Signaling Pathways Regulating Craniofacial Development in Mouse and Zebrafish
My lab is focused on elucidating the molecular signals involved in neural crest cell (NCC) patterning during craniofacial development. Cranial NCCs originate along the dorsal lip of the neural tube, migrating away around the time of tube closure to the pharyngeal arches on the ventral embryo surface. Once there, they form the bone, cartilage and connective tissue of the face and neck. Many signals contribute to NCC patterning, with these signals organized into hierarchical pathways. Within the mandibular portion of the first pharyngeal arch, one or more of these pathways is regulated by endothelin-A (Ednra) receptor signaling. Absence of Ednra signaling leads to defects in NCC patterning and subsequent neonatal lethality. Since birth defect syndromes affecting the face account for the majority of birth defects observed, understanding how Ednra signaling regulates genetic pathways during normal facial development is of significant interests to both basic and clinical scientists.
We have shown that targeted inactivation of the Ednra gene in mice causes loss of identity in mandibular arch NCCs, leading to a homeotic transformation of lower jaw structures into more maxilla-like structures. We have also identified Dlx5 and Dlx6 as downstream effectors of Ednra signaling, with these factors inducing expression of the gene encoding the basic helix-loop-helix transcription factor Hand2. Our recent data illustrate that a primary function of Hand2 is to act in a negative feedback loop to repress Dlx5/6 expression in the distal mandibular arch (the region from which the jaw and most of the middle ear arises). Failure to down regulate Dlx5/6 in this region leads to aglossia (absence of a tongue).
We are currently using a variety of approaches in both mice and zebrafish to further understand the role of Ednra signaling and Hand2 action during facial development. To better understand the function of Ednra signaling, we are using both conditional gene inactivation and over expression approaches in mice to assess the role of Ednra signaling in different sub-domains of the pharyngeal arches. To better understand Hand2 function, we are examining both the transcriptional regulation of Hand2 using zebrafish transgenesis and the interplay of Hand2 with other proteins within the pharyngeal arches that appear to be crucial for establishing the developmental plan of the lower jaw. In addition, we are probing the role of micro RNAs (miRNAs) in regulating lower jaw and midfacial development downstream of Hand2 using morpholino knockdown in zebrafish.
Abe, M., Ruest, L.-B. and Clouthier, D.E. (2007). Fate of cranial neural crest cells during craniofacial development in endothelin-A receptor deficient mice. Int. J. Dev. Biol. 51:97-105.
Hendershot, T.J., Liu, H., Sarkar, A.A., Giovannucci, D.R., Clouthier, D.E., Abe, M. and Howard, M.J. (2007). Expression of Hand2 is sufficient for neurogenesis and cell type-specific gene expression in the enteric nervous system. Dev. Dyn. 236:93-105.
Clouthier, D.E., Gray, J. and Artinger, K.A. (2008). Micromanaging Palate Development. Speech Sci. Oro. Dis. 18:62-72.
Hendershot, T.J., Liu, H., Clouthier, D.E., Shepherd, I.T., Coppola, E., Studer, M., Firulli, A.B., Pittman, D.L., Howard, M.J. (2008). Conditional deletion of Hand2 reveals critical functions in neurogenesis and cell type-specific gene expression for development of neural crest-derived noradrenergic sympathetic ganglion neurons. Dev Biol. 319:179-191.
Clouthier, D.E. (2008). MicroRNAs in facial development. Nat. Genet. 40:4-5.
Ruest, L.-B. and Clouthier, D.E. (2009). Elucidating timing and function of endothelin-A receptor signaling during craniofacial development using neural crest cell-specific gene deletion and receptor antagonism. Dev. Biol. 328:94-108.
Clouthier, D.E., Garcia, E. and Schilling, T.F. (2010). Regulation of facial morphogenesis by endothelin signaling: inisghts from mice and zebrafish. Am. J. Med. Genet Part A 152A:2962-2973.
Barron, F., Woods, C., Kuhn, K., Bishop, J., Howard, M.J. and Clouthier, D.E. (2011). Downregulation of Dlx5 and Dlx6 expression by Hand2 is essential for initiation of tongue morphogenesis. Development 138: 2249-2259. doi:10.1242/dev.056929
Ikle, J., Artinger, K.B. and Clouthier, D.E. (2012). Identification and characterization of the zebrafish pharyngeal arch-specific enhancer for the basic helix-loop-helix transcription factor Hand2. Dev. Biol., 368:118-126.