Alexandra Joyner - US grants

DESCRIPTION (provided by applicant): Fundamental to our understanding of mammalian development is the question of how spatial cues are established and interpreted. The mid/hindbrain region has become a paradigm for studying organizer driven development in the central nervous system, since a centrally located organizing center (isthmus) that expresses the secreted factor Fgf8 patterns the anterior/posterior axes of the midbrain and anterior hindbrain that gives rise to the cerebellum. Key to evolution of the brain, the position of the organizer ultimately determines the relative size of the midbrain and cerebellum, which control many human basic behaviors. Understanding how these regions normally develop is critical to our understanding of related congenital and neurodegenerative diseases. A basic knowledge of development could lead to disease prevention and cell based therapies. We have shown that patterning of the brain begins with division of the neural plate into separate Otx2 and Gbx2 domains. Fgf8 is then induced at the Otx2/Gbx2 junction, a lineage border. 2 isoforms of Fgf8, as well as Fgf17/18 then differentially induce midbrain and cerebellum. We propose to build on this framework of knowledge and address the following key questions. 1. What is the ultimate fate of cells in the isthmic organizer? 2. Is Fgf8a or Fgf8b sufficient for mid/hindbrain development and what is the contribution of Fgf17? 3. When is Fgf8 required for midbrain and cerebellum development? 4. Does Fgf8 induces different midbrain and hindbrain structures along the anterior/posterior axis directly through setting up a gradient of Fgf proteins? 5. Is Fgf8 or Wnt1 signaling required to maintain compartment borders?

DESCRIPTION (provided by applicant): The cerebellum (Cb) is implicated in contributing to cognitive and social functions, in addition to having a critical role in skilled motor performance. Accordingly, the Cb is associated with many debilitating developmental diseases including autism. One gene that regulates development of the Cb and has been implicated in autism is engrailed 2 (EN2), based on human studies and the finding that En2 null mice not only have deficits in motor control, but also in social behaviors and cognition. Before we can begin to understand higher order functions of the Cb, we must gain more insight into the basic cellular and genetic processes that regulate Cb development. Our approach is to use the two EN homeobox transcription factors as molecular entry points to study Cb development, as we discovered that En1/2 conditional mutants have defects in Cb morphology, molecular patterning and afferent circuitry. We will now direct our studies towards distinguishing the cellular processes regulated by En1/2 and identifying EN2 target genes critical for these processes that could be susceptibility loci for complex behavioral diseases. We will focus on the granule neurons (GNs) that comprise the main recipients of input to the Cb and the deep cerebellar nuclei (DCN) that generate the output. DCN neurons are consistently reduced in autistic patients, which could be a primary cause of some behaviors and also reflect defects elsewhere in the Cb circuit. We will apply a multi-facetted approach that combines novel genetic techniques in mice to study normal and mutant behaviors of GNs and DCN projection neurons, including a mosaic mutant analysis using our MASTR technique and a new method to precisely target over- expression of EN2 to GNs and the DCN to test sufficiency of EN2 to alter differentiation. We will then apply both mutant approaches to live imaging of GNs as a different approach to study Cb morphogenesis and cell proliferation/differentiation. We will also address the question of whether feed back loops ensure the correct proportion of cell types is produced by studying the interaction between En1/2 and the sonic hedgehog (SHH) pathway. Finally, in order to identify the first direct targets of EN2 in the brain, we are engineering new mouse strains expressing a tagged form of EN2. Aim 1. Study the cellular behaviors regulated by En1/2 in developing GNs and DCN projection neurons using conditional genetics and characterizing cellular behaviors in vivo, and in vitro with live imaging. Aim 2. Identify critical target genes o EN1/2 in GN precursors and DCN projection neurons using comparative microarray analysis and ChIP-seq.

