Kathleen Millen - US grants

DESCRIPTION (provided by applicant): We are studying dorsal CNS pattern formation in the mouse, as a paradigm for human congenital brain malformations based on the hypothesis that similar patterning defects underlie mouse and human malformations. Pattern formation is the term used to describe the emergence of spatial biological organization during development. Malformations of the dorsal midline of the human CNS are poorly understood congenital defects that include some forms of holoprosencephaly and megalencephaly. Both of these are primarily malformations of the dorsal cortex. An example of a dorsal midline malformation of the cerebellum is Dandy-Walker Malformation. The roof plate is a specialized dorsal midline structure in the embryonic CNS. It is a crucial regulator of dorsal patterning information in the developing spinal cord, directing the specification and differentiation of dorsal sensory interneurons via secreted molecules. We hypothesize that the roof plate performs a similar function in more anterior levels in the brain. Specifically, we hypothesize that the Lim-homeodomain encoding genes, Lmx 1a and Lmx 1b are required for normal roof plate development in the anterior CNS and that loss of these genes leads to loss of roof plate function and subsequent abnormal specification and differentiation of adjacent neurons in the developing cerebellum and cortex. We have previously demonstrated that the spontaneous neurological mouse mutant, dreher, harbors mutations in the Lmx 1a gene and that Lmx 1a is required for roof plate development in the mouse CNS. In the dreher spinal cord, no roof plate is generated. Consequently, the specification, patterning and differentiation of adjacent dorsal sensory interneurons are abnormal in the dreher spinal cord. At anterior levels of the developing CNS, a residual roof plate is still present in dreher mice, suggesting that roof plate in the brain has a different mechanism of genesis. This proposal makes use of gene targeting and transgenic technology in combination with extensive phenotypic analysis to examine the roles of Lmx 1a and the closely related gene, Lmx 1b, in roof plate formation and function adjacent to the developing cerebellum and cerebral cortex.

[unreadable] DESCRIPTION (provided by applicant): Dandy-Walker Malformation (DWM) is the most frequently occurring birth defect that causes developmental malformations of the cerebellum and hindbrain, leading to mental retardation or developmental delay. We have recently identified two members of the Zic transcription factor encoding gene family as strong DWM candidate genes. The overall goal of this proposal is to provide insight into the developmental roles of these genes in the pathogenesis of DWM. Our general strategy is to define the developmental roles of these DWM candidate genes in vertebrate cerebellar and hindbrain development. We will use vertebrate models, coupled with in vitro approaches, to study Zic gene function and redundancy. In preliminary experiments we have constructed targeted mouse mutants for these Zic genes, and demonstrated that mutant mice lacking either of the Zic genes have cerebellar malformation phenotypes. We have also demonstrated that compound mutant mice have more severe cerebellar malformation phenotypes than those observed in either single mutant alone. These data indicate that the two Zic genes have genetically redundant function and that gene dosage is critical for normal cerebellar development. In this proposal, we have outlined a series of experiments to assess the developmental basis of the cerebellar malformations in single and compound mutant mice, based on extensive morphological, in situ and immunohistochemical analysis of hindbrain development. We will use in vitro assays to assess and compare transcriptional function of the two Zic proteins. We will then extend these studies in vivo using both gain and loss of function approaches in the tractable zebrafish model system to investigate transcriptional function and to test whether these genes are functionally redundant. These experiments will also allow us to assess conservation of the roles of these genes across vertebrate hindbrain development. By taking a multi-pronged approach to the analysis of Zic1 and Zic4 gene function in CNS development, we will make more rapid and significant progress than by focusing on a single model system alone. Not only are these studies of importance to basic developmental biology, they also have direct relevance to understanding the pathogenesis of cerebellar malformation disorders in humans [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Developmental malformations of the cerebellum are common birth defects that often cause mental retardation or developmental delay, in addition to motor and visual handicaps. The most frequent cerebellar malformation is Dandy-Walker malformation (DWM), which is defined by hypoplasia of the cerebellar vermis, dilatation of the fourth ventricle, and often hydrocephalus. Although common, the specific causes of DWM remain undefined. While it is certain that environmental teratogens can cause DWM during embryogenesis, there is clear evidence that as yet undefined genetic factors are also responsible. The investigators have identified several DWM patients with chromosomal abnormalities. In particular, a subset of these patients has overlapping chromosomal deletions defining a small critical region that is hypothesized to identify a DWM locus. The experiments outlined in this application are focused on mutational and functional analysis of two excellent candidate genes, likely causative of DWM. To model DWM, the investigators will construct targeted mouse mutants for these two DWM candidate genes and assess the role of these genes during cerebellar development, based on the hypothesis that similar mechanisms underlie mouse and human CNS development. The identification of a DWM gene will represent a significant advance in the understanding of this common and often devastating brain malformation. It will also provide valuable diagnostic and prognostic information that can greatly influence treatment and genetic counseling. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Congenital caudal duplication, also known as caudal dipygus or parasitic posterior twinning, is a rare birth defect in humans and other vertebrates characterized by supernumerary lower limbs and pelvic structures. The phenotype is striking and presents a number of challenges to current models of vertebrate development. While there have been multiple case reports, there has never been a systematic analysis of the phenomenon, largely because all cases have been sporadic. To date, no heritable vertebrate model has been described for this extraordinary phenotype, and there is no understanding of its developmental basis. Recently a spontaneous mouse mutant arose in the investigator's breeding facility with this remarkable malformation. The trait is heritable as a single, autosomal dominant locus, with incomplete penetrance. There is no evidence that this represents an axial duplication, since the neural tube is not split. Rather, this mouse model appears to be a rare example of a contiguous mirror image segmental duplication of the sacral region, involving all three germ layers. Genetic and embryonic analyses are proposed to map the locus and define the disrupted developmental mechanisms underlying this phenotype, and are certain to provide new and exciting insights regarding vertebrate patterning. Further, an understanding of the nature of the genetic lesion will illuminate the mechanisms underlying variable expression and low penetrance that are characteristic of many birth defects in humans. [unreadable] [unreadable] [unreadable]

