Decision letter: A novel role for Ets4 in axis specification and cell migration in the spider Parasteatoda tepidariorum

Field: Agricultural and Biological Sciences (Life Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Organizers play important roles during the embryonic development of many animals. The most famous example is the Spemann organizer that sets up embryonic axes in amphibian embryos. In spiders, a group of BMP secreting mesenchymal cells (the cumulus) functions as an organizer of the dorsoventral axis. Similar to experiments performed with the Spemann organizer, transplantation of the cumulus is able to induce a secondary axis in spiders. Despite the importance of this structure, it is unknown which factors are needed to activate cumulus specific gene expression. To address this question, we performed a transcriptomic analysis of early embryonic development in the spider Parasteatoda tepidariorum. Through this work, we found that the transcription factor Pt-Ets4 is needed for cumulus integrity, dorsoventral patterning and for the activation of Pt-hunchback and Pt-twist expression. Furthermore, ectopic expression of Pt-Ets4 is sufficient to induce cell delamination and migration by inducing a mesoderm-like cell fate. https://doi.org/10.7554/eLife.27590.001 eLife digest At the earliest stages of animal development, embryos consisting of only a handful of cells must figure out where each part of their body will come from. The first step in this process is to determine what will be their head versus their tail, and what will be their front versus their back. Many animals use specialized groups of cells, called "organizers", to make this decision. This occurs in backboned animals – including humans – and also in distantly related animals such as spiders. In spiders, the developing embryo must form an organizer called the "cumulus" or the spiderling will not develop correctly. In order to form and maintain the cumulus, various genes must be turned on in a carefully controlled order in exactly the right cells. Pechmann et al. have now discovered the role of a previously unknown gene (called Pt-Ets4) that marks the spot where the cumulus forms. This gene is required for cumulus maintenance and it also helps to activate a number of other cumulus-specific genes. When this gene is disrupted, the spider embryo does not properly differentiate its front from its back. The findings presented by Pechmann et al. add to a growing foundation of studies aiming to understand how genes 'talk' to one another and organize embryos as they develop. In years to come, the unraveling of these gene pathways, where genes sequentially turn other genes on and off, will allow us to more fully understand how a single cell can grow into a complete adult animal. https://doi.org/10.7554/eLife.27590.002 Introduction The self-regulatory capacities of vertebrate embryos were most famously demonstrated by Spemann and Mangold. They found that by grafting the dorsal-lip of an amphibian embryo (now known as the Spemann Organizer) to the ventral side of the host gastrula embryo it was possible to induce a secondary body axis (Spemann and Mangold, 2001; De Robertis, 2009; Anderson and Stern, 2016). Intriguingly, spider embryos also have high self-regulatory capacities, even to the extent that twinning can occur spontaneously (Napiórkowska et al., 2016; Oda and Akiyama-Oda, 2008). During spider embryogenesis a group of migratory cells (the cumulus) is needed to break the radial symmetry of the early embryo and to induce the dorsoventral body axis (Oda and Akiyama-Oda, 2008; Akiyama-Oda and Oda, 2003, 2006; McGregor et al., 2008; Hilbrant et al., 2012; Mittmann and Wolff, 2012; Schwager et al., 2015). Similar to the vertebrate experiments, Holm showed that transplanting cumulus material was able to induce a secondary axis in spider embryos (Holm, 1952). Modern work has shown that the cumulus signals via BMP signaling (again, similar to vertebrates). The mesenchymal cumulus cells are the source of the BMP receptor ligand Decapentaplegic (Akiyama-Oda and Oda, 2006). Interfering with the BMP signaling pathway by gene knockdown results in the loss of dorsal tissue identity, which in turn leads to completely radially-symmetric and ventralized embryos (Akiyama-Oda and Oda, 2006). The cumulus forms in the center