Dirk Meyer

Guidance of primordial germ cell migration by the chemokine SDF-1.

The signals directing primordial germ cell (PGC) migration in vertebrates are largely unknown. We demonstrate that sdf-1 mRNA is expressed in locations where PGCs are found and toward which they migrate in wild-type as well as in mutant embryos in which PGC migration is abnormal. Knocking down SDF-1 or its receptor CXCR4 results in severe defects in PGC migration. Specifically, PGCs that do not receive the SDF-1 signal exhibit lack of directional movement toward their target and arrive at ectopic positions within the embryo. Finally, we show that the PGCs can be attracted toward an ectopic source of the chemokine, strongly suggesting that this molecule provides a key directional cue for the PGCs.

Neuromuscular synapses can form in vivo by incorporation of initially aneural postsynaptic specializations

Synapse formation requires the coordination of pre- and postsynaptic differentiation. An unresolved question is which steps in the process require interactions between pre- and postsynaptic cells, and which proceed cell-autonomously. One current model is that factors released from presynaptic axons organize postsynaptic differentiation directly beneath the nerve terminal. Here, we used neuromuscular junctions (NMJs) of the zebrafish primary motor system to test this model. Clusters of neurotransmitter (acetylcholine) receptors (AChRs) formed in the central region of the myotome, destined to be synapse-rich, before axons extended and even when axon extension was prevented. Time-lapse imaging revealed that pre-existing clusters on early-born slow (adaxial) muscle fibers were incorporated into NMJs as axons advanced. Axons were, however, required for the subsequent remodeling and selective stabilization of synaptic clusters that precisely appose post- to presynaptic elements. Thus, motor axons are dispensable for the initial stages of postsynaptic differentiation but are required for later stages. Moreover, many AChR clusters on later-born fast muscle fibers formed at sites that had already been contacted by axons, suggesting heterogeneity in the signaling mechanisms leading to synapse formation by a single axon.

The zebrafish forkhead transcription factor FoxH1/Fast1 is a modulator of Nodal signaling required for organizer formation

BACKGROUND Signaling molecules related to the Nodal protein play essential roles in the formation and patterning of the gastrula organizer and the germ layers during vertebrate development. The forkhead transcription factor FoxH1 (also known as Fast1) is a component of the Nodal signaling pathway. Although different roles have been suggested for FoxH1, its specific function during development is still unclear. RESULTS We report that the zebrafish locus schmalspur (sur) encodes a member of the FoxH1 family. Maternal sur transcripts were localized to the animal pole during oogenesis. Further expression was detected in a dorsoventral gradient at the onset of gastrulation and in specific domains in the organizer, notochord and lateral plate mesoderm. Embryos lacking zygotic sur function had variable deficiencies of prechordal plate and ventral neuroectoderm. In the absence of both maternal and zygotic sur function, embryos failed to form a morphologically distinct gastrula organizer and, later, developed severe defects in all axial structures. In these embryos, expression of nodal genes was initiated but not maintained. Unlike embryos lacking Nodal signaling, sur mutants formed endoderm and paraxial mesoderm. CONCLUSIONS FoxH1 is involved in regulatory feedback loops that control the duration and intensity of Nodal signals in early patterning. In zebrafish, FoxH1 is not essential to induce Nodal-dependent cell fates, but its function is central in modulating and enhancing morphogenetic Nodal signals.

Organization of cardiac chamber progenitors in the zebrafish blastula

Organogenesis requires the specification of a variety of cell types and the organization of these cells into a particular three-dimensional configuration. The embryonic vertebrate heart is organized into two major chambers, the ventricle and atrium, each consisting of two tissue layers, the myocardium and endocardium. The cellular and molecular mechanisms responsible for the separation of ventricular and atrial lineages are not well understood. To test models of cardiac chamber specification, we generated a high-resolution fate map of cardiac chamber progenitors in the zebrafish embryo at 40% epiboly, a stage prior to the initiation of gastrulation. Our map reveals a distinct spatial organization of myocardial progenitors: ventricular myocardial progenitors are positioned closer to the margin and to the dorsal midline than are atrial myocardial progenitors. By contrast, ventricular and atrial endocardial progenitors are not spatially organized at this stage. The relative orientations of ventricular and atrial myocardial progenitors before and after gastrulation suggest orderly movements of these populations. Furthermore, the initial positions of myocardial progenitors at 40% epiboly indicate that signals residing at the embryonic margin could influence chamber fate assignment. Indeed, via fate mapping, we demonstrate that Nodal signaling promotes ventricular fate specification near the margin, thereby playing an important early role during myocardial patterning.

Artemisinins Target GABAA Receptor Signaling and Impair α Cell Identity

Summary Type 1 diabetes is characterized by the destruction of pancreatic β cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional β-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic β cell mass from α cells.

Prep1.1 has essential genetic functions in hindbrain development and cranial neural crest cell differentiation

In this study we analysed the function of the Meinox gene prep1.1 during zebrafish development. Meinox proteins form heterotrimeric complexes with Hox and Pbx members, increasing the DNA binding specificity of Hox proteins in vitro and in vivo. However, a role for a specific Meinox protein in the regulation of Hox activity in vivo has not been demonstrated. In situ hybridization showed that prep1.1 is expressed maternally and ubiquitously up to 24 hours post-fertilization (hpf), and restricted to the head from 48 hpf onwards. Morpholino-induced prep1.1 loss-of-function caused significant apoptosis in the CNS. Hindbrain segmentation and patterning was affected severely, as revealed by either loss or defective expression of several hindbrain markers (foxb1.2/mariposa, krox20, pax2.1 and pax6.1), including anteriorly expressed Hox genes (hoxb1a, hoxa2 and hoxb2), the impaired migration of facial nerve motor neurons, and the lack of reticulospinal neurons (RSNs) except Mauthner cells. Furthermore, the heads of prep1.1 morphants lacked all pharyngeal cartilages. This was not caused by the absence of neural crest cells or their impaired migration into the pharyngeal arches, as shown by expression of dlx2 and snail1, but by the inability of these cells to differentiate into chondroblasts. Our results indicate that prep1.1 has a unique genetic function in craniofacial chondrogenesis and, acting as a member of Meinox-Pbc-Hox trimers, it plays an essential role in hindbrain development.

