Endodermal germ-layer formation through active actin-driven migration triggered by N-cadherin Florence A. Gigera,b,c and Nicolas B. Davida,b,c,d,1 aCNRS UMR8197, F-75005 Paris, France; bINSERM U1024, F-75005 Paris, France; cInstitut de Biologie de l’Ecole Normale Supérieure, F-75005 Paris, France; and dLaboratory for Optics and Biosciences, Ecole Polytechnique, 91128 Palaiseau, France Edited by Roeland Nusse, Stanford University School of Medicine, Stanford, CA, and approved August 16, 2017 (received for review May 16, 2017) Germ-layer formation during gastrulation is both a fundamental We show that cell internalization relies on an active, actin-driven step of development and a paradigm for tissue formation and migration process. Rather than being attracted to their destina- remodeling. However, the cellular and molecular basis of germ- tion, cells migrate away from their neighbors in a process medi- layer segregation is poorly understood, mostly because of the lack ated by Rac1 and triggered by cadherin-2 (hereafter referred to of direct in vivo observations. We used mosaic zebrafish embryos as “N-cadherin”). to investigate the formation of the endoderm. High-resolution live imaging and functional analyses revealed that endodermal cells Results reach their characteristic innermost position through an active, Endodermal Cells Emit Cytoplasmic Extensions Toward the Yolk oriented, and actin-based migration dependent on Rac1, which con- Syncytial Layer and Rapidly Migrate to Its Surface. To unravel the trasts with the previously proposed differential adhesion cell sorting. basis for germ-layer formation, we analyzed the cell behavior and Rather than being attracted to their destination, the yolk syncytial dynamics of endodermal cells during internalization. Naive cells layer, cells appear to migrate away from their neighbors. This migra- can easily be driven to adopt an endodermal fate (SI Experi- tion depends on N-cadherin that, when imposed in ectodermal cells, is mental Procedures and refs. 5, 6, and 11). Combined with cell sufficient to trigger their internalization without affecting their fate. transplants, this allows the creation of mosaic embryos, a pre- Overall, these results lead to a model of germ-layer formation in requisite to good imaging. At the late blastula stage, single en- which, upon N-cadherin expression, endodermal cells actively mi- dodermal progenitors expressing the actin-labeling construct grate away from their epiblastic neighbors to reach their internal Lifeact-GFP were transplanted close to the margin of embryos position, revealing cell-contact avoidance as an unexplored mech- expressing membrane-bound mCherry. Rapid 4D confocal im- anism driving germ-layer formation. aging was used to acquire entire volumes over time, and optical sections were reconstructed to analyze cell behavior in the plane gastrulation | endoderm | cell migration | zebrafish | cadherin of the internalization movement (Fig. 1A). When in the epiblast, endodermal cells emitted large, frequent, astrulation is the first developmental stage when different and short-lived cytoplasmic extensions enriched in actin and ori- Gprogenitors segregate and organize into distinct germ layers. ented toward the yolk syncytial layer (YSL) (n = 103 extensions, Large-scale cell movements set up the body plan of the embryo, Puniform/measured angle distribution < 0.001) (Fig. 1C). Cells internalized with endodermal and mesodermal cells becoming internalized through rapid migration to the surface of the YSL (mean speed: − beneath the ectoderm. Whereas an extensive body of literature 2.4 μm·min 1; n = 6cells)(Fig.1B, D,andE and Movie S1). They describes the pathways that specify endoderm and mesoderm later differentiated into endodermal derivatives (Fig. 1F and ref. identity (1), the cellular mechanisms that physically create these 6). To rule out artifacts due to cell transplants or endoderm in- layers in the embryo are much less understood. duction, we used mosaic expression of Lifeact-GFP in wild-type In frog, internalization is achieved by involution, in which the embryos to look at the behavior of endogenous untreated endo- prospective mesoderm and endoderm roll inward as a coherent derm. We focused on cells located in the four most marginal rows, tissue at the blastopore, driven by the vegetal rotation of the en- dodermal mass (2). In amniotes, as well as in the sea urchin and Significance urodeles, internalization is achieved via ingression of single cells, with endodermal and mesodermal progenitors undergoing an Construction of the adult body during development implies the epithelial-to-mesenchymal transition (EMT) (3, 4). In fish, cell separation of cells into tissues and organs. The first of these