genetics

[unreadable] DESCRIPTION (provided by applicant): Congenital caudal duplication, also known as caudal dipygus or parasitic posterior twinning, is a rare birth defect in humans and other vertebrates characterized by supernumerary lower limbs and pelvic structures. The phenotype is striking and presents a number of challenges to current models of vertebrate development. While there have been multiple case reports, there has never been a systematic analysis of the phenomenon, largely because all cases have been sporadic. To date, no heritable vertebrate model has been described for this extraordinary phenotype, and there is no understanding of its developmental basis. Recently a spontaneous mouse mutant arose in the investigator's breeding facility with this remarkable malformation. The trait is heritable as a single, autosomal dominant locus, with incomplete penetrance. There is no evidence that this represents an axial duplication, since the neural tube is not split. Rather, this mouse model appears to be a rare example of a contiguous mirror image segmental duplication of the sacral region, involving all three germ layers. Genetic and embryonic analyses are proposed to map the locus and define the disrupted developmental mechanisms underlying this phenotype, and are certain to provide new and exciting insights regarding vertebrate patterning. Further, an understanding of the nature of the genetic lesion will illuminate the mechanisms underlying variable expression and low penetrance that are characteristic of many birth defects in humans. [unreadable] [unreadable] [unreadable]

: Developmental defects of the cerebellum in humans have received less attention than other brain malformations such as neural tube defects and cortical malformations. Yet, cerebellar malformations are common, affecting approximately 1/5000 births. Dandy-Walker Malformation (DWM) is the most frequent cerebellar malformation. Affected individuals often have motor deficits, mental retardation, autism and some have hydrocephalus. Although the specific causes of this clinically important birth defect remain largely undefined, there is evidence for considerable genetic heterogeneity and complex inheritance. Based on physical mapping of chromosomal abnormalities in patients, we have identified 2 loci harboring human DWM on chromosomes 3q24 and 6p25. This proposal describes a series of experiments aimed at understanding the developmental mechanisms leading to DWM pathology through the study of several mouse models. We have previously demonstrated that heterozygous co-deletion of the closely linked ZIC1/4 genes on chromosome 3q24 causes DWM. Aim 1 of this proposal describes a series of genetic experiments in Zic1/4 mutant mice to assess the developmental pathways regulated by these Zic genes. The experiments in Aim 2 are designed to define the basis of 6p25 DWM, through phenotypic characterization of both null and conditional mouse mutants of a candidate gene influencing both posterior fossa mesenchymal and cerebellar development. Since similar mechanisms underlie both mouse and human CNS development, analysis of these mice will to determine the underlying molecular and developmental causes of human DWM. This information is critical to the identification of additional human DWM loci.

