Linda A. Barlow - US grants

taste buds; embryogenesis; developmental neurobiology; neuronal guidance; innervation; nonmammalian vertebrate embryology; oral pharyngeal; gastrointestinal epithelium; tissue /cell culture; Urodela; immunocytochemistry;

DESCRIPTION (provided by applicant): Brain Derived Neurotrophic Factor (BDNF) is the neurotrophin that appears to permit survival of gustatory ganglion cells during development. However, whether it also serves to direct axonal ingrowth, or cues ingrowing axons to invade particular target tissues is unclear. Furthermore, BDNF continues to be expressed by adult taste buds and its role in adult life is unknown. The proposed experiments utilize novel experimental approaches to investigate the role of BDNF in both developing and mature taste buds. In vitro techniques will be used to test whether axons emerging from the progenitor cells of gustatory ganglia require BDNF for either targeting or as a cue to invade target tissues. For these experiments the epibranchial placodes, which give rise to the gustatory ganglia of the VII and IX cranial nerves, will be cultured with appropriate and inappropriate epithelium taken from early embryos; i.e., before differentiation of gustatory primordia has begun. Cultures will be treated with function blocking BDNF or control antibodies to assess whether BDNF plays a role in either directing axonal outgrowth or in promoting invasion and innervation of target tissues. Other experiments will utilize ELISA in situ techniques to test whether mature taste buds release BDNF either constitutively or under conditions of stimulation. Finally, we will utilize conditional tissue-specific knockout transgenic mice to test whether elimination of BDN-F in adults has any effect on gustatory ganglion cell survival, or on maintenance of taste buds. For these experiments, we will cross two existing transgenic lines, one carrying a floxed BDNF construct, and the other carrying a tamoxifen-receptor-linked Cre, driven by the cytokeratin 14 promoter. Thus administration of tamoxifen to these adult mice will result in excision of the BDNF coding sequence in all epithelial tissues, including taste buds. By using conditional, tissue specific knockouts, we will be able to separate the effects of elimination of BDNF in adulthood from its trophic effects already documented for the embryonic period.

DESCRIPTION (provided by applicant): Brain Derived Neurotrophic Factor (BDNF) is the neurotrophin that appears to permit survival of gustatory ganglion cells during development. However, whether it also serves to direct axonal ingrowth, or cues ingrowing axons to invade particular target tissues is unclear. Furthermore, BDNF continues to be expressed by adult taste buds and its role in adult life is unknown. The proposed experiments utilize novel experimental approaches to investigate the role of BDNF in both developing and mature taste buds. In vitro techniques will be used to test whether axons emerging from the progenitor cells of gustatory ganglia require BDNF for either targeting or as a cue to invade target tissues. For these experiments the epibranchial placodes, which give rise to the gustatory ganglia of the VII and IX cranial nerves, will be cultured with appropriate and inappropriate epithelium taken from early embryos; i.e., before differentiation of gustatory primordia has begun. Cultures will be treated with function blocking BDNF or control antibodies to assess whether BDNF plays a role in either directing axonal outgrowth or in promoting invasion and innervation of target tissues. Other experiments will utilize ELISA in situ techniques to test whether mature taste buds release BDNF either constitutively or under conditions of stimulation. Finally, we will utilize conditional tissue-specific knockout transgenic mice to test whether elimination of BDN-F in adults has any effect on gustatory ganglion cell survival, or on maintenance of taste buds. For these experiments, we will cross two existing transgenic lines, one carrying a floxed BDNF construct, and the other carrying a tamoxifen-receptor-linked Cre, driven by the cytokeratin 14 promoter. Thus administration of tamoxifen to these adult mice will result in excision of the BDNF coding sequence in all epithelial tissues, including taste buds. By using conditional, tissue specific knockouts, we will be able to separate the effects of elimination of BDNF in adulthood from its trophic effects already documented for the embryonic period.

