The mutual repression between Pax2 and Snail factors regulates the epithelial/mesenchymal state during intermediate mesoderm differentiation

! 1! The mutual repression between Pax2 and Snail factors regulates the epithelial/mesenchymal state during intermediate mesoderm differentiation Juan M. Fons1,Ŧ, Oscar H. Ocaña1,Ħ,* and M. Angela Nieto1,2,* 1Instituto de Neurociencias (CSIC-UMH), Avda. Ramón y Cajal s/n, Sant Joan d’Alacant, Spain 2CIBERER, Centro de Investigación Biomédica en Red de Enfermedades Raras, ISCIII, Spain * Authors for correspondence [email protected] [email protected] Present addresses Ŧ Centre for Craniofacial and Regenerative Biology, King’s College London, Guy’s Hospital, SE1 9RT, London Ħ Cardiovascular Development Program, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain. Running title (61): Snail1 and Pax2 antagonism controls mesoderm differentiation Summary (19 words): The antagonism between Snail and Pax2 factors in the embryonic differentiation zone, tightly regulates the timing of mesodermal epithelialisation .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 30, 2021. ; https://doi.org/10.1101/2021.06.30.450631doi: bioRxiv preprint ! 2! ABSTRACT The pronephros is the first renal structure in the embryo, arising after mesenchymal to epithelial transition (MET) of the intermediate mesoderm, where Pax2 induces epithelialisation of the mesenchyme. Here we show that, in the early embryo, Snail1 directly represses Pax2 transcription maintaining the intermediate mesoderm in an undifferentiated state. Reciprocally, Pax2 directly represses Snail1 expression to induce MET upon receiving differentiation signals. We also show that BMP7 acts as one such signal by downregulating Snail1 and upregulating Pax2 expression. This, together with the Snail1/Pax2 reciprocal repression, establishes a regulatory loop in a defined region along the anteroposterior axis, the bistability domain within the transition zone, where differentiation of the neural tube and the somites are known to occur. Thus, we show that the antagonism between Snail1 and Pax2 determines the epithelial/mesenchymal state during the differentiation of the intermediate mesoderm and propose that the bistability zone extends to the intermediate mesoderm, synchronizing the differentiation of tissues aligned along the mediolateral embryonic axis. INTRODUCTION During embryonic development, most of the tissues are derived from the sequential activation of epithelial-mesenchymal (EMT) and mesenchymalepithelial transitions (MET) (Thiery et al., 2009). A prototypic example is the development of the sequential renal structures, pronephros and metanephros. The pronephros is the first epithelial structure of the urogenital system that arises from the intermediate mesoderm after MET. It encompasses the nephric duct and nephric cord running along both sides of the embryo adjacent to the somites. It elongates posteriorly until it reaches the metanephric mesenchyme where the adult kidney will develop (James and Schultheiss, 2003)(Dressler, 2009). Snail transcription factors are potent EMT inducers in the embryo at gastrulation (Acloque et al., 2011) and neural crest delamination (Nieto et al., 1994), among other embryonic processes. During the development of the renal system in vertebrates, they are expressed in the intermediate mesoderm .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 30, 2021. ; https://doi.org/10.1101/2021.06.30.450631doi: bioRxiv preprint ! 3! before pronephros epithelialisation and later in the metanephric mesenchyme prior to the MET that leads to the formation of the renal vesicle (Boutet et al., 2006). In both cases, epithelialisation concurs with the repression of Snail factors, which are maintained silent in the adult, although they are reactivated in pathological conditions like fibrosis, where they induce a partial EMT and instruct the interstitium to promote fibrogenesis and inflammation (Grande et al., 2015)(Lovisa et al., 2015). If the reactivation occurs in cancer cells, they disseminate from the primary tumour initiating the metastatic cascade (Ocaña et al., 2012)(Yang et al., 2020). By contrast, Pax2 is an essential promotor of MET and required for the specification of the renal epithelial lineage (Dressler et al., 1993)(Torres et al., 1995)(Bouchard et al., 2002). It is expressed in the intermediate mesoderm just prior to MET and continues to be expressed in the epithelial derivatives of the pronephros, mesonephros and metanephros (Dressler et al., 1990)(Dressler and Douglass, 1992)(Bouchard et al., 2000)(Bouchard et al., 2002)(Narlis et al., 2007). Another important molecule that promotes epithelialisation and MET during renal development and fibrosis is BMP7, a member of the TGFβ superfamily (Massagué, 2012) expressed in the pronephros, mesonephros and metanephros (Lyons et al., 1995)(Vukicevic et al., 1996). In agreement with this, Bmp7 null mutant mice display renal hypoplasia with a reduction of the epithelial compartment (Dudley et al., 1995)(Luo et al., 1995). Furthermore, BMP7 treatment prevents and reverses acute renal fibrosis restructuring the epithelial tubules of the kidney inducing MET by antagonizing TGFβ signalling (Zeisberg et al., 2003)(Sato et al., 2003)(Zavadil et al., 2004)(Zeisberg et al., 2005), a well known EMT inducer and Snail transcriptional activator (Peinado et al., 2003). We have examined the relationship between EMT and MET inducers Snail, Pax2 and Bmp7 in the intermediate mesoderm, as it is an excellent model to study epithelial plasticity in the embryo and found that a reciprocal repression between Snail1 and Pax2 controls the timing of differentiation. This negative regulatory loop is triggered by Bmp7, which simultaneous represses Snail1 and activates Pax2 expression. This loop is activated in the region were differentiation of the neural tube and the paraxial mesoderm also occurs, .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 30, 2021. ; https://doi.org/10.1101/2021.06.30.450631doi: bioRxiv preprint ! 4! indicating that it is integrated in the synchronization of differentiation processes along the mediolateral axis. RESULTS AND DISCUSSION Snail1 and Pax2 are expressed in mutually exclusive domains in the developing mesoderm Given that the silencing of Snail1 expression correlates with the epithelialisation of the metanephros (Boutet et al., 2006) and that Pax2 is required for the epithelialisation of pro and metanephros (Bouchard et al., 2002)(Narlis et al., 2007), we decide to compare the expression of both factors at early stages of mesoderm development in the chick embryo (Fig. 1). Snail1 is expressed in mesenchymal tissues including the undifferentiated intermediate and lateral mesoderm (Fig.1A-C) but absent from the nephric duct and cord of the pronephros (Fig.1B). By contrast, Pax2 is expressed in the epithelial structures of the pronephros but not in the undifferentiated intermediate mesoderm (Fig.1D-F). We performed a double FISH and confirmed the absence of Snail1/Pax2 co-expression (Fig.1G-I). These results show that Snail1 and Pax2 expression domains are mutually exclusive in the developing embryo and, in particular, in the differentiating intermediate mesoderm. Snail1 prevents epithelialisation of the intermediate mesoderm by directly repressing Pax2 transcription The expression pattern of Snail1 and Pax2 and their described roles in EMT/MET regulation already suggests a possible genetic interaction. To address this, we first performed gain of function experiments by coelectroporating plasmids coding for Snail1 and GFP and analysed Pax2 expression (Fig. 2A-D). The regions of the intermediate mesoderm with Snail1 ectopic expression, (Fig. 2A,B green), showed a downregulation of Pax2 expression, when compared with the control electroporation and the contralateral side (Fig. 2C,D arrow and asterisk; Fig. S1A-D). Analysis of the Pax2 promoter showed an E box compatible with Snail1 binding (Cano et al., 2000) (Fig. 2E). Luciferase assays in the Pax2-positive human renal embryonic epithelial cells HEK293T (Tamimi et al., 2008) show that Snail1 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 30, 2021. ; https://doi.org/10.1101/2021.06.30.450631doi: bioRxiv preprint ! 