Gage Dekoeyer Crump - US grants

[unreadable] DESCRIPTION (provided by applicant): How does the facial skeleton acquire its characteristic shape? What is the developmental basis of craniofacial birth defects in humans? The long-term goal of this proposal is to understand how epithelia communicate with skeletal precursors to generate precise cellular arrangements of cartilage and bone in the face. Zebrafish is an excellent system to model vertebrate development. This proposal uses strengths of the zebrafish system - powerful forward genetics, the ability to manipulate embryos, and in vivo imaging - to understand the tissue interactions, molecular signalling, and cell biology that specify cartilage shape. Development of the facial skeleton involves an interplay between intrinsic factors that give skeletal precursors a set identity and extrinsic signals from neighboring epithelia. The first aim is to examine how skeletal precursors acquire dorsal identity, and then how this dorsal identity allows them to respond to specific signals from neighboring epithelia. In a newly identified pucker mutant, the dorsal skeleton is transformed to a ventral character, and the expression of ventral dlx genes is expanded dorsally. The cloning and characterization of pucker will provide insights into how the dorsal skeleton is specified and shaped. In the second aim, the identity of the endodermal signals that act on skeletal precursors is investigated. Avian experiments demonstrate a role for endoderm in skeletal patterning, yet it is unclear whether the endoderm signals directly to skeletal precursors at pharyngeal arch stages. Ablation and graft experiments will test that the endoderm directly patterns facial cartilage at arch stages, and gain-of-function studies will test that the endoderm patterns cartilage by secreting distinct combinations of Fgfs and Hhs. In the third aim, the cell biology of epithelial-mesenchymal interactions in the face is investigated. Time-lapse imaging will test the model that endodermal epithelia initiate morphology and polarity changes in preskeletal mesenchyme that lead to the formation of cell condensations, a poorly understood developmental intermediate. The role of the endoderm in later cell rearrangements that refine cartilage shape will also be examined. The completion of these aims will further our understanding of facial skeleton development and lead to the better diagnosis, prevention, and treatment of human craniofacial disorders. In addition, the basic developmental knowledge obtained will be essential for the design of cell-based therapies aimed at regenerating the facial skeleton. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Facial epithelia, including the pharyngeal pouches, are important signaling centers that organize development of the head. Defects in pouch formation in human birth defects such as DiGeorge Syndrome result in a variety of developmental abnormalities of the facial skeleton, heart, and glands (e.g. parathyroid and thymus). However, we still know little about the genetic control and cellular behaviors underlying pouch formation. The long-term goal of this proposal is to understand how the DiGeorge Syndrome gene Tbx1 interacts with Fgf and Wnt signaling pathways to precisely control the epithelial transitions that drive pouch formation. In this proposal, we use innovative transgenic and mutant tools in zebrafish to assess the function of developmental genes in the pre-pouch endoderm. We combine this with time-lapse imaging of pouch development in living embryos, which allows us to understand how these genes control specific pouch cell behaviors. Zebrafish is ideally suited for these studies as pouch development is highly conserved with humans, yet zebrafish is the only vertebrate system in which high-throughout transgenic studies and single-cell- resolution time-lapse imaging are practical. Positive findings from this work will elucidate how Tbx1 acts upstream to activate Wnt and Fgf signaling cascades that drive pouch development. In particular, Wnt pathway genes will represent novel candidates for underlying and/or modifying human birth defects such as DiGeorge and Pfeiffer Syndromes. As branching of the embryonic endoderm generates not only pouches but also the liver, pancreas, lung, and other organs, lessons learned from our studies will also have general implications for understanding the initial formation of many important endodermal organs.

DESCRIPTION (provided by applicant): A major goal in human health is to improve the ability of large fractures and skeletal wounds to heal. In contrast to mammals, many amphibia and lizards do have a remarkable ability to reform entire limb and/or tail skeletons, yet the relative lack of genetic tools in these species have limited progress towards the underlying cellular and molecular mechanisms. Here, we present a new model of skeletal regeneration in the genetically tractable zebrafish. In a matter of just a few weeks, adult zebrafish can regenerate nearly two-thirds of their lower jawbone, and they appear to do so through an unusual chondrocyte population that directly produces woven bone. As potentially similar cells have been observed during mammalian fracture repair, a better understanding of these cells during skeletal repair, as well as how they contribute to more extensive regeneration in lower vertebrates, will aid in developing novel therapies for improving bone repair in patients. In the first aim, purification and expression profiling of regenerating chondrocytes, which express markers of both chondrocytes and osteoblasts, will determine the extent to which these cells are hybrid chondrocytes/ osteoblasts. Genes specifically upregulated in early regenerating chondrocytes will also indicate potential pathways that induce these cells in response to injury. Next, we use newly developed Cre/Lox transgenic lines to test the origins and long-term fate of regenerating chondrocytes. In particular, we test that the periosteum is a major source of regenerating chondrocytes, with these directly converting into the osteoblasts that produce woven bone. Using a novel intersectional transgenic strategy to specifically ablate regenerating chondrocytes, we then test that these cells are required for the large-scale regeneration of bone in the zebrafish jaw. During the development of endochondral bone, the majority of chondrocytes undergo hypertrophy and apoptosis, with bony matrix being produced by invading osteoblasts. Quite differently during regeneration, our preliminary data suggest that many chondrocytes directly differentiate into osteoblasts. Using an adult viable ihha mutant and a transgenic strategy to inhibit Hh signaling only in regenerating chondrocytes, we test in the second aim that persistently high Ihh signaling is essential for regenerating chondrocytes to differentiate into osteoblasts. The completion of these Aims will test a model that the ability of regenerating chondrocytes to directly make bone allows a rapid restoration of rigidity in a damaged body part, with the initial woven bone later being remodeled into mature bone. In the long-term, we plan to use lessons taken from this new zebrafish model to devise strategies to augment the inherent ability of the skeleton to repair critical size defects.

