Lee Niswander, Ph.D - US grants

DESCRIPTION (applicant's description): A key question in developmental biology is how growth and patterning are coordinated such that organs form with the appropriate three-dimensional structure. The vertebrate limb provides an excellent model system to study the cellular and molecular mechanisms that control embryonic growth, pattern formation and differentiation. The apical ectodermal ridge (AER) is critical for limb growth and patterning. Our studies, some of which were a direct outcome of the previous grant, have identified fibroblast growth factors (FGF) and bone morphogenetic proteins (BMP) as critical regulators of AER function. Here we will address how BMP signaling controls Fgf4 expression in the AER and the mechanism by which BMPs regulate AER function. We will also test the hypothesis that BMP acts at an earlier step to mediate formation of the AER through the transcription factor MSX and by interactions with the WNT signaling pathway. Finally, we will test the hypothesis that BMP acts through a different transcription factor, EN1, to control dorso-ventral patterning of the limb. Our knowledge of vertebrate limb development comes largely from studies in chick and mouse. The cellular and molecular processes are highly conserved between these organisms and we will take advantage of both of these systems to increase our understanding. The insight provided by these model organisms has proven directly applicable to understanding human limb development. Moreover, mutations in BMP, WNT, and FGF signaling pathways are all implicated in tumor formation, and tumor progression is associated with alterations in more than one of these pathways. A greater understanding of the manner in which BMP, WNT and FGF are coordinately regulated and how they cooperatively regulate gene expression in the limb should lead to a greater understanding of the etiology and consequences of their misregulated expression in cancers.

Funding is requested for the 2004 Santa Cruz meeting on Developmental Biology, to be held 5-9 August 2004 at the University of California, Santa Cruz, California. Previous Santa Cruz Developmental Biology (SCDB) meetings have provided a stimulating and focused forum for discussion of current research by developmental biologists. SCDB meetings are international in scope and are accessible to junior faculty, postdoctoral fellows, and graduate students. SCDB meetings emphasize the conceptual unity of developmental biology at the level of mechanism, while covering and eclectic range of current topics. The topics for the 2004 meeting include: pattern formation; morphogenesis and cell migration; evolution and development; stem cells; non-coding RNAs; cellular asymmetry; organogenesis; development of the nervous system, and disease. Speakers are invited from both plant and animal development fields. The 2004 meeting will have 40-50 platform speakers, of which 10-15 will be chosen from abstracts submitted to the meeting organizers. Platform sessions are organized to allow ample time for discussions; the extensive discussion periods have been cited as one of the best features of previous SCDB meetings. Posters are continuously displayed and several poster sessions are scheduled in the meeting, as well as time for informal discussions.

The 2004 SCDB meeting will build on the success of previous meetings in this series. SCDB meetings are hosted by the University of California, Santa Cruz (UCSC). The conference site is arranged so that platform sessions, posters, accommodation, and dining facilities are in close proximity; access for disabled participants is ensured by UCSC. The UCSC setting is cost effective. Conference logistics are provided by UCSC Conference Services. The UCSC campus is within an hour's drive of the San Francisco Bay Area; travel from nearby airports is simple, but the site is sufficiently isolated and pleasant that speakers and participants tend to stay in residence for the entire meeting. Thus, the SCDB meeting, although modeled on a Gordon Research Conference, has several additional advantages that have made it a valued venue for the Developmental Biology community.

Intellectual merit: The Santa Cruz meetings on Developmental Biology have become a well established venue for presentation and discussion of current results across the field of developmental biology. Unlike other comparably sized meetings, SCDB meetings emphasize interdisciplinary approaches, and are not focused on specific processes or organisms. One theme of the 2004 meeting will be the use of comparative and evolutionary approaches to understanding development; for example, in addition to classical model systems, the meeting will include presentations on mollusks, planarians, and stickleback fish. The SCDB meeting will emphasize the general implications of such studies for developmental mechanisms.

