William H. Klein - US grants

nucleic acid sequence; animal population genetics; nonmammalian vertebrate embryology; gene expression; messenger RNA; genetic transcription; genetic strain; ectoderm; saltwater environment; sperm; genetic mapping; immunofluorescence technique; monoclonal antibody; molecular cloning; genetic recombination;

The long term objective of this proposal is to understand the mechanisms involved in the differentiation of embryonic ectoderm in sea urchin development. The sea urchin Spec genes are the main focus of the proposed experiments. The Spec1 and Spec2 genes encode a family of calcium binding proteins whose accumulation is restricted to aboral ectoderm lineages. Spec3 encodes a protein hypothesized to be associated with ectodermal ciliogenesis. The specific aims are: (1) To investigate the mechanisms by which Spec1 and Spec2 genes are transcriptionally activated in aboral ectoderm cells. DNA sequences involved in expression will be identified using the sea urchin embryo expression system. Regions required for appropriate expression will be assayed for specific protein interactions using gel mobility shift and DNA footprinting techniques. Comparisons will be made among the different Spec genes and between the Spec genes and the CyIIIa actin gene whose expression is also limited to the same cells; (2) To obtain information on the properties of the Spec1 protein. Purified Spec1 protein will be used to determine calcium binding affinities. Interactions of Spec1 with other proteins will be examined by gel transfer techniques. The hypothesis that the Spec1 protein interacts with microfilaments will be tested by injection of FITC-labeled Spec1 protein into sea urchin eggs and observing whether the protein is localized to the cortex or cleavage furrows. The Spec1 protein also will be produced in mouse C2 myoblasts and its intracellular sites monitored by indirect immunofluorescence; (3) To compare the Spec1 gene of S. purpuratus to the homologous gene of its distant relative, L. pictus. Conserved sequences important for proper function and expression will be sought. The Spec1 genes will be compared by microinjection of the genes into homologous and heterologous eggs and monitoring proper expression; (4) To test the hypothesis that the Spec3 protein is associated with embryonic cilia and that its levels in the cell are coupled to levels of unpolymerized tubulin. Pools of unpolymerized tubulin will be increased by treating embryos with colcemid and Spec3 mRNA synthesis and stability will be monitored. Spec3 mRNA will be expressed in mouse L cells to determine whether the stability of the urchin mRNA is the same as endogenous mRNAs encoding microtubular components. Antibodies against the Spec3 protein will be generated from the Spec3 ORF and the location of the protein in urchin embryos will be determined by immunological detection.

We are interested in the regulation of gene expression in sea urchin embryogenesis. We have isolated two cDNA clones specifying mRNAs that are members of a small family of mRNAs. The mRNAs code for a group of approximately ten low molecular weight acidic proteins that accumulate in the embryonic ectoderm. The function of these proteins is unknown, but they are among the most actively synthesized proteins in later embryonic stages and undergo the most pronounced developmental changes observed during embryogenesis. The mRNAs accumulate in mass over 100 fold during embryogenesis and have a common repetitive element at their 3 foot ends. This element is present in thousands of sites in the sea urchin genome. In situ hybridization to sections of embryos suggest that the RNAs are present in a specific cell type. They appear in the dorsal ectoderm of the pluteus larva and in the future dorsal ectoderm of the gastrula stage embryo. We propose further in situ experiments to map this cell lineage to earlier developmental stages. Using recombinant DNA methodology, we plan to establish the number, arrangement, and structure of the genes coding for the ectoderm proteins. By preparing antibodies to two or three of the more abundant ectoderm proteins, we propose experiments to determine the location and possible function of these proteins in the embryo. Our ultimate objective is the isolation and characterization of embryonic regulatory elements involved in the expression of these genes. Using lysates of embryos at appropriate developmental stages, we will attempt to achieve accurate and specific in vitro transcription using the cloned ectoderm genes and their upstream sequences as templates.