DESCRIPTION (provided by applicant): Taste buds are multicellular receptor organs of the gustatory system, and are innervated by a discrete set of cranial nerves. For nearly a century, taste buds were believed to form late in embryonic development following induction by nerves. However, recent findings have shifted this view dramatically. Early taste bud development is not dependent upon nerves. Further, taste buds arise via processes intrinsic to the tongue, processes that occur long before taste buds differentiate. Most recently, we have demonstrated that epithelium destined to give to taste buds, the pharyngeal endoderm, receives signals very early, during gastrulation, that initially specify, or direct the fate of, this embryonic tissue. Further, cell-cell interactions within this specified endoderm regulate the subsequent patterning of this epithelium so that it gives rise to a distributed array of taste buds. We are now poised to expand substantially on our earlier model, and test detailed hypotheses concerning these key early events in taste bud development. In this proposal, molecular and cellular mechanisms that first specify (fate is directed but still reversible) and then determine (fate is irreversibly selected) the ability of pharyngeal endoderm to generate taste buds will be assessed. An additional question addressed in this aim is whether the same signals are responsible for both processes (Aim 1). Studies of taste bud patterning will be expanded to the molecular realm, to determine if, based upon suggestive gene expression patterns, the Notch signaling pathway is involved in patterning taste buds (Aim 2). Finally, to meld data from mammalian taste bud development with our own, genes known to be present in developing taste organs of mice will be cloned from axolotls and expression of these gene products will be explored in the developing taste epithelium of axolotls. By comparing gene expression patterns across species, we will identify genes associated in general with taste bud development (Aim 3).

[unreadable] DESCRIPTION (provided by applicant): The taste periphery comprises a distributed array of taste buds innervated by distinct cranial nerves. In recent years, theories of the embryonic development of this system have undergone radical revision. Previously, taste buds were thought to be induced from indifferent epithelium by in growth of taste nerves; but new results demonstrate that early events in taste bud development occur before, and in the total absence of, innervation, raising anew questions of how taste buds develop. We now propose to examine this problem by taking advantage of the excellent molecular genetics of the mouse model system. In mice, lingual taste buds are restricted to papillae, which form by mid-gestation and likely contain taste bud progenitors; yet taste buds proper do not appear until postnatal stages. Two signaling factors (Sonic Hedgehog [Shh] and Bone Morphogenetic Proteins [BMPs]) are expressed in a subset of papillary epithelial cells, and both pathways influence murine taste development. Our preliminary data suggest that the Shh- expressing cells may be taste bud stem cells. And our newest findings implicate yet another, crucial signaling system, the Wnt pathway, in taste organ development, as gain or loss of Wnt signaling in embryonic tongues significantly affects papillae. Interactions of these 3 signaling pathways (Wnt, Shh and BMP) are known to drive development of many other epithelial specializations, including hair, feather and teeth, but their precise roles in taste organ development are unknown. With a nerve-independent view of early taste organ development, and a molecular genetic approach, we are now poised to work out the molecular mechanisms governing development of this vital sensory system. In the following 3 aims, we will test the overall hypothesis that: Taste buds originate from embryonic Shh-expressing stem cells whose development is regulated by interactions of the Wnt, Shh and BMP pathways. Aim 1: Ascertain whether embryonic Shh-expressing taste placode cells are taste bud stem cells. Aim 2: Determine how Wnts interact with BMP and Shh to regulate taste organ development. Aim 3: Test if epithelial Wnt signaling is sufficient for taste organ development. Successful completion of these aims is important for issues of human health related to taste function, as well as more generally to enhance knowledge of embryonic development and its misregulation. [unreadable] [unreadable] [unreadable]

Over ttie past 2 decades, advances in mouse molecular genetics tiave lead to significant discoveries in neural function and development, findings that simply are not attainable without these valuable animal resources. The widespread use of genetically engineered mice has resulted in a huge array of available altered strains, which are shared widely among the research community. Additionally, the technology for generating genetically altered mice has continued to improve, and has become a conventional tool for manipulation of candidate genes. Importantly, conditional mouse genetic lines are now readily available that allow temporal and tissue specific manipulation of genes, either transiently, or indelibly. Researchers in the RMTSC employ mouse lines representing each of these aspects of cutting edge mouse molecular genetics, and in doing so, are advancing the field significantly. The primary function of Core A is to support RMTSC researcher use of mice, by maintaining breeding stocks of genetically altered mice, assisting in developing complex breeding strategies to produce offspring with the correct engineered alleles for genetic experiments, providing highly efficient genotyping of offspring, and imparting concise technical advice in the design of constructs for the genesis of new mouse lines. Given the success of our Core A in supporting mouse work through these services to RMTSC researchers in the past 9 years, the 3 overarching aims of this Core proposal remain unchanged from those ofthe previous submission. Aim 1. To maintain genetically altered mice. Aim 2. To genotype offspring of genetically altered mice.. Aim 3. To advise investigators in designing constructs for generation of genetically altered mice.

