Genetic analysis of vertebrate segmentation in the zebrafish, Danio rerio

Scott Holley, Ph.D. Associate Professor of Molecular, Cellular and Developmental Biology Room: KBT 1034 Phone: (203) 432-3230 Email: scott.holley@yale.edu B.S. Millsaps College, 1991 Ph.D. University of Chicago, 1997 Postdoctoral Fellow, Max Planck-Institut für Entwicklungsbiologie; Tübingen, Germany 1997-2002

Figure 1. Somites are the most prominent segmented structure in the zebrafish embryo. (a) a live, wild-type zebrafish embryo at the 15 somite stage. Anterior is left and posterior is right. The 5th and 10th somites indicated. (b) an after eight/deltaD mutant embryo in which the somites posterior fail to form. (c and d) show her1 expression is blue/purple and myoD expression in red. (c) in wild-type embryos, the oscillator generates stripes of her1 expression in the unsegmented presomitic mesoderm while myoD is expressed in a striped pattern in each somite. In c and d are dorsal views with anterior up and posterior down. (d) in after eight/deltaD mutant embryos, the oscillator is perturbed and no stripes of her1 expression are seen. The segmental expression of myoD is also abnormal in the posterior.

Segmentation is the developmental process by which the anterior-posterior body axis is divided into repeating elements. Somites are the segmented precursors to the vertebral column and skeletal muscle within the trunk and tail of vertebrate embryos. Somite formation, or somitogenesis, occurs sequentially from anterior to posterior as the embryo grows posteriorly. We study somitogenesis in zebrafish and have found that two mutant zebrafish strains defective in somite formation harbor mutations in the cell surface receptor notch1a and its ligand deltaD. These mutant zebrafish fail to make normal somites and thus have an abnormal vertebral column and disorganized trunk and tail. Mutations in the human or mouse orthologues of these genes result in a similar defect suggesting that the genetic circuitry that governs somite formation is the same in these different vertebrate species.

We are using the zebrafish as a model system to study somitogenesis and more generally to study how the sum of the function of many individual genes gives rise to higher levels of organization such as the dynamic yet stable cell behavior inherent in multicellular patterns/structures. To study somitogenesis, we use genetic, genomic, molecular and embryological techniques. Zebrafish embryos are transparent and thus are particularly well suited for microscopic imaging and embryological experiments. We also have roughly 20 mutant strains that contain specific mutations in at least 9 unique genes that are specifically required for normal somite formation.

Our previous work has shown that the Notch signaling pathway forms at least part of a circuit that creates rapid oscillations of gene expression within the field of cells that are about to undergo somitogenesis. These cells go through up to 7+ cycles in which they turn on and then turn off the transcription of a set of Notch target genes such as her1. These oscillations create stripes/waves of gene expression that repeatedly travel from posterior to anterior through the field of somite precursor cells. In the anterior of this field of somite precursor cells, the stripes of gene expression are stabilized and ultimately determine the position of the next segment border. We have shown that the stabilization of the oscillations requires a “wavefront” acting through the fused somites gene. Two areas interest in the lab are to understand the oscillator mechanism and to determine how the oscillator pattern is transformed into a morphological somite.

Different regions of the anterior/posterior axis require distinct genes to establish the somitic pattern: the Notch pathway mutations, in mouse, zebrafish and humans, mostly affect the posterior somites while the anterior somites are normal. We have identified a mutant with a segmentation defect in only the anterior somites and thus has a phenotype complementary to that of the Notch mutants. Another focus of the lab is to understand the differences between anterior and posterior somitogenesis.

			Figure 2 is a schematic of the oscillations of her1 expression during one full somite cycle. Anterior is up and posterior is down. Embryos I through IV represent a time series where I is the youngest and IV the oldest. As the somite cycle progresses, the waves of gene expression travel anteriorly at a rate of one cell diameter every 5-6 minutes through the field of somite precursor cells, the presomitic mesoderm. As time progresses, the embryo continues to grow or extend posteriorly and new somites are created in the anterior of the presomitic mesoderm: SI and S0 in embryo I become SII and SI in embryo IV while a new S0 created in the anterior PSM.
			Figure 3 Shown is an image of the forming somites in a live zebrafish embryo expressing Integrin alpha5-GFP (green) and a nuclear mRFP (red). The unsegmented cells to the left show Integrin alpha5-GFP along the entire cell cortex while the somite border cells show clustering along the basal surface of the epithelial border cells (arrowheads). Integrin alpha5-GFP clustering can also be seen along the nascent border cells (arrows).

Selected Publications

Lixia Zhang, Christina Kendrick, Dörthe Jülich and Scott A. Holley. 2008. Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function.  Development, 135, 2065-70.

Andrew Mara and Scott A. Holley. 2007. Oscillators and the Emergence of Tissue Organization during Zebrafish Somitogenesis.  Trends Cell Biol, 17, 593-9.

Andrew Mara, Joshua Schroeder, Cecile Chalouni, and Scott.A. Holley. 2007. Priming, Initiation and Synchronization of the Segmentation Clock by deltaD and deltaC. Nat Cell Biol, 9, 523-530.

Dörthe Jülich, Robert Geisler, Tübingen 2000 Screen Consortium and Scott A. Holley. 2005. Integrina5 and Delta/Notch Signalling have Complementary Spatiotemporal Requirements During Zebrafish Somitogenesis. Dev Cell, 8, 575-586.

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