Avian auditory hair cell regeneration is accompanied by JAK/STAT-dependent expression of immune-related genes in supporting cells.

The avian hearing organ is the basilar papilla that, in sharp contrast to the mammalian cochlea, can regenerate sensory hair cells and thereby recover from deafness within weeks. The mechanisms that trigger, sustain and terminate the regenerative response in vivo are largely unknown. Here, we profile the changes in gene expression in the chicken basilar papilla after aminoglycoside antibiotic-induced hair cell loss using RNA-sequencing. We identified changes in gene expression of a group of immune-related genes and confirmed with single-cell RNA-sequencing that these changes occur in supporting cells. In situ hybridization was used to further validate these findings. We determined that the JAK/STAT signaling pathway is essential for upregulation of the damage-response genes in supporting cells during the second day after induction of hair cell loss. Four days after ototoxic damage, we identified newly regenerated, nascent auditory hair cells that express genes linked to termination of the JAK/STAT signaling response. The robust, transient expression of immune-related genes in supporting cells suggests a potential functional involvement of JAK/STAT signaling in sensory hair cell regeneration.

Repurposing a novel anti-cancer RXR agonist to attenuate murine acute GVHD and maintain graft-versus-leukemia responses.

The nuclear receptor (NR) subclass, retinoid X receptors (RXRs), exert immunomodulatory functions that control inflammation and metabolism via homodimers and heterodimers, with several other NRs, including retinoic acid receptors. IRX4204 is a novel, highly specific RXR agonist in clinical trials that potently and selectively activates RXR homodimers, but not heterodimers. In this study, in vivo IRX4204 compared favorably with FK506 in abrogating acute graft-versus-host disease (GVHD), which was associated with inhibiting allogeneic donor T-cell proliferation, reducing T-helper 1 differentiation, and promoting regulatory T-cell (Treg) generation. Recipient IRX4204 treatment reduced intestinal injury and decreased IFN-γ and TNF-α serum levels. Transcriptional analysis of donor T cells isolated from intestines of GVHD mice treated with IRX4204 revealed significant decreases in transcripts regulating proinflammatory pathways. In vitro, inducible Treg differentiation from naive CD4+ T cells was enhanced by IRX4204. In vivo, IRX4204 increased the conversion of donor Foxp3- T cells into peripheral Foxp3+ Tregs in GVHD mice. Using Foxp3 lineage-tracer mice in which both the origin and current FoxP3 expression of Tregs can be tracked, we demonstrated that IRX4204 supports Treg stability. Despite favoring Tregs and reducing Th1 differentiation, IRX4204-treated recipients maintained graft-versus-leukemia responses against both leukemia and lymphoma cells. Notably, IRX4204 reduced in vitro human T-cell proliferation and enhanced Treg generation in mixed lymphocyte reaction cultures. Collectively, these beneficial effects indicate that targeting RXRs with IRX4204 could be a novel approach to preventing acute GVHD in the clinic.

RARγ is required for mesodermal gene expression prior to gastrulation in Xenopus.

The developing vertebrate embryo is exquisitely sensitive to retinoic acid (RA) concentration, particularly during anteroposterior patterning. In contrast to Nodal and Wnt signaling, RA was not previously considered to be an instructive signal in mesoderm formation during gastrulation. Here, we show in Xenopus that RARγ is indispensable for the expression of early mesoderm markers and is, therefore, an obligatory factor in mesodermal competence and/or maintenance. We identified several novel targets upregulated by RA receptor signaling in the early gastrula that are expressed in the circumblastoporal ring and linked to mesodermal development. Despite overlapping expression patterns of the genes encoding the RA-synthesizing enzyme Aldh1a2 and the RA-degrading enzyme Cyp26a1, RARγ1 functions as a transcriptional activator in early mesoderm development, suggesting that RA ligand is available to the embryo earlier than previously appreciated. RARγ1 is required for cellular adhesion, as revealed by spontaneous dissociation and depletion of ncam1 mRNA in animal caps harvested from RARγ1 knockdown embryos. RARγ1 knockdown obliterates somite boundaries, and causes loss of Myod protein in the presomitic mesoderm, but ectopic, persistent expression of Myod protein in the trunk. Thus, RARγ1 is required for stabilizing the mesodermal fate, myogenic commitment, somite boundary formation, and terminal skeletal muscle differentiation.

RARß2 is required for vertebrate somitogenesis.