Broader impact: The broader impacts of the 2004 SCDB meeting will be in fostering developmental biology research and in training. Previous SCDB meetings have stimulated collaborative research by bringing together an eclectic group of active investigators in a collegial setting. It is anticipated that the 2004 meeting will likewise foster new collaborations.

Previous SCDB meetings have also had broader impact in enhancing the training of graduate students, postdoctoral fellows, and junior faculty in the field of developmental biology. The size and organization of SCDB meetings allows all attendees to present their research and to fully participate in discussions. SCDB meeting attendees also regularly include representatives from major scientific journals. Active participation in meetings and exposure to prominent scientists in the field are both important training goals for scientists in the early stages of their careers.

[unreadable] DESCRIPTION (provided by applicant): Abnormalities of the human limb are one of the most common human birth defects, yet little is known as to the genetic or molecular changes underlying these defects. The developing mouse and chicken limb have provided excellent models to understand limb development and pattern formation in the vertebrate embryo. By defining the key molecular mechanisms by which the limb is patterned and bone formation is controlled, we will gain a better understanding of the possible causes of human limb defects. Many studies to date on these model organisms have explored the function of a relatively small set of patterning genes. However, there are many gaps in our knowledge as to what regulates these key players, what are their downstream targets and how is thr information translated into the formation of bones of correct number, shape and size. [unreadable] [unreadable] This proposal focuses on two different approaches to help to close these gaps in our knowledge. First, we will continue an unbiased forward genetic screen in the mouse initiated in the past granting period to identify new genes that regulate limb development, in particular those required for the formation of the correct number and size of skeletal elements. Second, we will use a novel live imaging system to follow the behaviors of cells as they undergo the early steps of cartilage formation. These approaches will allow us to elucidate where, when and how these novel genes function to regulate limb development and skeletal formation. In Aim 1 we will explore the genetics of polydactyly (extra digit formation) and syndactyly (digit and soft-tissue fusion and/or digit loss) and determine the molecular mechanisms by which novel genes regulate development of the hand and foot. In Aim 2 we will expand the mutagenesis screen to identify a set of new mutations that affect similar aspects of limb development to broaden our knowledge of the genes involved and to provide new models for understanding human limb defects. This will also allow for the testing of genetic links between different pathways that have not yet been realized. Finally, in Aim 3 we will use a novel live imaging system to begin to explore the cell dynamics of limb mesenchyme as it undergoes cartilage formation. This will provide unique insight into the critical events that occur as an undifferentiated mesenchyme cell undergoes the transition to a chondrogenic cell. We will test the hypothesis that cells along the A-P axis exhibit distinct cellular behaviors and that these behaviors are altered in new and classical mutants of polydactyly and syndactyly. Together, these studies will build a deeper foundation for understanding the key genetic, cellular and molecular events that regulate embryonic limb development and provide new animal models of limb development that will be useful for understanding the underlying causes of human birth defects. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): A major goal in understanding tissue and organ formation is to link key developmental signals to the fundamental molecular pathways responsible for guiding and controlling such development. The genomic era has created a tremendous body of knowledge and provided a global overview of the molecular regulators of the cell. However, there is a considerable gap in understanding the function of these regulators and even wider gap in understanding the links between these regulators and the mechanisms by which they generate complex three-dimensional tissues. The current state-of-the-art requires a comprehensive approach or systems approach in which the interaction between pathways and the complexity created by the convergence of different pathways must be addressed to further our understanding of the development of key organs relevant to human health and disease. The proposed research project aims at filling this gap by generating a comprehensive understanding of the functional relationship between kinase/phosphatase regulated molecular pathways and their roles in organizing the formation of the embryonic mouse lung. This proposal utilizes a variety of established as well as new and innovative cell biological and developmental techniques to pursue the following aims. Aim 1 will define the kinases and phosphatases involved in embryonic lung morphogenesis using a combination of a functional genomics (loss of function) screen with morphological analysis in lung explant cultures. Aim 2 will define the molecular pathways involved in lung morphogenesis, correlating known molecular pathway regulators among the kinome/phosphatome with hits from our loss of function screen. Within 2 years our approach will lead to a fundamental understanding of how lung morphogenesis is controlled by phosphoregulatory proteins and the cross-talk between key developmental signals. A long term goal is to provide potential targets for the treatment of human diseases including lung cancer, emphysema, asthma, and the immature lungs of premature infants. PROJECT NARRATIVE. The overall goal is to use a comprehensive systematic approach to provide a fundamental understanding of the essential molecular regulators of embryonic lung development. These findings have the potential to provide possible new targets for the treatment of the immature lungs of premature infants and of human diseases including lung cancer, emphysema, and asthma.