? DESCRIPTION (provided by applicant): Key to development of a healthy individual is precise scaling of growth of all the organs, as well as tissues within each organ. In order to develop therapies for congenital and environmentally induced diseases that result from disproportionate growth of organs, we must identify the pathways and cellular behaviors that act within and between organs to ensure coordinated growth. However, few studies have systematically addressed the genes and processes that ensure tissue scaling. We hypothesize that the peripheral nervous system (PNS), which interconnects all organs and communicates with the central nervous system, plays an important role in coordination of growth control. The paired limbs provide an excellent model to study growth regulation at multiple levels, including inter-organ coordination to ensure similar proportions of each limb are attained following temporary disruption of growth of one limb. A major obstacle to studies of organ growth coordination is a lack of animal models in which growth can be transiently perturbed. We propose to establish a robust mouse model that will enable future studies of how growth is coordinated between the left and right (LR) limbs, and test whether the PNS regulates LR symmetry. Of direct medical relevance to this model, Leg Length Discrepancy (LLD) in which the two lower limbs are >2cm different in length is prevalent in children, and is associated with abnormal gait, scoliosis and degenerative joint disease. Studies using mouse models to identify the factors that regulate limb symmetry should enable development of non-surgical methods for unilateral growth regulation in children. Our specific aims are: Aim 1. To develop a mouse model of LLD using intersectional genetics to alter chondrocyte growth in left limbs and to test whether different limb perturbations (cell death and altered growth rates) lead to a similar degree of recovery of symmetry. Aim 2. Test the role of the PNS in LR leg symmetry and identify potential growth regulatory genes using a transcriptome comparison of long bone growth plates undergoing recovery to that in paired unaffected limbs. Our new models will provide the basis for mechanistic studies to identify molecules involved in the inter-tissue and -organ communication responsible for proper scaling and that could be used for therapies.

? DESCRIPTION (provided by applicant): The cerebellum (CB), consisting of 80% of the neurons in the human brain, not only has a major role in balance and motor coordination, but also modulates language, reasoning and social processes via neural circuits that connect throughout the forebrain. The ratio of the number of neurons in the CB to the cerebral cortex is remarkably constant across mammalian species, indicating that interconnected circuits have scaled together during evolution. Since much of CB growth occurs in the third trimester and continues for a year after birth, the CB is particularly vulnerable to clinical and environmental factors. Granule cell production, stimulated Sonic Hedgehog (Shh) secreted by Purkinje cells, accounts for a majority of cerebellar growth during this period. Pre-term babies are at a significantly higher risk of developing cerebellar hypoplasia and neurological dysfunction, likely in part because they receive glucocorticoids. In rodent models, glucocorticoids increase death of granule cell precursors (GCPs) in the external granule cell layer (EGL), through a mechanism that involves altered SHH-GLI signaling. However, our preliminary results and other experimental models have demonstrated that the developing rodent CB has a large capacity to regenerate a depleted EGL. In order to enhance recovery from a transient insult to the developing CB, it is critical to identify the signaling pathways that stimulate compensatory expansion of cells, and ensure that all cell types scale together in order to have normally functioning circuits. We will utilize sophisticated mouse genetics approaches to identify such pathways. Our studies are based on the unique discovery we made that when the anterior EGL of the CB is depleted at birth, cells marked with Nestin-FlpoER in the white matter expand and populate the EGL and then differentiate, producing a major recovery. Our preliminary studies and novel genetic approaches provide a powerful approach for studying the cellular behaviors of normal and mutant Nestin-expressing white matter stem cells in response to depletion of the EGL and discovering the signals that stimulate expansion of white matter stem cells and their population of the EGL. Our studies should provide insights that can be used to develop new approaches for augmenting recovery of the infant CB in the face of premature birth, glucocorticoid treatment or other injuries including hemorrhage. Our specific aims are: Aim 1. To determine the regenerative potential of cerebellar white matter Nestin+ stem cells. Aim 2. To identify signaling pathways which are altered in Nestin+ stem cells during regeneration of the EGL. Aim 3. To test whether SHH signaling or some of the identified pathways enhance recruitment of Nestin+ stem cells to a depleted EGL.

