Gut endoderm takes flight from the wings of mesoderm Angela C. H. McDonald and Janet Rossant The endoderm layer destined to be primitive gut is a mosaic of earlier visceral endoderm and definitive endoderm that arises later, during gastrulation. Live imaging now reveals that in mouse embryos, definitive endoderm cells egress from underlying mesoderm and intercalate into the overlying cell layer. This process requires SOX17-mediated control of basement membrane organization. Embryonic development is a complex cascade of cell–cell interactions, cell fate specification, morphogenetic movements and cell rearrangements that together create a multilayered embryo with an organized body plan. During this process, embryonic epithelial cell layers display dynamic behaviour including rearrangement, coordinated movement and shape changes1. Cell intercalation into existing epithelia occurs in many developmental and disease contexts, directing cellular organization and driving morphogenesis2. In this issue of Nature Cell Biology, Viotti et al. investigate a cellular intercalation event driving gut endoderm morphogenesis in the mouse embryo, describing the migratory path that definitive endoderm (DE) cells travel prior to arrival in the visceral endoderm (VE). They also identify the transcription factor SOX17 as a critical regulator of this process3. The gut endoderm of the mammalian embryo forms the epithelial lining of the gastrointestinal tract and its associated organs, performing essential functions in the adult body including digestion, nutrient absorption, glucose homeostasis, gas exchange and detoxification4. DE is one of the three primary germ layers arising at gastrulation and has long been assumed to be the exclusive source of embryonic gut endoderm tissue. However, Angela C. H. McDonald and Janet Rossant are in the Program in Developmental and Stem Cell Biology, Hospital for Sick Children Research Institute, Peter Gilgan Centre for Research and Learning, 686 Bay Street, Toronto, Ontario M5G OA4, Canada. Angela C. H. McDonald is also in the Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto, Ontario M5S 3G9, Canada. Janet Rossant is also in the Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto Ontario M5S 1A8, Canada. e‑mail: [email protected]


prior to the onset of gastrulation, a layer of VE surrounds the embryo. VE arises from the primitive endoderm cell layer established earlier in the blastocyst. It performs important supportive functions for the embryo prior to placental formation and is also a key source of signals to pattern the developing body axis. It was not, however, thought to contribute to the later DE, as classical fate mapping studies suggested that DE cells displace VE cells during gastrulation5,6. DE cells were presumed to insert into the VE as a continuous sheet causing widespread displacement of VE cells, resulting in a gut endoderm layer exclusively derived of embryonic DE. Recently, higher resolution methodology utilizing live imaging and fluorescent reporter mice to mark all VE cells uncovered the persistence of VE cells within the DE (ref. 7). Instead of widespread VE displacement, DE cells were found to intercalate into the VE and form a mosaic epithelium, comprised of both extra-embyonic and embryonic tissue. This striking observation left many unanswered questions. What is the trajectory of DE cells as they travel from the primitive streak (the site of gastrulation) to the surface of the embryo? What molecular mechanisms drive this process? Do VE cells give rise to adult gut tissues? Viotti et al. set out to map the path of DE cells and uncover the molecular mechanisms driving intercalation3. To track cell movements, the authors used a double labelling technique to mark VE cells with GFP, and cells occupying the posterior mouse epiblast, the site of gastrulation, with RFP. They followed the relative movements of GFP+ and RFP+ cells using live imaging and found that RFP+ epiblast-derived cells migrated in an anteriorward stream, similar to the trajectory expected

