Gene burden analysis identifies genes associated with increased risk and severity of adult-onset hearing loss in a diverse hospital-based cohort.

Loss or absence of hearing is common at both extremes of human lifespan, in the forms of congenital deafness and age-related hearing loss. While these are often studied separately, there is increasing evidence that their genetic basis is at least partially overlapping. In particular, both common and rare variants in genes associated with monogenic forms of hearing loss also contribute to the more polygenic basis of age-related hearing loss. Here, we directly test this model in the Penn Medicine BioBank-a healthcare system cohort of around 40,000 individuals with linked genetic and electronic health record data. We show that increased burden of predicted deleterious variants in Mendelian hearing loss genes is associated with increased risk and severity of adult-onset hearing loss. As a specific example, we identify one gene-TCOF1, responsible for a syndromic form of congenital hearing loss-in which deleterious variants are also associated with adult-onset hearing loss. We also identify four additional novel candidate genes (COL5A1, HMMR, RAPGEF3, and NNT) in which rare variant burden may be associated with hearing loss. Our results confirm that rare variants in Mendelian hearing loss genes contribute to polygenic risk of hearing loss, and emphasize the utility of healthcare system cohorts to study common complex traits and diseases.

Genomic architecture of Shh-dependent cochlear morphogenesis.

The mammalian cochlea develops from a ventral outgrowth of the otic vesicle in response to Shh signaling. Mouse embryos lacking Shh or its essential signal transduction components display cochlear agenesis; however, a detailed understanding of the transcriptional network mediating this process is unclear. Here, we describe an integrated genomic approach to identify Shh-dependent genes and associated regulatory sequences that promote cochlear duct morphogenesis. A comparative transcriptome analysis of otic vesicles from mouse mutants exhibiting loss (Smoecko ) and gain (Shh-P1) of Shh signaling reveal a set of Shh-responsive genes partitioned into four expression categories in the ventral half of the otic vesicle. This target gene classification scheme provides novel insight into several unanticipated roles for Shh, including priming the cochlear epithelium for subsequent sensory development. We also mapped regions of open chromatin in the inner ear by ATAC-seq that, in combination with Gli2 ChIP-seq, identified inner ear enhancers in the vicinity of Shh-responsive genes. These datasets are useful entry points for deciphering Shh-dependent regulatory mechanisms involved in cochlear duct morphogenesis and establishment of its constituent cell types.

Genome-first approach to rare EYA4 variants and cardio-auditory phenotypes in adults.

While newborns and children with hearing loss are routinely offered genetic testing, adults are rarely clinically tested for a genetic etiology. One clinically actionable result from genetic testing in children is the discovery of variants in syndromic hearing loss genes. EYA4 is a known hearing loss gene which is also involved in important pathways in cardiac tissue. The pleiotropic effects of rare EYA4 variants are poorly understood and their prevalence in a large cohort has not been previously reported. We investigated cardio-auditory phenotypes in 11,451 individuals in a large biobank using a rare variant, genome-first approach to EYA4. We filtered 256 EYA4 variants carried by 6737 participants to 26 rare and predicted deleterious variants carried by 42 heterozygotes. We aggregated predicted deleterious EYA4 gene variants into a combined variable (i.e. "gene burden") and performed association studies across phenotypes compared to wildtype controls. We validated findings with replication in three independent cohorts and human tissue expression data. EYA4 gene burden was significantly associated with audiometric-proven HL (p = [Formula: see text], Mobitz Type II AV block (p = [Formula: see text]) and the syndromic presentation of HL and primary cardiomyopathy (p = 0.0194). Analyses on audiogram, echocardiogram, and electrocardiogram data validated these associations. Prior reports have focused on identifying variants in families with severe or syndromic phenotypes. In contrast, we found, using a genotype-first approach, that gene burden in EYA4 is associated with more subtle cardio-auditory phenotypes in an adult medical biobank population, including cardiac conduction disorders which have not been previously reported. We show the value of using a focused approach to uncover human disease related to pleiotropic gene variants and suggest a role for genetic testing in adults presenting with hearing loss.

Distinct temporal requirements for Sonic hedgehog signaling in development of the tuberal hypothalamus.

