Integration of vascular progenitors into functional blood vessels represents a distinct mechanism of vascular growth.

During embryogenesis, the initial vascular network forms by the process of vasculogenesis, or the specification of vascular progenitors de novo. In contrast, the majority of later-forming vessels arise by angiogenesis from the already established vasculature. Here, we show that new vascular progenitors in zebrafish embryos emerge from a distinct site along the yolk extension, or secondary vascular field (SVF), incorporate into the posterior cardinal vein, and contribute to subintestinal vasculature even after blood circulation has been initiated. We further demonstrate that SVF cells participate in vascular recovery after chemical ablation of vascular endothelial cells. Inducible inhibition of the function of vascular progenitor marker etv2/etsrp prevented SVF cell differentiation and resulted in the defective formation of subintestinal vasculature. Similar late-forming etv2+ progenitors were also observed in mouse embryos, suggesting that SVF cells are evolutionarily conserved. Our results characterize a distinct mechanism by which new vascular progenitors incorporate into established vasculature.

Single-cell transcriptome analysis of the zebrafish embryonic trunk.

During embryonic development, cells differentiate into a variety of distinct cell types and subtypes with diverse transcriptional profiles. To date, transcriptomic signatures of different cell lineages that arise during development have been only partially characterized. Here we used single-cell RNA-seq to perform transcriptomic analysis of over 20,000 cells disaggregated from the trunk region of zebrafish embryos at the 30 hpf stage. Transcriptional signatures of 27 different cell types and subtypes were identified and annotated during this analysis. This dataset will be a useful resource for many researchers in the fields of developmental and cellular biology and facilitate the understanding of molecular mechanisms that regulate cell lineage choices during development.

A multichannel computer-driven system to raise aquatic embryos under selectable hypoxic conditions.

The formation of a functional cardiovascular system is an essential step in the early vertebrate embryo. Nevertheless, the effect of hypoxia on the developmental program of organisms was studied rarely. In particular, this holds true for vertebrate embryos that depend on a functional placenta for proper development and had not been studied in this respect due to the obvious limitation. We established a protocol to culture aquatic embryos, which enabled us to culture a high number of Xenopus embryos until tadpole stage under defined hypoxic conditions in four hypoxia chambers simultaneously, employing a computerized system. In general, our results show that hypoxia results in delayed development and, in particular, we could show that oxygen availability was most crucial during gastrulation and organogenesis (early tailbud) phases during embryonic development of Xenopus laevis.

Heterozygous Pathogenic Variant in DACT1 Causes an Autosomal-Dominant Syndrome with Features Overlapping Townes-Brocks Syndrome.

A heterozygous nonsense variant was identified in dapper, antagonist of beta-catenin, 1 (DACT1) via whole-exome sequencing in family members with imperforate anus, structural renal abnormalities, genitourinary anomalies, and/or ear anomalies. The DACT1 c.1256G>A;p.Trp419* variant segregated appropriately in the family consistent with an autosomal dominant mode of inheritance. DACT1 is a member of the Wnt-signaling pathway, and mice homozygous for null alleles display multiple congenital anomalies including absent anus with blind-ending colon and genitourinary malformations. To investigate the DACT1 c.1256G>A variant, HEK293 cells were transfected with mutant DACT1 cDNA plasmid, and immunoblotting revealed stability of the DACT1 p.Trp419* protein. Overexpression of DACT1 c.1256G>A mRNA in Xenopus embryos revealed a specific gastrointestinal phenotype of enlargement of the proctodeum. Together, these findings suggest that the DACT1 c.1256G>A nonsense variant is causative of a specific genetic syndrome with features overlapping Townes-Brocks syndrome.

Xenopus laevis neuronal cell adhesion molecule (nrcam): plasticity of a CAM in the developing nervous system.

Neuron-glial-related cell adhesion molecule (NRCAM) is a neuronal cell adhesion molecule of the L1 immunoglobulin superfamily, which plays diverse roles during nervous system development including axon growth and guidance, synapse formation, and formation of the myelinated nerve. Perturbations in NRCAM function cause a wide variety of disorders, which can affect wiring and targeting of neurons, or cause psychiatric disorders as well as cancers through abnormal modulation of signaling events. In the present study, we characterize the Xenopus laevis homolog of nrcam. Expression of Xenopus nrcam is most abundant along the dorsal midline throughout the developing brain and in the outer nuclear layer of the retina.

Expressional characterization of mRNA (guanine-7) methyltransferase (rnmt) during early development of Xenopus laevis.

Methylation of the guanosine cap structure at the 5' end of mRNA is essential for efficient translation of all eukaryotic cellular mRNAs, gene expression and cell viability and promotes transcription, splicing, polyadenylation and nuclear export of mRNA. In the current study, we present the spatial expression pattern of the Xenopus laevis rnmt homologue. A high percentage of protein sequence similarity, especially within the methyltransferase domain, as well as an increased expression in the cells of the transcriptionally active stages, suggests a conserved RNA cap methylation function. Spatial expression analysis identified expression domains in the brain, the retina, the lens, the otic vesicles and the branchial arches.

Suppression of vascular network formation by chronic hypoxia and prolyl-hydroxylase 2 (phd2) deficiency during vertebrate development.

In the adult, new vessels and red blood cells form in response to hypoxia. Here, the oxygen-sensing system (PHD-HIF) has recently been put into focus, since the prolyl-hydroxylase domain proteins (PHD) and hypoxia-inducible factors (HIF) are considered as potential therapeutic targets to treat ischemia, cancers or age-related macula degeneration. While the oxygen-sensing system (PHD-HIF) has been studied intensively in this respect, only little is known from developing vertebrate embryos since mutations within this pathway led to an early decease of embryos due to placental defects. During vertebrate embryogenesis, a progenitor cell called hemangioblast is assumed to give rise to blood cells and blood vessels in a process called hematopoiesis and vasculogenesis, respectively. Xenopus provides an ideal experimental system to address these processes in vivo, as its development does not depend on a functional placenta and thus allows analyzing the role of oxygen directly. To this end, we adopted a computer-controlled four-channel system, which allowed us to culture Xenopus embryos under defined oxygen concentrations. Our data show that the development of vascular structures and blood cells is strongly impaired under hypoxia, while general development is less compromised. Interestingly, suppression of Phd2 function using specific antisense morpholinos or a chemical inhibitor resulted in mostly overlapping vascular defects; nevertheless, blood cell was formed almost normally. Our results provide the first evidence that oxygen via Phd2 has a decisive influence on the formation of the vascular network during vertebrate embryogenesis. These findings may be considered in certain potential treatment concepts.

Xenopus cadherin 5 is specifically expressed in endothelial cells of the developing vascular system.

Vasculogenesis is an important, multistep process leading to the formation of a functional primary network of blood vessels in the developing embryo. A series of interactions between secreted growth factors and their specific receptors leads to the specification of mesodermal cells to become hemangioblasts, which then differentiate into angioblasts. These subsequently proliferate, coalesce into cords and finally form tubular vascular structures. For proper function of these primary blood vessels, the close connection of endothelial cells is required. This is conferred by the interaction of an endothelium specific cadherin (Cadherin-5), starting during early vascular development. However, this interaction remains important throughout life and ageing. Therefore, cadherin-5 is a useful marker for late stages of vasculogenesis in several vertebrate species. To establish cadherin-5 as a marker for vascular studies in Xenopus, we cloned the Xenopus laevis ortholog and analyzed its expression pattern during embryogenesis.