for mesoderm cells, and subsequently found that some RFP+ epiblast-derived cells inserted themselves into the VE epithelium on the embryo surface. The authors then used a single labelling technique to mark the VE with GFP, and observed GFP– cells egressing into the VE, always posterior to the leading edge of the mesoderm wings (Fig. 1a). While double-labelling would have been more informative, the presence of GFP– cells posterior to the mesoderm leading edge is clear, suggesting that DE progenitor cells incorporate into, or travel alongside, mesodermal progenitors. Upon arrival on the surface epithelium, GFP– regions can be seen amongst dispersed GFP+ cells (Fig. 1b). The generation of 3D fate maps in the future outlining the full trajectories of DE cells by live imaging will be useful. However, the current data clearly show a population of presumptive DE cells travelling alongside migrating mesoderm prior to intercalation into the VE epithelium. These morphogenetic movements reported by Viotti et  al. are highly reminiscent of those that direct avian gut endoderm development. In the chick embryo, prospective epiblast-derived DE cells ingress through the primitive streak and migrate within the mesodermal layer prior to egressing into the extra-embryonic endoderm, known as the hypoblast 8–10. The molecular mechanisms underlying DE and VE intercalation are not understood. To address this question Viotti et al. explored the role of gastrulation and mesoderm migration in VE dispersal. They found that VE remained as a continuous epithelial sheet on the embryonic surface in mutant mouse strains where mesoderm invagination and migration fail to occur, demonstrating that the exit of cells from

NATURE CELL BIOLOGY VOLUME 16 | NUMBER 12 | DECEMBER 2014 © 2014 Macmillan Publishers Limited. All rights reserved

NEWS AND VIEWS the primitive streak and subsequent mesoderm migration are required for DE egression and VE dispersal. In an attempt to tie DE intercalation to DE gene regulatory network members, mutant mouse lines devoid of the key DE transcription factors SOX17 or FOXA2 were analyzed. Gut morphogenetic defects have been previously described in Foxa2–/– embryos11. However, live imaging in the current study demonstrated that FOXA2 is not required for initial DE migration, VE dispersal or assembly of a mixed endodermal-derived epithelium on the surface of the embryo, suggesting that FOXA2 may instead be required for infolding of the gut endoderm and subsequent formation of the primitive gut tube. In contrast, Viotti et al. show that SOX17 is required for VE dispersal and subsequent generation of a mosaic endodermal epithelium. In wild-type embryos, SOX17 was detected at low levels throughout the VE and at high levels in a subset of cells within the mesodermal layer and in contact with the VE — presumably DE cells undergoing egression. Upon completion of VE dispersal, all endoderm cells, regardless of origin, expressed equivalent levels of SOX17, suggesting that the phenotypes of VE and DE cells intermixed on the surface of the embryo converge, at least to some degree. Gastrulation and migration of mesoderm wings occurs normally in Sox17–/– embryos, but they show defective VE dispersal in the prospective foregut region and no dispersal in the mid- or hindgut regions. Interestingly, SOX17 and FOXA2 appear to play complementary roles in the regulation of gut endoderm morphogenesis: SOX17 regulates prospective mid- and hindgut development whereas FOXA2 regulates foregut development. Some clues as to the mechanism by which SOX17 regulates DE egression are provided by examining the cellular architecture of prospective DE cells in Sox17–/– mutants. Basement membranes provide rigidity to the structure of epithelial layers. In wild-type embryos, a basement membrane separates endoderm from mesoderm. Intriguingly, Sox17–/– embryos lacked a basement membrane at the mesoderm/endoderm interface. Despite the ability of SOX17 to regulate basement membrane protein synthesis in the extra-embryonic endoderm12, the expression of genes encoding basement membrane proteins was not affected by the loss of Sox17 in the prospective DE. Further analysis of Sox17–/– prospective DE cells demonstrated an inability of Sox17–/– cells


Pre-Streak Stage E6.25

Late Streak Stage E7.0

No Bud Stage E7.25

Late Bud Stage E7.5 A









Visceral endoderm



Definitive endoderm

Figure 1 DE and VE intercalation on the surface of the mouse embryo. (a) Transverse embryo sections illustrate mesoderm (purple) migration and DE (pink) egression into the VE (green). (b) During embryogenesis, DE cells egress into the VE, forming a mosaic embryonic and extraembryonicderived epithelium. Axis labels: A, anterior; P, posterior; D, distal; Pr, proximal; L, left; R, right.

