Sally Camper, Ph. D. - US grants

There are two mouse dwarfing mutations that provide good models for studying the basis for induction of tissue specific transcription during embryogenesis. Both mutants lack the three pituitary cell types which produce prolactin, growth hormone, and thyroid stimulating hormone (TSH). The similarity of the mutant phenotypes suggests that the three missing cell types may derive from a common precursor cell and that at least two genes are involved in this differentiation pathway. Classical genetic and molecular biology will clarify how the affected genes normally interact and whether they have overlapping functions. Specifically, double heterozygotes and double mutants will be produced and the phenotypes characterized in terms of neurological, development, immune dysfunction and ablation of additional pituitary cell types. An intersubspecific backcross will be used to screen candidate genes for linkage to the Ames dwarf mutation (df) on chromosome 11 and for fine mapping of the df gene. Closely linked molecular probes will be used to generate a long range map of the df region using pulsed field gel electrophoresis. The df mutant was generated by X-irradiation, therefore the long range map may reveal evidence for a deletion or rearrangement, which would facilitate locating and subsequent cloning of the df gene. Linked molecular probes will be used to screen a yeast artificial chromosome (YAC) library of the mouse. Pituitary cDNA and cross species homology will be used to identify the df gene within the YACs. Identification of the mutant gene by reverse genetics will clarify the basis for the failure of progenitor cells to differentiate into hormone producing cells and expand our understanding of the basic cellular commitment process. %%% The anterior pituitary is composed of a number of cell types, each specialized in the synthesis and secretion of different hormones. This study will determine if the products of genes essential for the development of the specific cell types act independently in each cell or whether their expression affects the development of neighbor cells.

DESCRIPTION (provided by applicant): Our overarching goal is to identify the genes that cause pituitary insufficiency (hypopituitarism) in humans and mice, and to understand their mechanism of action. Hypopituitarism affects 1/4000 children, causing short stature and risk of death. The rationale for this goal is that a molecular understanding of this common birth defect will yield 1) fundamental information about organogenesis, 2) diagnoses with value for predicting risk and monitoring progression, and 3) provide insight about therapeutics that could aid children with congenital problems and adults with acquired pituitary dysfunction. Mutations in ten genes cause hypopituitarism and growth insufficiency, yet approximately half the patients have no molecular diagnosis. Mutations in the pituitary specific transcription factor PROP1 are the most common known cause of hypopituitarism in humans. We hypothesize that understanding the mechanism of action of Prop1 will uncover genes that explain cases of hypopituitarism of unknown etiology and provide insight in the regulation of pituitary progenitors that initially establish the organ and replenish cells in adults. Prop1 deficiency causes pituitary hypoplasia and lack three cell types, including those that produce growth hormone. Gain of function alleles cause transient hypogonadism, delayed puberty, and increased risk of pituitary adenomas, the most common type of intracranial tumor in humans. Genes encoding HESX1 and POU1F1 are the only two known, direct, targets of PROP1, and mutations in these genes also cause hypopituitarism. We established a catalog of the developing pituitary transcriptome and carried out differential expression profiling of PROP1 and POU1F1 mutant pituitaries. We identified a collection of genes whose expression is altered specifically in Prop1 mutants including Otx2, a transcription factor that affects eye and pituitary gland development. The effects of PROP1 on Hesx1, Pou1f1 and Otx2 expression do not completely explain the Prop1 mutant phenotype. In particular, it is not clear how Prop1 regulates the proliferation vs. differentiation of progenitor cells. During the next grant cycle we propose to define the mechanism of PROP1 action and test the following hypotheses: 1) Prop1 is necessary for generation of precursor cells that contribute to multiple cell lineages during embryogenesis and for replenishment of cells during adult life by affecting Notch signaling and expression of the critical cell cycle regulator, cyclin E. 2) Prop1 repression of Otx2 is necessary for regulating growth of the pituitary primordium, and Otx2 stimulates hypothalamic production of BMP and FGF signaling. 3) Additional Prop1 target genes regulate changes in cell adhesion and migration that are akin to epithelial to mesenchymal transition, a process involved in normal organ development and in tumorigenesis. 4) Exome sequencing of DNA from patients with unexplained cases of hypopituitarism will identify variants of functional significance. Addressing each of these hypotheses will lead to better molecular diagnoses and provide fundamental information on pituitary precursor cell generation and proliferation. !