of the so-called germ-disc (the embryonic pole of the embryo) and migrates underneath the ectoderm towards the rim of the disc. Arrival of the cumulus at the rim induces the opening of the germ-disc (Oda and Akiyama-Oda, 2008; Akiyama-Oda and Oda, 2003, 2006; McGregor et al., 2008; Hilbrant et al., 2012; Mittmann and Wolff, 2012; Schwager et al., 2015). Cumulus migration is dependent on the Hh-signaling pathway (Akiyama-Oda and Oda, 2010) and it was shown that the knockdown of components of this signaling pathway results in cumulus migration defects and in the ectopic opening of the germ-disc (Akiyama-Oda and Oda, 2010). How the cumulus is specified and forms is still under debate. During the formation of the germ-disc a small cluster of cells ingress and form an indentation where the future center of the fully formed germ-disc will be located. This cluster of cells appears as a visible spot and is called the primary thickening (Akiyama-Oda and Oda, 2003; Hilbrant et al., 2012). However, it is not clear whether all or only a subset of the cells of the primary thickening give rise to the cumulus, or if cumulus cells arise from subsequent cell invagination at the site of the primary thickening (Oda and Akiyama-Oda, 2008; Akiyama-Oda and Oda, 2003). Cell tracing (Holm, 1952; Edgar et al., 2015), as well as the expression of the endodermal marker forkhead (Oda et al., 2007) within the primary thickening/cumulus cells led to the suggestion that the primary thickening/cumulus cells are central endodermal cells (Hilbrant et al., 2012; Oda et al., 2007). However, these studies could not completely rule out that the labeled cumulus cells develop into cells of the visceral mesoderm (Edgar et al., 2015). During the last 15 years, research focused on candidate genes known to be involved in development in Drosophila melanogaster has revealed several aspects of how spider embryos pattern their main body axis. However, there are many open questions regarding the early regulation of cumulus specific gene expression, cumulus establishment and maintenance. To overcome the limitations of the candidate gene approach, we have carried out transcriptome sequencing of carefully staged embryos to find new genes involved in cumulus and axial patterning in the spider Parasteatoda tepidariorum. From this work, we have identified the transcription factor Pt-Ets4 as a new gene expressed during early development and have found it to be expressed exclusively within the central primary thickening and the cells of the migrating cumulus. Our combined genetic and cellular analyses show that Pt-Ets4 is needed for the integrity of the cumulus. We found that the knockdown of this gene leads to embryos that show axis patterning defects reminiscent of BMP knockdown phenotypes, suggesting that an intact cumulus is needed to induce the formation of the bilaterally symmetric spider embryo. Importantly, Pt-Ets4 is necessary and sufficient for driving the early expression of twist (a gene involved in gastrulation and mesoderm formation in Drosophila) and hunchback, and the ectopic expression of Pt-Ets4 is sufficient to induce cell delamination. Results The formation of the germ-disc is one of the most important events during spider embryogenesis. While a regular blastoderm (with no visible axial polarity) is present at stage 2, the germ-disc condenses during stage 3 of embryonic development (Figure 1A and B, for detailed description see [Pechmann, 2016]). This event leads to the establishment of the anterior/posterior body axis (anterior: rim of the disc; posterior: center of the disc). Figure 1 with 3 supplements see all Download asset Open asset Early embryonic expression of Pt-Ets4. (A and B) Pt-Ets4 is expressed within the cluster of cells (arrow in A) that will develop into the central primary thickening (pt) (B). (C–D') Confocal scans (single optical slices (C and D'); maximum intensity projection (D)) of embryos stained for Pt-Ets4 (FastRed stain; red) and nuclei (DAPI; blue). Pt-Ets4 is expressed within the primary thickening and in the migrating cumulus (cu), which are covered by the surface epithelium of the germ-disc. Orthogonal views are boxed in green and magenta. The same embryo is depicted in D and D'. Scale bar is 100 µm. https://doi.org/10.7554/eLife.27590.003 To find new genes that are involved in