The UCCRC Transgenic Mouse/Embryonic Stem Cell Facility provides comprehensive genetic engineering services to alter the genome of the laboratory mouse. The UCCRC has many investigators who use mouse models as their primary tool of analysis, as well as a multitude of additional investigators who require the occasional mouse model in their studies of the causes and treatment of cancer. The generation and maintenance of transgenic animals through the microinjection of singlecelled mouse embryos, and the generation of genetically modified mice through the use of ES cells require specialized technical personnel. Furthermore, such efforts necessitate the acquisition of an array of devoted equipment. Thus, the availability of a shared facility greatly reduces research costs for the individual investigators of the UCCRC. The existence of this facility also greatly increases the accessibility of genetic engineering technology to investigators with limited related experience. The UCCRC Transgenic Mouse/Embryonic Stem Cell Facility was established in 1991, and has been tremendously productive and successful; the range of services provided has expanded considerably since its founding. Nine services are provided by the Transgenic Mouse/Embryonic Stem Cell Facility: (1) transgenic mouse production from founder through F1 Stage; (2) ES cell technology mouse production; (3) ES cell gene targeting and culturing; (4) mouse embryonic feeder (MEF) cell production; (5) Timed pregnancies of various strains and lines of mice; (6) embryo rederivation; (7) ES cell line development; (8) various breeding services and GEM model line maintenance; and (9) DMA preparation from ES cell lines for the construction of a gene targeting construct. In addition to the technical services, the Facility also offers assistance with the design of studies that require mouse molecular genetics and an annual course in Mouse Handling and Breeding. This course covers information regarding analysis and handling of the mice once the Facility has generated them, as well as general breeding and handling procedures for mice. In providing these comprehensive services, the UCCRC Transgenic Mouse/Embryonic Stem Cell Facility has generated mouse models that have led to advances in our understanding of cancer, including cancers of the breast, skin, colon, brain and hematopoietic system.

The mouse Genetic services aspect of this Model Organisms Core is widely used core service by MRDDRC investigators and serves an essential service to the MRDD community. The MRDDRC will partner with the existing Biological Sciences Divisional Core to provide transgenic and targeted mutagenesis services to MRDDRC investigators. By partnering with the existing Divisional core, the MRDDRC grant strengthens the already successful core. MRDDRC investigators will gain a subsidy services through this core. MRDDRC funding of the divisional core also allows the addition of ES cell culturing services and gene targeting - services not currently available in the Divisional Mouse Core. Further, the MRDDRC investigators will have available the expertise of both MRDDRC co-directors - both accomplished mouse geneticists with extensive neurobiological expertise. The co-directors will provide project design consultation, vector design expertise and advice for analysis. The co-directors are familiar with the research programs of all MRDDRC users and can facilitate interactions between investigators for reagent sharing and collaborations for further analysis. The codirectors will also refer users to other MRDDRC cores for additional analysis of mouse models generated, including the new Mouse Phenotyping Subcore.