The University of Michigan Multipurpose Arthritis Center Transgenic Animal Model Core provides genetically engineered mice to UM-UM-MAC investigators. Both conventional transgenic mice and mice with mutations induced by gene targeting in embryonic stem (ES) cells are produced for investigators. The Core guarantees that at least three transgenic mice will be produced for each transgene construct submitted. Core personnel provide quality tested reagents and work extensively with investigators to ensure the success for gene targeting experiments. The Core has an excellent track record. In the first four years of the preceding grant period, 687 transgenic mice were produced from 72 DNA constructs from 72 DNA constructs for 10 UM-MAC investigators. Mice with induced mutations in 12 genes were produced for 6 investigators. It is significant to note that 12 of the 13 gene targeted mice generated at the University of Michigan have been produced in the laboratories of UM-MAC investigators. Research of these animals by UM-MAC investigators has resulted in numerous publications in the fields of arthritis, musculoskeletal disease, inflammation, and gene regulation. We project that in the four years covered by the current proposal UM-MAC investigators will generate at least 80 requests for transgenic mice and 48 requests for production of germline ES cell-mouse chimeras for the generation of mouse strains with novel mutations. Dr. Sally Camper has used transgenic mice in her research since 1986 and is widely published in this field. Dr. Linda Samuelson established the technology of gene targeting in ES cells at the University of Michigan in 1990. Drs. Camper and Samuelson will work with the transgenic Core Steering Committee (composed of experienced users and a member of the UM- MAC Executive Committee) to monitor the Transgenic Core. Funding will be used to ensure that highly skilled personnel are available to meet the needs of UM-MAC investigators. Investigators will receive a 34% discount on orders for transgenic mice by pro-nuclear injection and a 34% discount on the production of gene targeted mice by blastocyst injection.

[unreadable] DESCRIPTION (provided by applicant): The pituitary gland contains five different cell types that are specialized in hormone production. Understanding the mechanism of cell specification is important because many body functions depend on it. Genetically engineered mice have proven the roles of several transcription factors and signaling molecules, and the correspondence with human pituitary disease is outstanding. We proved that the homeodomain transcription factor PITX2 has a dosage dependent role in development of the pituitary primordium and in activation of lineage specific transcription factor genes. In humans, PITX2 mutations are a cause of Rieger syndrome and isolated growth hormone deficiency. We propose to test the role of PITX2 in maintenance of differentiated functions of specialized pituitary cells by cell specific deletion in mice. The role of GATA2, a downstream target of PITX2, will be tested using a conditional null allele of Gata2. FOXL2 is a forkhead transcription factor that is one of the earliest markers of differentiated cells in the developing pituitary gland, and it activates gonadotropin releasing hormone receptor transcription. Humans haploinsufficient for FOXL2 have eye defects and premature ovarian failure. We propose that FoxI2 has roles in regulating the growth of committed anterior pituitary cells during development, in the function of mature gonadotropes, and in susceptibility to pituitary tumors. We will explore these ideas by characterizing Foxl2 expression, placing it in the genetic hierarchy of known transcription factors, and analyzing the consequences of an inducible loss of function allele in mice. Our understanding of pituitary cell specification would be advanced if we had markers to identify specialized cells prior to terminal differentiation and activation of hormone gene transcription. To generate such markers we propose to compare the transcriptomes of pituitary cell types using transgenic technology to mark cells for purification and gene array analysis. Transcripts unique to each differentiated cell type will be identified by bioinformatics and verified experimentally. During the proposed grant cycle we will have defined the roles of three pituitary transcription factors using well-established methods and initiated a new approach to studying cell specification. We expect that these studies will provide valuable insight for understanding the etiology of human pituitary hormone deficiency diseases and characterization of pituitary adenomas. [unreadable] [unreadable]