5! can repress Pax2 promoter activity (Fig. 2F) in a dose-dependent manner (Fig. S2A-C). Furthermore, the mutation of the E box into a sequence to which Snail1 does not bind (Batlle et al., 2000) restored promoter activity (Fig. 2F). Chromatin immunoprecipitation assays from chick embryo tissues obtained after electroporation of a plasmid encoding a Myc-tagged Snail1 version (Acloque et al., 2011) confirm the enrichment for Snail binding to the Pax2 promoter (Fig. 2G), indicating that Snail1 behave as a direct repressor of Pax2 expression in vivo. Thus, Snail1 expression can maintain the intermediate mesoderm in an undifferentiated and mesenchymal state by preventing Pax2 expression and with that, the premature differentiation of the pronephros. This is in agreement with the finding that the intermediate mesoderm fails to become epithelial and remains mesenchymal in Pax2 mutant mice (Torres et al., 1995). As Snail1 endogenous silencing in the metanephric mesenchyme correlates with MET and renal differentiation (Boutet et al, 2006), and differentiation relies on Pax2 upregulation (Narlis et al., 2007), it is likely that Snail1 also prevents premature metanephros differentiation by repressing Pax2 expression, in addition to its previously described role in repressing HNF1β and Cadherin 16 expression (Boutet et al., 2006). A Snail1/Pax2 reciprocal inhibitory loop regulates the differentiation of the intermediate mesoderm As Pax2 is required for MET and renal differentiation (Rothenpieler and Dressler, 1993)(Torres et al., 1995)(Bouchard et al., 2002) and Snail1 silencing is essential for pronephros differentiation (Fig. 2), we wondered whether Pax2 promoted MET in the intermediate mesoderm at least in part by repressing Snail1 transcription. We performed gain of function experiments for Pax2 as described above for Snail1 (Fig. 3A-D; Fig. S1E-H). The areas of Pax2 ectopic expression (Fig. 3A, green) are devoid of Snail1 transcripts (Fig. 3A-D, asterisks indicate the effect on the intermediate mesoderm). Interestingly, the electroporated mesoderm shows cellular aggregates compatible with a MET process (Fig. 3A, arrowheads), which was confirmed by the re-expression of E-cadherin in the electroporated area (Fig. S3). These results show that the sole activation of Pax2 expression in Snail1-positive .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 30, 2021. ; https://doi.org/10.1101/2021.06.30.450631doi: bioRxiv preprint ! 6! intermediate and lateral mesoderm is sufficient to induce epithelialisation. Thus, we examined whether Pax2 could also act as a Snail1 repressor. We identified a Pax2 binding site (GGGCATGG) 116bp upstream of the Snail1 TSS (JASPAR database) and a luciferase assay in a primary culture of chick embryonic fibroblasts showed that a fragment of 150 bp upstream the TSS co-transfected with a Pax2-encoding vector was sufficient to repress Snail1 promoter activity in a dose-dependent manner (Fig. 3 E,F and Fig. S2D-F). Deletion of the Pax2 binding site impaired this repression (Fig. 3F). We next performed ChIP analysis in chick embryo tissues and confirmed that Pax2 can bind to the Snail1 promoter in vivo (Fig. 3G). Altogether, these data show that, similar to Snail1 repressing Pax2 transcription, Pax2 represses Snail1 transcription by direct binding to its promoter. Thus, Snail1 and Pax2 establish a reciprocal negative loop, a mechanism widely used during development when fate decisions involve binary choices (Zhou and Huang, 2011). This is observed in the subdivision of embryonic territories, including the decision to become trophectoderm or inner cells mass (Cdx2/Oct4) or ectodermal versus mesendodermal (SoxB1/Snail) (Niwa et al., 2005)(Acloque et al., 2011)(Acloque et al., 2012). This reciprocal repression between Snail1 and Pax2 is also likely to occur during metanephros development and may also have a correlate in pathological conditions. Pax2 reactivation occurs in fibrotic or injured kidneys (Imgrund et al., 1999)(Hou et al., 2018), and this is thought to be an attempt to recover the normal renal