DESCRIPTION (provided by applicant): This grant proposes to significantly advance the current technology for generation of mouse models of human genetic disease. This addresses broad Challenge Area (15) Translational Science, and Specific Challenge Topic 15-OD(ORDR)-101: Pilot projects for prevention, early detection and treatment of rare diseases. Although recent advances in human genetic technologies have revolutionized our ability to identify disease candidate genes, we currently lack sufficient high-throughput technology to economically and efficiently assess the relevance of candidate genes in in vivo follow-up studies in mice. We propose a new strategy to rapidly and inexpensively generate mouse models of human structural birth defects of the cerebellum and cerebrum (Dandy-Walker malformation, microcephaly) and severe congenital heart defects (Holt-Oram Syndrome and Conotruncal malformations). Each specific disorder we are modeling in this pilot study has a prevalence of between 1/100,000 and 1/5,000 births and is genetically heterogeneous, qualifying these as Rare Disorders. Since we are generating mouse models of rare brain and heart congenital malformations, this proposal is of direct interest to ODS, NINDS, NICHD and NHLBI. Notably however, this research will have a high impact in general biomedical science and public health and is likely of interest to multiple NIH institutes, since the new technology proposed will be applicable to the generation of mouse models for any human genetic disorder. This technology is especially relevant to NHGRI and other institutes who are supporting large scale human Genome-wide Association and CNV studies currently underway to indentify human disease candidate genes. This proposal combines recent parallel advances in RNAi technology, mouse chimera generation and focused mouse phenotyping into a novel strategy allowing inexpensive and rapid generation of efficient knock-down mouse models. We predict that initial knock-down phenotypes can be assessed in 2 months for approximately $2,000 per gene. This is approximately 1/6 the time and at least 10% of the current cost of standard mouse knock-outs. Rapid, efficient and inexpensive production of knock-down mice will not replace, but rather complement ongoing large scale NIH mouse ENU and genome-wide gene targeting programs. The time and financial economies however, that result from this novel knock-down strategy will revolutionize disease candidate gene analysis. Funding will permit rapid advancement in our understanding the pathogenesis of human disease which in turn, will improve disease diagnosis and treatment and additionally accelerate drug discovery. As a direct result of funding 1.5 new full time employment (FTE) positions will be created and 2 FTE positions will be retained. Although the Human Genome Project has allowed identification of many human disease genes, current technology to model human diseases in mice is time consuming and expensive and cannot keep pace with the current rate of human genetic discoveries. Described in this proposal is a new, inexpensive and rapid strategy to generate mouse models of human disease. This technological advance will enable more rapid understanding of the biological basis of human disease, improve disease diagnosis and facilitate treatment and drug discovery.

DESCRIPTION (provided by applicant): Spontaneous and ENU-induced mouse mutants are a tremendously valuable resource for their ability to model human disease. The study of these mutants has provided new insights into basic clinically relevant biological mechanisms, which would not have been apparent through standard gene-targeting approaches. The key value of these mutants is the ability to associate novel phenotypes with the causative genotype. However, despite their significance, many mouse mutants have not been sufficiently phenotypically characterized to spark appropriate scientific interest. Further, many investigators are hesitant to invest resources into the required positional cloning efforts to identify the causative molecular lesions. As a result, many extant mouse mutant resources are significantly underutilized and of limited to value to the scientific community, despite the already significant resources invested into their generation and maintenance. One such mutant is the spontaneous neurological mouse mutant tippy. The tippy mutation arose at Jackson Labs in 1977 but has remained essentially uncharacterized, both phenotypically and genotypically since its first report in 1995. Homozygous tippy mutants exhibit ataxia and epilepsy and do not survive past weaning ages. Congenital ataxia and epilepsy are common human pediatric neurological disorders that are often co-morbid, and their developmental and pathogenesis poorly understood. We have determined that tippy mice have two very interesting neurological phenotypes, which are of broad interest to both basic and clinical neuroscientists. Our preliminary analysis has revealed novel cerebellar Purkinje cell dendritic abnormalities that likely underlie the ataxia in tippy mutants. Further, we have characterized a complex hippocampal malformation in these mutants likely causative for epilepsy. This malformation is similar to hippocampal dysplasias commonly observed in human temporal lobe epilepsy, a phenotype which has not been previously modeled in mice. Thus, we have demonstrated that the cerebellar and hippocampal phenotypes of tippy mutants are of broad interest to both basic and clinical neuroscientists. In this proposal, we present electrophysiological and immunohistochemical experiments to more completely define the cellular and functional basis of the observed tippy mutant morphological CNS abnormalities. We also propose to identify the underlying molecular genetic lesion in tippy mutants, which we have localized to a small critical region on distal mouse chromosome 9, to associate the tippy genotype with the clinically important tippy neurological phenotypes. Together these experiments will provide a comprehensive characterization of the tippy mutation which will provide new insights into the basic neurobiology of ataxia and epilepsy. These experiments will also significantly enhance the value and accessibility of tippy mutant mice, so that others may more readily exploit this valuable mouse reagent. PUBLIC HEALTH RELEVANCE: This proposal is focused on defining the etiology of congenital ataxia and epilepsy. These are two co-morbid neurodevelopmental disorders that are often associated with devastating cognitive and motor deficits. An improved understanding of the developmental mechanisms leading to the underlying cerebellar and hippocampal malformations will provide valuable diagnostic and prognostic information and influence treatment.