DESCRIPTION (provided by applicant): Taste dysfunction, either loss or alteration in sensation, is a common occurrence during radiotherapy for head and neck cancer. In addition to anti-tumor effects, however, ionizing irradiation frequently causes significant side effects in the surrounding oral mucosa (blistering/mucositis) and salivary glands (dry mouth/xerostomia). Although protocols have been developed to minimize some oral sequelae, mitigation of the impact of irradiation on taste function has remained elusive. Importantly, patients with reduced taste sensation tend to lack appetite and eat far less, leading to weight loss, as well as a significantly compromised quality of life. While radiotherapy induced taste loss is a well-documented side effect of cancer treatment, the precise target(s) responsible have not been determined. Plausible explanations have been put forth, including indirect effects, i.e., reduced salivation and oral blistering, and direct mechanisms such as disruption of taste bud innervation, loss of particular taste receptor cell type(s), as well as loss of taste progenitor cells. Our preliminary data favor the hypothesis that proliferating taste bud progenitor cells are the direct targets of radiation damage. Accordingly, in Aim 1 of this proposal, we will determine the kinetics of taste cell renewal by taste bud progenitors, and examine how irradiation affects this actively cycling progenitor population. We will also define the contribution of radiation-induced cell cycle arrest, DNA repair and apoptosis to taste cell renewal following injury. In Aim 2 we will investigate the role of the novel protein kinase C delta isoform (PKC4) in irradiation-induced taste epithelial injury. PKC4 is a key regulator of irradiation-induced apoptosis, i.e., suppression of PKC4 protects irradiated salivary gland cells from death. PKC4 has also been implicated in maintenance of cell cycle arrest required for DNA repair in UV damaged human keratinocytes. Thus, we will test the hypothesis that loss of PKC4 protects taste progenitor cells from death, and/or promotes their continued mitosis following irradiation injury. This model is suggested by our preliminary data, which indicate that, while PKC4 KO mice possess normal taste epithelia, in response to irradiation, their taste bud progenitor cell population appears to be protected and continues to proliferate. In sum, our taste model, where progenitors and differentiated taste receptor cells are distinct and readily identifiable, will allow us to better understand the precise cell population(s) targeted by head and neck radiation. Further, the defined cellular organization of taste epithelium will allow us to tease apart the functions of PKC4 in taste epithelium, and lead potentially to development of specific pharmacological inhibitors of PKC4 to protect oral tissues from irradiation-induced damage. PUBLIC HEALTH RELEVANCE: Radiation therapy of head and neck cancer frequently causes damage to nearby tissues in the oral cavity. In particular the majority of patients will experience an altered or reduced sense of taste, resulting in loss of appetite, weight loss, and a significantly compromised quality of life. Studies in this proposal will address novel and important questions regarding the cellular and molecular basis of taste loss in patients treated with irradiation for head and neck cancer. Understanding how taste loss occurs will enable the development of therapeutic strategies to reverse or prevent taste loss and improve the quality of life of patients undergoing head and neck irradiation.

Taste is a fundamental sense and is crucial for human health. Like our other primary senses, we consider our ability to appreciate sweet, sour, salty, bitter and umami tastes to be relatively constant, even though the taste bud cells that transduce these stimuli are renewed rapidly and regularly. The importance of the sense of taste is particularly evident for cancer patients receiving a range of chemotherapies, as these individuals often experience significant taste loss (ageusia) or dysfunction (dysgeusia). For patients with perturbed taste, simply eating a meal can be unpleasant or impossible; patients have significantly reduced quality of life including depression, lack of appetite and failure to maintain body weight, as well as poorer outcomes with cancer treatment. In particular, Basal Cell Carcinoma patients treated with drugs that target the Hedgehog (Hh) signaling pathway frequently experience dysgeusia. Interestingly Hh antagonists cause taste bud loss in mice, suggesting that functional taste loss in patients given these drugs is due to an effect on taste buds. Yet a mechanistic understanding of how taste bud homeostasis is impacted by Hh inhibition remains unexplored. And hence treatments to mitigate taste loss for cancer patients are yet to be imagined. Sonic hedgehog (Shh), one of 3 secreted Hh ligands, is implicated in taste bud cell renewal based on its expression pattern and that of its target gene, Gli1. A subset of taste bud cells is Shh+, while Gli1 expression is restricted to proliferating progenitor cells outside of buds, suggesting that Shh from within buds signals to adjacent progenitors to control taste cell turnover. However, the cellular and molecular processes regulated by Hh signaling are unknown, representing a significant gap in our knowledge of taste bud cell biology, and further limits our understanding of how Hh antagonists alter taste. Here, based on published reports and our pilot data, we propose the novel hypothesis that: Hedgehog signaling is required for taste receptor cell (TRC) differentiation and taste bud maintenance. We will test this overarching hypothesis using mouse models in 3 aims, and in completing these, greatly expand understanding of basic cellular and molecular mechanisms responsible for taste cell renewal, as well as define mechanistically how Hh pathway-targeted chemotherapy disrupts this process. Aim 1 Determine if Shh promotes TRC differentiation from taste progenitor cells. Aim 2 Identify the tissue source(s) of Shh required for TRC differentiation. Aim 3 Define cellular mechanisms underlying loss of differentiated TRCs in mice treated with Hh antagonist.