epithelial architecture either through MET of the renal mesenchyme or by increasing proliferation of the remaining epithelial cells (Lindoso et al., 2009). We propose that this role of Pax2 reactivation maybe achieved by counteracting the activation of Snail1 in renal epithelial cells upon injury (Grande et al., 2015). Bmp7 controls pronephros differentiation by repressing Snail1 and inducing Pax2 expression in the Bistability Domain Then we investigated signalling pathways that could be regulating these genes. We focused on BMP members as they induce pronephros differentiation. Specifically, we tested Bmp7, as it is also required for MET and .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 30, 2021. ; https://doi.org/10.1101/2021.06.30.450631doi: bioRxiv preprint ! 7! differentiation of the metanephros (Dudley et al., 1995)(Luo et al., 1995)(Vukicevic et al., 1996). Bmp7 is expressed in the ectoderm, dorsal neural tube, the notochord and in the pronephros (Fig. 4A,B) and we decided to challenge the embryo by administering BMP7 and assessing its impact on Snail1 and Pax2 expression. BMP7 represses Snail1 transcription in the mesoderm, including the intermediate mesoderm (Fig. 4C,D, asterisks, Fig. S4A-H). In addition, it induces Pax2 transcription in the paraxial/somatic mesoderm (Fig. 4E,F, arrows, see also Suppl. Fig. 4I-P), while represses Paraxis (Fig. S5). This is consistent with the gradient of BMP signalling in patterning the mesoderm along the mediolateral axis in the chick embryo among other vertebrates (James and Schultheiss, 2005) and with recent data showing that the embryo interprets Bmp7 concentrations thresholds (Greenfeld et al., 2021). Thus, Bmp7 induces pronephros differentiation through two independent mechanisms, by repressing an EMT inducer, Snail1, and by inducing a MET inducer, Pax2 in the intermediate mesoderm. The ectoderm is very likely the source of Bmp7, as ectodermal removal impairs pronephros differentiation (Obara-Ishihara et al., 1999). And again, these data and expression studies suggest that Bmp7 may also control metanephros epithelialisation and differentiation through the same mechanism. In addition, the mechanism behind the attenuation of renal fibrosis by BMP7 (Zeisberg et al., 2003)(Zeisberg et al., 2005) is likely mediated by the repression of Snail1 and the induction of Pax2. The ability of BMP7 to activate Pax2 expression in the paraxial mesoderm occurs in a specific region of the anteroposterior axis of the embryo. In addition, we did not observe Pax2 induction in territories that are naturally incompetent to express it, like the lateral mesoderm, or outside the bistability domain (Fig. 4E,F). This is compatible with the described competence of the cells to respond to BMP according to the embryonic anteroposterior level, synchronizing the differentiation in the different axes (Tucker et al., 2008). As such, the response is within the transition zone (TZ), and restricted to the bistability domain (BD)(Fig. 4E,F, arrows) (Fig. S4I-P). The bistability domain .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 30, 2021. ; https://doi.org/10.1101/2021.06.30.450631doi: bioRxiv preprint ! 8! is defined as a convergent area of two opposing gradients within the anterior one-third of the TZ, an anteroposterior gradient of retinoic acid and a posteroanterior gradient of FGF/Wnt (Del Corral and Storey, 2004)(OliveraMartinez and Storey, 2007)(Goldbeter et al., 2007)(Aulehla and Pourquié, 2010), where neurogenesis (Del Corral and Storey, 2004) and somitogenesis (Aulehla and Pourquié, 2010) occur. Differentiation at the bistability zone is perfectly compatible with the region where binary choices occur (Goldbeter et al., 2007), and particularly with the establishment of reciprocal negative loops as we describe here for Snail1 and Pax2 (Zhou and Huang, 2011). Interestingly, retinoic acid induces pronephros differentiation (Cartry et al., 2006)(Wingert et al., 2007) and it is known to repress Snail1 during somite epithelialisation (Morales et al, 2007). In addition, Snail1 is downstream of the Wnt/FGF signaling pathway