DESCRIPTION (provided by applicant): The goal of this proposal is to define the general principles of dorsal medial germinal zone organization and regulation, using Lmx1a as a molecular entry point. Dorsal brain germinal zones, such as the cerebellar rhombic lip and telencephalic cortical hem, contribute substantially to neuronal diversity in the CNS, but the mechanisms that drive their neurogenesis are ill-defined. Using genetic fate mapping and mutant analysis we have demonstrated that during cerebellar development, the LIM-homeodomain transcription factor Lmx1a 1) segregates the roof plate lineage from neuronal rhombic lip derivatives, 2) is required for maintenance of the entire late embryonic rhombic lip and 3) confers posterior vermis identity to a subset of rhombic lip cells. These experiments demonstrate that the cerebellar rhombic lip is a heterogeneous progenitor population with fates specified at very early stages, yet we know nearly nothing about the cellular and molecular characteristics of rhombic lip progenitors. In Aim 1 of this proposal we will conduct an extensive analysis of rhombic lip progenitor gene expression and cell cycle parameters in wild-type and Lmx1a-/- animals to characterize the organization, developmental mechanisms and molecular pathways conferring cell fate within the rhombic lip. Using expression microarray analysis of microdissected e13.5 cerebellar rhombic lip from wild-type and Lmx1a-/- animals, we have identified several Lmx1a-candidate effectors and we will characterize their roles in rhombic lip development through analysis of extant mouse mutants and manipulation of gene expression using in utero mouse electroporation. Our preliminary data demonstrate that Lmx1a also regulates neurogenesis in the telencephalic cortical hem, where loss of Lmx1a results in aberrant expression of the cortical selector gene Lhx2, which leads to excessive production of hippocampal cells instead of Cajal-Retzius cells. In Aim 2 we propose a series of genetic fate mapping and in vitro explant analyses to test if Lmx1a acts intrinsically in the cortical hem to regulate cortical hem neurogenesis and if the adjacent telencephalic choroid plexus, where Lmx1a is also expressed, also influences hem development. We will also conduct expression analyses to determine if Lmx1a-downstream effectors are conserved across dorso-medial germinal zones. Finally in Aim 3, we will explore the basis of dorsal midline abnormalities when both Lmx1a and Lmx1b function removed. These basic neurodevelopmental analyses have direct translational relevance to human structural malformations of the CNS and have potential to reveal new insights into the dramatic evolution of cortical size in mammals.

