The transformation of vertebrate mesoderm into muscle, cartilage, bone, notochord, kidneys, gonads, blood, and other tissues is a classic example of morphogenesis and cellular differentiation during embryonic development. During the past two decades, mutagenesis screens and positional cloning methods have revealed key developmental genes that control this process, including morphogens, cellular receptors, and their downstream transcription factors. In particular, studies of zebrafish development have demonstrated that several T-box (Tbx) transcription factors work in concert to pattern the mesoderm lineage, including no tail (ntl), spadetail (spt), and tbx6. Embryos lacking ntl function fail to develop a notochord and posterior mesoderm, and spt mutants exhibit severe deficits in trunk mesoderm. Although a tbx6 mutant has not yet been generated, tbx6 expression dynamics and overexpression phenotypes suggest that this T-box gene has an important role in mesoderm patterning as well. Based on these observations, it has been hypothesized that ntl, spt, and tbx6 act combinatorially to control the mesoderm morphogenesis and differentiation. While it is evident that these transcription factors regulate mesoderm development, precisely how they act in space and time to effect this transformation remains unclear. The constitutive and global loss of ntl and/or spt function in their corresponding zebrafish mutants masks the spatiotemporal complexity of this process. In addition, few transcription targets or downstream effectors of the T-box genes have been identified. Bridging these gaps in our knowledge will require an ability to control ntl, spt, and tbx6 function with spatiotemporal precision, and the applicant has developed a new chemical technology that will enable these genetic manipulations. This methodology involves caged synthetic reagents for light-controlled gene silencing and builds upon the extensive use of antisense morpholinos for targeted gene knockdowns by the developmental biology community. Preliminary studies with a caged morpholino targeting the ntl gene have demonstrated its requirement for morphogenetic movements, notochord fate choice, and notochord maturation. A caged morpholino-based strategy for transcription factor target discovery has also been established. The applicant now proposes to apply these technologies to elucidate the roles of ntl, spt, and tbx6 in zebrafish mesoderm development, focusing on spatiotemporal aspects of their activities and their transcriptional targets. The three transcription factors will be individually and combinatorially silenced in distinct embryonic tissues, and the resulting effects on cell movements and fate choice will be ascertained. Direct target genes and downstream effectors of these T-box factors will also be identified in a tissue-specific manner by combining caged morpholinos, fluorescence-activated cell sorting, and microarray analyses. Using this interdisciplinary approach, the applicant will decipher mesodermal patterning mechanisms that would be difficult to ascertain through conventional genetic methods.

Public Health Relevance

During fetal development, tissue patterning and organogenesis require precise spatiotemporal control of cell proliferation, differentiation, and movement. The proposed research investigates the molecular mechanisms that regulate this process, using the zebrafish as a model organism and a new chemical technology called caged morpholinos. These studies will reveal how the T-box transcription factors no tail, spadetail, and tbx6 act in space and time to create distinct mesodermal tissues during embryogenesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM087292-03
Application #
8292106
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Hoodbhoy, Tanya
Project Start
2010-07-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
3
Fiscal Year
2012
Total Cost
$331,592
Indirect Cost
$143,492
Name
Stanford University
Department
Biology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
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Moore, John C; Sheppard-Tindell, Sarah; Shestopalov, Ilya A et al. (2013) Post-transcriptional mechanisms contribute to Etv2 repression during vascular development. Dev Biol 384:128-40
Shestopalov, Ilya A; Pitt, Cameron L W; Chen, James K (2012) Spatiotemporal resolution of the Ntla transcriptome in axial mesoderm development. Nat Chem Biol 8:270-6