This is a proposal to investigate the role of stem cell regulation in cranial suture development and maintenance, and its pathophysiology in craniosynostosis. More broadly, this proposal focuses on how stem cells are controlled in space and time to promote the development and maintenance of vertebrate organs. Our recent results show that heterozygous loss of either of two related transcription factors, TWIST1 and TCF12, account for coronal suture defects in the majority of Saethre-Chotzen patients. Using sophisticated in vivo imaging and genetics in mice and zebrafish, we will test that Tcf12 modifies the function of Twist1 to maintain skeletal progenitors during both the specification and maintenance of sutures. A common role for Twist1 and Tcf12 in the developing and postnatal coronal suture would have the potential to explain both the initial synostosis and the high recurrence rate of postoperative synostosis in patients. A particular strength of our research plan is the complementary expertise of three accomplished investigators in craniofacial genetics. Rob Maxson has long-standing expertise in mouse models of synostosis, having contributed to the identification of TWIST1 and TCF12 as the two most affected genes in Saethre-Chotzen syndrome. Yang Chai recently identified a population of Gli1+ stem cells in the suture that are required for long-term suture patency and calvarial bone growth. Gage Crump has pioneered in vivo imaging techniques in zebrafish to examine the cellular basis of craniofacial defects. First, this team will test that Twist1 and Tcf12 function in the same tissues to repress the Ihh-driven differentiation of sutural progenitors into osteoblasts, as predicted if Tcf12 serves as a suture-specific dimerization partner for Twist1. Second, we will examine continuous requirements for Twist1 and Tcf12 in suture maintenance by conditionally deleting these genes in postnatal Gli1+ sutural stem cells. Third, we will use new knock-in tagged alleles of Twist1 and Tcf12 to identify the direct genomic targets of Twist1-Tcf12 dimers in postnatal sutural stem cells, as well as how Tcf12 modifies the ability of Twist1 to engage regulatory regions necessary for suture maintenance. Fourth, we will use powerful imaging techniques to reveal the in vivo spatial patterns of Twist1-Tcf12 dimers within sutures. Fifth, we will take advantage of the first zebrafish model of Saethre-Chotzen syndrome to directly visualize over time how changes in the pattern and timing of osteoblast differentiation result in later coronal suture defects. The results of these aims will test our model that Tcf12 functions as a suture-specific partner for Twist1, in part by guiding Twist1 to particular genomic regions necessary to inhibit premature osteoblast differentiation in suture mesenchyme. These new insights into the long-term requirements of synostosis genes in suture maintenance will have the potential to lead to new ways of preventing post-operative synostosis, thus reducing the number of risky operations currently performed on young children with Saethre-Chotzen syndrome.

Birth defects of the head and face are common in the human population. Skull injuries and joint disease, in particular affecting the temporomandibular joint of the jaw, are a major economic and societal burden. This proposal is to support the upward trajectory of a mid-career investigator, Dr. Gage Crump, who works at the interface of craniofacial development and stem cell biology. Specifically, he has developed powerful new zebrafish models of human craniofacial birth defects and disease, which are allowing him to unravel the developmental causes of common craniofacial birth defects and, in a bold new direction, to understand mechanisms of stimulating endogenous repair of the adult skull. Dr. Crump is Director of the PhD Program in Development, Stem Cells, and Regenerative Medicine at the University of Southern California and a founding member of the Eli and Edythe Broad Center for Stem Cell Research, a rapidly growing institute directed by Dr. Andrew McMahon with exceptional core resources and recently recruited junior faculty. He is currently PI on three R01's from NIDCR and has published featured articles in Developmental Cell, eLife, Development, and PLoS Genetics on diverse topics ranging from craniofacial development to jawbone repair and arthritis of the jaw. He has built up an exceptional research team, with several trainees receiving K99 and F31 fellowships from NIDCR, as well as prestigious private fellowships. His previous trainees have gone on to tenure-track faculty and industry positions, and postdocs in HHMI-funded labs. He also participates in local and national efforts to recruit under-represented minority students into stem cell science from high school to graduate levels. These efforts are reflected by a USC Mentoring Award to Dr. Crump in 2017. The research program focuses on the roles of progenitor cells in building the facial skeleton and then maintaining and repairing it in the adult. These studies exploit the unique genetic and imaging strengths of zebrafish, combined with its impressive capabilities of natural regeneration as adults. The first program uses new gene editing technology in zebrafish to analyse requirements for novel craniofacial patterning genes in progenitor regulation, as well as to directly image progenitor lineage commitment using time-lapse microscopy. As exemplified by studies of Jagged-Notch signaling from fish to man, efforts to validate zebrafish findings in mouse will be performed in latter years. The second program investigates the stem cell-based maintenance of two types of joints, the sutures of the skull and the synovial jaw joint. The third program builds on innovative models of bone, cartilage, and joint regeneration in the adult zebrafish jaw, as well as a highly collaborative network of basic researchers and clinicians at USC, to understand how different types of endogenous stem cells are activated to repair craniofacial tissues. Completion of these studies will reveal commonalities and differences between the stem cells that build and then maintain and repair the craniofacial skeleton, with foundational knowledge acquired in zebrafish informing future regenerative approaches towards improving skeletal healing in patients.