? DESCRIPTION (provided by applicant): Recent genome-wide analyses have changed the diagnosis of medulloblastoma (MB), the most common malignant brain tumor in children. It is now clear that MB is not a single disease of the cerebellum (CB), but encompasses a range of disease subgroups with diverse clinical presentations, histology, and pathway activation, cells of origin and cancer genetics. Given the complexity of MB, development of targeted therapies tailored to each patient will require deeper insight into the major pathways fueling the disease and further subdivision of the current 4 subgroups of patients. The sonic hedgehog (SHH) subgroup represents ~30% of MBs, has intermediate prognosis, and can arise from granule cell precursors (GCPs) that proliferate in response to SHH. The SHH subgroup of MB is diverse at all levels, and each factor is likely to influence prognosis and be critical for treatment. Loss-of function mutations are seen in genes that inhibit SHH signaling (PTCH1), activating mutations in the receptor SMO (e.g. SmoM2) and amplifications of the effector gene GLI2 and its targets associated with loss of TP53. Current HH inhibitors are only effective in a small percentage of MBs, and all patients develop resistance. We hypothesize that some of the intertumoral heterogeneity within SHH-MBs is due to their arising from different cell lineages, at different times, and in different anatomic regions of the CB. Additionally, that cellular and transcriptional states inherited from distinct cells of origin are maintained in the mature tumor, and could represent targets for therapy. Basic research approaches in mouse models are thus necessary as a foundation for translational studies. We have studied the SHH-GLI pathway in mammalian development for 20 years, with a focus on the CB. Recently we developed sporadic MB models with mutations in Ptch1 or Smo. Significantly, reproducible yet distinct histologies are seen during tumor progression in each model. Moreover, the majorities of lesions seen at early stages regresses and have different cellular phenotypes to lesions that progress to MB. We propose to: Aim 1. Marker analysis and longitudinal Mn-Enhanced MRI (Turnbull) of Ptch1 and SmoM2 models initiated at 2 developmental stages and lineages, and identify candidate intrinsic genes that influence progression of SHH-MBs (RNA-seq profiling). Aim 2. Define the microenvironment and test how tumor associated microglia and macrophages influence SHH-MB progression. Aim 3. Determine whether select candidate genes identified in Aims 1/2, mTOR signaling or NR2F2 alter SHH- MB progression and compare the results to human SHH-MB data sets (Taylor) and tumor samples. Our experimental studies of MB progression in mouse models will synergize with human MB studies and should aid in stratification of SHH subgroup patients, wherein new therapies targeted to essential pathways driving MB progression can be tested, improving both prognosis and quality of life for survivors.

PROJECT SUMMARY Prenatal or neonatal exposure to drugs of abuse such as cocaine and ethanol has been shown to disrupt neurogenesis and/or gliogenesis in the developing cerebral cortex, and induce functional abnormalities late in life. Proper formation of the cortex depends on the orderly production of a large number of diverse neurons, as well as glial cells. Radial glial cells have been shown to be a predominant population of neural progenitor cells in the developing cortex. In addition to their well-characterized role in supporting neuronal migration, radial glial progenitors (RGPs) actively divide to proliferate and to generate neurons and glial cells either directly or indirectly. The division mode and dynamics of RGPs essentially determine the number and types of neurons and glia in the cortex; however, the precise behavior and lineage progression of RGPs and the underlying molecular regulation are poorly understood. The long-term goal of this project is to systematically delineate RGP behavior and progeny output at the cellular and molecular levels. RGPs are neither synchronized in division dynamics nor homogenous in division pattern and progeny output. This calls for a systematic and quantitative analysis of the precise mitotic behavior and progeny output of RGPs in vivo at the single cell resolution. Recently, we exploited the unprecedented resolution of mosaic analysis with double markers (MADM) on progenitor division and progeny output, and performed a systematic and quantitative clonal analysis of RGP division and lineage progression. We revealed, for the first time, that RGPs progress through a remarkably deterministic and orderly program in proliferation, neurogenesis, and gliogenesis. Based on strong published and preliminary data, the central hypothesis of this application is that the behavior and output of individual RGPs are highly programmed at the cellular and molecular levels to produce a correct number and type of neurons and glia in the cortex. This hypothesis will be tested by 1) systematically and quantitatively examine the number, type, and organization of astrocytes and/or oligodendrocytes generated by individual RGPs at different embryonic stages using MADM, and 2) elucidate the molecular programs that regulate RGP lineage progression in proliferation, neurogenesis, and gliogenesis by performing in-depth real time single-cell transcriptome analysis of RGPs across different embryonic stages using CEL-seq in conjunction with loss-of- function studies using mouse genetics and/or CRISPR/CAS9 approaches. By integrating a battery of cutting- edge techniques, the proposed research will provide fundamental new molecular and cellular insights into RGP behavior and cortical neurogenesis and gliogenesis. This contribution will be significant because it will not only advance the basic knowledge of cortical histogenesis, but will also expand our understanding of the underlying cause of drugs of abuse-induced brain damage or other devastating developmental brain disorders with cortical abnormalities such as microcephaly, macrocephaly, and autism, and thereby potentially identify important molecular and cellular targets for diagnosis and treatment.