The long-term objective of this project is to gain a better understanding of mammalian muscle cell differentiation, in vivo, using mice carrying a targeted mutation at the gene encoding the myogenic regulatory protein myogenin. Myogenin is a muscle-specific transcription factor belonging to the helix-loop-helix (bHLH) class of proteins and is believed to play a crucial role in the differentiation of muscle. tissue in mammalian embryos. The role of myogenin in muscle cell differentiation will be tested directly by making use of a mouse model with a myogenin-null genotype. Using homologous recombination techniques, mice have recently been obtained that are heterozygous for the targeted mutation. The myogenin-null mice will be the starting point for a series of experiments to delineate myogenin function in vivo. The specific aims are: (l) To characterize the phenotypes associated with myogenin-null mice, by assessing the biochemical, histological and physiological state of muscle and associated tissues in embryos, fetuses, and adults; (2) To determine the role of myogenin in regulating the expression of muscle-specific genes, including other myogenic factors and genes encoding muscle-specific structural proteins and enzymes; (3) To determine if autoregulation is required for myogenin expression, making use of pre-existing transgenic mice harboring myogenin gene control regions fused to reporter genes; (4) To determine if mice that are doubly homozygous for null mutations at the myogenin locus and other myogenic factor loci exhibit stronger phenotypes than any of the single homozygous mutations; (5) To determine if other myogenic factors or modified forms of myogenin, expressed in the same temporal and spatial fashion, can rescue the myogenin-null phenotypes; and (6) To create myogenin-null fibroblast and myoblast lines to study the potential of these cells to differentiate muscle.

[unreadable] DESCRIPTION (provided by applicant): The objectives of this training program are to provide pre- and postdoctoral trainees with high quality training in fundamental aspects of cell growth, differentiation, and development, and to offer an interactive environment that extends beyond the individual laboratory experience. The program is designed to expose trainees to a wide range of research subjects and experimental systems that will be essential for their future career advancement. These include: embryo development, regulation of gene expression, cellular growth, differentiation and survival, chromatin structure and remodeling, and biophysical and structural analysis of macromolecules associated with cell regulation. Program faculty members are drawn from the Department of Biochemistry and Molecular Biology (seventeen) and the Department of Molecular Genetics (two) at The University of Texas M. D. Anderson Cancer Center. The Program benefits greatly from its location within the multi-institutional Texas Medical Center, a progressive and integrated graduate school, and a strong commitment from the M. D. Anderson leadership to educating future biomedical scientists. [unreadable] [unreadable] Predoctoral trainees begin the program in their second year and remain until the end of their fourth year. Students take formal coursework in biomedical ethics, experimental genetics, biochemistry and molecular biology, biomedical statistics, eukaryotic gene expression, developmental biology, and a series of seminar courses designed to develop skills in oral presentation. Postdoctoral trainees receive three year appointments and must document training in biomedical ethics and submit at least one postdoctoral fellowship application during their first year. Four pre- and one postdoctoral trainees participate in the program. A trainee orientation each fall, student rotation talks, Blaffer endowed lecture series, research and journal clubs, and an annual spring retreat are all designed to foster interactions and collaborations. A monthly trainee workshop focuses on the future of biomedical research. Trainees are selected from a large pool of well qualified applicants and many trainees advance to prestigious positions at major academic institutions following their training. [unreadable] [unreadable] [unreadable]