the process of axis specification we sequenced the embryonic transcriptomes of stage 1, stage 2 and stage 3 embryos (Figure 1—figure supplement 1) and searched for genes showing a similar expression profile as Pt-decapentaplegic (Pt-dpp), Pt-hedgehog (Pt-hh) and Pt-patched (Pt-ptc) (representing genes that show early transcription and are required for axis formation or cumulus migration, respectively (Akiyama-Oda and Oda, 2006, Akiyama-Oda and Oda, 2010); Figure 1—figure supplement 1). One of the candidates was an Ets-like gene with high similarity to Drosophila melanogaster Ets4/Ets98B (see Figure 1—figure supplement 2), henceforth called Parasteatoda tepidariorum Ets4 (Pt-Ets4). Pt-Ets4 is expressed in the migrating cumulus Prior to germ-disc condensation, Pt-Ets4 is expressed within the cluster of cells that will form the center of the future germ-disc at early stage 3 (Figure 1A). Expression persists throughout germ-disc formation and, at stage 4, Pt-Ets4 is strongly and exclusively expressed within the central cluster of cells (the so-called primary thickening, Figure 1B) that has delaminated during germ-disc formation (Figure 1C). During stage 5, the cumulus starts to migrate from the center of the germ-disc to its periphery (Oda and Akiyama-Oda, 2008; Akiyama-Oda and Oda, 2003, 2006; McGregor et al., 2008; Hilbrant et al., 2012; Mittmann and Wolff, 2012; Schwager et al., 2015). At this stage Pt-Ets4 is strongly and exclusively expressed in the migrating cumulus cells (Figure 1D and D'). We were not able to detect Pt-Ets4 transcripts in ovaries and early stage 1 embryos via RNA in situ hybridization (Figure 1—figure supplement 3A and B). In addition, our sequencing data shows that Pt-Ets4 is only expressed at a very low level during early stage 1, but is mildly up-regulated at late stage 2 and strongly up-regulated at early stage 3 (Figure 1—figure supplement 1). From this we conclude that Pt-Ets4 transcripts are not maternally provided. Pt-Ets4 is necessary for the integrity of the cumulus Time-lapse imaging and cross-sectioning revealed that the knockdown of Pt-Ets4 neither affected formation of the germ-disc nor of the primary thickening/cumulus (Video 1B; middle column in Figure 2, Figure 3A and B). However, during stage 5, cumulus integrity was affected in Pt-Ets4 knockdown embryos (Video 1B, middle column in Figure 2). While in control embryos the cumulus migrated towards the rim (Video 1A, left column in Figure 2), the cumulus of Pt-Ets4 RNAi embryos remained at the center of the germ-disc until early stage 5 and disappeared soon after gastrulation was initiated at the center and at the rim of the disc (Video 1B (15h onwards), middle column in Figure 2). Analysis of mid-stage 5 Pt-Ets4 RNAi embryos for the expression of the cumulus marker Pt-fascin (Akiyama-Oda and Oda, 2010) revealed that although the cells of the cumulus were still in the center of the germ-disc, they appeared to be more loosely organized (Figure 3C and D). Figure 2 with 1 supplement see all Download asset Open asset Pt-Ets4 is required for cumulus integrity. Stills from the embryos shown in Video 1. The cumulus (asterisk) migrates in the control, disappears in the Pt-Ets4 RNAi and stays in the center of the germ-disc in Pt-ptc RNAi embryo. Ectopic, central opening (induction of the dorsal field) of the germ-disc is depicted via the dotted line (Pt-ptc RNAi st. 6). Posterior (P) and anterior (A) tube formation in Pt-Ets4 and Pt-ptc knockdown embryos is also indicated. https://doi.org/10.7554/eLife.27590.007 Figure 3 with 1 supplement see all Download asset Open asset Cumulus integrity and signaling is affected in Pt-Ets4 knockdown embryos. (A and B) Cross-section through the central cumulus of ubiquitously stained (via Pt-arm RNA in situ hybridization) control (A) and Pt-Ets4 RNAi (B) embryos. (C and D) control and Pt-Ets4 RNAi embryos stained for the cumulus marker Pt-fascin. Cells of the cumulus are dispersing in the Pt-Ets4 knockdown embryo (D). (E and F) Single color double stain of anterior Pt-otd expression (anterior ring) and nuclear localized pMAD in the cells overlaying the cumulus. pMAD signal is absent in Pt-Ets4 RNAi embryos (F). In situ hybridization for the ventral fate marker Pt-sog (G, G', J and J') or the segmental marker Pt-en (H, I, K and L) in control (G–I) and Pt-Ets4 