during somitogenesis, and it needs to be repressed for epithelialisation to occur (Dale et al., 2006). Our results show that the bistability domain not only regulates neurogenesis and somitogenesis but also pronephros development. We propose a model for pronephros differentiation (Fig. 4G) in which Bmp7, represses Snail1 expression and, in combination with other signals, induces Pax2 transcription in the intermediate mesoderm. Snail1 maintains an undifferentiated state of the intermediate mesoderm in the posterior part of the embryo, and at the bistability domain, Snail1 and Pax2 stablish a negative reciprocal loop that leads to epithelialisation. Thus, we extend the bistability domain to the intermediate mesoderm, acting as the region in the anteroposterior axis for the initiation of differentiation, synchronizing the different cell populations along the mediolateral axis of the embryo. Material and Methods Chick embryonic fibroblast isolation Muscle fragments from HH35 chick embryos were dissected in PBS at 37ºC and disaggregated with trypsin for 5 minutes at RT. The homogenize were centrifuged at 1100 rpm for 6 minutes and the pellet were suspended in F12/HAM and seeded on p10. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 30, 2021. ; https://doi.org/10.1101/2021.06.30.450631doi: bioRxiv preprint ! 9! Cell culture HEK293T cells were cultured in DMEM (Sigma, D6429) with 10% FCS (invitrogen, 10106-169), 1% penicillin/streptomycin (Sigma, P4333) and 1% fungizone (Sigma, A2942). Chick embryonic fibroblasts (CEF) were cultured in F-12/HAM (Sigma, #N6658) with 10% FCS, 1% penicillin/streptomycin plus funginoze. Cell transient transfection and luciferase assays HEK293T cells and CEF were cultured at 105 cells/well in a 12 multiwell plate for 24hr prior to transfection. For luciferase assays, a reaction mix in a final volume of 50µl (for HEK293T) or 100µl (for CEF) in Opti-MEM (Invitrogen, #51985) was made respectively containing 400ng of pGL3basic-Pax2 promoter or 800ng of pGL3b-Snail1 promoter vectors and 40ng of CMVRenilla plasmid as endogenous control. To this mix, 50-100-200ng of pCXSnail1 or 20-50-100ng of pCX-Pax2 vectors were added to HEK293T cells or CEF, respectively. Fugene (Fugene, #E2312) was used for HEK293T cells and Lipofectamine (Lipofectamine, #18324-012) for CEF. After adjusting the culture medium to 500µl, the reaction mix for HEK293T was added and cells were assayed for luciferase activity after 48hr. For CEF, the reaction mix was added to 200µl of opti-MEM for 5hr and the medium was replaced with DMEM afterwards. After the transfection, cells were washed twice in PBS and assayed following the manufacturer´s instructions (Promega, Dual-Luciferase Reporter Assay System Part#TM040). Embryo electroporation The electroporation conditions were 5 pulses of 4 volts, 50ms and 1Hz frequency. Embryos at HH3-4 were electroporated with 2 µg/µl of pCX-Pax2 or pCX-Snail1 and 0.5µg/µl of pCX-EGFP and cultured until HH11 using the easy culture system (Ocaña et al., 2017). In situ hybridization We used a protocol previously described (Nieto et al., 1996) for nonradioactive in situ hybridization. For double fluorescence ISH we followed the method described in Acloque et al., 2008. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 30, 2021. ; https://doi.org/10.1101/2021.06.30.450631doi: bioRxiv preprint ! 16! expressed in embryonic kidney and Wilms tumor. Proc. Natl. Acad. Sci. U. S. A. 89, 1179–1183. Dressler, G. R., Deutsch, U., Chowdhury, K., Nornes, H. O. and Gruss, P. (1990). Pax2, a new murine paired-box-containing gene and its expression in the developing excretory system. Development 109, 787– 795. Dressler, G. R., Wilkinson, J. E., Rothenpieler, U. W., Patterson, L. T., Williams-Simons, L. and Westphal, H. (1993). Deregulation of Pax-2 expression in transgenic mice generates severe kidney abnormalities. Nature 362, 65–67. Dudley, A. T., Lyons, K. M. and Robertson, E. J. (1995). A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye. Genes Dev. 9, 2795–2807. Goldbeter, A., Gonze, D. and Pourquié, O. (2007). Sharp developmental thresholds