During vertebrate somitogenesis, retinoic acid is known to establish the position of the determination wavefront, controlling where new somites are permitted to form along the anteroposterior body axis. Less is understood about how RAR regulates somite patterning, rostral-caudal boundary setting, specialization of myotome subdivisions or the specific RAR subtype that is required for somite patterning. Characterizing the function of RARß has been challenging due to the absence of embryonic phenotypes in murine loss-of-function studies. Using the Xenopus system, we show that RARß2 plays a specific role in somite number and size, restriction of the presomitic mesoderm anterior border, somite chevron morphology and hypaxial myoblast migration. Rarß2 is the RAR subtype whose expression is most upregulated in response to ligand and its localization in the trunk somites positions it at the right time and place to respond to embryonic retinoid levels during somitogenesis. RARß2 positively regulates Tbx3 a marker of hypaxial muscle, and negatively regulates Tbx6 via Ripply2 to restrict the anterior boundaries of the presomitic mesoderm and caudal progenitor pool. These results demonstrate for the first time an early and essential role for RARß2 in vertebrate somitogenesis.

Active repression by RARγ signaling is required for vertebrate axial elongation.

Retinoic acid receptor gamma 2 (RARγ2) is the major RAR isoform expressed throughout the caudal axial progenitor domain in vertebrates. During a microarray screen to identify RAR targets, we identified a subset of genes that pattern caudal structures or promote axial elongation and are upregulated by increased RAR-mediated repression. Previous studies have suggested that RAR is present in the caudal domain, but is quiescent until its activation in late stage embryos terminates axial elongation. By contrast, we show here that RARγ2 is engaged in all stages of axial elongation, not solely as a terminator of axial growth. In the absence of RA, RARγ2 represses transcriptional activity in vivo and maintains the pool of caudal progenitor cells and presomitic mesoderm. In the presence of RA, RARγ2 serves as an activator, facilitating somite differentiation. Treatment with an RARγ-selective inverse agonist (NRX205099) or overexpression of dominant-negative RARγ increases the expression of posterior Hox genes and that of marker genes for presomitic mesoderm and the chordoneural hinge. Conversely, when RAR-mediated repression is reduced by overexpressing a dominant-negative co-repressor (c-SMRT), a constitutively active RAR (VP16-RARγ2), or by treatment with an RARγ-selective agonist (NRX204647), expression of caudal genes is diminished and extension of the body axis is prematurely terminated. Hence, gene repression mediated by the unliganded RARγ2-co-repressor complex constitutes a novel mechanism to regulate and facilitate the correct expression levels and spatial restriction of key genes that maintain the caudal progenitor pool during axial elongation in Xenopus embryos.

The unexpected teratogenicity of RXR antagonist UVI3003 via activation of PPARγ in Xenopus tropicalis.

The RXR agonist (triphenyltin, TPT) and the RXR antagonist (UVI3003) both show teratogenicity and, unexpectedly, induce similar malformations in Xenopus tropicalis embryos. In the present study, we exposed X. tropicalis embryos to UVI3003 in seven specific developmental windows and identified changes in gene expression. We further measured the ability of UVI3003 to activate Xenopus RXRα (xRXRα) and PPARγ (xPPARγ) in vitro and in vivo. We found that UVI3003 activated xPPARγ either in Cos7 cells (in vitro) or Xenopus embryos (in vivo). UVI3003 did not significantly activate human or mouse PPARγ in vitro; therefore, the activation of Xenopus PPARγ by UVI3003 is novel. The ability of UVI3003 to activate xPPARγ explains why UVI3003 and TPT yield similar phenotypes in Xenopus embryos. Our results indicate that activating PPARγ leads to teratogenic effects in Xenopus embryos. More generally, we infer that chemicals known to specifically modulate mammalian nuclear hormone receptors cannot be assumed to have the same activity in non-mammalian species, such as Xenopus. Rather they must be tested for activity and specificity on receptors of the species in question to avoid making inappropriate conclusions.

ERF and ETV3L are retinoic acid-inducible repressors required for primary neurogenesis.

Cells in the developing neural tissue demonstrate an exquisite balance between proliferation and differentiation. Retinoic acid (RA) is required for neuronal differentiation by promoting expression of proneural and neurogenic genes. We show that RA acts early in the neurogenic pathway by inhibiting expression of neural progenitor markers Geminin and Foxd4l1, thereby promoting differentiation. Our screen for RA target genes in early Xenopus development identified Ets2 Repressor Factor (Erf) and the closely related ETS repressors Etv3 and Etv3-like (Etv3l). Erf and Etv3l are RA responsive and inhibit the action of ETS genes downstream of FGF signaling, placing them at the intersection of RA and growth factor signaling. We hypothesized that RA regulates primary neurogenesis by inducing Erf and Etv3l to antagonize proliferative signals. Loss-of-function analysis showed that Erf and Etv3l are required to inhibit proliferation of neural progenitors to allow differentiation, whereas overexpression of Erf led to an increase in the number of primary neurons. Therefore, these RA-induced ETS repressors are key components of the proliferation-differentiation switch during primary neurogenesis in vivo.