to appropriately polarize, subsequently preventing deposition of polymerized basement membrane complexes at DE/mesoderm cell interfaces thereby preventing the generation of a basement membrane separating the emerging gut endoderm from mesoderm. Together, these data link the endoderm gene regulatory network member SOX17 to basement membrane assembly, a critical regulator of epithelial cell organization. The current study clearly demonstrates widespread intercalation of epiblast-derived DE cells into the VE, creating a mosaic endodermal layer on the embryonic surface, but the functional significance of this cellular behaviour is not yet clear. What purpose do VE cells serve during the formation of the embryonic gut? In the embryo, the VE secretes proteins involved in nutrient transport including apolipoproteins and, in combination with other extra-embryonic tissues, is transiently responsible for nutrient and waste exchange13,14. In the adult, gut endodermderived organs perform these metabolic functions. Given their functional similarity, it is intriguing to postulate that VE cells may contribute functionally to developing organs such as the liver and intestine. The VE also plays an important role in patterning the embryo, with the anterior VE producing inhibitory molecules that block posterior development and determine the future anterior–posterior body axis.15. Dispersed VE cells were previously shown to surround

NATURE CELL BIOLOGY VOLUME 16 | NUMBER 12 | DECEMBER 2014 © 2014 Macmillan Publishers Limited. All rights reserved

later embryonic signalling structures, such as the node7, at the anterior end of the primitive streak. Do VE cells continue to aid embryonic patterning by cooperating with embryonic signalling structures? In the chick, hypoblast cells persist underneath the developing primitive streak and are thought to do so to prolong their inductive influence. Our classical concepts of germ layer separation at gastrulation will continue to be challenged by the complexity of embryonic cell movements and interactions as revealed by live imaging. COMPETING FINANCIAL INTERESTS: The authors declare no competing financial interests. 1. Takeichi, M. Nat. Rev. Mol. Cell Biol. 15, 397–410 (2014). 2. Walck-Shannon, E. & Hardin, J. Nat. Rev. Mol. Cell Biol. 15, 34–48 (2014). 3. Viotti, M., Nowotschin, S. & Hadjantonakis, A‑K. Nat. Cell Biol. 16, 1146–1156 (2014). 4. Zorn, A. M. & Wells, J. M. Annu. Rev. Cell Dev. Biol. 25, 221–251 (2009). 5. Lawson, K. A., Meneses, J. J. & Pedersen, R. A. Dev. Biol. 115, 325–339 (1986). 6. Lawson, K. A. & Pedersen, R. A. Development 101, 627–652 (1987). 7. Kwon, G. S., Viotti, M. & Hadjantonakis, A‑K. Dev. Cell 15, 509–520 (2008). 8. Vakaet, L.  J.  Exmbryol. exp. Morph. 10, 38–57 (1962). 9. Azar, Y. & Eyal-Giladi, H. J. Embryol. Exp. Morph. 77, 143–151 (1983). 10. Kimura, W., Yasugi, S., Stern, C. D. & Fukuda, K. Dev. Biol. 289, 283–295 (2006). 11. Ang, S. L. & Rossant, J. Cell 78, 561–574 (1994). 12. Niakan, K.  K. et  al. Genes Dev. 24, 312–326 (2010). 13. Cross, J.  C., Werb, Z. & Fisher, S.  J. Science 266, 1508–1518 (1994). 14. Bielinska, M., Narita, N. & Wilson, D. B. Int. J. Dev. Biol. 43, 183–205 (1999). 15. Stern, C. D. & Downs, K. M. Development 139, 1059– 1069 (2012).


Gut endoderm takes flight from the wings of mesoderm.

The endoderm layer destined to be primitive gut is a mosaic of earlier visceral endoderm and definitive endoderm that arises later, during gastrulatio...
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