HOX genes encode for a group of homeodomain-containing transcription factors essential for patterning the skeleton in the developing embryo.  They are expressed in spatially restricted domains, where they regulate the patterning of specific bones that form within these expression domains.  The expression of Hox genes is continued into adulthood, however their function in the adult skeleton is relatively unknown.  We speculate that HOX genes regulate post-embryonic growth of bony elements which reside within their spatially-restricted expression domains.  In support of this hypothesis, we present three key findings.  First, we identify a subset of vertebrae in zebrafish (V2-V5) whose size and shape are under the control of hox5 genes.  These vertebrae reside in spatial domains associated with hox5 expression in embryonic zebrafish.  Second, we show that severe loss of hox5 induces gross changes in bone morphology, including loss of branching in the fourth ribs and sporadic fusion of the tripus with the fourth ribs.  Third, we note that fish with moderate loss of hox5 exhibit reduced size of bony elements but no obvious changes in bone morphology.  Our studies provide early evidence of the role of HOX genes in mediating post-embryonic growth of the skeleton.  They also help to elucidate potential vertebral homologies in zebrafish- an emerging model for orthapaedic research- and cervicothoracic vertebrae in mammals.

Ras is classified as a group of proteins that is involved in many signaling processes in the cell. Ras plays key roles in homeostatic cell proliferation, differentiation, and movement. However, the oncogenic point mutation from glycine to valine in the 12th position of H-Ras protein (denoted by RasV12) induces tumors and gives rise to metastatic cell behaviors. Cancer metastasis is characterized in collection of several steps, but the initial step – dissemination of cancer cells from the tumor of origin into the bloodstream – is not well studied. In this project, we use Drosophila Melanogaster as a model organism to screen for genes involved in the process of dissemination. Overexpression of RasV12 in intestinal epithelial cells caused them to disseminate from the intestine. Knockdown of genes known to be critical for the process suppressed the cell dissemination phenotype. Our image analysis in observing relative quantities of GFP-expressing RasV12 cells in the intestine allowed us to discern the importance of a gene on the cell dissemination process. In result, we identified several kinases and phosphatases involved in different steps of the cell dissemination process.

Osteoporosis is an orthopedic disease in which old bone begins to dissolve but is not replaced by new bone. This reduces overall bone density and increases a patient’s risk for fractures. One human gene associated with osteoporosis-related traits is WNT16, which is also expressed in zebrafish. Previous studies in our lab have shown that wnt16 mutant fish have skeletal defects. The goal of this project was to find the most effective method for knocking out genes associated with osteoporosis in zebrafish using CRISPR/Cas9 technology. Once Cas9 creates a double-strand break in the DNA, there are different methods of DNA repair, two of which are non-homologous end joining (NHEJ) and microhomology mediated end joining (MMEJ). In this study, we designed guide RNAs (gRNAs) targeting wnt16 to compare a more predictable, MMEJ-based CRISPR approach to the previous, NHEJ-based CRISPR approach used in the lab. Next, we assessed gene editing in zebrafish embryos injected with two new gRNAs biased toward MMEJ repair to determine their efficacy in knocking out wnt16. Using DNA sequencing and analysis, we found that injections of both MMEJ gRNAs caused high rates of insertions and deletions (indels) compared to a control group which was not injected with gRNA. Moreover, the predicted MMEJ indels were found to be in high abundance in DNA sequences from injected fish. Further, we detected anticipated morphological differences expected from loss of wnt16 in the fish injected with MMEJ gRNAs compared to the control fish, suggesting that the MMEJ gRNAs work as expected. Based on these results, MMEJ-biased gRNA design appears to be a promising approach to improve efficiency in knocking out zebrafish genes. This project may help optimize a rapid and effective screening of many zebrafish candidate genes using CRISPR/Cas9 technology to study skeletal phenotypes in zebrafish.