Obesogens: an emerging threat to public health.

Endocrine disrupting chemicals (EDCs) are defined as exogenous chemicals, or mixtures of chemicals, that can interfere with any aspect of hormone action. The field of endocrine disruption is historically rooted in wildlife biology and reproductive endocrinology where EDCs are demonstrated contributors to infertility, premature puberty, endometriosis, and other disorders. Recently, EDCs have been implicated in metabolic syndrome and obesity. Adipose tissue is a true endocrine organ and, therefore, an organ that is highly susceptible to disturbance by EDCs. A subset of EDCs, called "obesogens," promote adiposity by altering programming of fat cell development, increasing energy storage in fat tissue, and interfering with neuroendocrine control of appetite and satiety. Obesity adds more than $200 billion to US healthcare costs and the number of obese individuals continues to increase. Hence, there is an urgent, unmet need to understand the mechanisms underlying how exposures to certain EDCs may predispose our population to be obese. In this review, we discuss the history of obesogen discovery from its origins in reproductive biology to its latest role in the transgenerational inheritance of obesity in mice. We discuss the development of adipose tissue in an embryo, maintenance of adipocyte number in adults, how EDC disruption programs stem cells to preferentially make more adipocytes, the mechanisms by which chemicals can permanently alter the germline epigenome, and whether there are barriers to EDCs in the gametes.

Retinoic acid signaling and neuronal differentiation.

The identification of neurological symptoms caused by vitamin A deficiency pointed to a critical, early developmental role of vitamin A and its metabolite, retinoic acid (RA). The ability of RA to induce post-mitotic, neural phenotypes in various stem cells, in vitro, served as early evidence that RA is involved in the switch between proliferation and differentiation. In vivo studies have expanded this "opposing signal" model, and the number of primary neurons an embryo develops is now known to depend critically on the levels and spatial distribution of RA. The proneural and neurogenic transcription factors that control the exit of neural progenitors from the cell cycle and allow primary neurons to develop are partly elucidated, but the downstream effectors of RA receptor (RAR) signaling (many of which are putative cell cycle regulators) remain largely unidentified. The molecular mechanisms underlying RA-induced primary neurogenesis in anamniote embryos are starting to be revealed; however, these data have been not been extended to amniote embryos. There is growing evidence that bona fide RARs are found in some mollusks and other invertebrates, but little is known about their necessity or functions in neurogenesis. One normal function of RA is to regulate the cell cycle to halt proliferation, and loss of RA signaling is associated with dedifferentiation and the development of cancer. Identifying the genes and pathways that mediate cell cycle exit downstream of RA will be critical for our understanding of how to target tumor differentiation. Overall, elucidating the molecular details of RAR-regulated neurogenesis will be decisive for developing and understanding neural proliferation-differentiation switches throughout development.

RIPPLY3 is a retinoic acid-inducible repressor required for setting the borders of the pre-placodal ectoderm.

Retinoic acid signaling is a major component of the neural posteriorizing process in vertebrate development. Here, we identify a new role for the retinoic acid receptor (RAR) in the anterior of the embryo, where RAR regulates Fgf8 expression and formation of the pre-placodal ectoderm (PPE). RARα2 signaling induces key pre-placodal genes and establishes the posterolateral borders of the PPE. RAR signaling upregulates two important genes, Tbx1 and Ripply3, during early PPE development. In the absence of RIPPLY3, TBX1 is required for the expression of Fgf8 and hence, PPE formation. In the presence of RIPPLY3, TBX1 acts as a transcriptional repressor, and functions to restrict the positional expression of Fgf8, a key regulator of PPE gene expression. These results establish a novel role for RAR as a regulator of spatial patterning of the PPE through Tbx1 and RIPPLY3. Moreover, we demonstrate that Ripply3, acting downstream of RAR signaling, is a key player in establishing boundaries in the PPE.

Transcriptional and epigenetic mechanisms underlying enhanced in vitro adipocyte differentiation by the brominated flame retardant BDE-47.