Sonic hedgehog (Shh) plays well characterized roles in brain and spinal cord development, but its functions in the hypothalamus have been more difficult to elucidate owing to the complex neuroanatomy of this brain area. Here, we use fate mapping and conditional deletion models in mice to define requirements for dynamic Shh activity at distinct developmental stages in the tuberal hypothalamus, a brain region with important homeostatic functions. At early time points, Shh signaling regulates dorsoventral patterning, neurogenesis and the size of the ventral midline. Fate-mapping experiments demonstrate that Shh-expressing and -responsive progenitors contribute to distinct neuronal subtypes, accounting for some of the cellular heterogeneity in tuberal hypothalamic nuclei. Conditional deletion of the hedgehog transducer smoothened (Smo), after dorsoventral patterning has been established, reveals that Shh signaling is necessary to maintain proliferation and progenitor identity during peak periods of hypothalamic neurogenesis. We also find that mosaic disruption of Smo causes a non-cell autonomous gain in Shh signaling activity in neighboring wild-type cells, suggesting a mechanism for the pathogenesis of hypothalamic hamartomas, benign tumors that form during hypothalamic development.

The cochlear sensory epithelium derives from Wnt responsive cells in the dorsomedial otic cup.

Wnt1 and Wnt3a secreted from the dorsal neural tube were previously shown to regulate a gene expression program in the dorsal otic vesicle that is necessary for vestibular morphogenesis (Riccomagno et al., 2005. Genes Dev. 19, 1612-1623). Unexpectedly, Wnt1(-/-); Wnt3a(-/-) embryos also displayed a pronounced defect in the outgrowth of the ventrally derived cochlear duct. To determine how Wnt signaling in the dorsal otocyst contributes to cochlear development we performed a series of genetic fate mapping experiments using two independent Wnt responsive driver strains (TopCreER and Gbx2(CreER)) that when crossed to inducible responder lines (Rosa(lacZ) or Rosa(zsGreen)) permanently labeled dorsomedial otic progenitors and their derivatives. Tamoxifen time course experiments revealed that most vestibular structures showed some degree of labeling when recombination was induced between E7.75 and E12.5, consistent with continuous Wnt signaling activity in this tissue. Remarkably, a population of Wnt responsive cells in the dorsal otocyst was also found to contribute to the sensory epithelium of the cochlear duct, including auditory hair and support cells. Similar results were observed with both TopCreER and Gbx2(CreER) strains. The ventral displacement of Wnt responsive cells followed a spatiotemporal sequence that initiated in the anterior otic cup at, or immediately prior to, the 17-somite stage (E9) and then spread progressively to the posterior pole of the otic vesicle by the 25-somite stage (E9.5). These lineage-tracing experiments identify the earliest known origin of auditory sensory progenitors within a population of Wnt responsive cells in the dorsomedial otic cup.

Divergent roles for Wnt/ß-catenin signaling in epithelial maintenance and breakdown during semicircular canal formation.

The morphogenetic program that shapes the three semicircular canals (SSCs) must be executed with extreme precision to satisfy their complex vestibular function. The SSCs emerge from epithelial outgrowths of the dorsal otocyst, the central regions of which fuse and resorb to leave three fluid-filled canals. The Wnt/ß-catenin signaling pathway is active at multiple stages of otic development, including during vestibular morphogenesis. How Wnt/ß-catenin functionally integrates with other signaling pathways to sculpt the SSCs and their sensory patches is unknown. We used a genetic strategy to spatiotemporally modulate canonical Wnt signaling activity during SSC development in mice. Our findings demonstrate that Wnt/ß-catenin signaling functions in a multifaceted manner during SSC formation. In the early phase, Wnt/ß-catenin signaling is required to preserve the epithelial integrity of the vertical canal pouch perimeter (presumptive anterior and posterior SSCs) by establishing a sensory-dependent signaling relay that maintains expression of Dlx5 and opposes expression of the fusion plate marker netrin 1. Without this Wnt signaling activity the sensory to non-sensory signaling cascade fails to be activated, resulting in loss of vestibular hair and support cells and the anterior and posterior SSCs. In the later phase, Wnt/ß-catenin signaling becomes restricted to the fusion plate where it facilitates the timely resorption of this tissue. Mosaic recombination of ß-catenin in small clusters of canal pouch cells prevents their resorption, causing instead the formation of ectopic SSCs. Together, these disparate functions of the Wnt/ß-catenin pathway in epithelial maintenance and resorption help regulate the size, shape and number of SSCs.

Inhibition of Sox2-dependent activation of Shh in the ventral diencephalon by Tbx3 is required for formation of the neurohypophysis.