DESCRIPTION (provided by applicant): This application describes an interdisciplinary approach involving basic and clinical scientists employing new and innovative informatic, genetic and developmental strategies to identify the underlying pathogenesis and causative genes for Dandy-Walker malformation, the most common structural malformation of the cerebellum. Dandy-Walker malformation is common, affecting 1/3000 live births and causes significant motor and intellectual delay and yet is poorly understood. Our group has identified the only 2 characterized loci for this clinically and genetically heterogeneous birth defect. Our analysis of mouse models has lead us to the hypothesis that disruption of mesenchymal signaling to the developing cerebellum is critical to the developmental pathogenesis of this birth defect. The recognition that the meninges is a critical regulator of CNS development is a recent paradigm shift in the field of neurodevelopment and the basic biology and molecular pathways of these interactions is not known. Further, it has become apparent that disrupted meningeal signaling underlies not only the significant clinical phenotypes of posterior fossa disorders such as Dandy-Walker, but has broad implications for the pathogenesis of large group of neurodevelopmental disorders that also involve meningeal signaling including ACC and others. The experiments outlined in this proposal are designed to identify pathways and mechanisms for posterior fossa mesenchymal regulation of cerebellar development, using Foxc1, the most recently identified Dandy-Walker gene, as an entry point. Aims 1-3 use novel in vitro and in vivo assays including explant culture, electroporation, RNAi and BAC transgenesis together with extensive informatic analyses to identify and validate the signaling pathways from the posterior fossa to the adjacent developing cerebellum which modulate Dandy-Walker related phenotypes in mouse models. In Aim 4 we will then sequence the best Dandy-Walker candidates from the first 3 Aims, in a cohort of human Dandy-Walker patients to identify new disease-causative genes. Together these synergistic mouse and human experiments will define new biology regarding mesenchymal control of neural development and identify new DWM genes, which will immediately improve diagnosis for affected families and will be essential for future prognostic studies. PUBLIC HEALTH RELEVANCE: This application develops and implements innovative interdisciplinary approaches to identify and characterize the developmental and genetic disruptions that cause Dandy-Walker malformation spectrum birth defects. Dandy-Walker malformation spectrum birth defects are structural birth defects of the developing cerebellum of the brain. They are common, affecting approximately 1/3000 live births and cause intellectual and motor delays and hydrocephalus. Through gene discovery, we aim to develop badly needed effective diagnostic and prognostic tools for affected families.

PROJECT SUMMARY/ABSTRACT Numerous cerebellar malformations have been described in humans. Most cause cognitive, in addition to motor and sensory integration deficits. Surprisingly little is understood regarding the developmental basis of these malformations, particularly since little human specific data is available for normal or abnormal fetal cerebellar development. This proposal seeks to advance knowledge of human cerebellar development and malformations using human fetal samples and mouse models. The human-specific data will directly test the validity of our working mouse-derived hypotheses regarding the causes these disorders and strengthen the foundation of normal developmental data which will inform our ongoing genetic analyses of human cerebellar malformations. We will conduct the first in-depth analysis of normal human fetal cerebellar development from 4-23 Gestational Weeks, covering major developmental events. We will then examine the pathology of human fetal Dandy-Walker malformation the most common human cerebellar malformation, affecting ~1/3000 live births. Mouse models will be generated in conjunction with these experiments to assess the mechanisms of the developmental pathology. Finally, we will generate the first transcriptome data for normal human fetal cerebellar neurons. These cell-type specific data are critically missing from current publicly available brain resources. Our human fetal cerebellar neuron data will be compared to transcriptome data from existing datasets of endogenous mouse developing cerebellar neurons as well as mES and hPSC-derived cerebellar neurons to development to assess their validity as model systems. Further, the data will also be integrated with exome data from human cerebellar malformation patients to facilitate gene discovery for these important and understudied birth defects.

PROJECT SUMMARY This proposal outlines a comprehensive series of experiments to assess the basis of overgrowth phenotypes associated with gain of function mutations in PIK3CA, using the mouse as a model system. Although well studied in cancer, recent data has revealed a role for PIK3CA mutation in several developmental disorders including MCAP, DMEG, CLOVES syndrome, epidermal nevi and seborrheic keratosis. Here will assess the developmental and signaling pathway disruptions caused by 3 different Pik3ca gain of function mutations, with particular emphasis on the developing brain. To date, all reported PIK3CA mutations are postzygotic or mosaic rather than germline mutations. Accordingly, we hypothesize that PIK3CA phenotypes correlate with both the severity of the mutation and level (and distribution) of mosaicism. To test this and other hypotheses, we will use standard conditional genetic approaches to express 3 patient-related Pik3ca gof mutations in embryonic CNS neuronal progenitors and their descendants. We will then generate ES cell chimeras, injecting ES cells constitutively expressing the same mutations into wild-type blastocysts to assess phenotype-mosaicism relationships both within the brain and throughout the entire body. We also propose to use in utero electroporation technology to more specifically target mosaic expression to the developing brain. Our studies will assess the developmental pathogenesis of brain pathology and characterize the associated epilepsy, a pressing clinically relevant phenotype. Finally, we will use pharmacological approaches to assess the underlying mechanisms driving acutely Pik3ca-dependent seizures in post-natal mice. These assays represent the first step toward developing molecularly rational epilepsy therapy in PIK3CA segmental brain overgrowth syndrome patients.