defined through bistability by antagonistic gradients of retinoic acid and FGF signaling. Dev. Dyn. 236, 1495–1508. Grande, M. T., Sánchez-Laorden, B., López-Blau, C., De Frutos, C. A., Boutet, A., Arévalo, M., Rowe, R. G., Weiss, S. J., López-Novoa, J. M. and Nieto, M. A. (2015). Snail1-induced partial epithelial-tomesenchymal transition drives renal fibrosis in mice and can be targeted to reverse established disease. Nat. Med. 21, 989–997. Greenfeld, H., Lin, J. and Mullins, M. C. (2021). The BMP signaling gradient is interpreted through concentration thresholds in dorsal-ventral axial patterning. PLoS Biol. 19, e3001059. Hou, L., Du, Y., Zhao, C. and Wu, Y. (2018). PAX2 may induce ADAM10 expression in renal tubular epithelial cells and contribute to epithelial-tomesenchymal transition. Int. Urol. Nephrol. 50, 1729–1741. Imgrund, M., Gröne, E., Gröne, H. J., Kretzler, M., Holzman, L., Schlöndorff, D. and Rothenpieler, U. W. (1999). Re-expression of the developmental gene Pax-2 during experimental acute tubular necrosis in mice. In Kidney International 56,1423–1431. James, R. G. and Schultheiss, T. M. (2003). Patterning of the avian intermediate mesoderm by lateral plate and axial tissues. Dev. Biol. 253, 109–124. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 30, 2021. ; https://doi.org/10.1101/2021.06.30.450631doi: bioRxiv preprint ! 17! James, R. G. and Schultheiss, T. M. (2005). Bmp signaling promotes intermediate mesoderm gene expression in a dose-dependent, cellautonomous and translation-dependent manner. Dev. Biol. 288, 113–125. Lindoso, R. S., Verdoorn, K. S. and Einicker-Lamas, M. (2009). Renal recovery after injury: The role of Pax-2. Nephrol. Dial. Transplant. 24, 2628–2633. Lovisa, S., LeBleu, V. S., Tampe, B., Sugimoto, H., Vadnagara, K., Carstens, J. L., Wu, C. C., Hagos, Y., Burckhardt, B. C., PentchevaHoang, T., et al. (2015). Epithelial-to-mesenchymal transition induces cell cycle arrest and parenchymal damage in renal fibrosis. Nat. Med. 21, 998–1009. Luo, G., Hofmann, C., Bronckers, A. L. J. J., Sohocki, M., Bradley, A. and Karsenty, G. (1995). BMP-7 is an inducer of nephrogenesis, and is also required for eye development and skeletal patterning. Genes Dev. 9, 2808–2820. Lyons, K. M., Hogan, B. L. M. and Robertson, E. J. (1995). Colocalization of BMP 7 and BMP 2 RNAs suggests that these factors cooperatively mediate tissue interactions during murine development. Mech. Dev. 50, 71–83. Massagué, J. (2012). TGFβ signalling in context. Nat. Rev. Mol. Cell Biol. 13, 616–630. Narlis, M., Grote, D., Gaitan, Y., Boualia, S. K. and Bouchard, M. (2007). Pax2 and Pax8 regulate branching morphogenesis and nephron differentiation in the developing kidney. J. Am. Soc. Nephrol. 18, 1121– 1129. Nieto, M. A., Sargent, M. G., Wilkinson, D. G. and Cooke, J. (1994). Control of cell behavior during vertebrate development by Slug, a zinc finger gene. Science. 264, 835–839. Nieto, M. A., Patel, K. and Wilkinson, D. G. (1996). In Situ hybridization analysis of chick embryos in whole mount and tissue sections. Methods Cell Biol. 51, 219-235. Niwa, H., Toyooka, Y., Shimosato, D., Strumpf, D., Takahashi, K., Yagi, R. and Rossant, J. (2005). Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation. Cell 123, 917–929. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 30, 2021. ; https://doi.org/10.1101/2021.06.30.450631doi: bioRxiv preprint ! 18! Obara-Ishihara T, Kuhlman J, Niswander L, Herzlinger D. (1999). The surface ectoderm is essential for nephric duct formation in intermediate mesoderm. Development 126, 1103-1108. Ocaña, O. H., Córcoles, R., Fabra, Á., Moreno-Bueno, G., Acloque, H., Vega, S., Barrallo-Gimeno, A., Cano, A. and Nieto, M. A. (2012). Metastatic Colonization Requires the Repression of the EpithelialMesenchymal Transition Inducer Prrx1. Cancer Cell 22, 709–724. Ocaña, O. H., Coskun, H., Minguillón, C., Murawala, P., Tanaka, E. M., Galcerán, J., Muñoz-Chápuli, R. and Nieto, M. A. (2017). A righthanded signalling pathway drives heart looping in vertebrates. Nature 549, 86-90 Olivera-Martinez, I. and Storey, K. G. (2007). Wnt signals