Tbx2 and Tbx3 are two highly related members of the T-box transcription factor gene family that regulate patterning and differentiation of a number of tissue rudiments in the mouse. Both genes are partially co-expressed in the ventral diencephalon and the infundibulum; however, a functional requirement in murine pituitary development has not been reported. Here, we show by genetic lineage tracing that Tbx2(+) cells constitute the precursor population of the neurohypophysis. However, Tbx2 is dispensable for neurohypophysis development as revealed by normal formation of this organ in Tbx2-deficient mice. By contrast, loss of Tbx3 from the ventral diencephalon results in a failure to establish the Tbx2(+) domain in this region, and a lack of evagination of the infundibulum and formation of the neurohypophysis. Rathke's pouch is severely hypoplastic, exhibits defects in dorsoventral patterning, and degenerates after E12.5. In Tbx3-deficient embryos, the ventral diencephalon is hyperproliferative and displays an abnormal cellular architecture, probably resulting from a failure to repress transcription of Shh. We further show that Tbx3 and Tbx2 repress Shh by sequestering the SRY box-containing transcription factor Sox2 away from a Shh forebrain enhancer (SBE2), thus preventing its activation. These data suggest that Tbx3 is required in the ventral diencephalon to establish a Shh(-) domain to allow formation of the infundibulum.

Otic ablation of smoothened reveals direct and indirect requirements for Hedgehog signaling in inner ear development.

In mouse embryos lacking sonic hedgehog (Shh), dorsoventral polarity within the otic vesicle is disrupted. Consequently, ventral otic derivatives, including the cochlear duct and saccule, fail to form, and dorsal otic derivatives, including the semicircular canals, endolymphatic duct and utricle, are malformed or absent. Since inner ear patterning and morphogenesis are heavily dependent on extracellular signals derived from tissues that are also compromised by the loss of Shh, the extent to which Shh signaling acts directly on the inner ear for its development is unclear. To address this question, we generated embryos in which smoothened (Smo), an essential transducer of Hedgehog (Hh) signaling, was conditionally inactivated in the otic epithelium (Smo(ecko)). Ventral otic derivatives failed to form in Smo(ecko) embryos, whereas vestibular structures developed properly. Consistent with these findings, we demonstrate that ventral, but not dorsal, otic identity is directly dependent on Hh. The role of Hh in cochlear-vestibular ganglion (cvg) formation is more complex, as both direct and indirect signaling mechanisms are implicated. Our data suggest that the loss of cvg neurons in Shh(-/-) animals is due, in part, to an increase in Wnt responsiveness in the otic vesicle, resulting in the ectopic expression of Tbx1 in the neurogenic domain and subsequent repression of Ngn1 transcription. A mitogenic role for Shh in cvg progenitor proliferation was also revealed in our analysis of Smo(ecko) embryos. Taken together, these data contribute to a better understanding of the intrinsic and extrinsic signaling properties of Shh during inner ear development.

Spatial and temporal requirements for sonic hedgehog in the regulation of thalamic interneuron identity.

In caudal regions of the diencephalon, sonic hedgehog (Shh) is expressed in the ventral midline of prosomeres 1-3 (p1-p3), which underlie the pretectum, thalamus and prethalamus, respectively. Shh is also expressed in the zona limitans intrathalamica (zli), a dorsally projecting spike that forms at the p2-p3 boundary. The presence of two Shh signaling centers in the thalamus has made it difficult to determine the specific roles of either one in regional patterning and neuronal fate specification. To investigate the requirement of Shh from a focal source of expression in the ventral midline of the diencephalon, we used a newly generated mouse line carrying a targeted deletion of the 525 bp intronic sequence mediating Shh brain enhancer-1 (SBE1) activity. In SBE1 mutant mice, Shh transcription was initiated but not maintained in the ventral midline of the rostral midbrain and caudal diencephalon, yet expression in the zli was unaffected. In the absence of ventral midline Shh, rostral thalamic progenitors (pTH-R) adopted the molecular profile of a more caudal thalamic subtype (pTH-C). Surprisingly, despite their early mis-specification, neurons derived from the pTH-R domain continued to migrate to their proper thalamic nucleus, extended axons along their normal trajectory and expressed some, but not all, of their terminal differentiation markers. Our results, and those of others, suggest a model whereby Shh signaling from distinct spatial and temporal domains in the diencephalon exhibits unique and overlapping functions in the development of discrete classes of thalamic interneurons.

