Sharon L. Amacher
We are interested in how cells become sequentially determined to more precisely defined fates during vertebrate embryonic development, and how this process depends upon cell position and upon interactions among neighboring cells. To address these questions, we use genetics, molecular biology, time-lapse imaging, and embryology to investigate mesodermal patterning and segmentation in the zebrafish embryo.
Mesodermal patterning. The embryonic mesoderm is specified during gastrulation, with dorsal mesoderm becoming notochord, lateral mesoderm forming muscle, and ventral mesoderm becoming blood. We are characterizing the molecular and cellular events involved in patterning the gastrula mesoderm. Two T-box transcription factors, Spadetail (Spt) and No tail (Ntl)/Brachyury are required to specify specific mesodermal cell types, and together, the two T-box genes are required for development of all trunk and tail mesoderm. To understand how Spt and Ntl mediate mesodermal cell fate decisions, we have identified putative target genes using zebrafish microarrays, and are investigating the expression and function of putative targets, as well as characterizing regulatory regions that control their T-box-regulated expression. Intriguingly, several of the putative targets encode “cyclic” genes , implicating Spt and Ntl as important upstream regulators of the segmentation clock, a dynamic molecular oscillator with a periodicity equal to that of somite formation (30 minutes in the zebrafish).
Mesodermal segmentation. Following gastrulation, the trunk and tail mesoderm becomes segmented into a reiterated series of tissue blocks called somites. Somitogenesis is regulated both spatially and temporally and is controlled by the segmentation clock and by cell-cell interactions among presomitic cells. To uncover the molecular nature of the segmentation clock, we have performed genetic screens to identify and characterize mutations that disrupt cyclic gene expression. To understand how cyclic gene expression, thus segmentation clock function, is initiated, maintained, and eventually extinguished, we have constructed a transgenic line that allows us to follow oscillating gene expression in single cells in live wildtype and mutant embryos. Using this line, we are investigating the function of candidate regulators, like Spt and Ntl and their targets, in starting, stopping, and synchronizing the segmentation clock.
Cellular interactions during somitogenesis. In addition to studying the dynamic cell behaviors that occur prior to segmentation, we also use a variety of approaches to study somitic cell behaviors during and after segmentation. To understand mutant phenotypes, and thus gene function, at the level of single cells, we are using time-lapse microscopy of wild-type and mutant embryos to observe cell-cell contacts and interactions occurring before, during, and after somites form. In zebrafish, the majority of somitic cells form muscle, and we have discovered that a small population of early-differentiating muscle cells induces the morphogenesis of their neighbors as they migrate through the somite to their final position. Currently, we are pursuing the molecular nature of the trigger.
Role of muscle-specific splicing in muscle function. In collaboration with the Conboy lab (LBNL), we have uncovered a critical role for RRM domain-containing Fox proteins in skeletal and cardiac muscle function. We have identified multiple genes with alternative exons whose splicing is altered in the absence of Fox function, and our knockdown experiments have shown that Fox-deficient embryos, although quite normal by morphology, are completely paralyzed and have irregular and slow heartbeat. We are currently focusing on uncovering how the Fox-regulated muscle-splicing program creates specific isoforms critical for function and physiology of muscle.
Novel reverse genetic technologies. The ability to do forward genetic screens is a great strength of the zebrafish system. Reverse genetics, the ability to modify a specific locus of one's choosing, has lagged behind. In collaboration with Sangamo Biosciences, we have demonstrated that zinc finger nucleases (ZFNs) can be used to target double strand breaks (DSBs) to specific loci in the zebrafish genome. The subsequent repair of induced DSBs is often mutagenic, introducing small deletions and insertions in the targeted locus at high frequency in both somatic and germline tissue. Currently, we are investigating the feasibility of using ZFNs to facilitate homologous recombination.
Disrupting zebrafish genes efficiently with designed ZFNs. In “Zinc Finger Proteins: Methods and Protocols”, Humana Press (eds. Drs. Joel MacKay and David J. Segal) [J.M. McCammon and S.L. Amacher (2010) Methods Mol Biol 649, 281-298]
Visualizing enveloping layer glycans during zebrafish early embryogenesis. [J.M Baskin, K.W. Dehnert, S.T. Laughlin, S.L. Amacher, C.R. Bertozzi (2010) Proc Natl Acad Sci USA 107, 10360-10365]
Identification of direct T-box target genes in the developing zebrafish mesoderm. [A. T. Garnett, T. M. Han, M. J. Gilchrist, J. C. Smith, M. B. Eisen, F. C. Wardle, and S. L. Amacher (2009) Development, 136, 749-760]
Emerging gene knockout technology in zebrafish: zinc-finger nucleases. [S. L. Amacher (2008) Briefings in Functional Genomics and Proteomics, 7, 460-464]
Heritable targeted gene disruption in zebrafish using designed zinc finger nucleases. [Y. Doyon*, J. M. McCammon*, J. C. Miller, F. Faraji, C. Ngo, G. Katibah, R. Amora, T. D. Hocking, L. Zhang, E. J. Rebar, P. D. Gregory, F. D. Urnov, and S. L. Amacher (2008) Nature Biotechnology, 26, 702-708 (* Equal contributions)]
In vivo imaging of membrane-associated glycans in a developing vertebrate. [S. T. Laughlin*, J. M. Baskin*, S. L. Amacher, and C. R. Bertozzi (2008) Science, 320, 664-667 (* Equal contributions)]
A web based resource characterizing the zebrafish developmental profile of over 16,000 transcripts. [M. Ouyang, A. T. Garnett, T. M. Han, K. Hama, A. Lee, Y. Deng, N. Lee, H.-Y. Liu, S. L. Amacher, S. A. Farber, and S.-Y. Ho (2007) Gene Expression Patterns, 8, 171-180]
Control of morphogenetic cell movements in the early zebrafish myotome. [D. F. Daggett, C. R. Domingo, P. D. Currie, and S. L. Amacher (2007) Dev Biol, 309, 169-179]
Interactions between muscle fibers and segment boundaries in zebrafish. [C. A. Henry, I. M. McNulty, W. A. Durst, S. E. Munchel, and S. L. Amacher (2005) Dev Biol, 287, 346-350]
tortuga refines Notch pathway gene expression in the zebrafish presomitic mesoderm at the post-transcriptional level. [K. K. Dill and S. L. Amacher (2005) Dev Biol, 287, 225-236]
Zebrafish slow muscle cell migration induces a wave of fast muscle morphogenesis. [C. A. Henry and S. L. Amacher (2004) Dev Cell, 7, 917-923]
Developmentally restricted actin regulatory molecules control morphogenetic cell movements in the zebrafish gastrula. [D. F. Daggett, C. A. Boyd, P. Gautier, R. J. Bryson-Richardson, C. Thisse, B. Thisse, S. L. Amacher, and P. D. Currie (2004) Curr Biol, 14, 1632-1638]
Two linked hairy/enhancer-of-split-related zebrafish genes, her1 and her7, function together to refine alternating somite boundaries. [C. A. Henry, M. K. Urban, K. K. Dill, J. P. Merlie, M. F. Page, C. B. Kimmel, and S. L. Amacher (2002) Development, 129, 3693-3704]
Last Updated 2010-08-11