Nervous System Development in the Zebrafish
Summary: Cecilia Moens is interested in understanding how the vertebrate brain is shaped and patterned during early 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.
Our lab studies four fundamental questions in developmental biology: How does a planar epithelium, the neural plate, fold and then cavitate to form the neural tube? How does this apparently homogeneous neuroepithelium become patterned along its anterior-posterior axis into segments with distinct molecular and neuroanatomical identities? How are morphological boundaries formed between these segmental domains? How do cells move in a directed way through this complex, patterned environment? We address all four 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 and in which stereotyped migrations occur that position neurons in their functionally relevant contexts.
We use the zebrafish as a model system because all four processes, which take place in the first five weeks of human development, occur in rapid sequence during the first two days of zebrafish development. The zebrafish is also optically transparent, with an externally developing embryo that is exquisitely accessible to embryological manipulation and 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.
Neural Tube Morphogenesis
In fish, the neural tube forms secondarily, after the infolding of the neural plate into a neural keel in which the left and right sides of the epithelium contact each other at the forming midline. Progenitor cell divisions in the neural keel are apicobasal, distributing daughter cells to either side of the midline. Using live imaging of cells in the neural keel, we have found that the precise alignment of the mitotic spindle in a planar orientation and its subsequent 90˚ rotation prior to division across the forming midline are essential for normal tube formation. Mutants in which spindle orientation is randomized in the neural keel develop dramatic neural tube defects characterized by a disorganized, misaligned neural tube midline. We are seeking to understand what the cues are that regulate spindle orientation in the neural keel to enable neural tube morphogenesis.
Patterning the Hindbrain Neuroepithelium
Early in development, the neuroepithelium 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 special in that it 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. 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 segmental gene expression in the hindbrain. Retinoic acid (RA), a derivative of dietary vitamin A, is known to be the key morphogen in this context. RA is a potent teratogen in all vertebrates, including humans, so its levels in the embryo must normally be very tightly regulated. Our recent focus has therefore been on how RA levels are controlled along the anterior-posterior axis of the hindbrain. We have found that the Cyp26 RA-degrading enzymes, which are normally segmentally deployed in the hindbrain, are essential for normal hindbrain patterning, since in their absence the entire hindbrain expresses RA-responsive genes in an unpatterned manner. More recently, we have identified Dhrs3a, an enzyme that reduces the RA precursor retinaldehyde into vitamin A, as another key regulator of RA levels in the embryo. Microarray analysis reveals that cyp26, dhrs3a, and other genes involved in RA metabolism and RA signaling are themselves all strongly regulated by RA in the early embryo, revealing a complex feedback mechanism regulating the bioavailability and activity of this important vertebrate morphogen.
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 is preceded by the sharpening of rhombomere-specific domains of gene expression. This occurs by the sorting out of cells that are on the "wrong" side of a boundary, a process that is controlled by differential cell adhesion. Sorting out can be modeled in genetic mosaics, where mutant cells that are unable to take on particular rhombomere identities sort out from wild-type cells that can take on those identities. Cell sorting has been shown to involve local repulsive interactions between Eph and ephrin-expressing cells in alternating rhombomeres. We have found, however, that Eph receptors and ephrin ligands also play a role in promoting cell adhesion within the rhombomeres where they are expressed, since in mosaic embryos, cells lacking a particular Eph or ephrin sort out from the rhombomeres that normally express them. 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. By following the behaviors of cells in mosaic embryos in confocal time lapses, we have found that this within-rhombomere adhesion is particularly important in the neural keel to maintain rhombomere coherence during hindbrain morphogenesis. Our ongoing work in this area is to develop transgenic tools that allow us to visualize and manipulate cell-sorting events in live embryos at the single-cell level.
Once established, the regional patterning of the hindbrain manifests itself in the segment-specific differentiation of neuronal subtypes. 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. One such migration is the stereotyped 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 largely complete by 48 hours of development. We are using this migration as a model for neuronal migrations in general. Live imaging has shown that motor neurons migrate in contact with the basement membrane and with the axons of neurons that have migrated ahead of them. Forward genetic screens for motor neuron migration in our lab and others have identified multiple core components of the planar cell polarity (PCP) pathway, including Vangl, Prickle, Frizzled, and Celsr homologs, as well as Scribble. We identified Nhsl1a, a vertebrate homolog of Drosophila Gukh, as another component of the pathway that interacts physically and genetically with Scribble. Mosaic analysis suggests that these proteins function primarily within and between migrating motor neurons. To understand how this suite of proteins is deployed to regulate directed cell migration, we are developing tools to visualize the localization of PCP components in migrating motor neurons and their environment.
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 of interest in neural development, we have adapted to the zebrafish a method for reverse genetics termed TILLING, which detects chemically induced mutations in specific genes of interest in mutagenized genomes. By TILLING a library consisting of genomic DNA from a cryopreserved library of 8,600 ENU-mutagenized fish, we have identified and recovered loss-of-function mutations in more than 60 genes of interest to the zebrafish community, as well as several hundred missense mutations in these genes and others. We also continue to exploit this unique reverse genetics resource to identify mutations in genes that are predicted to function in the processes outlined above.
The National Institutes of Health also provided support for these projects.
As of May 30, 2012