Direct transcriptional regulation of Six6 is controlled by SoxB1 binding to a remote forebrain enhancer.

Six6, a sine oculis homeobox protein, plays a crucial and conserved role in the development of the forebrain and eye. To understand how the expression of Six6 is regulated during embryogenesis, we screened ~250 kb of genomic DNA encompassing the Six6 locus for cis-regulatory elements capable of directing reporter gene expression to sites of Six6 transcription in transgenic mouse embryos. Here, we describe two novel enhancer elements, that are highly conserved in vertebrate species and whose activities recapitulate Six6 expression in the ventral forebrain and eye, respectively. Cross-species comparisons of the Six6 forebrain enhancer sequences revealed highly conserved binding sites matching the consensus for homeodomain and SoxB1 transcription factors. Deletion of either of the binding sites resulted in loss of the forebrain enhancer activity in the ventral forebrain. Moreover, our studies show that members of the SoxB1 family, including Sox2 and Sox3, are expressed in the overlapping region of the ventral forebrain with Six6 and can bind to the Six6 forebrain enhancer. Loss of function of SoxB1 genes in vivo further emphasizes their role in regulating Six6 forebrain enhancer activity. Thus, our data strongly suggest that SoxB1 transcription factors are direct activators of Six6 expression in the ventral forebrain.

How Does the "Cookie-Bite" Audiogram Shape Perform in Discriminating Genetic Hearing Loss in Adults?

"Cookie-bite" or U-shaped audiograms-specifically, those showing midfrequency sensorineural hearing loss (HL)-are traditionally taught to be associated with genetic HL; however, their utility as a screening tool has not been reported. We aim to determine the performance of a cookie-bite audiogram shape in stratifying patients carrying putative loss-of-function variants in known HL genes from wild-type controls. We merged audiometric and exome sequencing data from adults enrolled in a large biobank at a tertiary care center. Of 321 patients, 50 carried a putative loss-of-function variant in an HL gene. The cookie-bite shape was present in 9 of those patients, resulting in low sensitivity (18%) and positive predictive value (15%) in stratifying genetic carrier status; 84% of patients with a cookie-bite audiogram did not carry a genetic variant. A cookie-bite audiogram should not be used to screen adults for possible genetic testing.

Developmental trajectories of thalamic progenitors revealed by single-cell transcriptome profiling and Shh perturbation.

The thalamus is the principal information hub of the vertebrate brain, with essential roles in sensory and motor information processing, attention, and memory. The complex array of thalamic nuclei develops from a restricted pool of neural progenitors. We apply longitudinal single-cell RNA sequencing and regional abrogation of Sonic hedgehog (Shh) to map the developmental trajectories of thalamic progenitors, intermediate progenitors, and post-mitotic neurons as they coalesce into distinct thalamic nuclei. These data reveal that the complex architecture of the thalamus is established early during embryonic brain development through the coordinated action of four cell differentiation lineages derived from Shh-dependent and -independent progenitors. We systematically characterize the gene expression programs that define these thalamic lineages across time and demonstrate how their disruption upon Shh depletion causes pronounced locomotor impairment resembling infantile Parkinson's disease. These results reveal key principles of thalamic development and provide mechanistic insights into neurodevelopmental disorders resulting from thalamic dysfunction.

A Genome-First Approach to Rare Variants in Dominant Postlingual Hearing Loss Genes in a Large Adult Population.

OBJECTIVE: To investigate the importance of rare variants in adult-onset hearing loss. STUDY DESIGN: Genomic association study. SETTING: Large biobank from tertiary care center. METHODS: We investigated rare variants (minor allele frequency <5%) in 42 autosomal dominant (DFNA) postlingual hearing loss (HL) genes in 16,657 unselected individuals in the Penn Medicine Biobank. We determined the prevalence of known pathogenic and predicted deleterious variants in subjects with audiometric-proven sensorineural hearing loss. We scanned across known postlingual DFNA HL genes to determine those most significantly contributing to the phenotype. We replicated findings in an independent cohort (UK Biobank). RESULTS: While rare individually, when considering the accumulation of variants in all postlingual DFNA genes, more than 90% of participants carried at least 1 rare variant. Rare variants predicted to be deleterious were enriched in adults with audiometric-proven hearing loss (pure-tone average >25 dB; P = .015). Patients with a rare predicted deleterious variant had an odds ratio of 1.27 for HL compared with genotypic controls (P = .029). Gene burden in DIABLO, PTPRQ, TJP2, and POU4F3 were independently associated with sensorineural hearing loss. CONCLUSION: Although prior reports have focused on common variants, we find that rare predicted deleterious variants in DFNA postlingual HL genes are enriched in patients with adult-onset HL in a large health care system population. We show the value of investigating rare variants to uncover hearing loss phenotypes related to implicated genes.

