Transcriptional regulation and biological Function of Zebrafish Myogenic Regulatory Factor, MYF5
Date Issued
2006
Date
2006
Author(s)
Lee, Hung-Chieh
DOI
en-US
Abstract
Myf5, one of the basic helix-loop-helix transcription factors, controls muscle differentiation and is expressed in somites during early embryogenesis. However, the transcription factors bound to the cis-elements of myf5 are poorly understood. In this study, we used the yeast onehybrid assay and found that Forkhead box d3 (Foxd3) interacted specifically with the -82/-62 cassette, a key element directing somite-specific expression of myf5. The dual-luciferase assay revealed that the expression of Foxd3 potently transactivated the myf5 promoter. Knocking down foxd3 with morpholino oligonucleotide (MO) resulted in a dramatic down-regulation of myf5 in somites and adaxial cells but not in the presomitic mesoderm. On the other hand, myod expression remained unchanged in foxd3 morphants. Foxd3 mediation of myf5 expression is stagedependent, maintaining myf5 expression in the somites and adaxial cells during the 7- to 18-somite stage. Furthermore, in the pax3 morphant, the expression of foxd3 was down-regulated greatly and the expression of myf5 was similar to that of the foxd3 morphant. Co-injection of foxd3 mRNA and pax3-MO1 greatly restored the expression of myf5 in the somites and adaxial cells, suggesting that pax3 induces foxd3 expression, which then induces the expression of myf5. This report is the first study to show that Foxd3, a well-known regulator in neural crest development, is also involved in myf5 regulation.
To understand whether myf5 plays other roles than myogenesis during embryogenesis. We first observed that myf5 was expressed in the non-axial mesoderm at the shield stage. Knockdown of Myf5 resulted in abnormal expansion and disorder of the dorsal organizer. We proved that the segmentation of the hindbrain were affected severely in the myf5 morphants due to either lost or defective expression of krox20 and pax6,. The expression of neural crests markers was dramatically reduced in the myf5 morphants; five-day-old myf5 morphants had serious chondrodysplasia in craniofacial cartilage. The TUNEL assay showed that apoptosis occurred significantly in the head of the myf5 morphants. These findings suggest that the reduction of head size and the absence of pharyngeal cartilage formation induced by myf5 inactivation were due primarily to apoptosis. Of interest, the pharyngeal arch defects found in the myf5 morphants were identical to those of the fgf3-MO-injected embryos, and the expression of fgf3 and its down-regulators erm and pea3 was greatly reduced in the myf5 morphants. The fgf3 transcripts also were reduced in the myf5 morphants, but co-injection of fgf3 mRNA and myf5-MO1 into the embryos rescued the hindbrain patterning and the ceratobranchial cartilage defects; the apoptotic signals were also reduced. This evidence suggests that, myf5 is involved in axial and non-axial mesoderm interaction during gastrulation. The disrupted hindbrain segmentation affects the fgf3 signaling, thus causing the CNC to undergo apoptosis. Myf5 is necessary for dorsal organizer patterning, hindbrain segmentation, CNC survival, and cranial cartilage formation.
Subjects
zebrafish
somitogenesis
development
gene regulation
Type
other
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