Slow muscles guide fast myocyte fusion to ensure robust myotome formation despite the high spatiotemporal stochasticity of fusion events.

Skeletal myogenesis is dynamic, and it involves cell-shape changes together with cell fusion and rearrangements. However, the final muscle arrangement is highly organized with striated fibers. By combining live imaging with quantitative analyses, we dissected fast-twitch myocyte fusion within the zebrafish myotome in toto. We found a strong mediolateral bias in fusion timing; however, at a cellular scale, there was heterogeneity in cell shape and the relationship between initial position of fast myocytes and resulting fusion partners. We show that the expression of the fusogen myomaker is permissive, but not instructive, in determining the spatiotemporal fusion pattern. Rather, we observed a close coordination between slow muscle rearrangements and fast myocyte fusion. In mutants that lack slow fibers, the spatiotemporal fusion pattern is substantially noisier. We propose a model in which slow muscles guide fast myocytes by funneling them close together, enhancing fusion probability. Thus, despite fusion being highly stochastic, a robust myotome structure emerges at the tissue scale.

The role of cellular active stresses in shaping the zebrafish body axis.

Tissue remodelling and organ shaping during morphogenesis are products of mechanical forces generated at the cellular level. These cell-scale forces can be coordinated across the tissue via information provided by biochemical and mechanical cues. Such coordination leads to the generation of complex tissue shape during morphogenesis. In this short review, we elaborate the role of cellular active stresses in vertebrate axis morphogenesis, primarily using examples from postgastrulation development of the zebrafish embryo.

Detection of mRNA by Whole Mount in situ Hybridization and DNA Extraction for Genotyping of Zebrafish Embryos.

In situ hybridization is used to visualize the spatial distribution of gene transcripts in tissues and in embryos, providing important information about disease and development. Current methods involve the use of complementary riboprobes incorporating non-radioactive labels that can be detected by immunohistochemistry and coupled to chromogenic or fluorescent visualization. Although recent fluorescent methods have allowed new capabilities such as single-molecule counting, qualitative chromogenic detection remains important for many applications because of its relative simplicity, low cost and high throughput, and ease of imaging using transmitted light microscopy. A remaining challenge is combining high contrast signals with reliable genotyping after hybridization. Dextran sulfate is commonly added to the hybridization buffer to shorten development times and improve contrast, but this reagent inhibits PCR-based genotyping. This paper describes a modified protocol for in situ hybridization in fixed whole mount zebrafish embryos using digoxigenin (DIG) labeled riboprobes that are detected with alkaline phosphatase conjugated anti-DIG antibodies and nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl-phosphate (BCIP) chromogenic substrates. To yield embryos compatible with downstream genotyping after hybridization without sacrificing contrast of the signal, this protocol omits dextran sulfate and utilizes a lower hybridization temperature.

Segmentation of the zebrafish axial skeleton relies on notochord sheath cells and not on the segmentation clock.

Segmentation of the axial skeleton in amniotes depends on the segmentation clock, which patterns the paraxial mesoderm and the sclerotome. While the segmentation clock clearly operates in teleosts, the role of the sclerotome in establishing the axial skeleton is unclear. We severely disrupt zebrafish paraxial segmentation, yet observe a largely normal segmentation process of the chordacentra. We demonstrate that axial entpd5+ notochord sheath cells are responsible for chordacentrum mineralization, and serve as a marker for axial segmentation. While autonomous within the notochord sheath, entpd5 expression and centrum formation show some plasticity and can respond to myotome pattern. These observations reveal for the first time the dynamics of notochord segmentation in a teleost, and are consistent with an autonomous patterning mechanism that is influenced, but not determined by adjacent paraxial mesoderm. This behavior is not consistent with a clock-type mechanism in the notochord.

Expression and localization of inhibitor of differentiation (ID) proteins during tissue and vascular remodelling in the human corpus luteum.

Members of the transforming growth factor-ß (TGF-ß) superfamily are likely to have major roles in the regulation of tissue and vascular remodelling in the corpus luteum (CL). There are four inhibitor-of-differentiation (ID1-4) genes that are regulated by members of the TGF-ß superfamily and are involved in the transcriptional regulation of cell growth and differentiation. We studied their expression, localization and regulation in dated human corpora lutea from across the luteal phase (n = 22) and after human chorionic gonadotrophin (hCG) administration in vivo (n = 5), and in luteinized granulosa cells (LGCs), using immunohistochemistry and quantitative RT-PCR. ID1-4 can be localized to multiple cell types in the CL across the luteal phase. Endothelial cell ID3 (P < 0.05) and ID4 (P < 0.05) immunostaining intensities peak at the time of angiogenesis but overall ID1 (P < 0.05) and ID3 (P < 0.05) expression peaks at the time of luteolysis, and luteal ID3 expression is inhibited by hCG in vivo (P < 0.01). In LGC cultures in vitro, hCG had no effect on ID1, down-regulated ID3 (P < 0.001), and up-regulated ID2 (P < 0.001) and ID4 (P < 0.01). Bone morphogenic proteins (BMPs) had no effect on ID4 expression but up-regulated ID1 (P < 0.01 to P < 0.005). BMP up-regulation of ID2 (P < 0.05) was additive to the hCG up-regulation of ID2 expression (P < 0.001), while BMP cancelled out the down regulative effect of hCG on ID3 regulation. As well as documenting regulation patterns specific for ID1, ID2, ID3 and ID4, we have shown that IDs are located and differentially regulated in the human CL, suggesting a role in the transcriptional regulation of luteal cells during tissue and vascular remodelling.