Wnt signaling inhibitors regulate the transcriptional response to morphogenetic Shh-Gli signaling in the neural tube.

Shh-Gli signaling controls cell fates in the developing ventral neural tube by regulating the patterned expression of transcription factors in neural progenitors. However, the molecular mechanisms that limit target gene responses to specific domains are unclear. Here, we show that Wnt pathway inhibitors regulate the threshold response of a ventral Shh target gene, Nkx2.2, to establish its restricted expression in the ventral spinal cord. Identification and characterization of an Nkx2.2 enhancer reveals that expression is directly regulated by positive Shh-Gli signaling and negative Tcf repressor activity. Our data indicate that the dorsal limit of Nkx2.2 is controlled by Tcf4-mediated transcriptional repression, and not by a direct requirement for high-level Shh-Gli signaling, arguing against a simple model based on differential Gli factor affinities in target genes. These results identify a transcriptional mechanism that integrates graded Shh and Wnt signaling to define progenitor gene expression domains and cell fates in the neural tube.

Genetic interaction between members of the Vangl family causes neural tube defects in mice.

Neural tube defects (NTDs) are very frequent congenital abnormalities in humans. Recently, we have documented independent association of Vangl1 and Vangl2 gene mutations with NTDs. In the Looptail mouse, homozygosity (but not heterozygosity) for loss-of-function alleles at Vangl2 causes the severe NTD craniorachischisis, whereas heterozygosity for mutant variants of VANGL1 is associated with NTDs in a human cohort of sporadic and familial cases. To understand the role of Vangl1 in normal development, we created a mouse mutant with an inactivating mutation at Vangl1 (Vangl1(gt)). Vangl1 shows a dynamic pattern of expression in the developing neural tube and notochord at the time of neural tube closure. Vangl1(gt/+) heterozygotes and Vangl1(gt/gt) homozygotes are viable and fertile, although Vangl1(gt/gt) display subtle alterations in polarity of inner hair cells of the cochlea. Remarkably, and as opposed to healthy Vangl1(gt/+) and Vangl2(lp/+) heterozygotes, Vangl1(gt/+);Vangl2(lp/+) double heterozygotes show profound developmental defects that include severe craniorachischisis, inner ear defects (disorganization of the stereociliary bundles of hair cells of the organ of Corti), and cardiac abnormality (aberrant right subclavian artery). These results show that genetic interaction between Vangl1 and Vangl2 genes causes neural tube defects and raise the possibility that interaction between individual Vangl genes and other genetic loci and/or environmental factors may additionally contribute to the etiology of NTDs.

Genetics of Postlingual Sensorineural Hearing Loss.

Literature and clinical practice around adult-onset hearing loss (HL) has traditionally focused on environmental risk factors, including noise exposure, ototoxic drug exposure, and cardiovascular disease. The most common diagnosis in adult-onset HL is presbycusis. However, the age of onset of presbycusis varies, and patients often describe family history of HL as well as individual variation in progression and severity. In recent years, there has been accumulating evidence of gene-environment interactions underlying adult cases of HL. Susceptibility loci for age-related HL have been identified, and genes related to postlingual nonsyndromic HL continue to be discovered through individual reports and genome-wide association studies. This review will outline main concepts in genetics as related to HL, identify implicated genes, and discuss clinical implications. Laryngoscope, 131:401-409, 2021.

Cochlear supporting cells require GAS2 for cytoskeletal architecture and hearing.

In mammals, sound is detected by mechanosensory hair cells that are activated in response to vibrations at frequency-dependent positions along the cochlear duct. We demonstrate that inner ear supporting cells provide a structural framework for transmitting sound energy through the cochlear partition. Humans and mice with mutations in GAS2, encoding a cytoskeletal regulatory protein, exhibit hearing loss due to disorganization and destabilization of microtubule bundles in pillar and Deiters' cells, two types of inner ear supporting cells with unique cytoskeletal specializations. Failure to maintain microtubule bundle integrity reduced supporting cell stiffness, which in turn altered cochlear micromechanics in Gas2 mutants. Vibratory responses to sound were measured in cochleae from live mice, revealing defects in the propagation and amplification of the traveling wave in Gas2 mutants. We propose that the microtubule bundling activity of GAS2 imparts supporting cells with mechanical properties for transmitting sound energy through the cochlea.

