Restricted expression of the zebrafish hsp90alpha gene in slow and fast muscle fiber lineages

Members of the heat shock protein 90 (Hsp90) family of molecular chaperones play important roles in allowing a select group of intracellular signaling molecules reach and maintain functionally active conformations. We have previously shown that hsp90alpha gene expression in early zebrafish embryos is restricted to a subgroup of paraxial-mesoderm derived somitic cells prior to muscle formation and that the gene is downregulated in mature trunk and tail muscle fibers. Here we have compared the expression of the hsp90alpha gene to muscle regulatory genes during development of slow and fast muscle fibers in normal embryos and in embryos carrying mutations which affect somitic muscle formation. We show that hsp90alpha is first expressed early during the development of slow somitic muscle progenitors shortly following myoD activation and at a point prior to or co-incident with the expression of other known muscle regulatory genes. Expression of hsp90alpha is also activated in the midline of flh mutants when these cells switch from a notochord to a muscle fate. Conversely, expression is not detectable in cells of the paraxial mesoderm lineage which fail to converge in spt mutants and which do not activate expression of other muscle specific marker genes. Finally, expression of hsp90alpha is downregulated in slow muscle fibers by 24 h of age but becomes detectable in the later developing fast fibers at this time. Thus, hsp90alpha is expressed in developing muscle progenitors during short temporal and spatial windows of both slow and fast fiber lineages in the zebrafish somite.     

Heat shock produces periodic somitic disturbances in the zebrafish embryo.

Environmental influences are known to produce segmental defects in a variety of organisms. In this paper we report upon segmental aberrations produced by brief heat shocks delivered to developing zebrafish embryos. The initial defects in the segmental pattern of somitic boundaries and motoneuron axon outgrowth were usually observed five somites caudal to the somite which was forming at the time of heat shock application. Segmental defects in zebrafish embryos exposed to a single heat shock treatment can occur in a periodic pattern similar to the multiple disturbances observed to occur in chick embryos. These data are discussed with regard to models involving cell cycle synchrony or 'clock and wavefront' schemes in the process of somitogenesis.     

Independent and cooperative action of Psen2 with Psen1 in zebrafish embryos

Presenilin1 (PSEN1) and presenilin2 (PSEN2) are involved in the processing of type-1 transmembrane proteins including the amyloid precursor protein (APP), Notch and several others. PSEN1 has been shown to be crucial for proteolytic cleavage of Notch in developing animal embryos. Mouse embryos lacking Psen1 function show disturbed neurogenesis and somite formation, resembling Notch pathway mutants. However, loss of Psen2 activity reveals only a minor phenotype. Zebrafish embryos are a valuable tool for analysis of the molecular genetic control of cell differentiation since endogenous gene expression can be modulated in subtle and complex ways to give a phenotypic readout. Using injection of morpholino antisense oligonucleotides to inhibit protein translation in zebrafish embryos, we show that reduced Psen2 activity decreases the number of melanocytes in the trunk but not in the cranial area at 2 days post fertilisation (dpf). Reduced Psen2 activity apparently reduces Notch signalling resulting in perturbed spinal neurogenin1 (neurog1) expression, neurogenesis and trunk and tail neural crest development. Similar effects are seen for reduced Psen1 activity. These results suggest that Psen2 plays a more prominent role in Notch signalling and embryo development in zebrafish than in mammals. Intriguingly, decreased Psen2 activity increases the number of Dorsal Longitudinal Ascending (DoLA) interneurons in the spinal cord while decreased Psen1 activity has no effect. However, the effect on DoLAs of reduced Psen2 can be ameliorated by Psen1 loss. The effects of changes in Psen2 activity on DoLA interneurons and other cells in zebrafish embryos provide bioassays for more detailed dissection of Psen2 function.     

Slow muscle regulates the pattern of trunk neural crest migration in zebrafish

In avians and mice, trunk neural crest migration is restricted to the anterior half of each somite. Sclerotome has been shown to play an essential role in this restriction; the potential role of other somite components in specifying neural crest migration is currently unclear. By contrast, in zebrafish trunk neural crest, migration on the medial pathway is restricted to the middle of the medial surface of each somite. Sclerotome comprises only a minor part of zebrafish somites, and the pattern of neural crest migration is established before crest cells contact sclerotome cells, suggesting other somite components regulate the pattern of zebrafish neural crest migration. Here, we use mutants to investigate which components regulate the pattern of zebrafish trunk neural crest migration on the medial pathway. The pattern of trunk neural crest migration is aberrant in spadetail mutants that have very reduced somitic mesoderm, in no tail mutants injected with spadetail morpholino antisense oligonucleotides that entirely lack somitic mesoderm and in somite segmentation mutants that have normal somite components but disrupted segment borders. Fast muscle cells appear dispensable for patterning trunk neural crest migration. However, migration is abnormal in Hedgehog signaling mutants that lack slow muscle cells, providing evidence that slow muscle cells regulate the pattern of trunk neural crest migration. Consistent with this idea, surgical removal of adaxial cells, which are slow muscle precursors, results in abnormal patterning of neural crest migration; normal patterning can be restored by replacing the ablated adaxial cells with ones transplanted from wild-type embryos.     