DESCRIPTION (provided by applicant): Peroxisomes are enzyme-containing membrane organelles that are involved in the detoxification of reactive oxygen species, the catabolism of very long chain fatty acids and the biosynthesis of plasmalogens. Patients with peroxisomal biogenesis disorders (PBD) show neurological defects ranging from the severe disease Zellweger syndrome that is lethal within six months of birth, to the progressive diseases of cerebellar ataxia and spinal ataxia. Despite the fact that neurological deficits can be observed at birth in some PBD patients, the embryological etiology of the disease has not been explored. Moreover, there are no effective therapies to prevent or ameliorate the neural degeneration that occurs in PBD patients. In mice we have identified a mutation in the peroxisomal gene Pex10. Biomarkers utilized for diagnosis of PBD in humans are similarly disrupted in the Pex10 mouse model. Moreover, Pex10 mutant mouse embryos show a progressive inability to move. Thus, our Pex10 mutant provides an excellent paradigm for the study of PBD neuropathology and the first vertebrate Pex10 model. Analysis of Pex10 mutant embryos shows defects in the connectivity between the motor nerves and the muscle. These preliminary data provide the basis for this R21 proposal to understand the embryological origin of the neurological deficits and to explore possible therapies to increase the lifespan and ameliorate the neuropathology in this peroxisomal animal model. Aim 1 will characterize the novel Pex10 mouse mutant as a model for PBD progressive ataxia. We will test the hypothesis that alterations in peroxisomal function in the embryo inhibit termination of axons at the synapse which disrupts the connectivity of the locomotor circuit. We will determine whether the progressive embryonic locomotor loss results from defects in myelination, axon guidance and/or synapse function of the spinal neurons. Aim 2 will provide the first definition of the peroxisomal proteome in the fetu and will determine the changes in the contents of the peroxisome in Pex10 mutants, in particular in the myelinating Schwann cells. Moreover, we will determine whether the point mutation in the PEX10 zinc RING finger disrupts ubiquitination activity, which could alter recycling of the peroxisomal receptor. Aim 3 will use the Pex10 mouse model to evaluate possible therapeutic strategies to increase the lifespan and rescue the neuronal phenotypes in mutant mice. Overall these studies will provide the first insight into the embryological origin of PBD neuropathies and will seek to define potential therapies to alleviate the profound neurodegenerative defects that underlie peroxisomal diseases.

