Henk Roelink - US grants

DESCRIPTION: (Adapted from the Applicant's Abstract) Cyclopamine is a teratogenic steroidal alkaloid which was isolated from the desert corn lily. Vertebrate embryos exposed to cyclopamine around the period of gastrulation develop brain and craniofacial malformations analogous to humans with holoprosencephaly. Holoprosencephaly has also been associated with loss of function mutations in the Sonic Hedgehog gene and abnormalities in cholesterol metabolism. Sonic Hedgehog is expressed in the notochord and prechordal plate and encodes a protein (SHH) that directs pattern formation in the ventral portion of the neural tube. SHH synthesis is followed by an autoproteolytic event whereby the amino-terminal cleavage product is attached to cholesterol, thereby affixed to the outer surface of the cell. Because cyclopamine is structurally similar to cholesterol, it is hypothesized that cyclopamine interferes with SHH-mediated signal transduction. Preliminary data support a model in which cyclopamine blocks SHH autoproteolytic processing. The effect of cyclopamine might be direct disruption of SHH association with cholesterol or indirect suppression of cholesterol biosynthesis. Hypocholesterolemic drugs (BM-15.766, AY-9944) which interfere with cholesterol biosynthesis produce similar teratogenic effects. The hypothesis will be tested by addressing the several aims. First, the effects of cyclopamine and hypocholesterolemic agents on SHH-mediated patterning events in the neural tubes of avian and hamster embryos will be determined. It is expected that each of these agents will induce abnormal morphological and biochemical patterns in the neural tube, comparable to those observed in SHH-deficient embryos. The teratogenic potential of various natural and synthetic structural analogues of cyclopamine will also be examined and the teratogenic potential of various natural and synthetic structural analogues of cyclopamine will be examined and the teratogenicity of all these agents will be correlated with their effects on SHH autoproteolysis and cholesterol biosynthesis, as measured in vitro. The goals are to elucidate the molecular events which underly the teratogenic effects of cyclopamine and related steroidal alkaloids, and to characterize the structural properties of teratogens which mediate these effects. The proposed research may have important environmental health implications since steroidal alkaloids are found in many agricultural products.

Description: The Sonic Hedgehog is a signaling molecule that is required for normal development of the central nervous system. The response to Shh is complex, and can be changed by environmental compounds like cyclopamine. Changes in the Shh response result in a specific type of embryo malformations characterized by defects of the neural midline, like holoprosencephaly, which can be reflected in the face as cyclopia or hypotelorism. All cell types in the ventral neural tube develop as a consequence of Shh signaling, and it is likely that small changes in the Shh response has subtle effects on the formation of ventral cell types. This in turn might result in congenital neurological defects. It has been determined that the Shh response is influenced by the cyclic nucleotide concentration within the responding cells. Increasing the camp concentration attenuates the Shh response, while loss of the camp dependent kinase (PKA) activates the Shh response. These authors showed that increasing the cGMP concentration also enhances the response to Shh, suggesting a model in which the Shh response is dependent on the cyclic nucleotide concentration within the responsive cells, and that camp and cGMP have opposite effects on the Shh response. Several compounds present in the environment can alter the intracellular cyclic nucleotide concentration, either by activating GTP/ATP cyclases, enzymes that generate cyclic nucleotides, or by blocking phosphodiesterases, enzymes that degrade cyclic nucleotides. It is hypothesized that environmental compounds that change the cyclic nucleotide complement of a cell, alter Shh response in such cells, resulting in embryo malformations and thus birth defects. The hypothesis will be tested using sensitive assays of the Shh response in the chick embryo. It will be determined if environmental compounds that change the cyclic nucleotide complement of a cell interfere with normal Shh signaling in the developing neural tube in vivo, or in neural explants in vitro. In humans, exposure to the compounds that will be tested is either voluntary, like forskolin, or involuntary, like bacterial enterotoxins, but in either case little is known about their possible adverse effects on early embryos and thus as a cause of birth defects.