Vg1 RBP intracellular distribution and evolutionarily conserved expression at multiple stages during development.

We have analyzed the expression and intracellular distribution, during oogenesis and embryogenesis, of Vg1 RBP, a protein implicated in the intracellular localization of Vg1 mRNA to the vegetal cortex of Xenopus oocytes. Vg1 RBP (protein) colocalizes with Vg1 RNA at all stages of oogenesis. Vg1 RBP RNA, however, localizes to the animal pole during late oogenesis, and remains in the animal blastomeres and ectodermal precursors until its zygotic transcription is activated, around stage 12. Vg1 RBP mRNA then becomes expressed throughout the neural epithelium. Vg1 RBP mRNA expression is also detected in what appears to be neural crest cells undergoing delamination and lateral migration. By tailbud stages, Vg1 RBP expression is present in the branchial arches, otic vesicle, pronephros, and along the neural tube. To examine the expression pattern in different species, we cloned the zebrafish homolog of Vg1 RBP by using a highly homologous EST clone to screen an embryonic cDNA library. In situ hybridization reveals that Vg1 RBP RNA localizes early in oogenesis to the animal pole. Although Vg1 RBP RNA is detected in all blastomeres of the early embryo, the expression pattern in the one day old zebrafish embryo is almost identical to that of the equivalent stage Xenopus embryo. These results indicate that the zygotic expression pattern is similar in frogs and fish, and that there is a conserved zygotic expression of Vg1 RBP distinct from its expression in the oocyte.

Factors Controlling Growth, Motility, and Morphogenesis of Normal and Malignant Epithelial Cells

Factors that control epithelial growth, motility, and morphogenesis play important roles in malignancy and in normal development. Here we discuss the molecular nature and the function of two types of molecules that control the development and maintenance of epithelia: Components that regulate epithelial cell adhesion; and soluble factors and their receptors that regulate growth, motility, differentiation, and morphogenesis. In development, the establishment of epithelial cell characteristics and organization is crucially dependent on cell adhesion and the formation of functional adherens junctions. The integrity of adherens junctions is frequently disturbed late in tumor progression, and the resulting loss of epithelial characteristics correlates with the metastatic potential of carcinoma cells. Various soluble factors that induce epithelial growth, motility, or differentiation in cell culture, function via tyrosine kinase receptors. We concentrate here on receptors that are expressed exclusively or predominantly on epithelia, and on ligands that are derived from the mesenchyme. In development, these receptors and their ligands function in mesenchymal-epithelial interactions, which are known to govern growth, morphogenesis, and differentiation of epithelia. During tumor development, mutations or overexpression of the receptors are frequently observed; these alterations contribute to the development and progression of carcinomas.

PlexinA3 Restricts Spinal Exit Points and Branching of Trunk Motor Nerves in Embryonic Zebrafish

The pioneering primary motor axons in the zebrafish trunk are guided by multiple cues along their pathways. Plexins are receptor components for semaphorins that influence motor axon growth and path finding. We cloned plexinA3 in zebrafish and localized plexinA3 mRNA in primary motor neurons during axon outgrowth. Antisense morpholino knock-down led to substantial errors in motor axon growth. Errors comprised aberrant branching of primary motor nerves as well as additional exit points of axons from the spinal cord. Excessively branched and supernumerary nerves were found in both ventral and dorsal pathways of motor axons. The trunk environment and several other types of axons, including trigeminal axons, were not detectably affected by plexinA3 knock-down. RNA overexpression rescued all morpholino effects. Synergistic effects of combined morpholino injections indicate interactions of plexinA3 with semaphorin3A homologs. Thus, plexinA3 is a crucial receptor for axon guidance cues in primary motor neurons.

Fast Homozygosity Mapping and Identification of a Zebrafish ENU-Induced Mutation by Whole-Genome Sequencing

Forward genetics using zebrafish is a powerful tool for studying vertebrate development through large-scale mutagenesis. Nonetheless, the identification of the molecular lesion is still laborious and involves time-consuming genetic mapping. Here, we show that high-throughput sequencing of the whole zebrafish genome can directly locate the interval carrying the causative mutation and at the same time pinpoint the molecular lesion. The feasibility of this approach was validated by sequencing the m1045 mutant line that displays a severe hypoplasia of the exocrine pancreas. We generated 13 Gb of sequence, equivalent to an eightfold genomic coverage, from a pool of 50 mutant embryos obtained from a map-cross between the AB mutant carrier and the WIK polymorphic strain. The chromosomal region carrying the causal mutation was localized based on its unique property to display high levels of homozygosity among sequence reads as it derives exclusively from the initial AB mutated allele. We developed an algorithm identifying such a region by calculating a homozygosity score along all chromosomes. This highlighted an 8-Mb window on chromosome 5 with a score close to 1 in the m1045 mutants. The sequence analysis of all genes within this interval revealed a nonsense mutation in the snapc4 gene. Knockdown experiments confirmed the assertion that snapc4 is the gene whose mutation leads to exocrine pancreas hypoplasia. In conclusion, this study constitutes a proof-of-concept that whole-genome sequencing is a fast and effective alternative to the classical positional cloning strategies in zebrafish.