DESCRIPTION (provided by applicant): Taste buds are found in a distributed array on the tongue surface, and are innervated by cranial nerves that convey taste information to the brain. For nearly a century, taste buds were thought to be induced by nerves late in embryonic development. Over the past decade, however, this view has shifted dramatically. A host of studies now indicate that taste bud development is initiated and proceeds via processes that are nerve- independent, occur long before birth, and are governed by cellular and molecular mechanisms intrinsic to the developing tongue. In mice, lingual taste buds reside in epithelial-mesenchymal specializations, termed taste papillae. The first indication of taste development is the appearance of taste placodes in the tongue surface at midgestation; it has been assumed that taste placodes morph into taste papillae, which then, around birth, produce taste buds from a subset of papilla epithelial cells. However, we have shown that taste placodes are actually taste bud precursor cells (TBPcs), which give rise to taste bud cells only, and not to taste papillae. These recent findings serve as the basis for our new model, one in which TBPc induction is the primary event in taste bud development, while taste papillae form in response to signals emitted from newly induced TBPcs. A number of signaling pathways are involved in the induction of TBPcs, most prominently Wnt/ß-catenin and Shh; these pathways also regulate later aspects of taste bud and papilla development. To date, we have focused on the functions of Wnt and Shh in lingual epithelium. However, because the developing tongue has both epithelial and mesenchymal compartments, and because taste papillae comprise a mesenchymal core surrounded by epithelium, interactions between lingual mesenchyme and epithelium also likely underlie development of the taste periphery. Using newly available conditional molecular genetic tools in mice, we propose to explore Shh and Wnt function in lingual mesenchyme and epithelium during precise and temporally separable aspects of taste organ development, testing the overarching hypothesis that: Development of taste buds is intrinsic to lingual epithelium, while taste papilla development is initiated by taste bud precursos and requires interactions between epithelium and mesenchyme. Aim 1. Define the roles of lingual epithelium and mesenchyme in the induction of taste bud precursors. Aim 2. Determine if morphogenesis of taste papillae requires receipt of signals from taste bud precursors. Aim 3. Test if autocrine signaling by taste bud precursors is required for taste cell differentiation. In elucidating these mechanisms, we will gain crucial insight into molecular genetic regulation of taste bud pattern and cell complement. In the long term, we will leverage these advances in our understanding to explore how taste bud variation influences taste preferences underlying healthy versus detrimental dietary choices.

PROJECT SUMMARY Building on the NIDCD Strategic Plan (2017-2022), we propose an interdisciplinary two-day conference to identify high-yield research investment opportunities on smell and taste disorders, with a specific focus on gene therapy and stem cell treatments. Our objectives are to (1) increase collaboration across fields by inviting scientists addressing similar topics in both the chemical senses and other tissues or systems, (2) produce a peer-reviewed, group-consensus recommendation for next steps in research, and (3) communicate conference findings to scientists, clinicians, and patients. Ensuring diversity among attendees is a priority with accommodations offered as needed for people with special needs. The conference will be face-to-face with time for small group discussion. We will also release edited videos of the outreach content.