DESCRIPTION (Adapted from applicant's abstract): The proposal is concerned with understanding how retinal ganglion cells are specified in the vertebrate retina. Although the pattern of retinal development has been well described, very little is known about the molecular events that control the arrangement, commitment, and differentiation of retinal neurons. One approach to understanding retinal development is to define specific components of essential genetic regulatory events. This proposal focuses on three closely related POU-domain genes, brn-3a, brn-3b, and brn-3c, which have been implicated in cell fate specification during neuronal development. These genes are expressed in the murine retina specifically within subsets of ganglion cells and also in dorsal root and trigeminal ganglia, and in selected regions of the brain. Targeted disruptions have shown that brn-3b is required for the development of a large set of retinal ganglion cells whereas brn-3a and brn-3c are essential for the development of somatosensory and auditory neurons, respectively. Initial experiments have led to the hypothesis that Brn-3b is part of a decision-making process that acts to control whether or not a post-mitotic neuroblast adopts a ganglion cell fate. Three specific aims are proposed to determine more precisely the role that Brn-3b plays in retinal ganglion cell development and its relationship to Brn-3a and Brn-3c: (1) the fate of brn-3b-expressing cells during retinal development will be determined in wild type and brn-3b-deficient embryos using a brn-3b(-/-)lacZ gene inserted into brn-3b locus as a marker for brn-3b positive cells; (2) the functional equivalence of Brn-3a, Brn-3b and Brn-3c in retina will be tested by homologous recombination of brn-3a and brn-3c sequences into brn-3b locus; and (3) the ability of Brn-3b to convert uncommitted retinal neuroblasts to a ganglion cell fate will be assessed by ectopic retroviral expression of brn-3b in the developing retinas of chick embryos.

DESCRIPTION (Applicant's abstract): The objective of this proposal is to use microarray technology to identify mouse genes whose products are involved in the differentiation, maintenance, and survival of retinal ganglion cells. Retinal ganglion cells are essential for normal vision and their loss contributes to major eye diseases in humans. For example, elevated intraocular pressure within the anterior chamber of the eye can trigger enhanced apoptosis in ganglion cells, which in turn leads to glaucoma. Despite their importance, however, the knowledge base of genes associated with retinal ganglion cell formation and survival is rudimentary. Using an arrayed embryonic retinal cDNA library that has recently been constructed, this application proposes to profile gene expression patterns in genetically altered retinas lacking the key transcription factors Brn-3b and Math5. Gene targeting has shown that loss of either Brn-3b or Math5 leads to major defects in retinal ganglion cell formation. The long-term goal is to generate comprehensive gene expression profiles for newly forming retinal ganglion cells and to determine the genetic regulatory mechanisms that lead to retinal ganglion cell differentiation. The specific aims are to: (1) Establish a comprehensive database of expressed retinal sequences from E14.5 mouse retina; (2) Prepare gene chips with known retinal sequences for use in profiling patterns of gene expression in the developing retina; (3) Generate gene expression profiles by comparing genetically altered mouse lines, and different stages of retinal development; and (4) Develop strategies to determine the genetic regulatory networks that lead to the differentiation of retinal ganglion cells.

DESCRIPTION (provided by applicant): The long-term objective of this application is to gain insights into the molecular events that lead to the differentiation of mammalian retinal ganglion cells. Retinal ganglion cells are essential for normal vision and their loss contributes to major eye diseases in humans. For example, elevated intraocular pressure within the eye can trigger enhanced apoptosis in ganglion cells, which in turn leads to glaucoma. Despite their importance, however, the knowledge base of genes associated with retinal ganglion cell formation and survival is rudimentary. In this application, experiments are proposed that focus on Brn-3b, a POU-domain transcription factor, in retinal ganglion cells. In the mouse, the brn-3b gene is among the first genes activated in postmitotic progenitor cells as ganglion cell differentiation begins. In spite of this early activation, brn-3b is not required for the initial specification of ganglion cells, but it is essential for their normal differentiation and survival. Mice with targeted deletions in brn-3b have defective retinas with a loss of most ganglion cells. Thus, brn-3b is a critical gene that marks the commitment to a ganglion cell fate and is essential for the survival of retinal ganglion cells. The first two aims of this application use the brn-3b locus to probe early events of retinal ganglion cell formation. The final two aims concern the transcriptional properties of Brn-3b. The Specific Aims will: (1) Test the hypothesis that as retinal ganglion cells form, they inhibit their further production. The proposed experiments will specifically ablate retinal ganglion cells using genetically targeted diphtheria toxin; (2) Identify the cis-regulatory elements within the brn-3b transcriptional control region that activate brn-3b in postmitotic ganglion cell progenitors. The cis-regulatory elements within the brn-3b transcriptional control region will be identified using BAC transgenic analysis; (3) Investigate the functional specificity of Brn-3b in retinal ganglion cell differentiation. The experiments will employ HSV-mediated gene transfer into cultured retinal explants; (4) Determine whether brn-3b can function in committing progenitor cells to a retinal ganglion cell fate. Math5 is a proneural bHLH gene required for retinal ganglion cell formation. Brn-3b will be misexpressed at the math5 locus to determine whether brn-3b is sufficient to promote ganglion cell differentiation in the presence and absence of math5.