DESCRIPTION (provided by applicant): Large-scale high-throughput mouse gene knockout studies have generated considerable data with respect to adult phenotypes of viable homozygous mouse strains. However, many strains do not survive into the post- natal period. The current KOMP2 initiative provides a tremendous opportunity to determine how the disruption of numerous genes impacts embryonic development and to provide insight into the gene networks responsible for normal embryogenesis and fetal development. These studies are especially relevant to the investigation of the underlying causes of human pregnancy loss and structural birth defects. Importantly, some of the most frequent developmental abnormalities observed in large-scale mouse knockout studies are also common human birth defects. Defects in neural tube closure and neural crest formation account for a high proportion of birth defects in humans and these defects can have severe consequences on viability. This reinforces how knowledge gained from the study of mouse models can impact our understanding of human pathology. This proposal brings together three experts - Williams, Clouthier, and Niswander - who have extensive and collaborative experience in the detailed characterization of embryonic mutations that disrupt neural tube closure, craniofacial formation, and heart development. Our experience in forward genetic screens and high- throughput characterization makes us cognizant of the necessity for rapid analysis of mutant strains in order to minimize the shelf-time of the live colonies. Our goal in Aim 1 is to expand and complement the phenotyping information for the KOMP2 strains by providing a detailed histological assessment of strains that would not otherwise undergo this gold standard of analysis at KOMP2. All data will be uploaded and available through a publicly accessible website, with annotations and ontologies established by IMPC, MGI and KOMP2. Aims 2 and 3 will delve much deeper into the cellular and molecular mechanisms responsible for the defects seen in neural tube (Aim 2) and neural crest (Aim 3) formation in a select number of KOMP2 strains. Aim 2 is relevant for understanding the causes of human neural tube defects such as exencephaly and spina bifida (1 in 1000 births). Molecular assays and genetic interaction studies will provide pathway information to elucidate the genetic networks that orchestrate NT closure. Innovative live-imaging will explore how changes in gene function alter the cell behaviors that underlie neural tube morphogenesis. Aim 3 is relevant to major abnormalities associated with neural crest pathology include craniofacial defects (1.5 in 1000 births), heart defects (1 in 125 births), and enteric nervous system defects (1 in 5000 births). Aim 3 will study formation, migration and differentiation of the neural crest ad its interactions with surrounding tissues. Furthermore, tissue-specific knockout studies will determine if the observed defects are autonomous to the neural crest or if they are caused by defective signaling interactions with other tissues, particular the ectoderm. Overall these proposed studies will provide biological insight into the function of new genes arising from the KOMP2 centers.

? DESCRIPTION (provided by applicant): There is a fundamental gap in our understanding of how defects in chromatin remodeling proteins, methyltransferases and acetyltransferases are causative for human craniofacial phenotypes. This represents an important problem, because craniofacial defects occur frequently in the human population, 1 in every 1000 live births annually in the U.S. (CDC, 2011) and many are associated with epigenetic regulators of the genome. Our long-term goal is to better understand the function of chromatin remodelers during cranial neural crest (cNCC) development. The objective of this application is to determine the mechanism by which two families of epigenetic regulators, KAT2a lysine acetyltransferase and PRDM lysine methyltransferases that regulate each other and act to modify the same H3K9 residue on histone 3, function in zebrafish and mouse cNCC development. We will use two excellent developmental model systems and combine genetic tools with live cell imaging of zebrafish and mouse cNCC behaviors and transcriptional studies to tackle the question of why mutations in Kat2a and Prdms lead to craniofacial abnormalities. The overall hypothesis is that these chromatin modifying enzymes act as opposing transcriptional regulators and function cell autonomously to regulate cNCC proliferation and migration. The rationale for this research is that understanding the mechanism of how KAT2a and PRDMs regulate cNCC development will have the potential to translate into a better understanding of the pathogenesis of craniofacial defects due to mutations in epigenetic regulators, including cleft lip and palate and various syndromes such as Kabuki and SBBYSS that affect the human population. From strong preliminary data, we have designed 3 specific aims: 1) Determine the developmental function of KAT2A and PRDMs in cranial neural crest development, 2) Examine the genetic interaction and regulation of gene targets by KAT2A and PRDMs, and 3) Determine the enzymatic regulation and chromatin state of KAT2A and PRDMs target genes. Under the first aim, we have determined that Prdm1, Prdm3, Prdm16 and Kat2a have craniofacial defects in both mouse and zebrafish. We have the tools and expertise to determine the specific craniofacial defects and to define abnormalities in proliferation and migration of cNCCs. For the second aim, we have generated and obtained most of the zebrafish and mouse strains, and performed transcriptional profiling in both zebrafish and mouse, demonstrating feasibility. For aim three, we have shown analysis of acetylation and methylation states in both tissue and biochemically. Our approach is conceptually innovative in testing a novel hypothesis and technically innovative in the use of live cell imaging and the interplay between two species that model human craniofacial development. The proposed research is significant because it is expected to advance an understanding of how cNCCs form the craniofacial skeleton, which has the potential to inform the treatment of neural crest associated birth defects and craniofacial syndromes.