The Transgenic Animal Model Core produces transgenic mice and gene targeted ("knockout") mice from embryonic stem (ES) cells carrying targeted genetic alterations. Transgenic mice are used in many ways, including the study of tissue specific gene expression, production of animal models of human disease, and testing efficacy of gene constructs for gene therapy. ES cell technology is used to establish animal models of recessive genetic disease and to characterize functional effects of gene loss and subtle changes in genes. ES cell technology is used to establish animal models of recessive genetic disease and to characterize functional effects of gene loss and subtle changes in genes. Other services provided include mouse embryo cryopreservation and recovery, derivation of pathogen free mice, and consultation. The Core maintains specialized reagents for transgenic and gene targeting research. The Core's specialized equipment is used to support the research efforts of multiple investigators who otherwise lack access to such equipment. The availability of a transgenic Core on site obviates the need for individual researchers to purchase costly equipment and invest personnel time in lengthy training in micromanipulation techniques, which would be technically and financially impossible for most investigators. Consultation on all aspects of transgenic and ES cell research is provided, from the design of DNA constructs to mouse husbandry. Production of Transgenic Mice: We deliver an average of ten transgenic founder mice and guarantee that at least three founders will be produced for each DNA construct submitted to the Core. Production of Gene Targeted ES Cells: The Core will electroporate totipotent ES cells with tar4getinbg vectors, select ES cell clones, and provide Center Members with DNA from the clones for genetic screening. Production of ES Cell-Mouse Chimeras: ES cell clones carrying the desired genetic mutation will be microinjected into mouse blastocysts which are surgically transferred to foster mothers to produce chimeras. Transgenic Technology Training: Researchers can also elect to be trained to 1) produce transgenic mice by pronuclear microinjection, 2) manipulate ES cells and produce gene targeted ES cell clones, and 3) produce ES cell-mouse chimeras by blastocyst injection. Commensurate with Center Member usage in the last four years, we project annual demand for 20 orders for transgenic mice, 6 orders for production of gene targeted ES cells, and 20 orders for ES cell-mouse chimeras. Center members will receive a 35% discount on the recharge rate for these services. Forty percent of all work done in the last four years by the Transgenic Core was devoted to Center members.

The Department of Human Genetics at the University of Michigan has historically been strong in the development and characterization of animal models of human genetic disease. Over the past ten years the number of researchers using mice in the department and the entire research community has increased dramatically. New technologies have radically changed the pace with which classic mouse mutants can be identified and revolutionized the use of the mouse for functional genomics. The possibilities for producing "designer mice" using transgenic and embryonic stem cells technologies are limited only by the imagination of the investigator. To meet the growing need for mouse facilities we have recently completed a renovation that modernizes and more than doubles our Human Genetics mouse housing space. To maintain high quality care and promote biomedical research we propose two improvements in outfitting our newly renovated mouse animal resource. 1. Upgrade from a conventional mouse colony to specific pathogen free facilities by purchasing laminar flow hoods for cage changing. 2. Reduce the expense of animal research by implementing automation where feasible. For example, the most expensive aspect of maintaining mice is the labor costs for cage changing. This can be reduced with automated bedding dispensers and by installing units that provide automatic watering and air filtration, reducing the frequency with which cages must be changed. These improvements will benefit animal projects totaling over $3.3 million annual direct costs (including over $2.9 million in support from NIH) and will add to the substantial improvements the University already has made to renovate the Human Genetics animal space, which cost approximately $1 million.

