Hazel L. Sive, Ph.D.
Department of Biology
Professor of Biology
Member, Whitehead Institute
Associate Dean, School of Science
Room WI 401
Ph.D 1986, Rockefeller University
We study vertebrate development, using frog and zebrafish embryos, since they are amenable to genetic, molecular and live imaging approaches. One focus of our research is on craniofacial development. In particular, we study signaling pathways that regulate formation of the primary mouth, the critical first opening between the gut and the outside of the embryo. A second broad focus is on formation of three-dimensional structure in the brain, including the brain ventricular system, an important circulatory system within the brain; and the “great bends” that pack the brain into the skull. A third focus is on use of the zebrafish as a tool to understand human mental health disorders. We are analyzing genes associated with schizophrenia, and the human 16p11.2 interval, associated with autism.
Development of the primary mouth
The primary mouth (also called the stomodeum) is the first opening between the outside and the developing pharynx, and is the earliest element of craniofacial development. It is critical for food ingestion. We previously defined multiple steps that occur during Xenopus primary mouth development, and isolated a set of regulatory genes whose expression is enriched in this region. An early, essential, step in primary mouth formation is local degradation of the basement membrane, which we have recently shown is mediated by local inhibition of the b-catenin-mediated Wnt pathway. This is the first demonstration that Wnt signaling is necessary for basement membrane synthesis.
Current focus is on the role of matrix metalloproteases (MMPs) that degrade the basement membrane. Function of MMPs expressed in the developing primary mouth will be analyzed by antisense loss of function assays, and gain of function assays, where effects are localized using “face transplants” and temporally regulated promoter constructs. In addition, we are examining the signaling systems that direct primary mouth formation. One focus is on the kinin pathway that is poorly characterized during embryogenesis. Preliminary data will be extended to analyze the role of BMP and non-canonical Wnt signaling in the primary mouth region.
Xenopus is an outstanding system for this project since tissue transplants can readily be used to localize effects of gene perturbation, whereas equivalent assays are difficult in mammalian models. Assays are rapid, as frog embryos develop rapidly; and frog genes are conserved with those of mammals. Steps occurring during formation of the mouse and chick primary mouth appear similar to those we have observed in Xenopus. Craniofacial birth defects appear with high frequency, in 1/700 live births, however the etiology of most is unknown, and the role of primary mouth perturbations has not been examined.
Development and function of the brain ventricular system
One unique feature of the vertebrate brain is that it is tubular. The lumen of the tube is filled with cerebrospinal fluid (CSF), and forms the brain ventricular system, a circulatory system within the brain. Ventricular abnormalities lead to devastating brain disorders such as anencephaly and hydrocephalus. The embryonic development and function of this system are poorly understood. We analyze embryonic brain ventricle development in the zebrafish, an excellent system, as imaging the brain at single cell resolution in living embryos is feasible, and as many brain ventricle mutants have been identified.
Brain ventricles form over a six-hour period, during mid-somitogenesis, and require normal junctions and ion pump activity. Thus, mutants in nagie oko, which encodes a MAGUK family protein, with no clear midline and disrupted epithelial junctional protein expression fail to inflate their ventricles. In the snakehead mutant brain, the ventricles also do not inflate due to a mutation in the NaK ATPase pump a subunit, Atp1a1, that leads to an absence of embryonic CSF (eCSF). Interestingly, Atp1a1 function is also required for normal junction formation, especially in conjunction with other pump subunits.
In addition to eCSF secretion, we have demonstrated that, in the zebrafish hindbrain, ventricular lumen expansion requires a “stretchy” epithelium. A mutation in the myosin phosphatase regulator, mypt1, results in a small hindbrain ventricle, due to inability of the surrounding neuroepithelium to stretch. 3D reconstruction of cell shape demonstrates that characteristic cell shapes within the hindbrain are also abnormal in mypt1 mutants. As wild type embryonic brain ventricles form, levels of phosphorylated myosin regulatory light chain (pMRLC) change dynamically; however, mutants show continuously high levels of pMRLC, with apical concentration of pMRLC and myosin II. Brain ventricle lumen expansion and cell shape are rescued by the inhibition of myosin II function, indicating that defects are a consequence of overactive myosin contraction. These results show that the epithelium must “relax”, via the activity of myosin phosphatase, in order to allow for normal expansion of the brain ventricular lumen. Epithelial relaxation may facilitate inflation of tubes in many organs.
