Chin Chiang - US grants

Morphogenesis of the hair follicle is driven by inductive interactions involving signals emanating from the developing epidermis and underlying dermis, but the molecular nature of these signals has not been identified. Our preliminary studies suggest that interaction between Sonic hedgehog (Shh) expressed in the epithelium and fibroblast growth factor 10 (Fgf-10) expressed in the mesenchyme may play a critical role during early follicle morphogenesis. The secreted molecule encoded by the Shh gene has recently emerged as a key signal in patterning during vertebrate development. Our genetic analysis of Shh null mutant suggests that hair follicle development is arrested at the hair germ stage and that epidermal development is enhanced. Throughout the initial stages of follicle formation, Shh is specifically expressed in the epithelial cells in close contact with the underlying mesenchyme, suggesting a direct role in induction and differentiation of the mesenchyme. Fgf10, the only member in the Fgf gene family found to be selectively expressed in the selectively expressed in the mesenchyme during early follicle morphogenesis, lead us to propose that Fgf10 may play an instructive in inducing the follicle. The objective of this research is to understand the role of Shh during early skin morphogenesis and to test a candidate mesenchymal signal for induction for epithelial differentiation. We will generate Shh conditional mutant in the skin to determine basal cell fates, and transgenic misexpressing Fgf10 in the basal cells to study Fgf10 function during hair follicle morphogenesis. The results obtained from the proposed studies will be useful for future genetic and biochemical endeavors and may provide insights into the potential use of Shh and Fgf10 as therapeutic proteins to treat baldness related diseases.

In this application, our goal is to understand the molecular and cellular mechanisms involved in the differentiation of the foregut. Preliminary studies have indicated that the Sonic hedgehog (Shh) gene is required in foregut development. Absence of Shh results in severe foregut defects that are reminiscent of a spectrum of human foregut malformations, including esophageal stenosis and atresia, tracheoesophageal fistula, and lung hypoplasia. The defective separation of the primitive foregut into the respiratory and digestive tracts in Shh mutant embryos suggests that Shh plays an important role in normal foregut partitioning. Shh is expressed in the primitive foregut endoderm, making it likely that the normal separation of the foregut endodermal derivatives, the tracheal primordium and the future esophagus, is mediated by inductive processes involving the adjacent splanchnic mesoderm. Our proposal is aimed at testing the following hypotheses: (1) Shh signaling is essential for normal development of the foregut, (2) localized Shh activity is essential for proper patterning of the foregut, (3) Shh genetically interacts with Gli3 in foregut development. To accomplish these aims, we will conduct genetic epistasis experiments and perform extensive morphological and molecular analyses of the mutant foregut. We will generate mouse mutants expressing the diffusible form of Shh using a novel genetic approach. We will also conduct functional studies using recombinant and organ cultures. The results obtained from these experiments will add greatly to our understanding of Shh inductive signaling in foregut patterning and differentiation, and shed new light on the etiology of foregut- associated anomalies in humans.

DESCRIPTION (provided by applicant): In this application, our goal is to define the role of cholesterol and Gli3 in the control of Shh activity and signaling in the neural tube. The secreted molecule encoded by the Shh gene has been shown to play a key role in the development of the vertebrate central nervous system. The activity of Shh is thought to be controlled by cholesterol covalently linked at its N-terminus, but the precise function of this lipid modification in neuronal patterning is not understood. The response to Shh is known to depend on transcription factors of the Gli family, but the detailed mechanism is not understood. Our genetic studies indicated that in the absence of Shh, Gli3 represses ventral neuronal cell fates in a dose-dependent manner. Whereas Shh mutant embryos show reduction in several classes of interneurons and a complete absence of motor neurons, these cell types are rescued in Shh/Gli3 double mutants. These observations indicate that Shh is required to antagonize Gli3, which would otherwise repress ventral neuronal cell fates. The ability of Shh/Gli3 double mutants to generate motor neurons and interneurons strongly suggests that factor(s), in addition to Shh, is involved in the generation of these ventral neurons. Our proposal is aimed at testing the following hypotheses:(l) Cholesterol-modified Shh is essential for normal differentiation of ventral neuronal cell types in the CNS. (2) Shh counteracts Gli3 repressor function in the generation of ventral neuronal progenitor cells by controlling Gli3 processing. (3) Gli3 repressor interferes with RA signaling in the ventral neural tube. To accomplish these aims, we have generated five strong chimeric mice with an altered endogenous Shh locus, designed to express Shh without the cholesterol adduct (Shh-N) in a Cre recombinase-dependent manner. The cellular distribution and neuronal patterning activities of Shh-N will be determined upon germline transmission. We will also determine Gli3 processing using N-terminal Gli3-specific antibody that we generated and analyze patterns of gene expression in mutants with loss-and gain-of-function in Shh signaling. We will also utilize RARE-lacZ reporter mice, RAR antagonist and neural explants to define the relationship between Gli3 repressor and RA. These studies will deepen our understanding of Shh function in the neural tube, and shed new light on the etiology of neural tube-associated anomalies in humans.