knockdown embryos (J–L). The same embryos in fluorescence vs. bright field channel are shown in G and G' as well as in J and J'. Nuclear stain (DAPI)/bright field overlay is shown in A-F, H, I, K and L. Flat mounted embryos in C-F. Lateral-ventral view (G, G', J–K), lateral view (H), ventral view (I). Abbreviations: ch: cheliceral segment; L1-L4: walking leg bearing segments 1–4; O1: opisthosomal segment 1. https://doi.org/10.7554/eLife.27590.009 Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Knockdown of Pt-Ets4 and Pt-ptc. Live imaging of a control (A), a Pt-Ets4 RNAi (B) and a Pt-ptc RNAi (C) embryo under transmitted light conditions. The video starts at stage 3 and ends at stage 9 of embryonic development. Cumulus migration and normal germ-band formation is visible in the control embryo (A). The cells of the cumulus disperse in the Pt-Ets4 knockdown embryo (B). The ventralized Pt-Ets4 RNAi embryo stays radially symmetric and posterior tube formation is initiated (30 hr onwards). The cumulus of the Pt-ptc RNAi embryo does not migrate (C). The germ-disc opens up at the central position and the radially symmetric embryo overgrows the yolk and anterior tube formation is initiated (48 hr onwards). https://doi.org/10.7554/eLife.27590.011 In Pt-Ets4 knockdown embryos the radial symmetry of the germ-disc was not broken (as shown by the formation of a tube-like germ band in Video 1B (30h onwards), and the middle column in Figure 2), presumably due to the loss of the cumulus. To investigate this phenotype in more detail, we knocked down another gene that also results in defects in radial symmetry breaking, Pt-ptc. In Pt-ptc knockdown embryos, cumulus migration is lost but the cumulus itself is otherwise unaffected (Akiyama-Oda and Oda, 2010). After pRNAi with Pt-ptc, the cumulus stays in the center of the germ-disc (Video 1C; right column in Figure 2) and BMP signaling is ectopically activated (as shown via antibody staining against the phosphorylated form of mothers against dpp (pMAD) (Akiyama-Oda and Oda, 2010)). As a result, the germ-disc ectopically opened at the center and the dorsal field was induced at the posterior pole of the Pt-ptc RNAi embryos (right column in Figure 2). This ectopic induction of the dorsal field in the center of the germ-disc was never observed in the Pt-Ets4 RNAi embryos. To test if the disappearing cumulus was at least partially able to activate BMP signaling in the germ-disc of Pt-Ets4 RNAi embryos, we performed a pMAD antibody staining in both control and Pt-Ets4 RNAi embryos. In control embryos the cumulus reached the periphery of the germ-disc at late stage 5 and a strong pMAD staining was visible in the overlying ectodermal cells (Figure 3E, Figure 3—figure supplement 1). At this stage, the anterior marker Pt-orthodenticle (Pt-otd) was expressed in a ring, which had a width of 3–5 cells (Akiyama-Oda and Oda, 2003; Pechmann et al., 2009; Akiyama-Oda and Oda, 2016) (Figure 3E). In Pt-Ets4 knockdown embryos Pt-otd expression was unaffected but nuclear pMAD was not detectable (Figure 3F). This lack of BMP signaling explains why Pt-Ets4 RNAi embryos do not induce the dorsal field and stay radially symmetric. Indeed, the knockdown embryos were completely ventralized; the expression of the ventral marker Pt-short-gastrulation (Pt-sog) was uniform around the embryonic circumference and the segmental marker Pt-engrailed (Pt-en) was expressed in symmetric rings demonstrating the radial symmetry of the embryo (Figure 3G–L). During later development the embryonic tissue either grew completely over the yolk (Figure 3J–L), or a tube like structure elongated at the posterior of the embryo (Video 1B, middle column Figure 2). This was in contrast to Pt-ptc RNAi embryos, where the germ-disc opened up centrally and a tube like structure formed at the anterior of the embryo (right column Figure 2; Video 1C). The Pt-Ets4 knockdown phenotype is rather similar to that of Pt-dpp, although cumulus migration/integrity is not affected in Pt-dpp RNAi embryos (Akiyama-Oda and Oda, 2006). This observation, plus the early and strong expression of Pt-Ets4 and the fact that BMP signaling disappeared upon Pt-Ets4 knockdown, led us to hypothesize that Pt-Ets4 functions as an activator of Pt-dpp transcription within the cells of the cumulus. However, there was no obvious