The objective of my project is to understand how chondrocytes, which make cartilage, and osteoblasts, which make bone, generate the specific elements of the zebrafish facial skeleton. During endochondral and dermal bone development, cells differentiate, organize into condensations, then proliferate and rearrange to generate the distinct shapes of skeletal elements.
My aim i s to understand how skeletogenic cells generate pattern by tracking cells from initial condensations at 50 hours post fertilization, to 4 days post fertilization when skeletal elements are easily identifiable. I will examine this process using 4D confocal in vivo imaging of wild-type and myocyte enhancing factor-2c (mef2ca) mutant (hoover) embryos, which have patterning defects in endochondral and dermal bones. Mef2c is a transcription factor in the endothelinl (ednl) pathway, disruption of which is associated with cleft palate defects in humans. With total loss of mef2c function in zebrafish (mef2cb morpholino-injected mef2ca mutants), ossification of bones does not occur. To track chondrocytes in endochondral bone formation (Aim 1), I will study embryos on the mCherry;fli1:EGFP reporter line which marks neural crest cells. Chondrocytes will be distinguished on the basis of morphology. The mCherry;fli1:EGFP line does not allow distinction of osteoblasts specifically, so to track osteoblasts in dermal bone formation (Aim 2), I will generate a fluorescent transgenic reporter line for osterix (osx), a transcription factor required for osteoblast differentiation. I hypothesize that correct morphology depends early cell position and subsequent stereotyped cell movements. I predict that in mef2ca mutants, either the initial organization of chondrocytes and osteoblasts in condensations or the subsequent rearrangement of cells during bone growth is abnormal, generating patterning defects. Recent analyses suggest that class II histone deacetylase-4 (Hdac4) represses Mef2c. Therefore, another aim of my project is to investigate the effects of Hdac4 in regulating bone shape by knocking down hdac4 using splice-blocking morpholinos in wild-type embryos, in mef2ca mutants, and in mef2cb morpholino-injected mef2ca mutants (Aim 3). I predict that loss of Hdac4 in zebrafish lacking mef2c orthologues will either have no effect on patterning or ossification defects, or will rescue defects by restoring normal cell behavior. More than half of all birth defects are associated with some sort of craniofacial malformation. Understanding how normal skeletal cell activity translates into shape is informative towards understanding how abnormal cell behavior can give rise to defects. By investigating the role of genes involved in palate formation, we can begin to test the function of specific genetic pathways in the generation of cleft palate deformities.