[unreadable] DESCRIPTION (provided by applicant): The long-term objective of this research is to elucidate the gene regulatory network that controls the formation of retinal ganglion cells (RGCs). Although many of the critical regulatory events that direct a retinal progenitor cell (RPC) towards a particular cell fate have been identified, RPCs still remain "black boxes" whose intrinsic properties are only vaguely defined. The proposed experiments focus on the mechanisms by which RPCs commit to an RGC fate. Previously, two key transcription factors were shown to be critical for RGC development. The proneural bHLH factor Math5 is essential for RPC competence to become an RGC, while the POU domain factor POU4f2 (Brn3b) is genetically downstream from Math5 and is required for competent, specified RPCs to differentiate into RGCs. Gene expression profiles of math5-null and pou4f2-null retinas obtained using microarrays generated from embryonic retinal cDNAs revealed numerous extrinsic and intrinsic regulatory factors that depend on Math5.or POU4f2 for their expression. The profiling analysis led to the construction of a gene regulatory network consisting of several hierarchial layers. Genetic ablation of RGCs during embryogenesis demonstrated that they are not required for the differentiation of other retinal cell types but are necessary for regulating the number of overlying RPCs by secreting growth factors such as Sonic hedgehog. The overriding hypothesis is that RPCs are a heterogeneous population of cells whose properties are defined largely by combinations of bHLH, homeobox, and other transcription factors. The specific aims are to: (1) use genetic ablation of RGCs to identify differences in gene expression in math5-null, pou4f2-null, and RGC-ablated retinas and to create an adult mouse model for RGC loss and optic nerve degeneration; (2) manipulate the properties of math5-expressing progenitor cells by replacing Math5 with other transcriptional regulators to determine the extent to which RPCs can adopt new fates; (3) elaborate the model for the RGC gene regulatory network by identifying cis-regulatory elements and transcription factors associated with RGC-specific gene expression; and (4) determine whether the ectopic expression of sonic hedgehog in RPCs restores normal numbers of RPCs in RGC-ablated retinas. The proposed experiments will lead to a better understanding of RPC behavior, which will eventually provide the means to manipulate RPCs for the purpose of retinal regeneration. [unreadable] [unreadable] [unreadable]