Wnt/beta-catenin signaling promotes expansion of Isl-1-positive cardiac progenitor cells through regulation of FGF signaling.

The anterior heart field (AHF), which contributes to the outflow tract and right ventricle of the heart, is defined in part by expression of the LIM homeobox transcription factor Isl-1. The importance of Isl-1-positive cells in cardiac development and homeostasis is underscored by the finding that these cells are required for cardiac development and act as cardiac stem/progenitor cells within the postnatal heart. However, the molecular pathways regulating these cells' expansion and differentiation are poorly understood. We show that Isl-1-positive AHF progenitor cells in mice were responsive to Wnt/beta-catenin signaling, and these responsive cells contributed to the outflow tract and right ventricle of the heart. Loss of Wnt/beta-catenin signaling in the AHF caused defective outflow tract and right ventricular development with a decrease in Isl-1-positive progenitors and loss of FGF signaling. Conversely, Wnt gain of function in these cells led to expansion of Isl-1-positive progenitors with a concomitant increase in FGF signaling through activation of a specific set of FGF ligands including FGF3, FGF10, FGF16, and FGF20. These data reveal what we believe to be a novel Wnt-FGF signaling axis required for expansion of Isl-1-positive AHF progenitors and suggest future therapies to increase the number and function of these cells for cardiac regeneration.

Neural crest stem cell maintenance by combinatorial Wnt and BMP signaling.

Canonical Wnt signaling instructively promotes sensory neurogenesis in early neural crest stem cells (eNCSCs) (Lee, H.Y., M. Kleber, L. Hari, V. Brault, U. Suter, M.M. Taketo, R. Kemler, and L. Sommer. 2004. Science. 303:1020-1023). However, during normal development Wnt signaling induces a sensory fate only in a subpopulation of eNCSCs while other cells maintain their stem cell features, despite the presence of Wnt activity. Hence, factors counteracting Wnt signaling must exist. Here, we show that bone morphogenic protein (BMP) signaling antagonizes the sensory fate-inducing activity of Wnt/beta-catenin. Intriguingly, Wnt and BMP act synergistically to suppress differentiation and to maintain NCSC marker expression and multipotency. Similar to NCSCs in vivo, NCSCs maintained in culture alter their responsiveness to instructive growth factors with time. Thus, stem cell development is regulated by combinatorial growth factor activities that interact with changing cell-intrinsic cues.

Single-cell transcriptomic analysis of adult mouse pituitary reveals sexual dimorphism and physiologic demand-induced cellular plasticity.

The anterior pituitary gland drives highly conserved physiologic processes in mammalian species. These hormonally controlled processes are central to somatic growth, pubertal transformation, fertility, lactation, and metabolism. Current cellular models of mammalian anteiror pituitary, largely built on candidate gene based immuno-histochemical and mRNA analyses, suggest that each of the seven hormones synthesized by the pituitary is produced by a specific and exclusive cell lineage. However, emerging evidence suggests more complex relationship between hormone specificity and cell plasticity. Here we have applied massively parallel single-cell RNA sequencing (scRNA-seq), in conjunction with complementary imaging-based single-cell analyses of mRNAs and proteins, to systematically map both cell-type diversity and functional state heterogeneity in adult male and female mouse pituitaries at single-cell resolution and in the context of major physiologic demands. These quantitative single-cell analyses reveal sex-specific cell-type composition under normal pituitary homeostasis, identify an array of cells associated with complex complements of hormone-enrichment, and undercover non-hormone producing interstitial and supporting cell-types. Interestingly, we also identified a Pou1f1-expressing cell population that is characterized by a unique multi-hormone gene expression profile. In response to two well-defined physiologic stresses, dynamic shifts in cellular diversity and transcriptome profiles were observed for major hormone producing and the putative multi-hormone cells. These studies reveal unanticipated cellular complexity and plasticity in adult pituitary, and provide a rich resource for further validating and expanding our molecular understanding of pituitary gene expression programs and hormone production.