[unreadable] DESCRIPTION (provided by applicant): Secreted molecules encoded by the Hedgehog (Hh) gene family have been recognized as key signals in regulating the growth and patterning of invertebrate and vertebrate embryos. Mutations in Shh genes encoding Shh signaling components have been associated with many clinical disorders found in humans including holoprosencephaly and various forms of cancer. One of the most salient features of Hh ligands is the ability to act as a classical morphogen in developing tissues. This is particularly evident in the developing vertebrate limbs, where Shh, normally expressed in the posterior limb margin of the zone of polarizing activity (ZPA), has ability to progressively specify increase in number of digits with more posterior identities in a dose-dependent manner. Previously, our studies identified the requirements of Shh in both growth and patterning of the skeletal elements of the limb. Additionally, we showed that this requirement is necessarily mediated by regulation of Gli3 processing. In this context, Shh secreted from the ZPA inhibits Gli3 processing into its repressor form (GN3R), hence promoting the accumulation of full-length GN3 protein (Gli3- FL or GH3-190), which can function as a transcriptional activator (GN3A) within the range of Shh signaling, thus, the relative balance between GN3A and GN3R likely plays a critical role in mediating the limb patterning function of Shh. Major gaps remain in understanding how the Shh activity gradient is regulated to generate defined patterns in the limb. Addressing this question requires a better understanding of factors affecting Shh movement, ligand-receptor interaction, patterning effects of paracrine activity, and transcriptional activity of effectors in the responsive tissue. Therefore, our proposed studies are aimed primarily at providing a better understanding of how Shh signaling is regulated and interpreted during limb development by addressing (1) the role of cholesterol moiety in regulating Shh movement/spatial range in a mammalian tissue environment, (2) the contribution of local and paracrine signaling in generating a complex pattern of digits, (3) the biological function of different forms of Gli3 in regulating tissue patterning. We believe this knowledge will be directly relevant to the role of Shh in human organogenesis, disease and cancer. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The cerebellum is essential for motor coordination and learning, and it is also implicated in contributing to cognitive function and social learning. Alterations in specific aspects of these functions are associated with various debilitating developmental diseases including ataxia and autism spectrum disorder in humans. The cerebellar circuitry is comprised of limited number of inhibitory and excitatory neurons that are integrated in the corticonuclear network, with Purkinje neurons (PN) being the sole output neurons. Precise assembly of this circuitry which governs cerebellar function is likely dependent on the balanced production of inhibitory and excitatory neurons from their precursors, yet these processes remain poorly understood. During the current funding period, we have shown that the PN is a central regulator of late-born neural precursors in the postnatal cerebellum, coupling the generation of excitatory and inhibitory interneurons and facilitating their coordinated integration into the emerging cerebellar circuit. PN accomplish these crucial tasks through disseminating Sonic hedgehog (Shh) to functionally and spatially distinct neurogenic niches, namely external granule layer and prospective white matter (PWM). Although Shh can travel far from its site of synthesis, the mechanisms remain unknown. The PWM consists of a heterogeneous population of precursors and the function of Shh signaling specifically in inhibitory neuronal precursors needs to be determined. Moreover, Shh-derived from PN also signal to neighboring Bergmann glia (BG), and how such regulation is integrated with the generation of excitatory neurons is also unknown. The goal of this proposal is to fill these critical gaps using a combination of multidisciplinary approaches.

Project Summary: The Shh pathway plays critical roles in development, stem cell maintenance and tissue homeostasis. Deregulated Shh signaling during cerebellar development causes medulloblastoma, the most common pediatric brain tumors in childhood with presumed cellular origin in granule cell precursors (GCPs). During postnatal development, GCPs undergo rapid and transient proliferation in the outer external granule layer (EGL) in response to Shh signaling before differentiating and migrating inward to become granule neurons. Persistent Shh signaling counters this stereotypic developmental pattern, resulting in disrupted differentiation and prolonged stay of GCPs in the outer EGL where cerebellar neoplasm is thought to initiate. Therefore, elucidating the mechanism by which CGP proliferation and differentiation are regulated is important to our understanding of cerebellar tumorigenesis. Deregulated cellular growth and proliferation are hallmarks of neoplasms that entail energy-consuming anabolic processes such as protein synthesis and lipogenesis. These anabolic processes are highly regulated and subject to stringent control by cellular energy sensors. The key energy sensor that is activated under condition of energy depletion is AMP-activated protein kinase (AMPK). We discovered that AMPK is a potent inhibitor of Shh signaling in GCPs and primary medulloblastoma cells. Moreover, AMPK is selectively activated in the inner EGL of the developing cerebellum where Shh signaling is downregulated and GCPs have begun differentiating. These observations suggest that AMPK may play a critical role in modulating Shh signaling in GCPs for differentiation, proliferation and tumorigenesis. We will test this hypothesis by three aims: 1) Elucidate the mechanism by which AMPK activation antagonizes Shh pathway activity; 2) Determine the role of AMPK in modulating GCP proliferation and differentiation in vivo; 3) Determine the effect of AMPK activation in Shh-driven medulloblastoma in vivo.

Project Summary The cerebellum is essential for motor coordination and learning, and it is also implicated in contributing to cognitive function and social learning. Alterations in specific aspects of these functions are associated with various debilitating developmental diseases including ataxia and autism spectrum disorder in humans. The cerebellar circuitry is comprised of limited number of inhibitory and excitatory neurons that are integrated in the corticonuclear network, with Purkinje neurons (PN) being the sole output neurons. Precise assembly of this circuitry which governs cerebellar function is likely dependent on the balanced production of inhibitory and excitatory neurons from their precursors, yet these processes remain poorly understood. we have previously shown that the PN is a central regulator of late-born neural precursors in the postnatal cerebellum, coupling the generation of excitatory and inhibitory interneurons. PN accomplish these crucial tasks through disseminating Sonic hedgehog (Shh) to functionally and spatially distinct neurogenic niches, namely external granule layer and prospective white matter (PWM). Although Shh can travel far from its site of synthesis, the mechanisms remain unknown. The PWM consists of a heterogeneous population of precursors and the function of Shh signaling specifically in inhibitory neuronal precursors needs to be determined. Moreover, Shh-derived from PN also signal to neighboring Bergmann glia (BG), and how such regulation is integrated with the generation of excitatory neurons is also unknown. The goal of this proposal is to fill these critical gaps using a combination of multidisciplinary approaches.