PROJECT SUMMARY Taste buds are composed of a heterogeneous collection of taste receptor cells (TRCs) that are continually renewed. Likely because of this regenerative capacity, taste distortion or dysgeusia is a common side effect of many anti-cancer drugs used to treat a host of malignancies. Crucially, patients report loss of taste as an extremely disruptive aspect of cancer treatment, dramatically affecting their quality of life and clinical outcomes. Current interventions for taste loss are minimally effective and consist primarily of dietary modifications and attention to oral hygiene. Here, we submit a revised proposal in response to PA-16-258 Mechanisms of Cancer and Treatment-related Symptoms and Toxicities, with the explicit rationale that understanding how cancer drugs impact taste homeostasis will generate future approaches to ameliorate taste dysfunction for cancer patients. Although a host of cancer drugs cause dysgeusia, we focus on a specific patient population, those suffering from malignant renal cell carcinoma (mRCC), treated with a one or more of 6 specific tyrosine kinase inhibitors (TKIs) used to inhibit receptor tyrosine kinases (RTKs) critical to tumor growth, i.e., VEGFR1-3 and PDGFRb. Importantly, these drugs, sunitinib, pazopanib, axitinib, cabozantinib, lenvatinib, and sorafenib, cause significant and troubling taste distortion for patients. However, how these drugs perturb taste is completely unknown. Intriguingly, interrogating both our own and published RNA sequence data reveals that neither VEGFR1-3 nor PDGFRb are expressed in taste epithelium. However, these TKIs also inhibit numerous other unintended RTKs including PDGFRa, c-Kit and Ret, which are expressed in taste epithelium, whose functions in mRCC are less essential, and whose roles in taste bud homeostasis are little explored. Thus, it is entirely plausible that TKIs used to treat mRCC act on intended VEGFR1-3 and PDGFRb to slow tumor progression, while in taste epithelium unintended RTKs are inhibited causing dysgeusia, thus providing us with a potential future avenue to mitigate taste dysfunction without interfering with cancer therapy. To assess how these targeted TKIs impact taste cell renewal, rather than using time-consuming and expensive mouse models, we propose to use cutting edge lingual organoid technology to rapidly and inexpensively screen for the effect of these drugs on discrete aspects of taste cell renewal. Our explicit hypothesis is that: TKIs used to treat mRCC affect discrete aspects of taste bud cell renewal, and act on RTKs expressed in taste epithelium that are distinct from their anti-tumor targets. This will be tested in 3 aims: Do mRCC TKIs affect activity and/or survival of taste bud progenitor cells (Aim 1); and/or differentiation of all or distinct subsets of TRCs (Aim 2); and which RTKs are inhibited in taste epithelium by TKIs used to treat mRCC patients (Aim 3).

PROJECT SUMMARY Taste receptor cells (TRCs) are continually replaced from adult stem/progenitor cells, and the fidelity of this process underlies the relative constancy of our sense of taste. However, a host of cancer therapies perturb taste and we posit this is due to perturbation of taste cell renewal. The Wnt/ß-catenin and Hedgehog pathways are implicated in scores of cancers, and many drugs have been and continue to be developed to target these pathways in tumors; these drugs invariably cause taste dysfunction for patients. Subsets of taste stem cells express the Wnt target gene Lgr5 and the Hedgehog target gene Gli1, and both Wnt and Hedgehog pathways have been shown to regulate taste cell renewal in vivo. Thus, in the long term, understanding the functional relationship of Wnt- and Hedgehog-sensitive stem cells in taste homeostasis, as proposed here, will shed light on how these progenitors are disrupted by chemotherapeutics that cause taste dysfunction, and allow development of strategies to mitigate dysgeusia. In our application, we propose to test explicit hypotheses of the functional relationship of LGR5+ and GLI1+ stem cells in the circumvallate taste papillae of mice. Hypothesis 1: Progenitors expressing high levels of LGR5 are slow cycling, multipotent stem cells that produce rapidly proliferating GLI1+/LGR5low/neg progenitors that give rise directly to TRCs. Hypothesis 2: Upon LGR5+ cell ablation, GLI1+ progenitors expand their potential to reconstitute circumvallate epithelium and give rise to new LGR5+ stem cells. To test these ideas, we combine in vivo molecular genetics, in vitro production of lingual organoids, and single cell transcriptome profiling ? all approaches with which we have become skilled. In Aim 1, we test the competency of LGR5 vs GLI1 progenitors to produce taste cell-replete organoids, and further assess the degree to which lineage production by each progenitor type is dependent upon Wnt signaling. In Aim 2, we explore the capacity of GLI1+ progenitors to regenerate both taste cells and LGR5+ stem cells following genetic ablation of LGR5+ cells. In Aim 3, we combine temporally fine-grained lineage tracing with single cell RNA sequencing to transcriptomically define the cell lineages that continually produce each of the functional taste cell types, i.e., glial-like cells and sweet, bitter, umami, salt and sour TRCs. In sum, our proposed studies will lead to significant advances in our understanding of the cellular and molecular mechanisms that maintain our sense of taste.