David E. Clouthier Associate Professor Craniofacial Biology/Cell and Developmental Biology PhD, University of Texas Southwestern Medical Center at Dallas, 1994 Craniofacial Biology RC1 South, Rm 11109 UCHSC, Box 8120 P.O. Box 6511 Aurora, CO 80045 Phone: 303-724-4565 Fax: 303-724-4580 Email: david.clouthier@uchsc.edu

Signaling Pathways Regulating Craniofacial and Cardiovascular Development

My lab is focused on elucidating the molecular signals involved in neural crest cell (NCC) patterning during craniofacial and cardiovascular development. NCCs originate along the dorsal lip of the neural tube, migrating away around the time of tube closure to the pharyngeal arches and arch arteries. Once there, they form the bone, cartilage and connective tissue of the face and neck and the smooth muscle surrounding the great vessels. In addition they contribute to septal and valve formation. Many signals contribute to NCC patterning, with these signals organized into hierarchical pathways. One or more of these pathways is regulated by endothelin-A (Ednra) receptor signaling. Absence of Ednra signaling leads to defects in cephalic and cardiac neural crest development and subsequent neonatal lethality. Since birth defect syndromes affecting the face and heart account for the majority of birth defects observed, understanding how Ednra regulates signaling pathways during normal facial and heart development is of significant interests to both basic and clinical scientists.

We have recently shown that targeted inactivation of the Ednra gene in mice leads to loss of identity in specific NCCs, leading to a homeotic transformation of lower jaw structures into more maxilla-like structures. While we have identified Dlx5 and Dlx6 as downstream effectors of Ednra signaling, the functional mechanism driving this transformation is not known. Our lab is focused on addressing three main questions:

1. What is the timing and function of Ednra signaling during NCC development?
2. Does Ednra signaling establish specific developmental domains within the pharyngeal arches?
3. What factors and signaling pathways work in conjunction with Ednra signaling to establish the identity of NCCs?

We are currently using a variety of approaches in both mice and chicks to address these questions. To understand the timing of Ednra action during development, we are using Cre/loxP technology in mice to generate conditional Ednra mutant mouse embryos, in which Ednra gene is inactivated at specific times during lower jaw development. We are also using conditional gene inactivation to assess the role of Ednra signaling in different subdomains of the pharyngeal arches. In addition, we are performing microarray analysis to identify downstream effectors of Ednra signaling.

In addition to the use of mice, we are also currently analyzing endothelin function during chick NCC development. Chick embryos are quite useful in studying NCC development, as genes or markers can be directly inserted into NCCs prior to their emigration from the neural tube and subsequent NCC behavior (migration rate, pathway, etc) examined. Using both mouse and chick approaches, we hope to achieve our goal of elucidating the signaling pathways regulating facial development.

Expression of green fluorescent protein (GFP) by chick neural crest cells. A GFP expression vector was electroporated into premigratory neural crest cells of a stage 10 chick embryo using in ovo electroporation. After incubating for 18 hours, GFP-expressing migratory neural crest cells can be observed extending from the midbrain and hindbrain towards the ventral embryo surface.

Selected Publications

Ruest, L.-B., Kedzierski, R., Yanagisawa, M. and Clouthier, D.E. (2005) Deletion of the endothelin-A receptor gene within the developing mandible. Cell Tissue Res. 319:447-453.

Clouthier, D.E. and Schilling, T.F. (2004). Understanding endothelin-1 function during craniofacial development in the mouse and zebrafish. Birth Defects Res. Part C, Embryo Today 72:190-199.

Ruest. L.-B., Xiang, X., Lim, K.-L., Levi, G. and Clouthier, D.E. (2004). Endothelin-A receptor-dependent and independent signaling pathways in establishing mandibular identity. Development 131:4413-4423.

Ruest, L.-B., Dager, M., Yanagisawa, H., Charité, J., Hammer, R.E., Olson, E.N., Yanagisawa, M. and Clouthier, D.E. (2003). dHAND-Cre transgenic mice reveal specific potential functions of dHAND during craniofacial development. Dev. Biol. 257:263-277.

Ruest, L.-B., Hammer, R.E., Yanagisawa, M. and Clouthier, D.E. (2003). Dlx5/6-enhancer directed expression of Cre recombinase in the pharyngeal arches and brain. Genesis, 37, 188-194.

Latest Publications in PubMed