Summary The mechanisms of tissue convergence and extension (CE) driving axial elongation in mammalian embryos, and in particular, the cellular behaviors underlying CE in the epithelial neural tissue, have not been identified. Here we show that mouse neural cells undergo mediolaterally biased cell intercalation and exhibit both apical boundary rearrangement and polarized basolateral protrusive activity. Planar polarization and coordination of these two cell behaviors is essential for neural CE, as shown by failure of mediolateral intercalation in embryos mutant for two proteins associated with planar cell polarity signaling: Vangl2 and Ptk7. Embryos with mutations in Ptk7 fail to polarize cell behaviors within the plane of the tissue, while Vangl2 mutant embryos maintain tissue polarity and basal protrusive activity, but are deficient in apical neighbor exchange. Neuroepithelial cells in both mutants fail to apically constrict, leading to craniorachischisis. These results reveal a cooperative mechanism for cell rearrangement during epithelial morphogenesis.

During gastrulation, an embryo acquires the three primordial germ layers that will give rise to all of the tissues in the body. In amniote embryos, this process occurs via an epithelial to mesenchymal transition (EMT) of epiblast cells at the primitive streak. Although the primitive streak is vital to development, many aspects of how it forms and functions remain poorly understood. Using live imaging and immunohistochemistry, we have shown that the murine primitive streak arises in situ by progressive initiation of EMT beginning in the posterior epiblast, without large-scale movement or convergence and extension of epiblast cells. Loss of basal lamina (BL) is the first step of this EMT, and is strictly correlated with ingression of nascent mesoderm. This is the first description of dynamic cell behavior during primitive streak formation in the mouse embryo, and reveals mechanisms that are quite distinct from those observed in other amniote model systems.

Despite being implicated as a mechanism driving gastrulation and body axis elongation in mouse embryos, the cellular mechanisms underlying mammalian convergent extension (CE) are unknown. Here we show, with high-resolution time-lapse imaging of living mouse embryos, that mesodermal CE occurs by mediolateral cell intercalation, driven by mediolaterally polarized cell behavior. The initial events in the onset of CE are mediolateral elongation, alignment and orientation of mesoderm cells as they exit the primitive streak. This cell shape change occurs prior to, and is required for, the subsequent onset of mediolaterally polarized protrusive activity. In embryos mutant for PTK7, a novel cell polarity protein, the normal cell elongation and alignment upon leaving the primitive streak, the subsequent polarized protrusive activity, and CE and axial elongation all failed. The mesoderm normally thickens and extends, but on failure of convergence movements in Ptk7 mutants, the mesoderm underwent radial intercalation and excessive thinning, which suggests that a cryptic radial cell intercalation behavior resists excessive convergence-driven mesodermal thickening in normal embryos. When unimpeded by convergence forces in Ptk7 mutants, this unopposed radial intercalation resulted in excessive thinning of the mesoderm. These results show for the first time the polarized cell behaviors underlying CE in the mouse, demonstrate unique aspects of these behaviors compared with those of other vertebrates, and clearly define specific roles for planar polarity and for the novel planar cell polarity gene, Ptk7, as essential regulators of mediolateral cell intercalation during mammalian CE.

12During vertebrate gastrulation, convergence & extension (C&E) of the primary anteroposterior (AP) embryonic 13 axis is driven by polarized mediolateral (ML) cell intercalations and is influenced by AP axial patterning. Nodal 14 signaling is essential for patterning of the AP axis while Planar Cell Polarity (PCP) signaling polarizes cells with 15 respect to this axis, but how these two signaling systems interact during C&E is unclear. We find that the 16 neuroectoderm of Nodal-deficient zebrafish gastrulae exhibits reduced C&E cell behaviors, which require 17 Nodal signaling in both cell-and non-autonomous fashions. PCP signaling is partially active in Nodal-deficient 18 embryos and its inhibition exacerbates their C&E defects. Within otherwise naïve zebrafish blastoderm 19 explants, however, Nodal induces C&E in a largely PCP-dependent manner, arguing that Nodal acts both 20 upstream of and in parallel with PCP during gastrulation to cooperatively regulate embryonic axis extension. 21 22 48in explants by different doses of the TGF ligand Activin (28), which signals largely via the Nodal signaling 49 pathway during early vertebrate embryogenesis (31). These results demonstrate that AP patterning is required 50 for axial extension ex vivo and implies a crucial role for Nodal signaling at this intersection of tissue patterning 51 and morphogenesis in vivo. 52Nodal is a TGF-superfamily morphogen whose graded signaling within the embryo produces discrete 53 developmental outcomes depending on a cell's position within that gradient and the resulting signaling 54 level/duration to which it is exposed (32)(33)(34)(35)(36). Upon binding of Nodal-Gdf3 (Vg1) heterodimers (37-39), the 55 receptor complex -comprised of two each of the Type I and Type II serine-threonine kinase receptors Acvr1b 56 and Acvr2b and the co-receptor Tdgfis activated and phosphorylates the downstream transcriptional 57 effectors Smad2 and/or Smad3 (40, 41). Nodal signaling is essential for specification of endoderm and 58 mesoderm germ layers and their patterning along the AP axis, with the highest signaling levels producing 59 3 endoderm and the most dorsal/anterior mesoderm fates (42)(43)(44)(45)(46)(47). Mouse embryos mutant for Nodal signaling 60 components fail to gastrulate, resulting in early embryonic lethality (48). Nodal-deficient zebrafish undergo 61 highly abnormal gastrulation, failing to specify endoderm and most mesoderm (35, 40, 46), resulting in 62 embryos comprised largely of neuroectoderm and displaying severe neural tube and axis extension defects 63 (49, 50). 64Restoration of mesoderm to maternal-zygotic one-eyed pinhead (MZoep) zebrafish mutants, which lack 65 the essential Tdgf Nodal co-receptor (40), improves AP length and morphology of the neural tube (51), 66 implying that Nodal promotes C&E of the neuroectoderm non-autonomously via specification of mesoderm. 67However, additional evidence points to a more direct role for Nodal signaling in C&E cell behaviors. First, 68Activin signaling via Nodal receptors is sufficient...

It is during gastrulation that the primordial germ layers are specified, embryonic axes become morphologically manifest, and the embryonic body plan begins to take shape. As morphogenetic movements push and pull nascent tissues into position within the gastrula, new interactions are established between neighboring cells and tissues. These interactions represent an emergent property within gastrulating embryos, and serve to regulate and promote ensuing morphogenesis that establishes the next set of cell/tissue contacts, and so on. Several recent studies demonstrate the critical roles of such interactions during gastrulation, including those between germ layers, along embryonic axes, and at tissue boundaries. Emergent tissue interactions result from - and result in - morphogen signaling, cell contacts, and mechanical forces within the gastrula. Together, these comprise a dynamic and complex regulatory cascade that drives gastrulation morphogenesis.