difference in the expression of Pt-dpp in stage 4 control and Pt-Ets4 RNAi embryos (Figure 4A and E). Vice versa, Pt-dpp appears not to be regulating the expression of Pt-Ets4 (Figure 4—figure supplement 1A). In addition, Hh signaling seems not to be involved in the regulation of Pt-Ets4 expression and Pt-Ets4 appears not to regulate the expression of Pt-ptc within the primary thickening (Figure 4—figure supplement 1B and C). Figure 4 with 1 supplement see all Download asset Open asset Analysis of 'cumulus marker' genes. Flat mount preparation of in situ stained stage 4 embryos. While Pt-dpp and Pt-fkh expression is unaffected (compare A to E and B to F), Pt-fascin expression is slightly (compare C to G) and Pt-hb expression is strongly down regulated in Pt-Ets4 RNAi embryos (compare D to H). https://doi.org/10.7554/eLife.27590.012 In order to determine what other genes Pt-Ets4 may regulate, we studied the expression of genes normally expressed in the primary thickening in Pt-Ets4 RNAi embryos. We found that Pt-forkhead (Pt-fkh) expression was unaffected (Figure 4B and F), while the cumulus marker Pt-fascin was slightly down regulated, and Pt-hunchback (Pt-hb) was strongly down regulated in Pt-Ets4 knockdown embryos (Figure 4C,D,G and H). Ectopic expression of Pt-Ets4 induces cell migration As Pt-Ets4 is strongly expressed in the cumulus and is required for cumulus integrity, we wondered how ectopic expression of Pt-Ets4 would affect cell behavior. To generate small cell clones ectopically expressing Pt-Ets4 within the germ-disc, we micro-injected late stage 1 embryos (via single cell/blastomere injections [Hilbrant et al., 2012; Kanayama et al., 2010]) with in vitro synthesized capped mRNA coding for an EGFP-Pt-Ets4 fusion protein (see Figure 5—figure supplement 1). Our first observation was that the EGFP-Pt-Ets4 fusion protein localizes to the nuclei of the injected cells, suggesting that the nuclear-localization-signal of Pt-Ets4 is functioning normally (Figure 5A). EGFP-Pt-Ets4 marked cell clones resembled wild-type until stage 4. As soon as a dense germ-disc formed, however, the cell clones expressing EGFP-Pt-Ets4 seemed to move beneath the germ-disc and the EGFP signal became occluded by the opaque cells of the germ-disc (Figure 5B). We have never seen such behavior following injection of EGFP-NLS constructs alone (Figure 5F–F'''), indicating that the change in cell behavior is due to the ectopic expression of Pt-Ets4. Figure 5 with 1 supplement see all Download asset Open asset Ectopic expression of Pt-Ets4 causes the delamination and migration of cells. (A and A') A stage 4 embryo in which a cell clone has been marked via the ectopic expression of an EGFP-Pt-Ets4 fusion construct. The fusion protein localizes to the nuclei. (B and B') The cell clone has delaminated four hours later. As the overlaying epithelium is highly light-scattering, the nuclear EGFP signal is no longer visible. The inset shows the magnification of the boxed region in A and B. (C and D) Stills from Video 2A and D (magnifications of the lynGFP Cells expressing lynGFP alone stay at the surface epithelium of the germ-disc. Cells expressing and EGFP antibody staining intensity projection is shown in E and the view is shown in of a embryo ectopically expressing Stills from Video 3A A cell clone marked via the ectopic expression of nuclear localized EGFP marks the ectoderm during germ-band Stills from Video expression of A cell clone ectopically expressing EGFP-Pt-Ets4 in with EGFP-NLS after germ-disc formation The cells of this cell clone to disperse during later stages of development and Scale bar is 100 in To the process of cell clone we marked cell by capped mRNA coding for EGFP-Pt-Ets4 with capped mRNA coding for lynGFP and In control embryos expression of lynGFP lynGFP strongly marked the cell and cell clones at the surface epithelium of the germ-disc (Video Figure of the position and the of the cell cells expressing and delaminated after the formation of the germ-disc (Video Figure The of EGFP-Pt-Ets4 cell clones in embryos an antibody against EGFP that the labeled cells were the epithelium of the germ-disc (Figure and Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Ectopic expression of Pt-Ets4 causes the delamination of cells. Ectopic expression of lynGFP (A) and lynGFP in with The lynGFP