Bmp Signal Gradient Modulates Convergent Cell Movement via Xarhgef3.2 during Gastrulation of Xenopus Embryos

Gastrulation is a critical step in the establishment of a basic body plan during development. Convergence and extension (CE) cell movements organize germ layers during gastrulation. Noncanonical Wnt signaling has been known as major signaling that regulates CE cell movement by activating Rho and Rac. In addition, Bmp molecules are expressed in the ventral side of a developing embryo, and the ventral mesoderm region undergoes minimal CE cell movement while the dorsal mesoderm undergoes dynamic cell movements. This suggests that Bmp signal gradient may affect CE cell movement. To investigate whether Bmp signaling negatively regulates CE cell movements, we performed microarray-based screening and found that the transcription of Xenopus Arhgef3.2 (Rho guanine nucleotide exchange factor) was negatively regulated by Bmp signaling. We also showed that overexpression or knockdown of Xarhgef3.2 caused gastrulation defects. Interestingly, Xarhgef3.2 controlled gastrulation cell movements through interacting with Disheveled (Dsh2) and Dsh2-associated activator of morphogenesis 1 (Daam1). Our results suggest that Bmp gradient affects gastrulation cell movement (CE) via negative regulation of Xarhgef3.2 expression.

The Adaptor Protein Lurap1 Is Required for Cell Cohesion during Epiboly Movement in Zebrafish

Cell adhesion and polarized cellular behaviors play critical roles in a wide variety of morphogenetic events. In the zebrafish embryo, epiboly represents an important process of epithelial morphogenesis that involves differential cell adhesion and dynamic cell shape changes for coordinated movements of different cell populations, but the underlying mechanism remains poorly understood. The adaptor protein Lurap1 functions to link myotonic dystrophy kinase-related Rac/Cdc42-binding kinase with MYO18A for actomyosin retrograde flow in cell migration. We previously reported that it interacts with Dishevelled in convergence and extension movements during gastrulation. Here, we show that it regulates blastoderm cell adhesion and radial cell intercalation during epiboly. In zebrafish mutant embryos with loss of both maternal and zygotic Lurap1 function, deep cell multilayer of the blastoderm exhibit delayed epiboly with respect to the superficial layer. Time-lapse imaging reveals that these deep cells undergo unstable intercalation, which impedes their expansion over the yolk cell. Cell sorting and adhesion assays indicate reduced cellular cohesion of the blastoderm. These defects are correlated with disrupted cytoskeletal organization in the cortex of blastoderm cells. Thus, the present results extend our previous works by demonstrating that Lurap1 is required for cell adhesion and cell behavior changes to coordinate cell movements during epithelial morphogenesis. They provide insights for a further understanding of the regulation of cytoskeletal organization during gastrulation cell movements.

Glypican 4 mediates Wnt transport between germ layers via signaling filopodia

Glypicans influence signaling pathways by regulating morphogen trafficking and reception. However, the underlying mechanisms in vertebrates are poorly understood. In zebrafish, Glypican 4 (Gpc4) is required for convergence and extension (C&E) of both the mesoderm and endoderm. Here, we show that transgenic expression of GFP-Gpc4 in the endoderm of gpc4 mutants rescued C&E defects in all germ layers. The rescue of mesoderm was likely mediated by Wnt5b and Wnt11f2 and depended on signaling filopodia rather than on cleavage of the Gpc4 GPI anchor. Gpc4 bound both Wnt5b and Wnt11f2 and regulated formation of the filopodia that transport Wnt5b and Wnt11f2 to neighboring cells. Moreover, this rescue was suppressed by blocking signaling filopodia that extend from endodermal cells. Thus, GFP-Gpc4–labeled protrusions that emanated from endodermal cells transported Wnt5b and Wnt11f2 to other germ layers, rescuing the C&E defects caused by a gpc4 deficiency. Our study reveals a new mechanism that could explain in vivo morphogen distribution involving Gpc4.