There is a long-standing scientific interest in the field of developmental biology, as development is a central biological problem, as well as an increasing public interest in issues such as evolution, stem cells and regenerative biology, highlighting the importance of the discipline both in terms of scientific advancement and public education. The Society for Developmental Biology (SDB) annual meetings provide a forum to address these broader aspects, and to foster young scientists coming into the field, especially the under-represented and underserved populations. The scientific sessions contribute to furthering our understanding of the processes used by a single fertilized cell to reach adult form with many cell types, in all organisms. The education sessions offer opportunities for bench researchers to find out about more effective ways to teach and mentor the next generation. The meetings also provide an informed discussion forum on topics that are relevant to establishing national policies for research and public understanding of science. The advances in developmental biology have contributed to beneficial practices in agriculture and animal (including human) health, to understanding evolution and preservation of species, and to the well-being of our planet and its biodiversity.

The SDB is the largest society devoted to the field of developmental biology, with over 2,000 members worldwide, including undergraduate and graduate students, postdoctoral fellows, junior and established investigators, many of whom hold NSF grants or fellowships. Participants present their latest, mostly unpublished findings at these annual meetings. Thus, the meeting contributes to the advancement of knowledge in the discipline and cross-fertilization of ideas. In recent years, other national societies have joined SDB in our annual meetings, confirming the leadership SDB has in the field worldwide. The proposed meetings will continue the tradition of mixing poster presentations, plenary and concurrent sessions, a postdoctoral symposium, an education symposium and workshops on new technologies, publishing, teaching and outreach, as well as on current issues. In all sessions, a special effort is made to have a diversity of speakers in terms of: model organisms, experimental approaches, career stages, gender and racial/ethnic background. The 2015 meeting, to be held in Snowbird, Utah, will cover a variety of themes and approaches, including: Epigenetic Mechanisms of Development; Imaging: Seeing is Believing; Gene Networks; Tissue Patterning/ Organogenesis; Evo-Devo Mechanisms; Quantitative Biology; Biomechanical Influences in Development; Signal Integration and Timing in Development; Growth and Metabolism.

? DESCRIPTION (provided by applicant): Failure of neural tube closure is a devastating birth defect. Research in the Niswander lab has provided significant insights into the molecular and cellular regulation of NT closure. We have created and studied mouse models with neural tube defects (NTDs) to elucidate the molecular foundations of NT closure. Moreover, we have created robust and novel technology to visualize NT closure in a living mammalian embryo. Our dynamic imaging and key genetic mutants have focused attention on the little studied but critical role for the non-neural ectoderm (NNE) in NT closure. In addition we developed methods to specifically isolate NNE cells to provide a refined and robust platform to study the biology of the NNE. Here we will build upon our unique perspective and turn our attention to spinal NTDs, to provide insight into the most common type of NTD in humans, and to two genetic pathways that are associated with spina bifida in mice and humans. Aim 1 will test the hypothesis that the two closely related Grainyhead-like (GRHL) transcription factors, GRHL2 and GRHL3, differentially control cranial and spinal NT closure through unique and differential activation of target genes, in part mediated by interaction with the JNK signaling pathway that activates the AP1 (cJUN/cFOS) transcription factor. Aim 2 will extend our live platform to test the hypothesis that GRHL-regulated NNE transcriptional programs drive NT closure by controlling cell adhesion, recycling of membrane components, cell shape changes, and/or actin dynamics. Aim 3 will combine our comprehensive molecular and cellular insights with novel unpublished analyses of hundreds of NTD samples to test the hypothesis that mutations identified in GRHL3 and the JNK pathway are causative for spinal NTDs in humans. Relevance of research to public health: The proposed experiments will lead to new cellular and molecular insights into the causes of caudal NTDs, the most common type of NTD and which leads to a profoundly important and frequently disrupted aspect of mammalian embryogenesis. Moreover, our studies will impart novel insights into the general mechanisms of embryonic tissue fusion including the face and body wall. The insights gained here may lead to therapies of general application for treatment of embryonic tissue closure defects that together represent a significant percentage of human birth defects. Abbreviations used in proposal: CDH1 Cadherin1 or E-cadherin EMT Epithelial-to-mesenchymal transition GRHL Grainyhead-like (GRH is the fly homolog) KD Knock-down mT/mG Membrane tomato/membrane GFP fluorescent reporter, GFP expression is activated by Cre NNE Non-neural or surface ectoderm NT Neural tube NTD Neural tube defect Grhl2-null: We will use Grhl21Nisw allele that we isolated in our ENU-screen and which has the same phenotype as other Grhl2 null alleles. Grhl3-Cre: We will use Grhl3-Cre which generates a null allele (obtained from S. Coughlin; Grhl3tm1(cre)Cgh).

