Differential requirement of bone morphogenetic protein receptors Ia (ALK3) and Ib (ALK6) in early embryonic patterning and neural crest development

Background Bone morphogenetic proteins regulate multiple processes in embryonic development, including early dorso-ventral patterning and neural crest development. BMPs activate heteromeric receptor complexes consisting of type I and type II receptor-serine/threonine kinases. BMP receptors Ia and Ib, also known as ALK3 and ALK6 respectively, are the most common type I receptors that likely mediate most BMP signaling events. Since early expression patterns and functions in Xenopus laevis development have not been described, we have addressed these questions in the present study. Results Here we have analyzed the temporal and spatial expression patterns of ALK3 and ALK6; we have also carried out loss-of-function studies to define the function of these receptors in early Xenopus development. We detected both redundant and non-redundant roles of ALK3 and ALK6 in dorso-ventral patterning. From late gastrula stages onwards, their expression patterns diverged, which correlated with a specific, non-redundant requirement of ALK6 in post-gastrula neural crest cells. ALK6 was essential for induction of neural crest cell fate and further development of the neural crest and its derivatives. Conclusions ALK3 and ALK6 both contribute to the gene regulatory network that regulates dorso-ventral patterning; they play partially overlapping and partially non-redundant roles in this process. ALK3 and ALK6 are independently required for the spatially restricted activation of BMP signaling and msx2 upregulation at the neural plate border, whereas in post-gastrula development ALK6 exerts a highly specific, conserved function in neural crest development

PAPC and the Wnt5a/Ror2 pathway control the invagination of the otic placode in Xenopus

<p>Abstract</p> <p>Background</p> <p>Paraxial protocadherin (PAPC) plays a crucial role in morphogenetic movements during gastrulation and somitogenesis in mouse, zebrafish and Xenopus. PAPC influences cell-cell adhesion mediated by C-Cadherin. A putative direct adhesion activity of PAPC is discussed. PAPC also promotes cell elongation, tissue separation and coordinates cell mass movements. In these processes the signaling function of PAPC in activating RhoA/JNK and supporting Wnt-11/PCP by binding to frizzled 7 (fz7) is important.</p> <p>Results</p> <p>Here we demonstrate by loss of function experiments in Xenopus embryos that PAPC regulates another type of morphogenetic movement, the invagination of the ear placode. Knockdown of PAPC by antisense morpholinos results in deformation of the otic vesicle without altering otocyst marker expression. Depletion of PAPC could be rescued by full-length PAPC, constitutive active RhoA and by the closely related PCNS but not by classical cadherins. Also the cytoplasmic deletion mutant M-PAPC, which influences cell adhesion, does not rescue the PAPC knockdown. Interestingly, depletion of Wnt5a or Ror2 which are also expressed in the otocyst phenocopies the PAPC morphant phenotype.</p> <p>Conclusions</p> <p>PAPC signaling via RhoA and Wnt5a/Ror2 activity are required to keep cells aligned in apical-basal orientation during invagination of the ear placode. Since neither the cytoplasmic deletion mutant M-PAPC nor a classical cadherin is able to rescue loss of PAPC we suggest that the signaling function of the protocadherin rather than its role as modulator of cell-cell adhesion is required during invagination of the ear placode.</p

Author Correction: Gap junction protein Connexin-43 is a direct transcriptional regulator of N-cadherin in vivo

Correction to: Nature Communications (2018); https://doi.org/10.1038/s41467-018-06368-x, published online 21 September 2018. The original version of this Article contained an error in the spelling of the author Alexandra Schambony, which was incorrectly given as Alexandra Schambon. This has now been corrected in both the PDF and HTML versions of the Article

The deleted in brachydactyly B domain of ROR2 is required for receptor activation by recruitment of Src

The transmembrane receptor 'ROR2' resembles members of the receptor tyrosine kinase family of signalling receptors in sequence but its' signal transduction mechanisms remain enigmatic. This problem has particular importance because mutations in ROR2 are associated with two human skeletal dysmorphology syndromes, recessive Robinow Syndrome (RS) and dominant acting Brachydactyly type B (BDB). Here we show, using a constitutive dimerisation approach, that ROR2 exhibits dimerisation-induced tyrosine kinase activity and the ROR2 C-terminal domain, which is deleted in BDB, is required for recruitment and activation of the non-receptor tyrosine kinase Src. Native ROR2 phosphorylation is induced by the ligand Wnt5a and is blocked by pharmacological inhibition of Src kinase activity. Eight sites of Src-mediated ROR2 phosphorylation have been identified by mass spectrometry. Activation via tyrosine phosphorylation of ROR2 receptor leads to its internalisation into Rab5 positive endosomes. These findings show that BDB mutant receptors are defective in kinase activation as a result of failure to recruit Src