This Core provides a complete set of services to the University of Michigan Nathan Shock Center (UM-NSC) scientists. These services include the production of transgenic mice, transgenic rats, and gene targeted ("knockout") mice from embryonic stem (ES) cells. Transgenic mice are used in many ways, including the tissue specific gene overexpression, production of animal models of human disease, and correction of disease phenotypes. ES cell technology is used to establish animal models of recessive genetic disease and to characterize functional effects of gene loss and subtle changes in genes. Other available services are rederivation of pathogen free mice and rats, and mouse embryo cryopreservation and recovery. The Core maintains specialized reagents for transgenic and gene targeting research. The availability of this Core obviates the need for Center members to invest in expensive equipment and specialized training in transgenic technology. Consultation on all aspects of transgenic and gene targeting research is provided, from the design of DNA constructs to gene expression analysis. Guaranteed Production of Transgenic Mice and Rats: We deliver an average of nine transgenic founder mice and nine founder rats per transgenic order. We guarantee that at least three founders will be produced for each DNA construct submitted to the Core. Production of Gene Targeted ES Cells: The Core will electroporate pluripotent mouse ES cells with gene targeting vectors, pick 480 ES cell clones, and provide DNA from the clones for genetic screening. The Core will expand gene targeted ES cell clones and prepare them for the blastocyst microinjection. Blastocyst Microinjection: The Core guarantees that at least 50 blastocysts will be microinjected to produce ES-cell mouse chimeras. Investigators will breed the chimeras for germline transmission. Commensurate with Center Member usage in the last five years, we project annual demand of 20 orders for transgenic mouse production, one order for transgenic rats, 6 orders for production of gene targeted ES cells, and 15 orders for blastocyst microinjections to make chimeras. In exchange for Center support, UM-NSC scientists will receive a 40% discount on the recharge rate for these services.

neoplasm /cancer

Abstract Hereditary inner ear disease is prevalent and has significant implications for quality of life. There is currently no available clinical cure for hereditary inner ear disease. The mouse serves as an ideal mammalian model for understanding genetic inner ear disease and for developing therapeutic measures. Mouse models have facilitated the discovery of genes that underlie hereditary disease in humans, have made it possible to study the role of these genes in inner ear development and function, and hold great promise as models for developing treatments for hereditary inner ear disease. This grant application builds on our discovery that mutations in the unconventional myosin gene, Myo15, are responsible for profound congenital deafness and vestibular dysfunction in two spontaneous mouse mutants: shaker 2 and shaker 2J, and in humans with DFNB3. We used these mouse models to demonstrate the long-term structural and functional phenotypic correction of deafness with a transgene expressing Myo15. We characterized the development of pathology in Myo15, Myo6, Myo7a, pirouette, and whirlin deficient mutants, double heterozygotes and double mutants. Although there is no enhanced risk of age related hearing loss in double heterozygotes, these studies revealed unique functions of each myosin gene, and suggested the possibility that MYO15 has other functions besides transportation of whirlin to the stereocilia tips. We established adenoviral vectors for gene therapy and a database of genes exhibiting differential expression in the cochlea between weaning and adulthood in normal and Myo15 mutant mice. These studies laid a sound foundation for the goals of this grant. There are multiple isoforms of MYO15 that are generated by alternative splicing, including the presence or absence of a large proline-rich region N-terminal to the motor domain of MYO15. We hypothesize that this proline-rich region is important for protein-protein interactions necessary for hearing. We have generated a mouse model that recapitulates a human mutation in the proline-rich domain using knock-in technology. These mutants have profound congenital deafness, hair bundle pathology that is distinct from shaker 2 and shaker 2J mice, and apparently normal vestibular function. We propose a structure-function analysis that will reveal the importance of MYO15 isoforms in the development and function of the cochlea using mutant alleles, cell culture and cochlear explant assays. We will conduct a classical genetic analysis to evaluate interactions between mutant alleles and identify interacting proteins. Our investigative team has a track record for accomplishments resulting from cross-disciplinary collaboration, bringing together experts in otolaryngology, microscopy, physiology, and developmental genetics. This team will enable us to exploit the animal models fully to understand the mechanisms of inner ear disease and has the potential to identify novel genes essential for normal hearing.