Strain maps characterize the symmetry of convergence and extension patterns during zebrafish gastrulation

During gastrulation of the zebrafish embryo, the cap of blastoderm cells organizes into the axial body plan of the embryo with left–right symmetry and head–tail, dorsal–ventral polarities. Our labs have been interested in the mechanics of early development and have investigated whether these large-scale cell movements can be described as tissue-level mechanical strain by a tectonics-based approach. The first step is to image the positions of all nuclei from mid-epiboly to early segmentation by digital sheet light microscopy, organize the surface of the embryo into multi-cell spherical domains, construct velocity fields from the movements of these domains and extract strain rate maps from the change in density of the domains. During gastrulation, tensile/expansive and compressive strains in the axial and equatorial directions are detected as anterior and posterior expansion along the anterior–posterior axis and medial–lateral compression across the dorsal–ventral axis and corresponds to the well characterized morphological movements of convergence and extension. Following gastrulation strain is represented by localized medial expansion at the onset of segmentation and anterior expansion at the onset of neurulation. In addition to linear strain, symmetric patterns of rotation/curl are first detected in the animal hemispheres at mid-epiboly and then the vegetal hemispheres by the end of gastrulation. In embryos treated with C59, a Wnt inhibitor that inhibits head and tail extension, the axial extension and vegetal curl are absent. By analysing the temporal sequence of large-scale movements, deformations across the embryo can be attributed to a combination of epiboly and dorsal convergence-extension.

A Time-Saving Strategy to Generate Double Maternal Mutants by an Oocyte-Specific Conditional Knockout System in Zebrafish

Maternal products are those mRNAs and proteins deposited during oogenesis, which play critical roles in controlling oocyte formation, fertilization, and early embryonic development. However, loss-of-function studies for these maternal factors are still lacking, mainly because of the prolonged period of transgenerational screening and technical barriers that prevent the generation of maternal (M) and maternal and zygotic (MZ) mutant embryos. By the transgenic expression of multiple sgRNAs targeting a single gene of interest in the background of a transgenic line Tg(zpc:zcas9) with oocyte-specific cas9 expression, we have successfully obtained maternal or maternal–zygotic mutant for single genes in F1 embryos. In this work, we tandemly connected a maternal GFP marker and eight sgRNA expression units to target dvl2 and dvl3a simultaneously and introduced this construct to the genome of Tg(zpc:zcas9) by meganuclease I-Sce I. As expected, we confirmed the existence of Mdvl2;Mdvl3a embryos with strong defective convergence and extension movement during gastrulation among outcrossed GFP positive F1 offspring. The MZdvl2;MZdvl3a embryos were also obtained by crossing the mutant carrying mosaic F0 female with dvl2+/−;dvl3a−/− male fish. This proof-of-principle thus highlights the potential of this conditional knockout strategy to circumvent the current difficulty in the study of genes with multiple functionally redundant paralogs.

Glypican 4 regulates planar cell polarity of endoderm cells by controlling the localization of Cadherin 2

Non-canonical Wnt/Planar Cell Polarity (Wnt/PCP) signaling has been implicated in endoderm morphogenesis. However, the underlying cellular and molecular mechanisms of this process are unclear. We found that during convergence and extension (C&E) in zebrafish, gut endodermal cells are polarized mediolaterally, with GFP-Vangl2 enriched at the anterior edges. Endoderm cell polarity is lost, and intercalation is impaired, in the absence of glypican 4 (gpc4), a heparan-sulfate proteoglycan that promotes Wnt/PCP signaling, suggesting that this signaling is required for endodermal cell polarity. Live imaging revealed that endoderm C&E is accomplished by polarized cell protrusions and junction remodeling, which are impaired in gpc4-deficient endodermal cells. Furthermore, in the absence of gpc4, Cadherin 2 expression on the endodermal cell surface is increased due to impaired Rab5c-mediated endocytosis, which partially accounts for the endodermal defects in these mutants.These findings indicate that Gpc4 regulates endodermal planar cell polarity during endoderm C&E by influencing localization of Cadherin 2. Thus, our study uncovers a new mechanism by which Gpc4 regulates planar cell polarity and reveals the role of Wnt/PCP signaling in endoderm morphogenesis.