cell clone (A) stays in the ectodermal cell of the germ-disc. of the of the cell Pt-Ets4 cells and The nuclear signal of the EGFP-Pt-Ets4 fusion the signal of the is visible after the delamination we capped mRNA for EGFP-Pt-Ets4 with capped mRNA for nuclear localized EGFP see Figure 5—figure supplement a that we have found to a very bright and This in a strong nuclear localized EGFP signal within the marked cell which we to While in the control embryos with EGFP-NLS the marked cell clones at the surface of the germ-disc (Video Figure and the cell clone expressing both EGFP-NLS and EGFP-Pt-Ets4 delaminated after the formation of the germ-disc (Video Figure and As previously shown et al., Kanayama et al., germ-disc cells to and during the formation of the which causes cell clones to and elongated as seen in our control EGFP-NLS clones (Video Figure and In contrast to cell clones expressing both EGFP-NLS and EGFP-Pt-Ets4 as soon they In addition, the cumulus to the delaminated cells of the marked cell clone lost with each other and out underneath the epithelium (Video Figure and This observation was and in embryos (Video Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Ectopic expression of Pt-Ets4 causes the delamination and migration of cells. Ectopic expression of EGFP-NLS (A) and EGFP-NLS in with (B). The cell clone for EGFP-NLS (A) stays in the ectodermal cell of the germ-disc. Cells and form a and cell clone (via et al., at stage of embryonic development. The Pt-Ets4 cell clone at stage 4 (B). Cells and to disperse as soon as the cells of the cumulus to migrate Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Ectopic expression of EGFP-NLS and EGFP-Pt-Ets4 in embryos. Ectopic expression of EGFP-NLS and EGFP-NLS in with EGFP-NLS and ectopic clones expressing Pt-Ets4 have within the germ-disc. While all of the control cell clones form and cell clones at stage of embryonic development, the Pt-Ets4 cells clones and the cells disperse from stage 5 the EGFP channel is shown for all embryos. results that Ets4 expression is sufficient to induce cell delamination and migration in embryos of tepidariorum. Ectopic expression of Pt-Ets4 induces mesoderm-like fate As Pt-Ets4 seems to have no on the early expression of Pt-dpp itself (Figure 4A and E). Furthermore, we were not able to detect Pt-dpp transcripts in cells ectopically expressing Pt-Ets4 (Figure and In contrast to Pt-dpp, we find that Pt-Ets4 is regulating the expression of While Pt-hb expression was absent in Pt-Ets4 RNAi embryos (Figure Pt-hb transcripts were present in the ectopically Pt-Ets4 cell clone (Figure and Figure with 2 supplements see all Download asset Open asset Pt-Ets4 Pt-hb and expression within the primary Live stage 4 embryos in which a cell clone is marked via the of are depicted in The same embryos have been and for their expression of Pt-dpp Pt-hb and Expression of in a control and a Pt-Ets4 knockdown embryo D and E are of in situ hybridization The behavior of the Pt-Ets4 cells is reminiscent of migrating cells that the germ-disc from the center and from the rim of the germ-disc and Wolff, 2012; Kanayama et al., et al., (see Video 1A). As Pt-Ets4 the expression of Pt-hb (a gene that is known to be expressed in cells in animals et al., 2009; and et al., we wondered if Pt-Ets4 is inducing a mesoderm-like cell fate. this we whether the ectopic expression of Pt-Ets4 is also inducing the expression of the marker Pt-twist Indeed, was detectable within the cell clone that ectopically expressed Pt-Ets4 (Figure and to in Figure supplement 1). in the stage 4 embryos ectopically expressing we could not only detect transcripts within the ectopic Pt-Ets4 expressing cell but also within the central primary thickening (Figure This as a as it was that expression is not initiated the of stage 5 et al., this we the expression of in wild-type embryos and we were able to that is expressed in the developing primary thickening of stage 3 and 4 embryos (Figure supplement we the regulation of via Pt-Ets4 by the expression of in Pt-Ets4 knockdown embryos. transcripts were no longer detectable in the primary thickening of stage 4 Pt-Ets4 RNAi embryos (Figure and E). However, late segmental mesoderm specification was as the expression of was in stage Pt-Ets4 knockdown embryos (Figure supplement This that the formation and