DESCRIPTION (provided by applicant): Sonic Hedgehog is a morphogen. To fulfill this role, it is necessary that it is able to travel many cell diameters away from its source of synthesis through the responding tissue. This requirement appears to be in conflict with the biological properties of Shh; it both contains an Acyl and a cholesterol moiety, which severely curtail its solubility in a hydrophilic intercellular environment. Although a functional, soluble hexameric form of Shh has been identified, medium conditioned by Shh expressing cells do not accumulate Shh to levels high enough to elicit a response in neural plate explants. However, when such responsive tissue is in contact with Shh producing cells, long-range Shh signaling can be observed in the explant, indicating that Shh is actively transported in the plane of the responding tissue. It is hypothesized that this long-range transport is mediated in part by transcytosis, which is supported by observation in Drosophila that export of cholesterol-associated Shh homolog Hedgehog (Hh) requires the activity of a dedicated molecule, Dispatched (Disp), while the uptake of Shh and Hh is mediated by the Disp homolog Patched (Ptc). In addition, the Ptc1-mediated trafficking of Shh into late endosomes is required to initiate the Shh response. To complete transcytosis in these responding cell it is necessary that Shh is transported from the late endosomes to the cell surface and made available to neighboring cells, and it is hypothesized that this is mediated by Disp. The hypotheses will be tested determining if Ptc1-mediated and Disp1-mediated trafficking of Shh are required in concert for long-range signaling. Determine the long-range signaling efficacy of alternatively anchored forms of Shh. It will be determined if the acidic environment in late endosomes favors Shh binding to Dispatched, while the neutral environment in the intercellular space allows Shh to bind to Ptc1, creating a continuous chain of events resulting in transcytosis. The requirement for intracellular transport in generating a long-range signal will be tested by blocking the function of Rabs and Dynamin. The Shh response coupled to transcytosis allows the generation of a graded Shh response, in a tissue actively responding to Shh, without the requirement for long-range transport in the intercellular space, and provides an explanation for the unusual activities of Ptc1 and Disp1. Better understanding of the mechanism by which the long-range Shh signal forms is important. Inappropriate activation of the Shh response is the cause of a wide variety of cancers, including a high fraction of pancreatic tumors, while normal Shh signaling is required for the development of many organ systems.

[unreadable] DESCRIPTION (provided by applicant): The enclosed proposal represents a competitive renewal of an established graduate training program in Genetics at UC Berkeley. We request funds to support 17 graduate trainees in the areas of Developmental Genetics and Genomics, Cell & Systems Genetics, and Population Genetics and Evolution. Students enter this inter-disciplinary training program after gaining admission into one of four Departments on campus. Two of the Departments, Molecular and Cell Biology (MCB) and Integrative Biology (IB), are located within the College of Letters and Sciences. A third Department, Plant and Microbial Biology (PMB), is located in the College of Natural Resources. Students can also enter the training program through the School of Public Health (SPH). Students are selected into the program based on an interest in one of the broadly defined areas of genetics that are sponsored by this training grant. They are also selected based on merit and future promise as independent investigators. Despite their diverse backgrounds, the training program includes a number of mechanisms to ensure that all of the graduate students obtain rigorous training in genetics. Once they enter the program, students are free to select any one of 41 different faculty mentors located in the four aforementioned performance sites. These faculty employ a variety of genetic and genomic methods to study a broad spectrum of problems in cell, developmental, and evolutionary biology, including metazoan and plant patterning, embryogenesis, sex determination, cell determination and differentiation, morphogenesis, and organogenesis. The study of cellular processes in the areas of signal transduction, DNA replication, transcription, cell trafficking and the cytoskeleton, transcription, and DNA replication are the focus of many faculty. Other faculty use genetic approaches to study evolutionary processes such as the evolution of chordates and patterning in invertebrates. In the first year of the program, trainees complete advanced lecture, laboratory, and seminar courses. Students in PMB and MCB are also required to complete three 10-week laboratory rotations with potential dissertation mentors. All students commence their dissertation research by the beginning of the second year. They are also required to complete an oral qualifying exam that is administered by four faculty from at least two different departments within the training program. All students are required to complete at least two semesters of teaching as graduate instructors during the second and third years of the training program. In addition to formal course offerings in the different areas of genetics, students are expected to participate in a variety of seminar programs, joint lab meetings, journal clubs, and retreats that are sponsored by the Genetics Training Program. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Sonic Hedgehog (Shh) is a signaling molecule that undergoes post-translational modifications resulting in the addition two lipophilic moieties to the protein, rendering it obligatory membrane associated. Despite these modifications, during development, in stem cell niches, and in tumors Shh signals to cells often several cell diameters away from the sites of synthesis. Two related molecules, Disp1 and Ptc1, are required for the secretion of Shh and its uptake in neighboring cells respectively. It is hypothesized that transport of Shh involves the reiterated and complementary actions of Disp1 and Ptc1 generating and absorbing Shh containing complexes (either lipoprotein particles or exosomes) thus mediating long-range transport while generating a Shh activity gradient. Three central observations support this hypothesis, i/ cells without Disp1 do not secrete Shh, ii/ Ptch1 in surrounding cells is required for the uptake of Shh and iii/ tissues without Disp1 are less able to transport Shh. Ptch1 and Disp1 are members of a large family of trimeric, proton driven antiporters, and in the first aim it is tested if Ptch1 and Disp1 function is required for planar transcytosis of Shh. In aim 2 the apicobasal polarity of the transport as well as the response is addressed, to reconcile basolateral transport with the apical localization of the primary cilium. Aims 3 and 4 address the Shh itinerary as well as the role of the putative proton driven pump activity of Ptch1 and Disp1 in the transport of Shh, and the associated relocation of Smoothened.

