Characterization of an Enhancer Upstream of Msx3 and its Role in Development of the Neural Tube of Embryonic and Larval Zebrafish

Abstract

The vertebrate nervous system arises during embryogenesis from an epithelial sheet of cells called the neural plate that subsequently folds to become a rod of cells called the neural tube. Several signaling pathways act on the neural progenitors of the neural tube to give rise to the diverse set of neurons and glia that will make up the spinal cord and brain in adulthood. In vertebrates, Muscle segment homeobox (Msx) genes are expressed in the dorsal neural tube during development, and pattern dorsal neural progenitors to give rise to dorsal neuronal subtypes. Additionally, Msx genes are involved in the regulation of neurogenesis and proliferation in the neural tube. In zebrafish, three msx genes are expressed in the neural tube: msx1a, msx1b, and msx3. The Akimenko lab has identified a potential enhancer of msx3 called Fragment C that drives expression in the dorsal neural tube. We hypothesized that Fragment C is a bona fide enhancer of msx3 specifically in the neural tube, and that this enhancer contributes toward proper patterning and neurogenesis/proliferation in the developing neural tube of zebrafish. To test this hypothesis, I have generated zebrafish mutants with a deletion of the Fragment C enhancer using CRISPR/Cas9 that also have a transgenic Fragment C enhancer driving reporter expression of enhanced green fluorescent protein (Egfp). The deletion of Fragment C abolishes msx3 expression in the neural tube excluding the dorsal-most cells likely corresponding to the roof plate. The spatial domain in which msx3 is lost corresponds to where Fragment C drives expression in the neural tube, suggesting that Fragment C contains an enhancer of msx3. This domain of expression corresponds to where dorsal neural progenitors reside. Analysis of markers for the cells with Fragment C-driven Egfp expression shows that at least some of these cells are indeed neural progenitors, many of which give rise to neurons during embryonic and larval development. The deletion of Fragment C and loss of msx3 expression in neural progenitors did not affect the numbers of neurons or neural progenitors amongst cells with Fragment C-driven expression, nor did it affect the dorsoventral location of cells in the neural tube. Taken together, we conclude that Fragment C-driven msx3 expression does not contribute to the dorso-ventral position of neural progenitors nor the balance of proliferation and neurogenesis in the developing neural tube. However, a role for msx3 in regulating neural progenitor identity along the dorso-ventral axis without affecting progenitor position cannot be ruled out.