Control of Ectodermal Development in Vertebrate Embryos
- Thomas D. Sargent, PhD, Head, Section on Vertebrate Development
- Mariam Awad, BA, Postbaccalaureate Fellow
- Sebastian Bilitsa, BA, Postbaccalaureate Fellow
- Neal MacDonald, AB, Postbaccalaureate Fellow
- Allisan Aquilina-Beck, BA, Technician (part time)
The lab focuses on mechanisms regulating the differentiation of cranial neural crest cells that give rise to the bone and cartilage of the vertebrate jaw, neurocranium, and other structures of the face and head. Our approach is to manipulate transcriptional control mechanisms in the intact zebrafish embryo using gain and loss of function strategies, with the aim of identifying the regulatory networks that control craniofacial development. Disruption of these developmental programs is the most common source of birth defects in humans, the detection, prevention, and treatment of which is a central aspect of NICHD's mission. Equally important, understanding the regulation of gene expression in this complex embryonic tissue represents a challenging and fascinating problem in basic molecular and developmental biology.
The origin of the lab’s neural crest research was our discovery in 2003 that, in the frog Xenopus, the transcription factor TFAP2a is both necessary and sufficient to trigger the conversion of cells at the neural plate border from neural to neural crest identity. We went on to show that TFAP2a mediates the transcriptional response to bone morphogenetic protein (BMP) signaling in neural crest induction. The lab also carried out pioneering research on the homeodomain factor Dlx3, performing the first mouse knock-out of the gene encoding this factor and demonstrating the factor's function in the development of mammalian epidermis and placenta. We were also the first to demonstrate the phylogenetic conservation of Dlx gene–regulatory elements by transferring Xenopus Dlx enhancers into the mouse genome, showing that enhancers were conserved between these two distantly related vertebrates, including those active in tissues such as hair follicle and mammary gland that have no counterpart in amphibians. We also investigated the role of Dlx3 and other members of this family in establishing the boundary of the neural crest in Xenopus. The Dlx family remains our current focus of research.
Dlx gene function in cranial neural crest development
In the previous year, the lab completed-gain-of function experiments based on over-expression of Dlx factors by injecting synthetic mRNA into fertilized eggs. Injected embryos were harvested early, at the beginning of gastrulation, and RNA isolated and subjected to RNAseq analysis. The hypothesis is that, because early embryonic cells are pluripotent, over-expressing a given transcription factor will result in precocious activation of cognate target genes, revealing potential downstream components of the regulatory network based on the factor injected (i.e., a Dlx factor). This was confirmed by the strong activation of several genes already known to be downstream of Dlx genes. We also confirmed numerous additional candidate targets. The data are being integrated with genomics studies on mouse Dlx mutants by our collaborating lab headed by Maria Morasso.
Most recently, we focused on loss-of-function experiments. Following reports from numerous labs, including our own, indicating that "knock down" analysis of gene function using antisense morpholinos frequently yielded results that were artifacts or otherwise at variance with chromosomally based gene inactivation strategies, we set out to apply the CRISPR/Cas targeting approach to the Dlx family in zebrafish. The objective of this project is to identify and characterize embryonic craniofacial phenotypes resulting from loss of individual Dlx genes or combinations thereof. The targeting is being conducted in a line of fish carrying a GFP fluorescent marker expressed in cranial cartilage, enabling high-resolution imaging of affected tissues using confocal microscopy. Until the writing of this report, we had identified in G0 founders individual mutations in all six major Dlx genes, as well as fish in which pairs of linked Dlx genes had been deleted (or inverted) in tandem. Out-crossing to GFP reporter individuals was initiated and is expected to be completed shortly, followed by in-crossing to yield Dlx nulls. We anticipate that these studies will reveal some of the earliest events that lead to Dlx-dependent craniofacial dysmorphology, with significant impact on the diagnosis of and ultimately on developing therapy for affected human fetal and neonatal patients.
Publications
- Law SHW, Sargent TD. The serine-threonine protein kinase PAK4 is dispensable in zebrafish: identification of a morpholino-generated pseudophenotype. PLoS One 2014; E100268.
Collaborators
- Maria Morasso, PhD, Laboratory of Skin Biology, NIAMS, Bethesda, MD