Basal constriction during brain morphogenesis
The vertebrate brain develops from a tube, which becomes bent at stereotypical positions, to pack the brain into the skull. This packing protects the brain as a compact and stable structure. The first major bend to form separates the midbrain and hindbrain forming the midbrain-hindbrain boundary constriction (MHBC). Morphogenesis of the MHBC requires basal constriction, a novel cellular mechanism we described. This is in contrast to apical constriction, which has been extensively studied. Basal constriction occurs stepwise, in a group of cells that first basally constrict, before apically expanding to form wedge-shaped cells. An intact basement membrane is required, as sleepy mutants, corresponding to a Laminin chain, fail to undergo basal constriction. We have recently shown that focal adhesion kinase (FAK) is essential for basal constriction. Additionally, intact microtubules are essential for this process. We are presently addressing the connection between FAK, microtubules and other signaling modulators in basal constriction.
The zebrafish as a tool to analyze mental health disorders
Our goals are to use the zebrafish as a tool to study the function of genes associated with human mental health disorders, including schizophrenia, bipolar disorder and autism. We use the term animal “tool” in contrast with the more familiar notion of an animal “model”. While a model has the requirement of phenocopying a human disorder, a tool can provide insight into the disorder without phenocopying it. Our approach uses the attributes of the zebrafish, including ability to perform rapid loss and gain of function assays, and ability to perform chemical screens in whole embryos. The approach also rests on the hypotheses that 1) zebrafish homologs of human mental health disorder risk genes can be identified; 2) these genes function early during brain development and 3) human and zebrafish genes will show orthologous functions.
We have focused on function of the DISC1 gene (Disrupted In Schizophrenia), a schizoprenia risk gene. We have shown that DISC1 modulates Wnt signaling during zebrafish brain and somite development, and that the fish and human genes have interchangeable function. We are also analyzing zebrafish homologs of human genes corresponding to the human 16p11.2 region, associated with autism. In loss of function assays, 12/14 genes assayed show a brain phenotype, indicating that this region is rich in genes active during brain development. Future assays include use of chemical screening to identify modulators of selected mental health disorder risk gene activity.
- Gutzman, J.H. and Sive, H. Epithelial relaxation, mediated by the myosin phosphatase regulator mypt1, is required for brain ventricle lumen expansion, and hindbrain morphogenesis. Development 137, 795-804, 2010.
- Dickinson, A. and Sive. H. The Wnt antagonists, Frzb-1 and Crescent locally regulate basement membrane dissolution in the developing primary mouth. Development 136, 1071-81, 2009.
- Lowery, L.A. and Sive, H. Totally tubular: the mystery behind function and origin of the brain vetricular system. BioEssays 31, 446-58, 2009.
- Blaker, A.. DeRienzo, G. and Sive, H. Zebrafish as a tool to study autism. In “Autism Spectrum Disorders” (eds. D. Amaral, G. Dawson and D. Geschwind) Oxford University Press, 2009, in press.
- Gutzman, J.H.*, Graeden, E.G.*, Lowery, L.A., Holley, H.S. and Sive, H. Formation of the zebrafish midbrain-hindbrain boundary constriction requires laminin-dependent basal constriction. Mech.Dev. 125, 974-983, 2008. * equal contribution.
- Dickinson, A. and Sive, H. Positioning the extreme anterior in Xenopus: Cement gland, primary mouth and anterior pituitary. Seminars in Cell and Developmental Biology 18, 525-533, 2007.
Last Updated: January 16, 2011