Genetically engineered mice have become the "gold standard" for animal models in cancer. They are widely used to mimic human cancers with specific point mutations in tumor suppressor genes, as well as tissuespecific expression of mutations in somatic tissues. By combining several mutations in one animal, it is now possible to phenocopy human cancers that were difficult to reproduce previously in animal models. The Genetically Engineered Mouse Facility (GEMF) provides valuable resources to Cancer Center members. It generates transgenic and gene-targeted mice, performs embryo rederivations to generate pathogen-free mice, and provides cryopreservation services to archive animal models. The Mouse Resource Facility (MRF), as part of the GEMF, provides vectors for construct development, northern blots of adult and embryonic tissues, genomic DNA and cDNA libraries, and BAG filter sets. The MRF also has a small colony of Cre and lacZ transgenic mice essential for generating conditional deletions in mice. The GEMF thus provides unique opportunities to faculty to develop sophisticated animal models to study a variety of cancer problems. The GEMF has been extremely successful in generating and maintaining mouse models. In the last five years, the GEMF has generated animal models for more than 300 projects for MDACC investigators and has cryopreserved more than 120 mouse lines. A >1000% increase has been achieved in utilization of mouse resources through the MRF. The lead facility coordinator has developed new, more efficient methods of generating embryos for use by the facility. She is responsible for training faculty and their staff, and provides genetic and technical expertise to many investigators in many different programs. In addition to the facility coordinator, the GEMF is staffed by 7 highly-trained technicians. In the past five years, the GEMF has served 96 different users who represent 19 of the 20 sponsored programs;97% of the investigators served are MDACC faculty. To better serve the needs of our investigators and add needed capacity, a small satellite facility was added recently to the South Campus. This expansion will enable the GEMF to develop more models and offer additional services. The GEMF is currently funded from multiple sources, including 53% provided by the CCSG and 45% recovered through user fees, with the remainder provided by MDACC.

DESCRIPTION (provided by applicant): Chronic hepatitis B virus (HBV) infection remains a major problem, with 400 million chronic carriers in the world for whom, to date, there is no reliable treatment. The HBV X gene is a major factor in hepato-carcinogenesis and is essential for viral replication and infection in vivo. The X gene product deregulates various cellular functions, including the Ca2+dependent signal transduction pathways, transcription and apoptosis pathways, and eventually leads to transformation of liver cells. However, detection of X gene expression is technically difficult and thus the regulatory mechanisms of X gene transcription have been unclear. The goal of this project is to identify and characterize the transcription factors important for X gene transcription. Recent investigations revealed the importance of general transcription factor (GTF) diversity and core promoter-enhancer specificity in transcriptional regulation. Therefore, features of the X gene core promoter that may explain the unique pattern of the X gene transcription will be particularly studied. X mRNA transcription starts at multiple sites, and the promoter region contains no commonly used core promoter elements such as the TATA box. The X gene promoter may use previously unknown core promoter elements, core promoter-binding factors, and a nontraditional set of GTFs. The X core promoters may preferentially interact with particular classes of transcriptional regulators and coregulators to establish unique expression patterns. In this work, the core promoter elements will be defined by extensive mutagenesis and their binding proteins will be purified by using DNA-protein interaction (Specific Aim 1). The GTFs required for X gene transcription will be determined by immunodepletion and in vitro reconstituted transcription (Specific Aim 2). Regulation of the X gene promoter will be investigated by transient transfection, in vitro transcription, and various biochemical interaction assays (Specific Aim 3). By thoroughly characterizing the transcription factors involved in X gene transcription, this research will serve as a foundation for identifying potential targets for therapeutics and strategies to control HBV gene expression. It will also contribute to the general understanding of transcription from TATA-less promoters, which covers about two thirds of protein-coding genes in humans. Combined with the functional studies of the X protein, our research will improve understanding of the role that the X gene plays in hepatocarcinogenesis.

