from stage 10/11 onwards.
SUPPLEMENTAL MOVIE LEGENDS
In the wild type, GFP fluorescence can be seen at the dorsal edge during dorsal closure while in the mutant widespread fluorescence becomes detectable as development proceeds. In this and in movie S2, embryos are oriented with anterior on the left and dorsal at the top. for 10 min at 4°C in 100 µl of 100 mM Tris (pH 7.5), 1 mM DTT, 0.5% TritonX100 and 1%
SUPPLEMENTAL MOVIE LEGENDS
NP40. Radioactively labeled probes were generated by annealing and filling in partially overlapping oligonucleotides in the presence of α-32 dATP. Binding reactions were carried out in a total volume of 25 µl containing 12.5 µl 2x binding buffer (200 mM KCl, 40 mM HEPES (pH 7.9), 40% glycerol, 2 mM DTT, 0.6% BSA and 0.02% NP40), 10000 cpm of radioactively labeled probe, 1 µl poly dIdC (1mM) and 7 µl of cleared S2 cell extracts. After incubation for 30 min at 4°C, the reactions were analyzed by non-denaturing 4% polyacrylamide gel electrophoresis followed by autoradiography .
SUPPLEMENTAL MOVIE LEGENDS
Live imaging by classical confocal microscopy. After dechorionation in 50% bleach for 2 minutes, embryos were washed with water. Live embryos of the appropriate stage and genotype were selected under a fluorescence microscope. They were then mounted on a thin strip of glue at the bottom of a Petri dish, covered with PBS, and imaged at 22°C in the Live Data mode of an upright Leica SP5 microscope equipped with a resonant scanner. A 20x (NA=0.8) water immersion objective (Leica) was used. Typical acquisition time was 40-55 seconds/embryo. The time-lapse series were assembled and analysed with ImageJ/Fiji.
SUPPLEMENTAL MOVIE LEGENDS
Live imaging by MuVi-SPIM. Embryos were imaged with a custom-built MuVi-SPIM setup with two illumination objective lenses (Nikon 10x 0.3NA) and two detection lenses (Nikon 25x
SUPPLEMENTAL MOVIE LEGENDS
1.1NA), as previously described . Two custom-modified Hamamatsu Flash 4 cameras were used to record the opposing views of the sample. These cameras were operated in the Lightsheet Readout Mode™ to reject scattered photons (De Medeiros et al., 2014) . Embryo preparation and mounting in transparent gels was performed as previously described . Every three minutes, two perpendicular oriented z-stacks (each with 200 planes and 1 µm spacing) covering the entire embryo were recorded. These stacks were computationally fused into a single 3D dataset for each time point. Slight shifts of the embryo in the gel over the 14hrs image period were compensated by a generic particle image velocimetry method written in Matlab (www.mathworks.com). Epidermis projections: for in toto analysis and visualization of development of the embryonic epidermis, the dimensionality of the entire dataset (3D images and time) was reduced by projecting the surface of the embryo to a plane; first, for each time point, we determined the convex hull of the 3D surface of the embryo based on the fluorescent intensity distribution in the 3D image. A standard Mercator projection was then applied to map the embryonic surface to a plane. Despite this projection having an effect on size and shape of objects at the embryo poles, the analysis was done in the central locations where it yields an unaltered representation of the tissue away from the poles. A multi-slice Mercator projection was generated, covering a thin layer around the embryos surface using Python (www.python.org). A secondary maximum projection of the multiple Mercator slices into a single 2D image provides a complete view of the fluorescent intensity distribution of the embryos surface, thus the epidermis. For the movies, volumetric rendering of the 3D fluorescent intensity distributions were performed using Fiji (www.fiji.sc) with the 3D-Viewer plugin. The rendering yields a 2D
SUPPLEMENTAL MOVIE LEGENDS
view of the fluorescence distribution as observed from a viewpoint (e.g. dorsal and ventral) outside of the embryo. Viewpoints and time points were rendered independently and then stitched together.

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