This administrative supplement request through the Office of Dietary Supplements (PA-16-319) takes advantage of the expertise of one member of the multi-PI group of scientists on the Parent Award ?Phenotyping embryonic lethal knockout mice with neural crest and neural tube defects?. The Niswander lab has extensive experience in the detailed characterization of embryonic mutations that disrupt neural tube closure leading to neural tube defects (NTDs) like spina bifida. NTDs are a common birth defects (1 in 1000 births) and the mouse represents an excellent model of human neural tube closure and has led to the discovery of numerous genes that control neural tube development and that are now associated with human NTDs. The original goal in Aim 2 under Dr. Niswander?s direction is to delve deeply into the cellular and molecular mechanisms responsible for defects in neural tube formation in mice arising from the Knock-Out Mouse Project (KOMP2) to better understand the genetic networks that orchestrate neural tube closure. Folate deficiency has long been associated with increased risk for NTD-affected pregnancies. Recommendations to increase folate levels in women of child-bearing age and subsequent fortification of flour with folic acid have decreased the incidence of NTDs by nearly 30%. Yet, still very little is known as to how folic acid acts to decrease the NTD incidence. Moreover, the few studies in mice suggest that there is a range of responses to folic acid fortification, depending on the genetic risk factors, from beneficial to no response to detrimental ? highlighting the importance of understanding which genetic variants and perhaps molecular pathways are favorably influenced by folic acid fortification. Furthermore, despite the decades-long folic acid fortification campaign, the possible consequences of long-term and potential epigenetic changes on NTD risks and mechanisms have not been studied. The request for an administrative supplement will allow these outstanding questions to be addressed. The requested experiments take advantage of two novel mutant mouse lines characterized in the parent grant, one that results in spina bifida and the other in cranial NTD, in conjunction with mouse lines that show altered NTD risk dependent on the dose and duration of FA exposure. Within two new Aims, the goals here are to gain insight into the phenotypic and molecular changes induced by folic acid fortification over multiple generations in NTD models. The ultimate goal is to discover expression biomarkers that may help guide individualized recommendations for optimal outcomes.