This application is for renewal of a Predoctoral Training Program in Developmental Biology, Stem Cells, and Regeneration (DSR), which is administered at the University of Southern California (USC) Keck School of Medicine, in conjunction with affiliated faculty at the Children's Hospital Los Angeles. This training program leverages recent and dramatic growth in stem cell biology and regenerative medicine at USC, which is reflected in the creation of a new Department of Stem Cell Biology and Regenerative Medicine and a university-wide USC Stem Cell initiative that is investing large sums of money to recruit world leaders and promising junior faculty. Particular strengths of training-related research, which benefit from interactions with closely situated institutes and one of the largest public hospitals in the country, include skeletal biology, neural and sensory systems, kidney, digestive and metabolic organs, and cancer stem cells. The rationale of this training program is that cohesive, structured training in basic developmental and stem cell biology, coupled with training-grant-specific courses and activities that provide in-depth exposure to clinical problems, will best train the future generation of scientists in the field of regenerative medicine. A major strength of this training program is that it provides added value, in the form of clinical exposure, on top of a newly created DSR PhD program. In particular, each trainee is paired with a Clinical Co-Mentor, who guides the student in learning about the diseases to which their primary research relates. During the first four years of the training program, the ten funded trainees, plus an additional four trainees funded by the Dean's office, will have published 30 first-author manuscripts, received an NIH F31 fellowship, and won a number of honors and awards. The majority of trainees first enter USC through a Program in Biomedical and Biological Sciences (PIBBS) umbrella admissions program, which has seen a steady rise in the quality of its training-grant-eligible applicant pool. At the end of their first year, students join the lab of one of our 26 training faculty, matriculate in the DSR PhD program, and take an intensive summer core course in developmental and stem cell biology, followed by journal club and research presentation courses in their second year. Following a formal call for applications and external review, select students join the T32 training program in their second and sometimes third years, at which point they take a trainee-specific Clinical Perspective of Regenerative Medicine course and engage in a number of trainee-specific activities including an annual retreat, a student-led symposium, frequent interactions with their clinical co-mentor, and monthly lunch gatherings. The cohesive structure of this training program provides an extra level of clinical fluency that the trainees would not otherwise have obtained through the DSR program alone. By leveraging the unique clinical resources within the neighboring Los Angeles area with the recent growth in basic stem cell research at USC, this training program provides a focused educational experience for those promising young scientists who wish to make a future impact in regenerative medicine.

Project Summary In order for the jaw to properly function, the jaw skeleton must be integrated to the underlying muscles through tendons. The prevalence of tendon injuries is high and in some cases can contribute to temporomandibular joint disorders, yet both the development and repair of tendons are largely understudied. The skeleton and tendons of the jaw are derived from cranial neural crest cells (CNCCs), in contrast to the skeleton and tendons of the fins/limbs and spine that are derived from mesoderm. Do tendons derived from different lineages depend on similar or distinct upstream signals for their specification? This proposal tests the innovative idea that tendon specification is modular, with head- and trunk-specific transcription factors initiating tendon development through head- and trunk-specific enhancers of critical tenocyte factors such as Scleraxis. By conducting single-cell RNA expression profiling of CNCC derivatives in the developing zebrafish face, we have found that expression of the nr5a2 orphan nuclear receptor marks cells along a developmental trajectory toward jaw tendon fate. Utilizing mutant and transgenic zebrafish models, we find that Nr5a2 is necessary and sufficient for specification of jaw tendon at the expense of cartilage fates. In addition, by assaying open chromatin indicative of active enhancers in CNCCs, as well as head versus trunk tenocytes, we find a number of putative head- and trunk-specific enhancers of scleraxis-a. In this proposal, we use transgenic and cutting- edge genomics techniques to test that Nr5a2 directly binds and activates jaw-specific scleraxis-a enhancers. We also use conditional genetics in mouse to test that Nr5a2 has a conserved role in specifying jaw and middle ear tendons derived from the mandibular arch in mammals. Completion of these aims will reveal the regulatory logic by which tendons are specified in different parts of the body, as well as a highly specific role for the Nr5a2 orphan nuclear receptor in promoting jaw tendon formation.