Hazel Sive, PhD - US grants

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

The aims of this proposal are to identify the genes that regulate formation of the anteroposterior (A/P) axis in the ectoderm of the frog, Xenopus laevis. The ectoderm gives rise to both neural tissues (including the brain and spinal cord) and to non-neural tissues (such as the cement- and hatching glands). These tissues are induced by signals arising from the mesoderm and by intraectodermal interactions. Previous results showed that the ectoderm acquired an A/P pattern many hours before terminal differentiation begins. Very little is known about the molecular basis for this patterning in any vertebrate. This information is crucial to understanding the early genetic events that regulate ectodermal patterning during vertebrate development. Two approaches to analyzing A/P ectodermal patterning will be taken. 1. The function of the gene Xhox.lab1 will be analyzed by misexpression and functional ablation. Xhox.lab1 is a novel homeodomain containing gene that is expressed in posterior ectoderm early during A/P patterning. 2. Other genes expressed soon after ectodermal induction will be isolated by subtractive hybridization between microdissected induced and uninduced ectoderm.

The research plan that the PI has initiated will answer a long-standing puzzle of embryology- how do inducers transmit their effects to cause particular patterns of organ formation in vertebrates? The PI is involved in several innovative teaching projects, and is committed to increasing the standard of science education in the United States. This NYI award will free time that she would otherwise devote to grant applications and allow her attention to focus on performing research of the highest quality and on implementing changes in both the curriculum of her home institution and in the involvement of scientists in school-level education.

vertebrate embryology; developmental neurobiology; histogenesis; neurogenesis; gene expression; developmental genetics; early embryonic stage; ectoderm; subtraction hybridization; chimeric proteins; Xenopus; molecular cloning; in situ hybridization;

The goals of this project are to determine how the anterior of a vertebrate
embryo becomes correctly positioned. The extreme anterior ectoderm in the frog
Xenopus forms the cement gland, a mucus-secreting organ that is an excellent
positional indicator. The focus of this project is to define ectodermal genes
that regulate cement gland determination. One transcription factor expressed
in the cement gland that is key for cement gland formation is the homeobox
gene otx2. Ectopically expressed otx2 activates ectopic cement gland
development, while ablation of otx2 activity using a dominant negative protein
prevents cement gland formation. A hormone-inducible otx2 protein, otx2GR,
activates the cement gland marker XAG1, however only with ongoing protein
synthesis, suggesting that otx2 activates expression of downstream genes which
in turn activate XAG1 expression. In this proposal, genes activated by otx2GR
that may directly activate XAG1 expression will be isolated using a
subtractive cloning strategy. In order to further analyze cement gland
positioning, dissection of the XAG1 promoter has been carried out. A 200bp
XAG1 fragment directs expression of a reporter to the cement gland. This
fragment is also responsive to otx2GR, although it contains no otx2 binding
sites. Further analysis of the XAG1 promoter will be performed to define
promoter elements necessary for cement gland formation. Factors that interact
with XAG1 regulatory sequences will be identified and characterized. Since the
cement gland is an indicator of normal head development, and since genes and
developmental strategies are evolutionarily conserved, what we learn in
Xenopus is likely to have relevance for understanding normal and abnormal
development of human embryos.

DESCRIPTION (Applicant's abstract reproduced verbatim): The goals of this proposal are to understand how posterior regions of the nervous system (hindbrain and spinal cord) are initially determined in vertebrates, using the zebrafish as a paradigm. The zebrafish is a wonderful model, allowing analysis of the early, crucial stages of nervous system development, by both embryological and genetic analysis. Integral to this study is a set of genes that we isolated by subtractive cloning, and that is expressed exclusively in the posterior of the embryo during gastrulation, when neural patterning has begun but before neural differentiation has occurred. Some of the genes isolated contain DNA binding motifs, while some are novel, suggesting that we have enriched for regulatory genes. This application has three parts. First, using these genes as markers, the tissues responsible for posterior neural induction will be determined, using explant assays that were developed for zebrafish in my laboratory. Second, the factors required for induction of posterior neural tissue will be analyzed using zebrafish mutants and dominant negative proteins. Third, the function of a subset of genes isolated by subtraction will be addressed. Genes will be placed on the zebrafish genetic map to determine whether they correspond to known mutations. Gain of function assays will ask how the novel nlz gene modulated posterior neural patterning. This application will define the cell interactions and genes required for normal hindbrain and spinal cord development. Normal brain development and health require integration of all parts of the nervous system, suggesting that deficits in the posterior nervous system may contribute to or exacerbate malfunction of other brain regions. The genes to be studied here may also help define the genetic basis of neural tube birth defects.