provide a timing mechanism for the FGF-retinoid differentiation switch during vertebrate body axis extension. Development 134, 2125–2135. Peinado, H., Quintanilla, M. and Cano, A. (2003). Transforming growth factor β-1 induces Snail transcription factor in epithelial cell lines. Mechanisms for epithelial mesenchymal transitions. J. Biol. Chem. 278, 21113–21123. Rothenpieler, U. W. and Dressler, G. R. (1993). Pax-2 is required for mesenchyme-to-epithelium conversion during kidney development. Development 119, 711–720. Sato, M., Muragaki, Y., Saika, S., Roberts, A. B. and Ooshima, A. (2003). Targeted disruption of TGF-β1/Smad3 signaling protects against renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction. J. Clin. Invest. 112, 1486–1494. Tamimi, Y., Ekuere, U., Laughton, N. and Grundy, P. (2008). WNT5A is regulated by PAX2 and may be involved in blastemal predominant wilms tumorigenesis. Neoplasia 10, 1470–1480. Thiery, J. P., Acloque, H., Huang, R. Y. J. and Nieto, M. A. (2009). Epithelial-Mesenchymal Transitions in Development and Disease. Cell 139, 871–890. Torres, M., Gómez-Pardo, E., Dressler, G. R. and Gruss, P. (1995). Pax-2 controls multiple steps of urogenital development. Development 121, 4057–4065. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 30, 2021. ; https://doi.org/10.1101/2021.06.30.450631doi: bioRxiv preprint ! 19! Tucker, J. A., Mintzer, K. A. and Mullins, M. C. (2008). The BMP Signaling Gradient Patterns Dorsoventral Tissues in a Temporally Progressive Manner along the Anteroposterior Axis. Dev. Cell 14, 108–119. Vukicevic, S., Koppt, J. B., Luyten, F. P. and Sampath, T. K.(1996). Induction of nephrogenic mesenchyme by osteogenic protein 1 (bone morphogenetic protein 7). Dev. Biol. 93, 9021-9026. Wingert, R. A., Selleck, R., Yu, J., Song, H. D., Chen, Z., Song, A., Zhou, Y., Thisse, B., Thisse, C., McMahon, A. P., et al. (2007). The cdx genes and retinoic acid control the positioning and segmentation of the zebrafish pronephros. PLoS Genet. 3, 1922–1938. Yang, J. H., Kim, N. H., Yun, J. S., Cho, E. S., Cha, Y. H., Cho, S. B., Lee, S. H., Cha, S. Y., Kim, S. Y., Choi, J., et al. (2020). Snail augments fatty acid oxidation by suppression of mitochondrial ACC2 during cancer progression. Life Sci. Alliance 3, e202000683. Zavadil, J., Cermak, L., Soto-Nieves, N. and Böttinger, E. P. (2004). Integration of TGF-β/Smad and Jagged1/Notch signalling in epithelial-tomesenchymal transition. EMBO J. 23, 1155–1165. Zeisberg, M., Hanai, J. I., Sugimoto, H., Mammoto, T., Charytan, D., Strutz, F. and Kalluri, R. (2003). BMP-7 counteracts TGF-β1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury. Nat. Med. 9, 964-968. Zeisberg, M., Shah, A. A. and Kalluri, R. (2005). Bone morphogenic protein7 induces mesenchymal to epithelial transition in adult renal fibroblasts and facilitates regeneration of injured kidney. J. Biol. Chem. 280, 8094– 8100. Zhou, J. X. and Huang, S. (2011). Understanding gene circuits at cell-fate branch points for rational cell reprogramming. Trends Genet. 27, 55–62. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 30, 2021. ; https://doi.org/10.1101/2021.06.30.450631doi: bioRxiv preprint ! 20! Figures Fig. 1. Snail1 and Pax2 are expressed in a mutually exclusive pattern in the developing embryo. Dorsal view of chicken embryos at HH11 and their transversal sections. (A-C) Snail1 is expressed in the ingressing mesendoderm (me), undifferentiated intermediate mesoderm (im), lateral plate mesoderm (lm) and somites (s) (n=10). (D-F) Pax2 is expressed in the pronephros (p), somitomeres (sm) and in the tail bud (tb) (n=10). (G) 3D reconstruction of a double in situ hybridization (FISH) for Snail1 (red) and Pax2 (green) (n=3). Longitudinal (H) and Transversal (I) optical sections from the indicated level in a representative embryo in G. Note the mutually exclusive expression pattern. n: notochord, nc: nephric cord, nd: nephric duct, nt: neural tube, pm: paraxial mesoderm. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 30, 2021. ; https://doi.org/10.1101/2021.06.30.450631doi: bioRxiv preprint ! 