DESCRIPTION (provided by applicant): Our overarching goal is to identify the genes that cause pituitary insufficiency (hypopituitarism) in humans and mice, and to understand their mechanism of action. Hypopituitarism affects 1/4000 children, causing short stature and risk of death. The rationale for this goal is that a molecular understanding of this common birth defect will yield 1) fundamental information about organogenesis, 2) diagnoses with value for predicting risk and monitoring progression, and 3) provide insight about therapeutics that could aid children with congenital problems and adults with acquired pituitary dysfunction. Mutations in ten genes cause hypopituitarism and growth insufficiency, yet approximately half the patients have no molecular diagnosis. Mutations in the pituitary specific transcription factor PROP1 are the most common known cause of hypopituitarism in humans. We hypothesize that understanding the mechanism of action of Prop1 will uncover genes that explain cases of hypopituitarism of unknown etiology and provide insight in the regulation of pituitary progenitors that initially establish the organ and replenish cells in adults. Prop1 deficiency causes pituitary hypoplasia and lack three cell types, including those that produce growth hormone. Gain of function alleles cause transient hypogonadism, delayed puberty, and increased risk of pituitary adenomas, the most common type of intracranial tumor in humans. Genes encoding HESX1 and POU1F1 are the only two known, direct, targets of PROP1, and mutations in these genes also cause hypopituitarism. We established a catalog of the developing pituitary transcriptome and carried out differential expression profiling of PROP1 and POU1F1 mutant pituitaries. We identified a collection of genes whose expression is altered specifically in Prop1 mutants including Otx2, a transcription factor that affects eye and pituitary gland development. The effects of PROP1 on Hesx1, Pou1f1 and Otx2 expression do not completely explain the Prop1 mutant phenotype. In particular, it is not clear how Prop1 regulates the proliferation vs. differentiation of progenitor cells. During the next grant cycle we propose to define the mechanism of PROP1 action and test the following hypotheses: 1) Prop1 is necessary for generation of precursor cells that contribute to multiple cell lineages during embryogenesis and for replenishment of cells during adult life by affecting Notch signaling and expression of the critical cell cycle regulator, cyclin E. 2) Prop1 repression of Otx2 is necessary for regulating growth of the pituitary primordium, and Otx2 stimulates hypothalamic production of BMP and FGF signaling. 3) Additional Prop1 target genes regulate changes in cell adhesion and migration that are akin to epithelial to mesenchymal transition, a process involved in normal organ development and in tumorigenesis. 4) Exome sequencing of DNA from patients with unexplained cases of hypopituitarism will identify variants of functional significance. Addressing each of these hypotheses will lead to better molecular diagnoses and provide fundamental information on pituitary precursor cell generation and proliferation.

Abstract The forebrain, midbrain, hindbrain, five facial prominences, and pituitary gland develop between wk 4-7 of gestation in humans. Genetic defects that disrupt these processes cause a spectrum of developmental disorders with life-long consequences that range in severity from holoprosencephaly (HPE) to septo-optic dysplasia (SOD) to pituitary hormone deficiency (congenital hypopituitarism, CH). HPE patients have variable defects in forebrain, eyes, and pituitary, and severe cases are embryonic lethal. The triad of features diagnostic of SOD include optic nerve hypoplasia, midline brain abnormalities, and CH. Patients diagnosed with CH, but not HPE or SOD, sometimes have features associated with those disorders, including vision, hearing, and/or brain anomalies. The genetic causes of these disorders are highly heterogeneous and overlapping. Prominent amongst the genetic causes are several genes that affect sonic hedgehog (SHH) signaling, including CDON, GLI2, GLI3, HHIP, SHH, SIX3, and TGIF1. We screened a cohort of ~ 200 unrelated probands with CH and various associated features and identified rare, likely pathogenic variants and variants of uncertain significance (VUS) in the transcription factors GLI2 and SIX3. We confirmed pathogenicity of several GLI2 and SIX3 variants using a SHH signaling sensor cell line assay and transient transfection assay, respectively. VUS are a major impediment to delivering on the promise of genetic testing for molecular diagnosis, and it is daunting for individual laboratories to establish the variety of functional testing assays necessary for genetically heterogenous disorders. We propose to create a catalog of the functional effects of all possible variants in GLI2 and SIX3 using multiplexed assays of variant effects (MAVEs). This approach scales up the assays we have already developed for testing one variant at a time so that thousands of variants can be tested simultaneously, yielding quantitative functional information that assigns variants as gain of This high throughput system addresses the problem of variant interpretation by providing comparable information about the phenotypic consequences of single nucleotide variants, which will improve the translation of genetic information into diagnosis. MAVEs have been applied to understand the function of diverse genes and types of pathogenicity, from splicing to amino acid substitution and from signaling pathways to transcription factors. function, tolerated, or loss of function. Completing these aims will further our knowledge of GLI2 and SIX3 structure and function in disease and set the stage for using MAVEs to generate catalogs of functional annotation for other genes in the SHH pathway that cause craniofacial defects.