[unreadable] DESCRIPTION (provided by applicant): Brain ventricles are a highly conserved system of cavities that contain cerebrospinal fluid and are believed to protect the brain from injury, remove waste, and carry chemical signals. Blockage of free fluid circulation leads to hydrocephalus, one of the most common birth defects. Additionally, abnormalities in brain ventricle structure and size have been extensively documented in individuals affected with schizophrenia and autism. This exploratory proposal seeks to understand the genetic basis for brain ventricle formation, using the zebrafish as a model, and definition of a set of brain ventricle mutants as the foundation for the study. The longer term goal is to understand the role that genes corresponding to these mutants play in formation of the brain ventricular system, and to address whether their misfunction contributes to the etiology of autism, schizophrenia and related disorders. The zebrafish has proven an excellent genetic and molecular model for issues in developmental biology including brain formation and function, and has served as a model for many human diseases. It is hypothesized that ventricle morphology and function may alter brain function, and conversely, that ventricle formation and maintenance is dependent on normal brain function. In preliminary studies, the timecourse of brain ventricle formation in the zebrafish has been described and 3 mutants with defects in brain ventricle development have been examined. It is proposed, firstly, to continue characterization of these mutants by analyzing (i) molecular and cell biological changes relative to wild type fish embryos, (ii) cell autonomy of their function and (iii) epistatic interactions between them (hierarchy of function). Secondly, 28 mutants reported to have brain ventricle phenotypes, but otherwise unstudied, that were derived from chemical mutagenesis screens will be further analyzed. Additional mutants will be defined in collaboration with Dr. Nancy Hopkins, MIT, by screening through 275 retroviral insertional mutants for which candidate genes have been identified. Brain ventricle mutants will be categorized as a prelude to cloning corresponding genes. This study will define a large collection of genes required for normal brain ventricle formation, and which may display abnormal activity in patients with mental health disorders. [unreadable] [unreadable]

The goals of this new project are to examine the mechanism by which the primary mouth of deuterostomes forms at the extreme anterior of the embryo. A particular emphasis is on evolutionary conservation, or lack of it, in determining this organ. The primary mouth is the initial opening from the gut of the embryo to the outside, allowing ingestion of food. In most deuterostome species, the primary mouth remains the mouth opening. In vertebrates, the neural crest grows around the primary mouth to form the face and a secondary mouth opening must therefore form. The primary mouth then becomes the pharyngeal opening. The region from which the primary mouth develops is unusual as here ectodermal and endodermal germ layers are directly juxtaposed. Both ectoderm and endoderm are required for primary mouth formation. The broad hypothesis of this proposal is that the mechanisms by which the primary mouth is determined are evolutionarily conserved. There are three specific aims. 1. Markers of the primary mouth region in Xenopus will be isolated. 2. Mechanisms of primary mouth determination in Xenopus will be examined, focusing on candidate transcription factors and signaling systems involved. 3. Comparison of the expression of selected Xenopus markers will be made with expression of homologs in another deuterostome, the zebrafish, Danio. The intellectual merit of this proposal will be the increased understanding of the mechanisms and evolution of primary mouth formation. This region is important and its development poorly understood. Part of the broad impact of the proposal is that it spans the fields of Developmental and Evolutionary Biology. It will allow investigators involved to work with more than one species, which is an unusual training opportunity. Further, this study is a wonderful teaching tool, that will allow graduate, undergraduate and high school students a hands-on opportunity to think about evolution, and to compare formation of distinct animals.