Wnt5b–Ryk pathway provides directional signals to regulate gastrulation movement

The soluble ligand Wnt5b repels cells expressing the Ryk (related to tyrosine kinase) receptor, establishing directional motility during gastrulation

Live Imaging of Xwnt5A-ROR2 Complexes

Secreted molecules of the Wnt family regulate key decisions in embryogenesis and adult tissue homeostasis by activating a complex network of Wnt signaling pathways. Although the different branches of Wnt signaling have been studied for more than 25 years, fluorophore tagged constructs for live cell imaging of Wnt molecules activating the Wnt/β-catenin pathway have become available only recently. We have generated a fluorophore tagged Wnt construct of the Xenopus Wnt5a protein (Xwnt5A) with the enhanced green fluorescent protein (EGFP), Xwnt5A-EGFP. This construct activates non-canonical Wnt pathways in an endocytosis dependent manner and is capable of compensating for the loss of endogenous Xwnt5A in Xenopus embryos. Strikingly, non-canonical Wnt pathway activation was restricted to short-range signaling while an inhibitory effect was observed in transwell cell cultures taken as long-range signaling model sytem. We used our Xwnt5A-EGFP construct to analyze in vivo binding of Wnt5A to its co-receptor ROR2 on the microscopic and on the molecular level. On the microscopic level, Xwnt5A-EGFP clusters in the membrane and recruits ROR2-mCherry to these clusters. Applying dual-colour dual-focus line-scanning fluorescence correlation spectroscopy on dorsal marginal zone explants, we identified membrane tethered Xwnt5A-EGFP molecules binding to ROR2-mCherry molecules. Our data favour a model, in which membrane-tethered Wnt-5A recruits ROR2 to form large ligand/receptor clusters and signals in an endocytosis-dependent manner

Sulf1 has ligand-dependent effects on canonical and non-canonical Wnt signalling

Wnt signalling plays essential roles during embryonic development and is known to be mis-regulated in human disease. There are many molecular mechanisms that ensure tight regulation of Wnt activity. One such regulator is the heparan-sulfate-specific 6-O-endosulfatase Sulf1. Sulf1 acts extracellularly to modify the structure of heparan sulfate chains to affect the bio-availability of Wnt ligands. Sulf1 could, therefore, influence the formation of Wnt signalling complexes to modulate the activation of both canonical and non-canonical pathways. In this study, we use well-established assays in Xenopus to investigate the ability of Sulf1 to modify canonical and non-canonical Wnt signalling. In addition, we model the ability of Sulf1 to influence morphogen gradients using fluorescently tagged Wnt ligands in ectodermal explants. We show that Sulf1 overexpression has ligand-specific effects on Wnt signalling: it affects membrane accumulation and extracellular levels of tagged Wnt8a and Wnt11b ligands differently, and inhibits the activity of canonical Wnt8a but enhances the activity of non-canonical Wnt11b

Coordination of Cell Polarity during Xenopus Gastrulation

Cell polarity is an essential feature of animal cells contributing to morphogenesis. During Xenopus gastrulation, it is known that chordamesoderm cells are polarized and intercalate each other allowing anterior-posterior elongation of the embryo proper by convergent extension (CE). Although it is well known that the cellular protrusions at both ends of polarized cells exert tractive force for intercalation and that PCP pathway is known to be essential for the cell polarity, little is known about what triggers the cell polarization and what the polarization causes to control intracellular events enabling the intercalation that leads to the CE. In our research, we used EB3 (end-binding 3), a member of +TIPs that bind to the plus end of microtubule (MT), to visualize the intracellular polarity of chordamesoderm cells during CE to investigate the trigger of the establishment of cell polarity. We found that EB3 movement is polarized in chordamesoderm cells and that the notochord-somite tissue boundary plays an essential role in generating the cell polarity. This polarity was generated before the change of cell morphology and the polarized movement of EB3 in chordamesoderm cells was also observed near the boundary between the chordamesoderm tissue and naïve ectoderm tissue or lateral mesoderm tissues induced by a low concentration of nodal mRNA. These suggest that definitive tissue separation established by the distinct levels of nodal signaling is essential for the chordamesodermal cells to acquire mediolateral cell polarity