Recent work has provided evidence for functional connections between the cerebellum and cerebral cortex, with the hemispheres and central zone of the vermis housing the main cerebro-cerebellar circuits. Cerebellar neuropathologies have been detected in cognitive disorders including autism spectrum disorder (ASD), schizophrenia and dementias, and modulation of Purkinje neurons in one lobule in the right lateral cerebellum results in altered social and cognitive behaviors. The excitatory neurons of the cerebellar nuclei (eCN) are the output neurons of the cerebellum that interconnect with the cerebral cortex via the pons and thalamus, but their development, electrophysiology and molecular genetics are poorly understood. In particular, functional relationships between the cerebellar nuclei and cerebral cortex have not been demonstrated. In order to understand how the cerebellum functions and modulates the activity of the cerebral cortex, it is necessary to define the molecular diversity of the eCN, understand how eCN subpopulations (subP) develop, map their circuitry and relate cerebro-cerebellar functions to specific eCN subP. A major problem for such studies is a lack of genetic tools for specifically marking and modulating eCN. This project will define molecular subP of the eCN and determine how and when the subP form during normal development and in developmental mutants in which a subset of eCN are lost (En1/2 mutants) using single cell RNA sequencing (scRNA-seq). Mouse lines will then be developed to manipulate eCN subP and applied to mapping the synaptic partners of subP of eCN and to optogenetics to inhibit/activate specific eCN. Electrophysiology and cognitive/social behavior assays will be used to determine the functional interactions between specific subP of the eCN and regions of the cerebral cortex. This project represents the first definition of the molecular subP of the cerebellar nuclei and determination of their functional impact on cerebral cortex functions. RELEVANCE (See instructions): The cerebellum, in addition to the cerebral cortex, is involved in cognitive and social disorders including autism and schizophrenia, however little is know of the neural circuits that connect the two brain structures. We will map how distinct subpopulations of neurons in the cerebellum project to the cerebral cortex during development and determine how they influence cerebro-cerebellar functions. Our results will provide insights into how cerebro-cerebellar circuits function and might be therapeutically modified in diseases.

Project Summary/Abstract Epithelial-mesenchymal transitions (EMTs) are highly regulated dynamic processes in which cells in stable epithelia acquire the ability to migrate and organize new tissues. EMTs are essential for establishment of tissues layers in developing embryos and for the development of many different organs, and disrupted EMTs can cause birth defects. In the adult, abnormal EMTs can cause fibrosis in the kidney, liver and lung, and they can also drive tumor progression and metastasis. Despite their importance, the dynamics and regulation of the cellular events of mammalian EMTs in vivo are poorly understood. The mouse gastrulation EMT provides an unparalleled context to combine the tools of genetics, imaging and cell biology to define the cellular, tissue and mechanical mechanisms that control cell behavior during mammalian epithelial-to- mesenchymal transitions in vivo. The mouse gastrulation EMT, which generates the three body layers of the animal, provides a uniquely advantageous context to dissect the molecular and cellular processes that regulate an EMT. The gastrulation EMT is relatively rapid: individual cells move from the epithelium to the mesenchymal layer in less than an hour. Signaling pathways and transcription factors that are required for this EMT have been identified, and the EMT can be visualized using fluorescent transgenic reporters in real time. This proposal uses a novel set of mouse mutations to define the cell biological events required for the EMT. Genetic and cell biological experiments will test the novel hypothesis that a self-organizing network of interactions among a set of apical epithelial proteins controls the stochastic ingression of cells during gastrulation. Experiments will test whether FGF signaling directly promotes the cell biological changes that drive cells to exit the epithelium, in addition to its established role in the regulation of gene expression. The final step of the EMT is the organized and directed migration of the newly formed mesenchymal cells to generate the organs of the animal. Experiments will test the hypothesis that regulated migration of mesoderm cells drives elongation of the anterior-posterior body axis and that directed mesoderm migration depends on the Striatin Interacting Phosphatases and Kinases (STRIPAK) protein complex. This work will provide the first dynamic analysis of a genetic network that controls the cellular events of a mammalian EMT in vivo and will provide a foundation for understanding other normal EMTs, as well as how aberrant EMTs cause human disease.