[unreadable] DESCRIPTION (provided by applicant): The goals of this proposal are to define the molecular mechanisms involved in vertebrate primary mouth formation, using the frog Xenopus tropicalis as a model. The primary mouth (also called the stomodeum, buccopharyngeal membrane) is the initial opening from the outside of the embryo to the gut. Primary mouth formation is an essential step in cranoiofacial development, but is not well understood. In vertebrates, the neural crest grows around the primary mouth to form the jaws and face and a secondary mouth opening must therefore form. The primary mouth then becomes the pharyngeal opening. The primary mouth forms from a unique region at the extreme anterior of the embryo where ectoderm and endoderm are directly juxtaposed. In order to define the molecular mechanisms involved in this process, we began to characterize this process in X. laevis. We fate mapped the presumptive primary mouth, and defined tissues required for its induction. In order to define genes required for primary mouth formation, we used database mining and expression microarray analysis, to identify genes whose expression is enriched in the presumptive primary mouth region. Two genes that came out of this analysis are fgf8 and the Wnt pathway inhibitor, frzb1, and preliminary data indicates that both are essential for primary mouth formation. We propose to extend this project to X. tropicalis. Compared to X. laevis, this species has the advantage of a diploid genome, making antisense knockdowns more effective, and offering the ability to rapidly prepare stable transgenic lines. Initial data suggests that primary mouth formation in X. laevis and X. tropicalis is very similar. I hypothesize that the process of primary mouth formation is highly conserved. I further hypothesize that primary mouth formation requires the coordinate and sequential input of multiple regulatory genes expressed from several inducing regions. We will examine the temporal and spatial requirement for Fgf and Frzbl function during primary mouth formation using transgenic lines expressing inducible dominant negative or RNAi constructs, and transplant assays. We will begin to examine the requirement for other genes using antisense approaches. This proposal is exploratory since the study is new and unpublished, since it proposes extension into a new species and since it requires reagent development. Reagents developed will be important not only for this project, but for the X. tropicalis community. The primary mouth is an essential structure required for normal eating and speaking. Craniofacial development is severely impacted by an abnormal or absent primary mouth, however the molecular mechanisms underlying this process are not understood. This study will add insight into the genetic basis of craniofacial abnormalities, focusing on a crucial structure. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The broad goals of this proposal are to understand the mechanisms underlying formation of the vertebrate primary mouth, using the frog Xenopus laevis as a model. 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, and may contribute to the tongue and teeth. 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 is mediated by local inhibition of the 2-catenin-mediated Wnt pathway. There are two Specific Aims. Aim 1 analyses regulation of basement membrane metabolism in the primary mouth region. 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. The hypothesis that basement membrane synthesis and degradation are balanced, and coordinately activated by Wnt signaling will be tested. Steps in primary mouth formation that are altered if the basement membrane does not break down will be defined. Aim 2 examines the signaling systems that direct primary mouth formation. One focus is on the kinin-kallikrein pathway that is required for primary mouth formation, but is poorly characterized during embryogenesis. The function during primary mouth formation of enzymes, receptors and modulators in this pathway will be tested by antisense loss of function and in vitro assays. 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, indicating that our analyses will be useful for suggesting comparative studies in mammals. This study has broad significance. Craniofacial birth defects appear with high frequency, in 1/700 live births, however the etiology of most is unknown, and primary mouth perturbations have not generally been considered as causal. Both the interplay of Wnt and MMP signaling during basement membrane remodeling, and the kinin-kallikrein pathway are of likely significance in other embryonic organs.

DESCRIPTION (provided by applicant): The Whitehead Institute requests $500,000 for the purchase of a Zeiss LSM710 scanning confocal microscope, to be located in the W.M. Keck Core Facility, and made available to both Whitehead and MIT investigators. Scanning confocal microscopes allow optical sectioning of living and fixed material, so that investigators can image deep into a solid sample, and make a 3D reconstruction of the sample. The instrument requested is of great importance, as it will replace an out-dated, nine year old Zeiss LSM510, currently the only scanning confocal microscope at the Whitehead Institute. Deterioration of the current LSM510 has begun to hamper research, as repair times on this instrument have become excessive, replacement parts cannot be obtained, and the system cannot support upgrades. Our choice of the Zeiss LSM710 as a replacement for the LSM510 is due to the large number of outstanding features on the LSM710, its ease of use, and Zeiss'excellent customer support. Some of the new features offered by the LSM710 include a 405nm wavelength laser, which, in conjunction with the LSM710's five other lasers, allows simultaneous multi-imaging of up to ten different dyes;a new spectral recycling loop and a twin gate beam splitter which will increase sensitivity significantly, enhancing our ability to perform live imaging without phototoxicity. A tunable pulsed laser with a capacity from 488-640nm will be purchased for the LSM710 through a financial commitment of $212,587 from the Whitehead Institute. This tunable laser allows Fluorescence Lifetime Imaging (FLIM), which enables study of molecular lifetime, and facilitates FRET (Fluorescence Resonance Energy Transfer) analyses, to determine molecular interactions. The LSM710 can perform FRAP (Fluorescence Recovery After Photobleaching) to analyze molecular dynamics;calcium imaging is simple and targeted uncaging of fluorescent indicators or bioactive molecules is readily feasible. New Zen software that comes with the LSM710 will simplify previously arduous tasks, such as deconvolution, ratio imaging, and photobleaching analysis. In addition to purchasing FLIM equipment, the Whitehead Institute will contribute to the yearly service contract for the LSM710, and to salary support for two technicians who train users and oversee use of the instrument. By expanding the experimental approaches available to researchers, the LSM710 will contribute greatly to the success of many NIH-funded projects, including the analysis of neural degeneration, nervous system development and birth defects, mental health disorders, immune function, meiosis and associated chromosomal abnormalities, cancer, metabolic regulation, regeneration and identification of stem cells. Acquisition of the LSM710 will allow researchers to address these important biomedical issues, and the outcomes of this research will include new diagnostics and therapeutics, in accord with the mission of the NIH.