? DESCRIPTION (provided by applicant): Failure of neural tube closure is a devastating birth defect. Research in the Niswander lab has provided significant insights into the molecular and cellular regulation of NT closure. We have created and studied mouse models with neural tube defects (NTDs) to elucidate the molecular foundations of NT closure. Moreover, we have created robust and novel technology to visualize NT closure in a living mammalian embryo. Our dynamic imaging and key genetic mutants have focused attention on the little studied but critical role for the non-neural ectoderm (NNE) in NT closure. In addition we developed methods to specifically isolate NNE cells to provide a refined and robust platform to study the biology of the NNE. Here we will build upon our unique perspective and turn our attention to spinal NTDs, to provide insight into the most common type of NTD in humans, and to two genetic pathways that are associated with spina bifida in mice and humans. Aim 1 will test the hypothesis that the two closely related Grainyhead-like (GRHL) transcription factors, GRHL2 and GRHL3, differentially control cranial and spinal NT closure through unique and differential activation of target genes, in part mediated by interaction with the JNK signaling pathway that activates the AP1 (cJUN/cFOS) transcription factor. Aim 2 will extend our live platform to test the hypothesis that GRHL-regulated NNE transcriptional programs drive NT closure by controlling cell adhesion, recycling of membrane components, cell shape changes, and/or actin dynamics. Aim 3 will combine our comprehensive molecular and cellular insights with novel unpublished analyses of hundreds of NTD samples to test the hypothesis that mutations identified in GRHL3 and the JNK pathway are causative for spinal NTDs in humans. Relevance of research to public health: The proposed experiments will lead to new cellular and molecular insights into the causes of caudal NTDs, the most common type of NTD and which leads to a profoundly important and frequently disrupted aspect of mammalian embryogenesis. Moreover, our studies will impart novel insights into the general mechanisms of embryonic tissue fusion including the face and body wall. The insights gained here may lead to therapies of general application for treatment of embryonic tissue closure defects that together represent a significant percentage of human birth defects. Abbreviations used in proposal: CDH1 Cadherin1 or E-cadherin EMT Epithelial-to-mesenchymal transition GRHL Grainyhead-like (GRH is the fly homolog) KD Knock-down mT/mG Membrane tomato/membrane GFP fluorescent reporter, GFP expression is activated by Cre NNE Non-neural or surface ectoderm NT Neural tube NTD Neural tube defect Grhl2-null: We will use Grhl21Nisw allele that we isolated in our ENU-screen and which has the same phenotype as other Grhl2 null alleles. Grhl3-Cre: We will use Grhl3-Cre which generates a null allele (obtained from S. Coughlin; Grhl3tm1(cre)Cgh).

Defects in neuroepithelial progenitor self-renewal and differentiation can result in profound neurodevelopmental disorders including devastating birth defects such as microcephaly. The long-term objective of the proposed studies is to understand how neuroepithelial progenitor cell self-renewal and differentiation are coordinated. This proposal specifically focuses on a long non-coding RNA (lncRNA) that is expressed early in mouse neuroepithelial progenitor cells and, as differentiation proceeds, the lncRNA transcript is processed to yield a microRNA that is involved in neuronal differentiation. Moreover, the lncRNA physically interacts with key microcephaly proteins but the functional relationship between the lncRNA and these proteins is unknown. The major questions addressed in three Aims are as follows. Aim 1 will test the hypothesis that the lncRNA functions - independent of the miRNA - in regulating neuroepithelial progenitor proliferation and survival. Aim 1 creates cell lines and mouse models with specific deletion mutations, including a small deletion of this locus observed in a patient with microcephaly, for functional studies. Aim 2 will explore the cellular mechanism underlying the microcephaly phenotype, preliminary data which suggests a mitotic arrest. Moreover, Aim 2 will address the hypothesis that the lncRNA functions as a scaffold to help maintain sister chromatid cohesion through its interactions with the Cohesin complex, which is also implicated in microcephaly. Aim 3 will explore the mechanism underlying the temporal-spatial difference between the lncRNA host transcript and the embedded miRNA. Our overall goal is to discover new mechanisms that coordinate neuroepithelial progenitor cell proliferation and differentiation, as well as to decipher how this unexplored lncRNA mechanistically acts to allow normal brain growth. Harnessing the potential of neuroepithelial progenitor cells holds promise for the treatment of neuronal injury and neurodegenerative diseases, and dysfunction of neuroepithelial progenitors is at the root of numerous neurological disorders. Our studies will provide mechanistic links between a novel lncRNA and known microcephaly proteins to greatly extend our knowledge of this profound brain disorder.