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Nervous System Development in the Zebrafish
Summary: Cecilia Moens is interested in understanding how the vertebrate brain achieves its neuromeric organization early in development. She uses a combination of zebrafish genetics and experimental embryology to identify the genes that control early brain development, to learn where and when they function, and by what molecular mechanism.
We study three fundamental questions in developmental biology: How does an apparently homogeneous epithelium become patterned along its anterior-posterior axis? How are morphological boundaries formed between groups of cells with different identities? How do cells move in a directed way through a complex patterned environment? We address these three questions in the context of the developing zebrafish hindbrain, which has a distinct and well-characterized anterior-posterior polarity divided into morphological segments called rhombomeres (Figure 1), and in which stereotyped migrations occur that position neurons in their functionally relevant contexts. In the zebrafish, all three processes occur during the first three days of development—in an optically transparent, externally developing embryo that is exquisitely accessible to live imaging. The availability of mutants generated through forward and reverse genetic approaches makes it possible for us to identify the genes and the genetic pathways that regulate these important events in the development of the vertebrate brain.
Patterning the Hindbrain Neuroepithelium
The central nervous system begins its development as an epithelium—the neuroepithelium—which becomes regionalized along its anterior-posterior and dorsoventral axes. Differentiating neurons acquire unique identities that are dictated by their coordinates in this "Cartesian grid" of positional information. Regionalization of the hindbrain is linked to a segmental pattern: the hindbrain is divided into seven reiterated units, the rhombomeres, whose boundaries correlate with domains of Hox gene expression and function (Figure 2). Hox genes encode homeobox transcription factors that have an evolutionarily conserved role in specifying segment identities. We wish to understand the genetic hierarchy leading to the spatial deployment of Hox gene expression during hindbrain development. Retinoic acid, a derivative of dietary vitamin A, plays a central role in this process; eliminating it results in severe patterning defects. Produced posterior to the hindbrain, retinoic acid is thought to diffuse into the hindbrain, establishing a posterior-to-anterior morphogen gradient, specifying different rhombomere identities at different concentration thresholds. However, the lack of endogenous retinoic acid can be rescued by a uniform concentration of exogenous retinoic acid, suggesting that a diffusion gradient is not strictly required for normal development. Our recent focus has therefore been on how retinoic acid signaling is controlled along the anterior-posterior axis of the hindbrain.
We have found that a family of cytochrome P450 enzymes, the Cyp26 enzymes, is essential for normal hindbrain patterning. In their absence the entire hindbrain expresses retinoic acid–responsive genes in an unpatterned manner and is thereby transformed to the posterior-most rhombomere identity (Figure 3). The cyp26 genes are expressed in dynamic domains whose boundaries correspond with future rhombomere boundaries. We propose a "gradient-free" model for retinoic acid patterning of the hindbrain, in which Cyp26 enzymes establish successive boundaries of retinoic acid responsiveness, even in the absence of a diffusion gradient of retinoic acid. Our ongoing effort is focused on understanding how cyp26 genes are themselves regulated, and how other factors that modulate retinoic acid signaling contribute to the overall patterning process.
Boundary Formation
Boundaries that prevent cell movement allow groups of cells to maintain their identity and follow independent developmental trajectories without the need for ongoing instructive signals from surrounding tissues. The appearance of rhombomere boundaries corresponds with the sharpening of rhombomere-specific domains of gene expression (Figure 4). Boundary sharpening can occur by a number of possible mechanisms: cells on the "wrong" side of a boundary can use a mechanism based on differential cell adhesion to move across it, they can change their identity to match that of their neighbors, or they can be eliminated by programmed cell death.
We have observed the first of these mechanisms in the developing zebrafish hindbrain. In genetic mosaics, mutant cells that are unable to take on particular rhombomere identities sort out from wild-type cells that do take on those identities. Cell sorting involves local repulsive interactions between Eph and ephrin-expressing cells. Eph receptors and their ephrin ligands are expressed in complementary rhombomere-restricted domains in the hindbrain, and interactions between Eph- and ephrin-expressing cells cause a repulsive response that is thought to drive cell sorting. We have found, however, that EphA4 and ephrinB2 also promote cell adhesion within the rhombomeres where they are expressed, since cells lacking either protein sort out from cells that express them in mosaic embryos. We hypothesize that two Eph and ephrin-dependent mechanisms—cell repulsion between cells with different Eph-ephrin expression and cell adhesion between cells with the same Eph-ephrin expression—lead to a robust boundary formation process. Current work in our lab is directed toward understanding at the cellular level how Ephs and ephrins promote cell adhesion in the hindbrain neuroepithelium, and how their distinct adhesive and repulsive functions separately contribute to boundary formation.
Neuron Migration
Once established, the regional patterning of the hindbrain manifests itself in the segment-specific differentiation of neuronal subtypes (Figure 1). Differentiated neurons then exhibit behaviors determined by their segmental identity and by cues that they perceive in their environment. These behaviors include the elaboration of axons toward specific targets as well as the migration of neuronal cell bodies through a complex, patterned environment. Stereotyped neuronal migrations occur at many times and places during brain development and are vital for the establishment of functional neural circuits. They include the radial migrations of cortical neurons and the tangential migrations of GABAergic neurons into the cortex from extracortical regions of the forebrain. Another stereotyped tangential migration is the posterior migration of the motor neurons of the seventh cranial nerve, which in the zebrafish occurs over a distance of about 100 microns and is complete by 72 hours of development. We are using this migration as a model for neuronal migrations in general, and to that end we have performed a forward genetic screen to identify mutations that disrupt the normal process (Figure 5).
We have also studied the migration in mutants that function cell autonomously to control segment identities. To migrate, motor neurons must acquire the identity appropriate for their segment of origin, but they can migrate irrespective of whether the cells they migrate through have their appropriate segmental identities. This suggests that the extracellular cues that drive neuronal migration in the hindbrain are not linked to its segmental organization. To elucidate what these cues are and how they are deployed, as well as how cells respond to them, we are continuing to map and clone migration mutants.
Zebrafish Reverse Genetics
Zebrafish forward genetic screens have been extraordinarily successful at identifying important developmental genes. However, many genes have not been identified in forward genetic screens because of redundancy with other genes and/or because their mutant phenotypes are subtle. To study the functions of particular genes in neural development, we are using TILLING, a method for reverse genetics, in the zebrafish. This approach detects chemically induced mutations in specific genes in mutagenized genomes. By TILLING genomic DNA from 8,600 fish, we have identified loss-of-function mutations in over 20 genes of interest. By preserving our library as frozen sperm, we will have a long-term resource for the identification of mutations in many zebrafish genes.
The National Institutes of Health provided support for these projects.
Last updated: January 4, 2008
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