The three-dimensional structure of human organs is essential for function, and understanding how organs are constructed from cells is a huge challenge in biology. One important mechanism involves bending sheets of connected cells. A new type of cell sheet bending called 'basal constriction' has recently been discovered in the brain. This project will uncover genes necessary for basal constriction, using the zebrafish. The zebrafish is an excellent model as the embryo is transparent, allowing microscopic imaging of single cells in the living animal. It is hypothesized that a cellular communication system called the 'Wnt-PCP pathway' is essential for basal constriction. The function(s) of genes in this pathway, including one called focal adhesion kinase will be examined. A hypothesis that membrane around part of the cell is removed by a process called 'endocytosis' to constrict the cell and bend the cell sheet will be tested. This project is groundbreaking, since it brings together the disciplines of tissue structural engineering and molecular biology. Basal constriction occurs in many organs, and knowledge obtained from these studies will be pertinent for understanding organ formation and regeneration.

The broader impacts of the project include trainee mentoring, training, and communicating with the public. Mentorship will include middle school students through Boston Science Club for Girls. Undergraduates will participate in this research, while graduate students and postdoctoral researchers will be trained as effective supervisors. The PI has a special interest in encouraging young women to consider a research career, and will mentor and lead discussions for this group. The PI also has a strong interest in mentoring postdoctoral researchers, and set up an annual review process for Whitehead/MIT postdocs. Lectures on the importance of scientific inquiry and the excitement of research will be offered to lay audiences in various venues.

? DESCRIPTION (provided by applicant): This proposal will define signaling mechanisms associated with the extreme anterior domain (EAD) and face formation using the frogs Xenopus laevis and X. tropicalis as models. The EAD is a conserved embryonic region where ectoderm and endoderm are juxtaposed, that develops into the mouth, anterior pituitary and nostrils. In the previous funding period, we made several novel findings. (1) The EAD is an organizing center necessary for cranial neural crest (CNC) development, using the Kinin-Kallikrein pathway and nitric oxide (NO); (2) Incoming CNC induces the EAD to undergo convergent extension, a novel step in mouth formation, using the Wnt/PCP pathway; (3) Wnt antagonists frzb1+crescent that are localized in the EAD act globally, in other facial regions. Our data give insight into the earliest stages of facial development and suggest two hypotheses with high impact that will be addressed in this proposal. Wnt antagonists from the EAD regulate frontonasal prominence (FNP) and first arch CNC development. The EAD signals through Bradykinin and nitric oxide to regulate CNC migration, in a concentration-dependent manner. There are two Aims. The first Aim will delineate the role of Frzb1+Crescent derived from the EAD in neural crest development. The role(s) of EAD-derived Frzb1+Crescent in FNP and first arch CNC determination, migration, proliferation and survival will be assessed after local loss of function (LOF). X. tropicalis mutants will complement and extend analyses. Frzb1+Crescent will be assayed for sufficiency to direct neural crest development, using heterologous cells expressing Frzb1 or beads soaked in Frzb-IgG protein. Ability of Frzb1+Crescent to inhibit ?-catenin-mediated Wnt reporter activity in the FNP and first arch CNC will be assessed. Signaling pathways and candidate target genes modulated by frzb1+crescent will be determined. The second Aim will define the role of the EAD and the Kinin-Kallikrein pathway in modulating nitric oxide (NO) signaling and CNC migration. Cells in the developing facial region which produce NO will be identified, using the NO sensor DAF2 and NO-sensor nanotubes. A requirement for the EAD and EAD Kinin-Kallikrein factors in facial NO production will be assessed by extirpation and local LOF. cCNC cells will be assayed for ability to respond directly to Bdk peptides or NO in the embryo or in culture. Downstream effects of NO on target cells will be examined focusing on changes in signaling pathways and the cytoskeleton. The face is the defining feature of the individual human. Facial abnormalities are frequent (~1/700 births), resulting in physical and psychological disturbances. Much of the face may be impacted by reduced activity of the EAD organizer. The data obtained here will inform pre-natal diagnosis and correction, and are highly significant in the craniofacial field.