transplantation experiments have demonstrated that cells inter- events occurs during gastrulation, when cells segregate in germ nalize individually but in a coordinated manner, termed “syn- ” – layers, setting the bases of the body plan. Despite its impor- chronized ingression, at the very margin of the blastoderm (5 7). tance, the molecular and cellular basis of germ-layer formation The general movements leading to germ-layer formation have has remained elusive. This work uses live imaging and functional thus been described in many species. However, how these move- approaches to reveal how the endodermal layer forms in ments are driven at the cellular scale remains poorly understood zebrafish, leading to an original model of cell segregation by (4). Sixty years ago, Townes and Holtfreter established that, when active migration away from neighboring cells, a process trig- dissociated and mixed, embryonic cells would sort into their pre- gered by N-cadherin. We propose this may be a core conserved viously specified germ layers (8). Following this original observa- mechanism driving germ-layer formation and might be ex- BIOLOGY tion, it was proposed that the mechanism underpinning germ-layer tended to other processes of cell segregation, highlighting gas- DEVELOPMENTAL formation in vivo could be cell sorting based on differential ad- trulation as a paradigm for tissue formation. hesion (9, 10) and/or differential cortical tension (11). However, this hypothesis has not been validated in the embryo (12). Author contributions: F.A.G. and N.B.D. designed research, performed research, analyzed Our limited understanding of the mechanisms driving cell in- data, and wrote the paper. ternalization and germ-layer formation most likely stems from the The authors declare no conflict of interest. difficulty and hence the limited number of direct in vivo obser- This article is a PNAS Direct Submission. vations, in particular in vertebrates (4, 13, 14). Here, we focused 1To whom correspondence should be addressed. Email: [email protected]. on endodermal cells in the zebrafish embryo to decipher the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. molecular and cellular mechanisms driving germ-layer separation. 1073/pnas.1708116114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1708116114 PNAS | September 19, 2017 | vol. 114 | no. 38 | 10143–10148 Downloaded by guest on September 29, 2021 Fig. 1. Endodermal cells rapidly migrate to the YSL. (A) Lifeact-GFP–expressing endodermal cells were transplanted to the margin of embryos expressing membrane-bound mCherry, just beneath the EVL. 4D stacks were acquired, and sagittal sections were reconstructed. (B) Sagittal sections showing endoder- mal cell internalization (Movie S1). Arrowheads point to actin-rich cytoplasmic extensions. The dashed line delineates the surface of the embryo; the dotted line represents the limit between the blastoderm and the YSL. Animal pole is to the top. (C)Orientationof actin-rich protrusions. Dots represent measurements. (D) Movement of internalizing cells along the surface– YSL axis. The spatial and temporal origin is defined as the beginning of migration. (E) Instant speed of in- ternalizing cells along the surface–YSL axis. Mean speed for each cell is represented as a colored seg- ment. (F) Transplanted endodermal cells contribute to endodermal derivatives at 24 hpf (arrowhead). (Scale bar: 20 μm.) which contain endodermal precursors (15). These cells exhibited cells into their animal pole (Fig. 2C). We checked that transplanted the same behavior as transplanted ones, extending actin-rich cyto- and surrounding cells expressed the endodermal marker sox32 (Fig. plasmic extensions toward the YSL and migrating to its surface 2 H and I) and displayed similar levels of cadherin-1 (hereafter re- − (mean speed: 1.7 μm·min 1; n = 7cells)(Movie S2). ferred to as “E-cadherin”)(Fig.2J and K), which accounts for about We noticed that endodermal cells internalized either inde- 80% of cell adhesion at this stage (11). Contrary to ectodermal cells pendently of neighboring cells or in coordination with them (Fig. that stay at the surface when surrounded by identical neighbors, S1 A, A′, B, and B′ and compare Movies S3 and S4), which is about half of the transplanted endodermal cells internalized (n = consistent with previous reports showing that internalization of 21 embryos) (Fig. 2 F and G). Live analysis of these internalizing hypoblastic cells is a more coherent process at the ventral than at cells revealed that they emitted actin-rich protrusions toward the the dorsal margin (16). Coordinated internalization likely cor- YSL, as observed at the margin of the embryo (n = 6 cells for each relates with nonautonomous effects that were first