PROJECT SUMMARY Our goal is to develop Acomys cahirinus as a new alternative mammalian model system, since they represent the only known mammalian species that retains a remarkable capacity for increased regenerative healing potential across multiple systems as adults. As mammals, the molecular pathways for regenerative repair in Acomys are unique, yet are not likely to be distant from those required for human therapy. Although Acomys may be the best key for unlocking our own capacity for regeneration and untangling the relationship between homeostatic repair versus pathological fibrosis in mammals, Acomys animals for research purposes are more stringently regulated than other rodent models, and hence are currently in limited use in biomedical research. Our innovative approach to enable rapid research across multiple systems has been to first sequence, assemble and annotate a high quality Acomys cahirinus Genomic Resource. Our second step is to develop Acomys Cell and Transgenic Resources to more rapidly facilitate their utilization in biomedical research. Our multipisciplinary Acomys Research Team has extensive evidence that cells from different adult Acomys tissues grow surprisingly well in primary cultures. In fact, isolated Acomys fibroblasts actively resist cytokine-mediated myofiboblast induction by both changing gene expression and by differentially regulating signal transduction kinetics in homeostatic and regenerative pathways. We propose to develop Acomys cell lines from multiple adult tissues, genetically encode them with an array of fluorescent biosensor tools that report activity of key signaling pathways, generating essential tools to unravel the important mechanistic details of Acomys regenerative biology. We also aim to develop transgenic Acomys technology and animals to rapidly facilitate use of these remarkable animals by the broader scientific community. We believe that enabling access and facilitating research into this fascinating experiment of nature offers truly novel possibilities for discovery with far-reaching implications for human regenerative medicine.

Abstract of the funded parent grant Numerous cerebellar malformations have been described in humans. Most cause cognitive, in addition to motor and sensory integration deficits. Surprisingly little is understood regarding the developmental basis of these malformations, particularly since little human specific data is available for normal or abnormal fetal cerebellar development. This proposal seeks to advance knowledge of human cerebellar development and malformations using human fetal samples and mouse models. The human-specific data will directly test the validity of our working mouse-derived hypotheses regarding the causes these disorders and strengthen the foundation of normal developmental data which will inform our ongoing genetic analyses of human cerebellar malformations. We will conduct the first in-depth analysis of normal human fetal cerebellar development from 4-23 Gestational Weeks, covering major developmental events. We will then examine the pathology of human fetal Dandy-Walker malformation the most common human cerebellar malformation, affecting ~1/3000 live births. Mouse models will be generated in conjunction with these experiments to assess the mechanisms of the developmental pathology. Finally, we will generate the first transcriptome data for normal human fetal cerebellar neurons. These cell-type specific data are critically missing from current publicly available brain resources. Our human fetal cerebellar neuron data will be compared to transcriptome data from existing datasets of endogenous mouse developing cerebellar neurons as well as mES and hPSC-derived cerebellar neurons to development to assess their validity as model systems. Further, the data will also be integrated with exome data from human cerebellar malformation patients to facilitate gene discovery for these important and understudied birth defects. Abstract of Requested Supplement This application is being submitted for PA-19-056 in accordance with NOT-OD-19-071. The purpose of this research supplement is to define the cellular, molecular and morphological cerebellar developmental trajectories in human Down Syndrome samples. Developmental profiles will be generated through a combination of single cell sequencing, histological and immunohistochemical analyses and complemented with cell culture assays defining the mitogenic properties of cerebellar granule progenitors. Data from Down Syndrome samples will then be directly compared to profiles from normal and Dandy-Walker malformation developmental cerebellar samples available in the lab and generated under the parent R01. Cerebellar hypoplasia is one of the most consistent phenotypes in Down Syndrome patients that is a significant contributor to neurological phenotypes in these patients. Yet, very little is understood about the developmental disruption of cerebellar development that underlies the congenital hypoplasia. We will produce a multi-modal description of human cerebellar development in Down Syndrome, comparable to data we are already generating to define normal cerebellar development. An understanding how and when Down Syndrome cerebellar developmental trajectories differ from normal and other cerebellar malformations will elucidate the cellular and circuit underpinnings of pediatric and adult Down Syndrome neurological phenotypes. The studies are of high impact with considerable translational potential to identify new therapeutic approaches for neurological deficits in Down Syndrome. They will also generate baseline data human data to the developmental stage-, cell type-, and molecular-specificity of model systems (hiPSCs, organoids, animal models). These experiments specifically address Component 1 and Component 2 of the INCLUDE Project research objectives.

