The Hedgehog signaling pathway is a critical regulator of tissue patterning and homeostasis. First discovered for its role in fruit fly segmentation, the pathway contributes to digit specification, neural tube differentiation, hair follicle cycling, and a variety of other processes in mammals. Dysregulated Hedgehog pathway activation also promotes tumorigenesis in children and adults, including basal cell carcinoma, medulloblastoma, and rhabdomyosarcoma. Small-molecule modulators of the Hedgehog pathway are therefore useful tools for studying its embryonic and physiological functions and for dissecting the molecular mechanisms that regulate pathway activity. Hedgehog pathway inhibitors also constitute a new class of targeted anti-tumor drugs, as Smoothened inhibitors have demonstrated remarkable efficacy against certain human cancers. Our laboratory has been involved in the discovery and/or mechanistic characterization of several Hedgehog pathway antagonists and agonists, including the cyclopia-inducing natural product cyclopamine, the SANTs (Smoothened ANTagonists), SAG (Smoothened AGonist), purmorphamine, robotnikinin, JK184, and the HPIs (Hedgehog Pathway Inhibitors) (Figure 1). Many of these compounds directly target canonical Hedgehog pathway components, whereas others disrupt cellular processes required for Hedgehog signal transduction. For example, HPI-4 (ciliobrevin A) is the first specific inhibitor of cytoplasmic dyneins.
Our current studies are focused on inhibitors that act downstream of Smoothened, since how this transmembrane protein regulates Gli function remains enigmatic and tumor resistance to Smoothened-targeting drugs is an emerging challenge. In collaboration with the Stanford High-Throughput Bioscience Center and the Conrad Prebys Center for Chemical Genomics at the Sanford-Burnham Medical Research Institute, we have identified several small molecules that are epistatic to Suppressor of Fused, a direct negative regulator of the Gli transcription factors. We are now investigating how these compounds perturb Gli function and evaluating their efficacies in mouse tumor models.
Current models of Hedgehog signal transduction are largely derived from organism-based genetic screens, which have revealed canonical signaling proteins conserved across species and vertebrate-specific modulators. The vertebrate Hedgehog pathway also utilizes a specialized signaling center called the primary cilium to regulate specific molecular interactions (Figure 2). Within this general framework, several aspects of Hedgehog pathway regulation remain enigmatic, such as the mechanisms by which Patched1 inhibits Smoothened and Smoothened regulates Gli function. To bridge these gaps in our knowledge, we have conducted genome-scale cDNA overexpression, siRNA knockdown, and CRISPR knockout screens to discover new Hedgehog pathway modulators.
For example, we have identified the atypical Rho GAP family member Arhgap36 as a potent Hedgehog pathway activator. This signaling protein acts in Smoothened-independent manner to drive the activation of Gli transcription factors, and it requires ciliary transport proteins such as Ift88 and Kif3a for its function. We also observe that Arhgap36 can functionally and biochemically interact with Suppressor of Fused. Interestingly, Arhgap36 is upregulated in murine medulloblastomas that have acquired resistance to Smoothened antagonists, and a subset of human medulloblastomas overexpress this putative Rho GAP protein as well. Our current model is that Arhgap36 promotes oncogenic Gli activation, and we are now investigating the biochemical mechanisms that underlie this process and how they contribute to medulloblastoma etiology and progression.
While many aspects of developmental signaling can be studied in cultured cells, model organisms are required to gain a comprehensive understanding of how these pathways regulate embryonic patterning, tissue regeneration, and tumorigenesis. Our laboratory uses the zebrafish as a model of vertebrate biology, taking advantage of its facile aquaculture and husbandry, rapid development, optical transparency during embryogenesis, compatibility with forward-genetic approaches, and accessibility to chemical perturbations. Realizing the full potential of zebrafish in biomedical research, however, will require new technologies for manipulating and visualizing the genetic programs within this model organism.
Toward this goal, we have developed caged morpholinos that enable light-or enzyme-controlled gene silencing in zebrafish embryos (Figure 3). With these reagents, we can inhibit in vivo gene function with spatial and temporal precision, creating the chemical equivalent of genetically mosaic organisms. We have also integrated caged morpholinos (MOs), photoactivatable fluorophores, fluorescence-activated cell sorting, and RNA profiling technologies to investigate how transcription factors dynamically regulate embryonic patterning (Figure 4). Our findings have demonstrated a remarkable degree of functional plasticity during No tail-a (Ntla)-dependent notochord development and elucidated the mechanisms by which Ntla and T-box gene 16 (Tbx16/Spadetail) regulate medial floor plate formation. We have also characterized the Tbx16-dependent transcriptome that drives paraxial mesoderm development, revealing an unexpected role for this transcription factor in hox gene activation (Figure 5).
Optogenetic tools are an emerging interest in our laboratory, particularly those that enable light-dependent of developmental signaling pathways. Our work in this area has primarily focused on light-oxygen-voltage (LOV) domains, compact flavin-binding photoreceptors that undergo conformational changes in response to blue light. For example, we have used a photoactivatable transcription factor (GAVPO) to control gene expression and cell ablation in zebrafish embryos (Figure 6). GAVPO is composed of the VIVID LOV domain, GAL4 DNA-binding domain, and p65 activation domain, and it forms a transcriptionally active dimer in a light-dependent manner. We are currently using LOV domains from various microbial and plant species to engineer new optogenetic variants of tissue-patterning genes.
Finally, we have developed methods for imaging photoluminescent lanthanide chelates in vivo. These metal complexes have large Stokes shifts, narrow emission lines, and long-lived luminescence that can be differentiated from background autofluorescence. Lanthanide-based probes are widely used for ultrasensitive photometric detection (e.g., in microplate assays); however, their application in optical microscopy has been relatively limited. In collaboration with Pehr Harbury, we have integrated Q-switched laser (QSL) transreflected illumination, luminescence resonance energy transfer, and time-resolved microscopy to achieve ultrasensitive lanthanide imaging (Figure 7). This new approach (QSL trLRET) is compatible with both live and fixed zebrafish embryos, and our laboratory is now developing lanthanide-based probes for visualizing vertebrate development at the molecular level.
Our research group is also interested in the germ cell biology that precedes embryonic development. We recently identified homeodomain-interacting protein kinase 4 (HIPK4) as an essential driver of spermiogenesis, the process by which spermatids differentiate into mature spermatozoa. Hipk4 knockout male mice are completely sterile but otherwise appear have normal physiology. Our studies show that HIPK4 is predominantly expressed in round and elongating spermatids and required for spermatid head shaping. This remarkable morphological transformation involves external Sertoli cell-generated forces and the acroplaxome, an F-actin-scaffolded plate that circumscribes the anterior pole of spermatid nucleus (Figure 8). Our findings support a role for HIPK4 in remodeling the acroplaxome during spermatid differentiation, and we are now seeking to identify the direct substrates and downstream effectors of this kinase. We are concurrently pursuing the development of HIPK4-specific inhibitors, with the hope that these small-molecule probes could lead to new strategies for reversible, non-hormonal male contraception.