21! Fig. 2. Snail1 is a direct repressor of Pax2 transcription. (A-D) Chick embryos were co-electroporated on the right side with a vector encoding GFP (pCX-GFP) and another containing the Snail1 coding region (pCX-Snail1). Dorsal views and their respective sections showing GFP immunofluorescence (A,B) and Pax2 expression (C,D). Snail1 ectopic expression (GFP in A,B) represses endogenous Pax2 expression (C,D) in the intermediate mesoderm (A, B, white arrows; C, D, black asterisks) (n=9/9). (E) Scheme of Pax2 promoter fragment (1.8 kb upstream of TSS) cloned into pGL3-Luc vector. A Snail1 binding site (E box) is showed in red. (F) Luciferase assay in HEK293T cells transfected with a vector containing a fragment of the Pax2 promoter bearing the wildtype or a mutated version of the E box plus either pCX (black bars) or pCX-Snail1 (grey bars) vectors. Snail1 decreases Pax2 promoter activity and the mutation of the E box significantly restores the activity (n=3, average representation. T-Test; pValue<0.01; two tails, unequal variance). (G) ChIP assay in chick embryos electroporated with a Myc-tagged Snail1 coding plasmid (Myc-Snail1) or an empty Myc plasmid (Myc) (n=3, representative experiment shown). (1) input; (2) IgG antibody (IgG), negative control; (3) Myc antibody (anti-Myc); (4) Histone3 antibody (anti-H3), positive control. There is amplification of DNA fragments containing the E box in the presence of Myc-Snail1. Therefore, Snail1 represses Pax2 transcription through direct binding to its promoter. im: intermediate mesoderm, n: notochord, nt: neural tube, p: pronephros, s: somites. tb: tail bud. ** pValue <0.001. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 30, 2021. ; https://doi.org/10.1101/2021.06.30.450631doi: bioRxiv preprint ! 22! Fig. 3. Pax2 is a direct repressor of Snail1 transcription. (A-D) Electroporation on the right side of a chick embryo with pCX-GFP plus pCX-Pax2 vectors showing IF for GFP (A,B) and ISH for Snail1 (C,D). (A,C) Dorsal views. (B,D) Sections taken at the level of the dashed lines. Pax2 ectopic expression (GFP) represses Snail1 expression in the intermediate mesoderm (asterisks) (n=18/18). (E) Scheme of Snail1 promoter fragment (0.15kb upstream of TSS) cloned into pGL3-Luc vector. A Pax2 binding box is shown in red. F) Luciferase assay in CEF transfected with a Snail1 promoter vector containing or not the Pax2 binding box plus pCX (black bars) or pCXPax2 (grey bars) plasmids. Pax2 decreases Snail1 promoter activity and the deletion of the box restores it (n=3, average representation. T-Test; two tails, unequal variance). G) ChIP assay. Scheme showing the PCR fragments analyzed, one in the promoter region and another one from a Non Coding Region (NCR). (1) input; (2) IgG antibody (IgG), negative control; (3) Pax2 antibody (anti-Pax2); (4) Histone3 antibody (anti-H3), positive control. (n=3, representative experiment shown). Pax2 represses Snail1 transcription through direct binding to the promoter. im: intermediate mesoderm, n: notochord, nt: neural tube, pm: paraxial mesoderm. ***pValue<0.0001 .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 30, 2021. ; https://doi.org/10.1101/2021.06.30.450631doi: bioRxiv preprint ! 23! Fig. 4. BMP7 represses Snail1 and induces Pax2 expression. (A,B) ISH for Bmp7 and the respective transverse sections. Bmp7 is expressed in the ectoderm (e), dorsal neural tube (nt), notochord (n), pronephros (p) and tail bud (tb). (C-F) Chick embryos bearing a bead (dotted circle) previously soaked in BMP7 (50µg/ml) and subjected to ISH for Snail1 (C,D) or Pax2 (E,F). BMP7 represses Snail1 in the mesoderm (C,D, asterisks,n=3/5) and induces Pax2 expression in the bistability domain (BD) (E,F, arrows, n=3/5). G) Model for pronephros differentiation. Bmp7 directs the flow of information by repressing Snail1 and inducing Pax2 to promote renal differentiation through MET in the intermediate mesoderm within the BD. s: somites, lm: lateral mesoderm, im: intermediate mesoderm. .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted June 30, 2021. ; https://doi.org/10.1101/2021.06.30.450631doi: bioRxiv preprint