Project Summary Many of the major biological discoveries of the 20th century were made using very few model species. They were chosen for historical tractability, rather than biological attributes relevant to critical biological questions or relevance to pressing global health issues. However, the advent of new sequencing and genome engineering technologies makes establishment of new genetic models feasible. History has demonstrated that the success of model organisms is self-perpetuating: as the community of researchers grows, new methodologies and resources are developed and shared, and the body of specific knowledge and access to powerful tools to manipulate, observe and experiment upon these model organisms further lowers the bar to entry so that the cycle can repeat (18). Here we propose to develop transgenic infrastructure for Acomys cahinirus an emerging model of adult mammalian non-fibrotic regenerative healing and healthy aging. We have propose a methodical comprehensive and feasible plan to achieve transgenesis in Acomys. We first propose the generation of interspecies Mus musculus Acomys leveraging Acomys ES cells which we have generated and CRISPR tagged with a fluorescent marker. We will then work to generate whole animal transgenic Acomys animals using a variety of ex vivo and in situ methodologies. Acomys transgenesis will unlock the genetics of the extraordinary regenerative biology of Acomys certain to yield novel therapeutic approaches to human health and disease.

PROJECT SUMMARY While the cerebellum's role in motor function is well recognized, the cerebellum also plays cardinal roles in affective regulation, cognitive processing, and linguistic function (1). Indeed, there is a growing recognition that disruptions of cerebellar development cause considerable cognitive, behavioral, and social deficits (2-6). Yet, though cerebellar malformations are amongst the most commonly recognized structural brain malformation in prenatal imaging (7-10). Reliable information about their cause is sparse (11, 12). Diagnosis is based on imaging studies which are often unreliable, a problem amplified during fetal development (13, 14). In stark contrast to the wealth of knowledge gained over the decades regarding the mechanisms and genes driving cerebellar development in mice and other model organisms (15-19), we actually know very little about human cerebellar development. We recently reported multiple aspects of human cerebellar development significantly differing from mice and even rhesus macaque, a non-human primate. These discoveries challenge our current mouse-centric models of normal cerebellar development and the pathogenesis human cerebellar developmental disorders (20). This proposal seeks to advance knowledge of normal developing human cerebellum and cerebellar birth defects, leveraging 1) our unique access to normal and abnormal human fetal cerebellar tissue and 2) our extensive, specific expertise of mouse and human cerebellar development and our deep knowledge of human cerebellar malformations. Our detailed characterization of normal and abnormal cerebellar development, combined with humanized mouse models will improve our understanding of the biology of normal human cerebellar development and the pathogenesis of a clinically important human cerebellar birth defect, Dandy-Walker malformation (DWM). They will provide gold standard histological and transcriptomic datasets to assess model systems of human cerebellar development, generate the first ?humanized? mouse models of human cerebellar development and finally, enable improved and sorely needed prenatal diagnostic information for families affected by cerebellar malformations.