Guidance by followers ensures long-range coordination of cell migration through α-Catenin mechanoperception

Morphogenesis, wound healing and some cancer metastases depend upon migration of cell collectives that need to be guided to their destination as well as coordinated with other cell movements. During zebrafish gastrulation, extension of the embryonic axis is led by the mesendodermal polster that migrates towards the animal pole, followed by axial mesoderm that is undergoing convergence and extension. We here investigate how polster cells are guided towards the animal pole. Using a combination of precise laser ablations, advanced transplantations and functional as well as silico approaches, we establish that the directional information guiding polster cells is mechanical, and is provided by the anteriorward migration of the following cells. This information is detected by cell-cell contact through E-Cadherin/α-Catenin mechanotransduction and propagates from cell to cell over the whole tissue. Such guidance of migrating cells by followers ensures long-range coordination of movements and developmental robustness.

Anterior expansion and posterior addition to the notochord mechanically coordinate embryo axis elongation

During development the embryo body progressively elongates from head-to-tail along the anterior-posterior (AP) axis. Multiple tissues contribute to this elongation through a combination of convergence and extension and/or volumetric growth. How force generated by the morphogenesis of one tissue impacts the morphogenesis of other axial tissues to achieve an elongated axis is not well understood. The notochord, a rod-shaped tissue possessed by all vertebrates, runs across the entire length of the somitic compartment and is flanked on either side by the developing somites in the segmented region of the axis and presomitic mesoderm in the posterior. Cells in the notochord undergo an expansion that is constrained by a stiff sheath of extracellular matrix, that increases the internal pressure in the notochord allowing it to straighten and elongate. Therefore, it is appropriately positioned to play a role in mechanically elongating the somitic compartment. Here, we use multi-photon mediated cell ablation to remove specific regions of the developing notochord and quantify the impact on axis elongation. We show that anterior notochord cell expansion generates a force that displaces notochord cells posteriorly relative to adjacent axial tissues and contributes to the elongation of segmented tissue during post-tailbud stages of development. Crucially, unexpanded cells derived from progenitors at the posterior end of the notochord provide resistance to anterior notochord cell expansion, allowing for force generation across the AP axis. Therefore, notochord cell expansion beginning in the anterior, and addition of cells to the posterior notochord, act as temporally coordinated morphogenetic events that shape the zebrafish embryo AP axis.

Glypican4 mediates Wnt transport between germ layers via signaling filopodia

Glypicans influence signaling pathways by regulating morphogen trafficking and reception. However, the underlying mechanisms in vertebrates are poorly understood. In zebrafish, Glypican 4 (Gpc4) is required for convergence and extension (C&E) of both the mesoderm and endoderm. Here we show that transgenic expression of GFP-Gpc4 in the endoderm of gpc4 mutants rescues C&E defects in all germ layers. The rescue of mesoderm was likely mediated by Wnt5b and Wnt11f2, and depended on signaling filopodia rather than on cleavage of the Gpc4 GPI anchor. Gpc4 bound Wnt5b and regulated formation of the filopodia that transport Wnt5b to neighboring cells. Blocking signaling filopodia that extend from endodermal cells suppressed this rescue. Thus, endodermal signaling filopodia that expressed GFP-Gpc4 transported Wnt5b, and likely Wnt11f2, to other germ layers, rescuing the C&E defects caused by a gpc4 deficiency. Our study reveals a new mechanism that could explain in vivo morphogen distribution involving Gpc4.

Nodal and planar cell polarity signaling cooperate to regulate zebrafish convergence and extension gastrulation movements

During vertebrate gastrulation, convergence and extension (C and E) of the primary anteroposterior (AP) embryonic axis is driven by polarized mediolateral (ML) cell intercalations and is influenced by AP axial patterning. Nodal signaling is essential for patterning of the AP axis while planar cell polarity (PCP) signaling polarizes cells with respect to this axis, but how these two signaling systems interact during C and E is unclear. We find that the neuroectoderm of Nodal-deficient zebrafish gastrulae exhibits reduced C and E cell behaviors, which require Nodal signaling in both cell- and non-autonomous fashions. PCP signaling is partially active in Nodal-deficient embryos and its inhibition exacerbates their C and E defects. Within otherwise naïve zebrafish blastoderm explants, however, Nodal induces C and E in a largely PCP-dependent manner, arguing that Nodal acts both upstream of and in parallel with PCP during gastrulation to regulate embryonic axis extension cooperatively.