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 Lab Website B.S. Millsaps College Ph.D. University of Chicago Postdoctoral Fellow, Max Planck-Institut für Entwicklungsbiologie; Tübingen, Germany

Figure 1. In zebrafish and humans, somites become the skeletal muscle and both the bone and cartilage of the vertebral column, which retains the segmental organization.

Somites are the most obvious metameric structures in the vertebrate embryo, and along with a dorsal neural tube, the notochord and pharyngeal pouches, they are the defining features of the vertebrate phylotype. Somites are mesodermal segments that form sequentially, in an anterior to posterior progression, concomitant with the posterior growth of the embryo. Somites ultimately give rise to the vertebral column and ribs (Figure 1), skeletal muscle, and dermis. We study somitogenesis in zebrafish and have found that zebrafish Notch pathway mutants are defective in somite formation. 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. Furthermore, mutation of these genes in adults can lead to cancer as the genes regulate cell behavior in both the developing embryo and the adult.

We study pattern formation and morphogenesis during zebrafish somitogenesis. More generally, we are interested in how the collective behavior of individual cells gives rise to emergent, tissue-levels of organization. To study somitogenesis, we use genetic, molecular, computational and imaging techniques. Zebrafish embryos are transparent and thus are particularly well suited for microscopic imaging and embryological experiments.

The segmentation clock

Oscillations are common in biology, e.g. the circadian clock, cardiac pacemaker, cortical rhythms, etc, and have been observed in cell signaling networks as a result of feedback loops. These cellular and physiological oscillations maintain homeostasis, and analysis of these rhythms is an emerging area in biomedical science. During vertebrate segmentation, ultradian oscillations govern the formation of somites. In zebrafish, the segmentation clock causes cells to undergo repeated cycles of expression and repression of Notch target genes. The oscillations can be visualized by high-resolution fluorescent in situ hybridization which reveals the phase in each cell via the subcellular distribution of the mRNA (Figure 2).

			Figure 2. High-resolution fluorescent in situ hybridization of the oscillating expression of two segmentation clock genes her1 (green) and deltaC (red). Nuclei are blue. These stripes of gene expression sweep though the tissue in a reiterated, wave-like fashion from posterior (right) to anterior (left). This striped pattern presages the segmental pattern of morphological somites.
			Figure 3 Cell movement in the posterior tailbud (right) contributes to axis elongation. Cells are labeled with a nuclear-localized green-to-red photoconvertible protein. Inititally all nuclei are green but two clusters of nuclei were photoconverted to red. Note the more extensive cell movement in the posterior group of cells (right) than the more anterior group of cells (left).

Somite and tail morphogenesis

The clock generates a segmental pattern as the embryonic body axis extends posteriorly. Growth of the embryo is driven by a combination of cell migration and cell proliferation (Figure 3). The segmental pattern instructs nascent somite boundary cells to undergo a mesenchymal to epithelial transition during somite morphogenesis. We identified mutants for zebrafish integrin α5, the primary receptor for the extracellular matrix (ECM) protein Fibronectin (FN). The mutant was originally called before eight as loss of either the zygotic or maternal-zygotic functions leads to defects in the morphogenesis of the only first 3-8 somite borders, yet the segmentation clock and polarity within each segment appears normal.

ECM coats and subdivides animal tissues, but it is unclear how ECM formation is restricted to tissue surfaces and specific cell interfaces (Figure 4). Using in vivo imaging and genetic mosaics, we found that incipient Integrin α5 clustering along the nascent somite border precedes matrix formation and is independent of FN binding. Integrin clustering can be initiated by Eph/Ephrin signaling with Ephrin reverse signaling being sufficient for clustering. Prior to activation, Integrin α5 expressed on adjacent cells reciprocally and non cell-autonomously inhibit spontaneous Integrin clustering and assembly of an ECM. Surface derepression of this inhibition provides a self-organizing mechanism for formation and maintenance of ECM along the tissue surface. Within the tissue, interplay between Eph/Ephrin signaling, ligand-independent Integrin clustering, and reciprocal Integrin inhibition restricts de novo ECM production to somite boundaries. Part of the lab seeks to further elucidate the mechanisms of Integrin regulation and ECM remodeling during somite morphogenesis and axis elongation.

			Figure 4. (A) Immunostaining of Fibronectin matrix in the zebrafish trunk. The somites show strong matrix deposition at their boundaries, but the tissue itself is also coated in extracellular matrix. (B) Integrin α5 heterodimerizes with Integrin α1 to form the Fibronectin receptor. Fibronectin is secreted as a dimer and converted into a matrix by the Integrin receptor. Integrin signaling also affects cell polarity and the cytoskeleton as well as gene expression.

Selected Publications

Jülich, D., Geisler, R., Consortium, T. S. and Holley, S. A. 2005. Integrin α5 and Delta/Notch Signalling have Complementary Spatiotemporal Requirements during Zebrafish Somitogenesis. Dev Cell, 575-86.

Mara, A., Schroeder, J., Chalouni, C. and Holley, S. A. 2007. Priming, Initiation and Synchronization of the Segmentation Clock by deltaD and deltaC. Nat Cell Biol 9, 523-30.

Zhang, L., Kendrick, C., Jülich, D. and Holley, S. A. 2008. Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function. Development 135, 2065-70.

Jülich, D., Mould, A. P., Koper, E. and Holley, S. A. 2009. Control of extracellular matrix assembly along tissue boundaries via Integrin and Eph/Ephrin signaling. Development 136, 2913-21.

			Holley Lab 2010. Back row from left: Dörthe Jülich, Anna Trofka, Mike Stulberg, Jamie Schwendinger-Schreck. Front row: Nicolas Dray, Patrick McMillen, Andrew Lawton, Scott Holley

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