DESCRIPTION (provided by applicant): The overall objective of this revised R21 application is to determine the potential of mouse retinal progenitor cells (RPCs) to differentiate into retinal ganglion cells (RGCs) outside of their normal embryonic environment. The potential of embryo- or embryonic stem cell-derived RPCs to differentiate into functional RGCs in adult retinas will be examined in vivo by transplanting them into adult host retinas in which RGCs have been genetically ablated. A novel genetic mouse model has been developed in which some or all RGCs can be ablated at any time during adult life with the consequent degeneration of the optic nerve. Retinas from these mice provide a unique microenvironment for cell transplantation approaches to restore RGCs and regenerate the optic nerve or prevent its further degeneration. The experimental plan depends on understanding the key regulatory events that control the specification and differentiation of RGCs during retinogenesis. The proneural basic helix-loop-helix factor Math5 occupies a central node in the gene regulatory network that controls RGC development because it is responsible for endowing RPCs with the competence to acquire a RGC fate. The hypothesis to be tested is that Math5-expressing RPCs are a distinct, isolatable RPC subpopulation and that Math5-expressing RPCs will retain their developmental potential even when placed into the microenvironment of the adult retina. To investigate the interactions between Math5-expressing RPCs and the adult retinal microenvironment, the potential of purified Math5-expressing RPCs to differentiate into functional RGCs after transplantation into RGC- ablated adult retinas will be investigated. Determining the developmental potential of embryonic RPCs to adapt to the microenvironment of the adult retina will contribute towards an understanding of retina development as well as establishing more robust methods to prevent, repair and regenerate damaged retinas. Genetic ablation of RGCs in adult mice provides a new model for retinal regeneration. Knowledge of the mechanisms that allow RPCs to differentiate into RGCs in their normal environment provides the underpinning for generating large numbers of functional RGC progenitors from embryonic retinas or pluripotent embryonic stem cells. PUBLIC HEALTH RELEVANCE: This project will reveal new insights into the properties of the embryonic retinal progenitor cells that give rise to retinal ganglion cells in the adult neural retina. Experiments are designed to determine the potential of retinal progenitor cells to adapt to the microenvironment of the adult retina rather than their normal embryonic microenvironment. The project will contribute to an understanding of retinal development as well as establishing new approaches to regenerate damaged retinas and prevent or repair optic nerve degeneration in retinal pathologies such as optic neuritis, ischemic optic neuropathy, and glaucoma.

Project Summary Our long-term objective in the proposed experiments is to obtain new knowledge on the basic mechanisms that control retinal development and to apply this knowledge to develop novel ways to treat retinal degenerative diseases. Our strategy is to use genetically engineered mouse models that we have already created or that we will create. Although an impressive amount of information has accumulated on the mechanisms that control retinal development, large gaps still remain. In particular, the mechanisms that control a progenitor cell's decision whether to proliferate or differentiate are only vaguely understood. A better understanding is of great importance for finding new ways to repair damaged retinas. Retinal ganglion cells (RGCs) are the first cell type to differentiate from retinal progenitor cells (RPCs) during development and are the retinal neurons that connect to the brain. We focus on the regulatory events that cause RPCs to commit to a RGC fate. In related experiments, embryonic RPCs will be used to repopulate RGCs in adult retinas that have been depleted of their endogenous RGCs. Our underlying hypothesis is that for RPCs to differentiate into RGCs, Atoh7 must integrate with other regulatory factors to achieve a balance between proliferation and differentiation. To address the hypothesis, we proposed three specific aims. The first aim will determine whether Atoh7 is sufficient to convert non-RGCs to a RGC fate. Preliminary work indicates that replacing Neurod1 with Atoh7 leads to ectopic RGC gene expression in the inner nuclear layer. We will determine whether Atoh7 can drive RGC differentiation in non-RGC neurons in developing and adult retinas. The second aim will determine whether Atoh7 regulates Notch signaling to control the balance between RPC proliferation and RGC commitment. In preliminary experiments, we found that Atoh7 binds to E-box elements upstream of Notch1 and that Atoh7 negatively regulates Notch1 expression. We will identify the time at which Atoh7 appears relative to Notch signaling. We will determine whether Atoh7 and Notch1 participate in a negative feedback loop and whether RPC proliferation is perturbed when the Atoh7 binding sites on Notch1 are deleted. In the third aim, we will optimize our experiments on repopulating RGC-depleted retinas by transplanting Atoh7-expressing RPCs into the retinas of RGC-depleted mice along with neuroprotective factors. We will also determine whether Atoh7-expressing RPCs can regenerate optic nerves in optic nerve crush and other mouse models. Our knowledge of the factors controlling retinal development allows us to apply developmental concepts to adult retinas. We have developed realistic genetic models for human optic nerve degeneration that will have ultimate use in stem cell replacement therapy to repair damaged optic nerves.