Integrinalpha5-dependent fibronectin accumulation for maintenance of somite boundaries in zebrafish embryos

Boundary formation and epithelialization are crucial processes in the morphological segmentation of vertebrate somites. By a genetic screening procedure with zebrafish, we identified two genes, integrinalpha5 (itga5) and fibronectin (fn), required for these processes. Fibronectin proteins accumulate at somite boundaries in accordance with epithelialization of the somites. Both Fibronectin accumulation and the epithelialization are dependent on itga5, which is expressed in the most medial part of somites. Although somite boundaries are initially formed, but not maintained, in the anterior trunk of the mutant embryos deficient in either gene, their maintenance is defective at all axial levels of embryos deficient for both of these genes. Therefore, Integrinalpha5-directed assembly of Fibronectin appears critical for epithelialization and boundary maintenance of somites. Furthermore, with an additional deficiency in ephrin-B2a, the segmental defect in itga5 or fn mutant embryos is expanded posteriorly, indicating that both Integrin-Fibronectin and Eph-Ephrin systems function cooperatively in maintaining somite boundaries.     

Generality of vertebrate developmental patterns: evidence for a dermomyotome in fish

The somitic compartment that gives rise to trunk muscle and dermis in amniotes is an epithelial sheet on the external surface of the somite, and is known as the dermomyotome. However, despite its central role in the development of the trunk and limbs, the evolutionary history of the dermomyotome and its role in nonamniotes is poorly understood. We have tested whether a tissue with the morphological and molecular characteristics of a dermomyotome exists in nonamniotes. We show that representatives of the agnathans and of all major clades of gnathostomes each have a layer of cells on the surface of the somite, external to the embryonic myotome. These external cells do not show any signs of terminal myogenic or dermogenic differentiation. Moreover, in the embryos of bony fishes as diverse as sturgeons (Chondrostei) and zebrafish (Teleostei) this layer of cells expresses the pax3 and pax7 genes that mark myogenic precursors. Some of the pax7-expressing cells also express the differentiation-promoting myogenic regulatory factor Myogenin and appear to enter into the myotome. We therefore suggest that the dermomyotome is an ancient and conserved structure that evolved prior to the last common ancestor of all vertebrates. The identification of a dermomyotome in fish makes it possible to apply the powerful cellular and genetic approaches available in zebrafish to the understanding of this key developmental structure.     

斑马鱼pax2a(-3.1 kb):eGFP转基因品系的构建

为了更好地了解脊椎动物肾脏的早期发育,本研究利用斑马鱼这一研究脊椎动物器官发育的理想模式生物,通过构建肾脏特异性pax2a荧光标记转基因品系以实现实时在体地观察斑马鱼前肾的发育过程。我们分析了斑马鱼pax2a基因的启动子,扩增出其翻译起始位点上游3.1 kb的基因组DNA,利用Tol2转座子系统,经胚胎显微注射及三代筛选,成功构建了一个稳定的pax2a(-3.1 kb):eGFP转基因鱼品系。此转基因品系的绿色荧光蛋白在3体节时期就可以标记出中间中胚层,并在后续体节时期的中间中胚层前端(第3体节至第5体节对应的部分),及24 hpf的肾管前端和中段表达,基本模拟了pax2a在斑马鱼早期前肾发育中的时空表达模式。本研究所获得的pax2a(-3.1 kb):eGFP弥补了已有pax2a转基因品系中报告基因无法准确标记出中间中胚层和前肾管前端的缺陷,是研究斑马鱼前肾早期发育的良好材料。     

Zebrafish Embryonic Slow Muscle Is a Rapid System for Genetic Analysis of Sarcomere Organization by CRISPR/Cas9, but Not NgAgo

Zebrafish embryonic slow muscle cells, with their superficial localization and clear sarcomere organization, provide a useful model system for genetic analysis of muscle cell differentiation and sarcomere assembly. To develop a quick assay for testing CRISPR-mediated gene editing in slow muscles of zebrafish embryos, we targeted a red fluorescence protein (RFP) reporter gene specifically expressed in slow muscles of myomesin-3-RFP (Myom3-RFP) zebrafish embryos. We demonstrated that microinjection of RFP-sgRNA with Cas9 protein or Cas9 mRNA resulted in a mosaic pattern in loss of RFP expression in slow muscle fibers of the injected zebrafish embryos. To uncover gene functions in sarcomere organization, we targeted two endogenous genes, slow myosin heavy chain-1 (smyhc1) and heat shock protein 90 α1 (hsp90α1), which are specifically expressed in zebrafish muscle cells. We demonstrated that injection of Cas9 protein or mRNA with respective sgRNAs targeted to smyhc1 or hsp90a1 resulted in a mosaic pattern of myosin thick filament disruption in slow myofibers of the injected zebrafish embryos. Moreover, Myom3-RFP expression and M-line localization were also abolished in these defective myofibers. Given that zebrafish embryonic slow muscles are a rapid in vivo system for testing genome editing and uncovering gene functions in muscle cell differentiation, we investigated whether microinjection of Natronobacterium gregoryi Argonaute (NgAgo) system could induce genetic mutations and muscle defects in zebrafish embryos. Single-strand guide DNAs targeted to RFP, Smyhc1, or Hsp90α1 were injected with NgAgo mRNA into Myom3-RFP zebrafish embryos. Myom3-RFP expression was analyzed in the injected embryos. The results showed that, in contrast to the CRISPR/Cas9 system, injection of the NgAgo-gDNA system did not affect Myom3-RFP expression and sarcomere organization in myofibers of the injected embryos. Sequence analysis failed to detect genetic mutations at the target genes. Together, our studies demonstrate that zebrafish embryonic slow muscle is a rapid model for testing gene editing technologies in vivo and uncovering gene functions in muscle cell differentiation.