Recent studies suggest that exposure to endocrine-disrupting compounds (EDCs) may play a role in the development of obesity. EDCs such as the flame retardant 2,2',4,4'-tetrabrominated diphenyl ether (BDE-47) have been shown to enhance adipocyte differentiation in the murine 3T3-L1 model. The mechanisms by which EDCs direct preadipocytes to form adipocytes are poorly understood. Here, we examined transcriptional and epigenetic mechanisms underlying the induction of in vitro adipocyte differentiation by BDE-47. Quantitative high content microscopy revealed concentration-dependent enhanced adipocyte differentiation following exposure to BDE-47 or the antidiabetic drug troglitazone (TROG). BDE-47 modestly activated the key adipogenic transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) in COS7 cells, transiently transfected with a GAL4 reporter construct. Increased gene expression was observed for Pparγ2, leptin (Lep), and glucose-6-phophatase catalytic subunit (G6pc) in differentiated 3T3-L1 cells after BDE-47 exposure compared to TROG. Methylation-sensitive high resolution melting (MS-HRM) revealed significant demethylation of three CpG sites in the Pparγ2 promoter after exposure to both BDE-47 and TROG in differentiated 3T3-L1 cells. This study shows the potential of BDE-47 to induce adipocyte differentiation through various mechanisms that include Pparγ2 gene induction and promoter demethylation accompanied by activation of PPARγ, and possible disruption of glucose homeostasis and IGF1 signaling.

An essential signaling cascade for avian auditory hair cell regeneration.

Hearing loss is a chronic disease affecting millions of people worldwide, yet no restorative treatment options are available. Although non-mammalian species can regenerate their auditory sensory hair cells, mammals cannot. Birds retain facultative stem cells known as supporting cells that engage in proliferative regeneration when surrounding hair cells die. Here, we investigated gene expression changes in chicken supporting cells during auditory hair cell death. This identified a pathway involving the receptor F2RL1, HBEGF, EGFR, and ERK signaling. We propose a cascade starting with the proteolytic activation of F2RL1, followed by matrix-metalloprotease-mediated HBEGF shedding, and culminating in EGFR-mediated ERK signaling. Each component of this cascade is essential for supporting cell S-phase entry in vivo and is integral for hair cell regeneration. Furthermore, STAT3-phosphorylation converges with this signaling toward upregulation of transcription factors ATF3, FOSL2, and CREM. Our findings could provide a basis for designing treatments for hearing and balance disorders.

High resolution mapping of the tumor microenvironment using integrated single-cell, spatial and in situ analysis.

Single-cell and spatial technologies that profile gene expression across a whole tissue are revolutionizing the resolution of molecular states in clinical samples. Current commercially available technologies provide whole transcriptome single-cell, whole transcriptome spatial, or targeted in situ gene expression analysis. Here, we combine these technologies to explore tissue heterogeneity in large, FFPE human breast cancer sections. This integrative approach allowed us to explore molecular differences that exist between distinct tumor regions and to identify biomarkers involved in the progression towards invasive carcinoma. Further, we study cell neighborhoods and identify rare boundary cells that sit at the critical myoepithelial border confining the spread of malignant cells. Here, we demonstrate that each technology alone provides information about molecular signatures relevant to understanding cancer heterogeneity; however, it is the integration of these technologies that leads to deeper insights, ushering in discoveries that will progress oncology research and the development of diagnostics and therapeutics.

Cell-type identity of the avian utricle.

The avian utricle, a vestibular organ of the inner ear, displays turnover of sensory hair cells throughout life. This is in sharp contrast to the mammalian utricle, which shows limited regenerative capacity. Here, we use single-cell RNA sequencing to identify distinct marker genes for the different sensory hair cell subtypes of the chicken utricle, which we validated in situ. We provide markers for spatially distinct supporting cell populations and identify two transitional cell populations of dedifferentiating supporting cells and developing hair cells. Trajectory reconstruction resulted in an inventory of gene expression dynamics of natural hair cell generation in the avian utricle.

Mutation of SLC7A14 causes auditory neuropathy and retinitis pigmentosa mediated by lysosomal dysfunction.

Lysosomes contribute to cellular homeostasis via processes including macromolecule degradation, nutrient sensing, and autophagy. Defective proteins related to lysosomal macromolecule catabolism are known to cause a range of lysosomal storage diseases; however, it is unclear whether mutations in proteins involved in homeostatic nutrient sensing mechanisms cause syndromic sensory disease. Here, we show that SLC7A14, a transporter protein mediating lysosomal uptake of cationic amino acids, is evolutionarily conserved in vertebrate mechanosensory hair cells and highly expressed in lysosomes of mammalian cochlear inner hair cells (IHCs) and retinal photoreceptors. Autosomal recessive mutation of SLC7A14 caused loss of IHCs and photoreceptors, leading to presynaptic auditory neuropathy and retinitis pigmentosa in mice and humans. Loss-of-function mutation altered protein trafficking and increased basal autophagy, leading to progressive cell degeneration. This study implicates autophagy-lysosomal dysfunction in syndromic hearing and vision loss in mice and humans.