? DESCRIPTION (provided by applicant): Sonic Hedgehog (Shh) signaling is important both during development and in adults. Defects in Shh signaling is associated with severe birth defects such as holoprosencephaly, while inappropriate activation of the Shh response contributes to the formation of common and deadly tumors such as medulloblastomas and pancreatic cancer. Shh is a morphogen, and it is crucial that the Shh response is precisely regulated for normal Shh-mediated patterning to occur. The central players in the Shh response are Patched1 (Ptch1) and Smoothened (Smo). Ptch1 is a potent inhibitor of Smo, and it is shown that this inhibition requires the proton-driven antiporter activity of Ptch1. It has been suggested that binding of Shh to Ptch1 inhibits the antiporter activity of Ptch1, thus releasing th inhibition of Smo resulting in its activation. This model is incomplete as demonstrated by our finding that forms of Ptch1 mutant for its antiporter activity can mediate the activation of Smo. Further complicating the assessment of Ptch1 activity is our finding that its paralog Ptch2 can mediate Shh signaling, in particular in Ptch1-/- cells. Based on our preliminary results it is hypothesized that the proton-driven antiporter activity of Ptch1/2 is required for inhibition of Smo, while independently, the loop2 of Ptch1 can mediate the activation of Smo. The first Aim will examine the extent of functional overlap between Ptch1 and Ptch2 in their ability to inhibit Smo. It will test the distinct roles of Ptch1/2 in the activation of Smo by assessing if proton antiporter-mediated inhibition of Smo and the Ptch1- mediated activation of Smo can be mediated by distinct Ptch1/2 mutants, that together can restore a normal Shh response to Ptch1-/-;Ptch2-/- cells. It is thought that the substrate of the proton-driven antiporter activity f Ptch1/2 inhibits Smo. As many proton driven antiporters mediate the efflux of their cargo, it is tested in Aim 2 if Ptch1/2 containing cells can inhibit Smo in adjacent cells and the nature of the Ptch1/2 antiporter substrate is further assessed. Besides changes in transcription, cells can also respond to Shh by chemotaxis, and under these conditions Smo acts as a G-Protein Coupled Receptor. In Aim 3 it is addressed how Ptch1/2 affect Smo activity while it relays the Shh chemotactic signal. All aims take advantage of a unique panel of mouse embryonic stem cell lines that are deficient for most combinations of Ptch1, Ptch2, Smo and Shh in addition to Disp1, Boc and Cdo. This panel of cells has been critical to demonstrate the overlapping functions of Ptch1 and Ptch2, and will provide an ideal cellular background to test the activity of Ptch1 and Ptch2 mutants without interference by the endogenous proteins. The unraveling of the events leading to the inhibition and activation of Smo will not only help gaining a deeper understanding of birth defects cause by an aberrant Shh response, but will be particularly important in providing new targets for Shh pathway inhibition when inappropriately activated in tumors.

Hedgehog (Hh) signaling plays a central role during development in most animals, including mammals, while aberrant activation of the Hh response is associated with common cancers. Hhs are synthesized as pro-proteins that undergo an autoproteolytic event yielding the active N-terminal domain (HhN), and a C-terminal domain (HhC). HhN has 3 domains: its N-terminus binds the Hh receptor Patched (Ptch), the middle domain binds calcium (Ca) and the third domain coordinates zinc (Zn). The Ca- and Zn-binding domains (the bulk of HhN) are up to 65% identical to some bacterial peptidases (BacHhs), including all residues predicted to mediate catalysis and allosteric regulation. Many of these conserved residues are found mutated in holoprosencephaly, a congenital birth defect, emphasizing their importance for normal Hh function. Preliminary studies show that the peptidase activity intrinsic to Shh (a vertebrate Hh) is required for its release into the extracellular matrix (ECM) and the supernatant and thus for non-cell autonomous signaling. The inclusion of Shh in a highly conserved family of peptidases that spans multiple domains of life, combined with observations that the peptidase activity of Shh is required for Shh distribution and signaling form the premise of the hypothesis that ShhN is a Ca- regulated Zn-peptidase that mediates its release to allow non-cell autonomous signaling. Using Shh mutants in addition to BacHh/Shh mosaics, Aim1 will examine Shh residues that are required for catalysis, substrate recognition, and Ca regulation by assessing Shh distribution and signaling. Aim 2 will determine the substrates of Shh-mediated catalysis. Two plausible, non-mutually exclusive, substrates for Shh are Shh itself, including the removal of hydrophobic anchors or the release of the Ptch1-dinding domain, and ECM proteoglycans. In particular, heparan sulphate proteoglycans (HSPGs) that affect Shh signaling and extracellular distribution are assessed as substrates. Further characterization of the Shh-associated peptidase activity and identification of substrates will have major ramification in understanding the role of Shh as a developmental morphogen and will likely provide new targets to suppress non-cell autonomous Shh signaling that is a driving force in several cancers.