Expression of the adhesion G protein-coupled receptor A2 (adgra2) during Xenopus laevis development.

The adhesion G protein-coupled receptor A2 (Adgra2) is a seven transmembrane receptor that has been described to be a regulator for angiogenesis in mice. Furthermore, the zebrafish ouchless mutant is unable to develop dorsal root ganglia through a disrupted trafficking of Adgra2. Besides RNA sequencing data, nothing is reported about Adgra2 in the south African crawled frog Xenopus laevis. In this study, we investigated for the first time the spatio-temporal expression of adgra2 during early Xenopus embryogenesis in detail. In silico approaches showed that the genomic adgra2 region as well as the Adgra2 protein sequence is highly conserved among different species including Xenopus. RT-PCR experiments confirmed that embryonic adgra2 expression is primarily detected at the beginning of neurulation and is then present throughout the whole Xenopus embryogenesis until stage 42. Whole mount in situ hybridization approaches visualized adgra2 expression in many tissues during Xenopus embryogenesis such as the cardiovascular system including the heart, the migrating neural crest cells and the developing eye including the periocular mesenchyme. Our results indicate a role of Adgra2 for embryogenesis and are a good starting point for further functional studies during early vertebrate development.

Developmental neurogenesis in mouse and Xenopus is impaired in the absence of Nosip.

BACKGROUND: Genetic deletion of Nosip in mice causes holoprosencephaly, however, the function of Nosip in neurogenesis is currently unknown. RESULTS: We combined two vertebrate model organisms, the mouse and the South African clawed frog, Xenopus laevis, to study the function of Nosip in neurogenesis. We found, that size and cortical thickness of the developing brain of Nosip knockout mice were reduced. Accordingly, the formation of postmitotic neurons was greatly diminished, concomitant with a reduced number of apical and basal neural progenitor cells in vivo. Neurospheres derived from Nosip knockout embryos exhibited reduced growth and the differentiation capability into neurons in vitro was almost completely abolished. Mass spectrometry analysis of the neurospheres proteome revealed a reduced expression of Rbp1, a regulator of retinoic acid synthesis, when Nosip was absent. We identified the homologous nosip gene to be expressed in differentiated neurons in the developing brain of Xenopus embryos. Knockdown of Nosip in Xenopus resulted in a reduction of brain size that could be rescued by reintroducing human NOSIP mRNA. Furthermore, the expression of pro-neurogenic transcription factors was reduced and the differentiation of neuronal cells was impaired upon Nosip knockdown. In Xenopus as well as in mouse we identified reduced proliferation and increased apoptosis as underlying cause of microcephaly upon Nosip depletion. In Xenopus Nosip and Rbp1 are similarly expressed and knockdown of Nosip resulted in down regulation of Rbp1. Knockdown of Rbp1 caused a similar microcephaly phenotype as the depletion of Nosip and synergy experiments indicated that both proteins act in the same signalling pathway. CONCLUSIONS: Nosip is a novel factor critical for neural stem cell/progenitor self-renewal and neurogenesis during mouse and Xenopus development and functions upstream of Rbp1 during early neurogenesis.

Frizzled 3 acts upstream of Alcam during embryonic eye development.

Formation of a functional eye during vertebrate embryogenesis requires different processes such as cell differentiation, cell migration, cell-cell interactions as well as intracellular signalling processes. It was previously shown that the non-canonical Wnt receptor Frizzled 3 (Fzd3) is required for proper eye formation, however, the underlying mechanism is poorly understood. Here we demonstrate that loss of Fzd3 induces severe malformations of the developing eye and that this defect is phenocopied by loss of the activated leukocyte cell adhesion molecule (Alcam). Promoter analysis revealed the presence of a Fzd3 responsive element within the alcam promoter, which is responsible for alcam expression during anterior neural development. In-depth analysis identified the jun N-terminal protein kinase 1 (JNK1) and the transcription factor paired box 2 (Pax2) to be important for the activation of alcam expression. Altogether our study reveals that alcam is activated through non-canonical Wnt signalling during embryonic eye development in Xenopus laevis and shows that this pathway plays a similar role in different tissues.

An Epha4/Sipa1l3/Wnt pathway regulates eye development and lens maturation.

The signal-induced proliferation-associated family of proteins comprises four members, SIPA1 and SIPA1L1-3. Mutations of the human SIPA1L3 gene result in congenital cataracts. In Xenopus, loss of Sipa1l3 function led to a severe eye phenotype that was distinguished by smaller eyes and lenses including lens fiber cell maturation defects. We found a direct interaction between Sipa1l3 and Epha4, building a functional platform for proper ocular development. Epha4 deficiency phenocopied loss of Sipa1l3 and rescue experiments demonstrated that Epha4 acts upstream of Sipa1l3 during eye development, with both Sipa1l3 and Epha4 required for early eye specification. The ocular phenotype, upon loss of either Epha4 or Sipa1l3, was partially mediated by rax We demonstrate that canonical Wnt signaling is inhibited downstream of Epha4 and Sipa1l3 during normal eye development. Depletion of either Sipa1l3 or Epha4 resulted in an upregulation of axin2 expression, a direct Wnt/ß-catenin target gene. In line with this, Sipa1l3 or Epha4 depletion could be rescued by blocking Wnt/ß-catenin or activating non-canonical Wnt signaling. We therefore conclude that this pathomechanism prevents proper eye development and maturation of lens fiber cells, resulting in congenital cataracts.