Endocrine disrupting chemicals and the developmental programming of adipogenesis and obesity.

Obesity and related disorders are a burgeoning public health epidemic, particularly in the U.S. Currently 34% of the U.S. population is clinically obese (BMI > 30) and 68% are overweight (BMI > 25), more than double the worldwide average and 10-fold higher than Japan and South Korea. Obesity occurs when energy intake exceeds energy expenditure; however, individuals vary widely in their propensity to gain weight and accrue fat mass, even at identical levels of excess caloric input. Clinical, epidemiological, and biological studies show that obesity is largely programmed during early life, including the intrauterine period. The environmental obesogen hypothesis holds that prenatal or early life exposure to certain endocrine disrupting chemicals can predispose exposed individuals to increased fat mass and obesity. Obesogen exposure can alter the epigenome of multipotent stromal stem cells, biasing them toward the adipocyte lineage at the expense of bone. Hence, humans exposed to obesogens during early life might have an altered stem cell compartment, which is preprogrammed toward an adipogenic fate. This results in a higher steady state number of adipocytes and potentially a lifelong struggle to maintain a healthy weight, which can be exacerbated by societal influences that promote poor diet and inadequate exercise. This review focuses on the developmental origins of the adipocyte, the relationship between adipocyte number and obesity, and how obesogenic chemicals may interfere with the highly efficient homeostatic mechanisms regulating adipocyte number and energy balance.

Cell-type identity of the avian cochlea.

In contrast to mammals, birds recover naturally from acquired hearing loss, which makes them an ideal model for inner ear regeneration research. Here, we present a validated single-cell RNA sequencing resource of the avian cochlea. We describe specific markers for three distinct types of sensory hair cells, including a previously unknown subgroup, which we call superior tall hair cells. We identify markers for the supporting cells associated with tall hair cells, which represent the facultative stem cells of the avian inner ear. Likewise, we present markers for supporting cells that are located below the short cochlear hair cells. We further infer spatial expression gradients of hair cell genes along the tonotopic axis of the cochlea. This resource advances neurobiology, comparative biology, and regenerative medicine by providing a basis for comparative studies with non-regenerating mammalian cochleae and for longitudinal studies of the regenerating avian cochlea.

Transcriptomic characterization of dying hair cells in the avian cochlea.

Sensory hair cells are prone to apoptosis caused by various drugs including aminoglycoside antibiotics. In mammals, this vulnerability results in permanent hearing loss because lost hair cells are not regenerated. Conversely, hair cells regenerate in birds, making the avian inner ear an exquisite model for studying ototoxicity and regeneration. Here, we use single-cell RNA sequencing and trajectory analysis on control and dying hair cells after aminoglycoside treatment. Interestingly, the two major subtypes of avian cochlear hair cells, tall and short hair cells, respond differently. Dying short hair cells show a noticeable transient upregulation of many more genes than tall hair cells. The most prominent gene group identified is associated with potassium ion conductances, suggesting distinct physiological differences. Moreover, the dynamic characterization of >15,000 genes expressed in tall and short avian hair cells during their apoptotic demise comprises a resource for further investigations toward mammalian hair cell protection and hair cell regeneration.

Stem Cells and the Bird Cochlea-Where Is Everybody?

In sharp contrast to the adult mammalian cochlea, which lacks regenerative ability, the mature avian cochlea, or basilar papilla (BP) is capable of complete recovery from hearing loss after damage. Avian sensory hair cell regeneration relies on rousing quiescent supporting cells to proliferate or transdifferentiate after hair cell death. Unlike mammalian cochlear supporting cells, which have clearly defined subtypes, avian BP supporting cells are deceptively indistinguishable and molecular markers have yet to be identified. Despite the importance of supporting cells as the putative stem cells in avian regeneration, it is unknown whether all supporting cells possess equal capability to give rise to a hair cell or if a specialized subpopulation exists. In this perspective, we reinvigorate the concept of a stem cell in the BP, and form comparisons to other regenerating tissues that show cell-cycle reentry after damage. Special emphasis is given to the structure of the BP and how anatomy informs both the potential, intrinsic heterogeneity of the supporting cell layer as well as the choice between mitotic and nonmitotic regenerative strategies.