Doctor of Philosophy, University of California Irvine (2013)
Bachelor of Science, University of California Irvine (2008)
Stefan Heller, Postdoctoral Faculty Sponsor
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.
View details for PubMedID 30249599
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 RAR? 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, RAR?1 functions as a transcriptional activator in early mesoderm development, suggesting that RA ligand is available to the embryo earlier than previously appreciated. RAR?1 is required for cellular adhesion, as revealed by spontaneous dissociation and depletion of ncam1 mRNA in animal caps harvested from RAR?1 knockdown embryos. RAR?1 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, RAR?1 is required for stabilizing the mesodermal fate, myogenic commitment, somite boundary formation, and terminal skeletal muscle differentiation.
View details for DOI 10.1242/dev.147769
View details for Web of Science ID 000447270800001
View details for PubMedID 30111657
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 RAR? has been challenging due to the absence of embryonic phenotypes in murine loss-of-function studies. Using the Xenopus system, we show that RAR?2 plays a specific role in somite number and size, restriction of the presomitic mesoderm anterior border, somite chevron morphology and hypaxial myoblast migration. Rar?2 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. RAR?2 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 RAR?2 in vertebrate somitogenesis.
View details for DOI 10.1242/dev.144345
View details for Web of Science ID 000402276800011
View details for PubMedID 28432217
In ToxCast? Phase I, the U.S. EPA commissioned screening of 320 pesticides, herbicides, fungicides, and other chemicals in a series of high-throughput assays. The agency also developed a toxicological prioritization tool, ToxPi, to facilitate using ToxCast? assays to predict biological function.We asked whether top-scoring PPAR? activators identified in ToxCast? Phase I were genuine PPAR? activators and inducers of adipogenesis. Next, we identified ToxCast? assays that should predict adipogenesis, developed an adipogenesis ToxPi, and asked how well the ToxPi predicted adipogenic activity.We used transient transfection to test the ability of ToxCast? chemicals to modulate PPAR? and RXR?, and differentiation assays employing 3T3-L1 preadipocytes and mouse bone marrow-derived mesenchymal stem cells (mBMSCs) to evaluate the adipogenic capacity of ToxCast? chemicals.Only 5/21 of the top scoring ToxCast? PPAR? activators were activators in our assays, 3 were PPAR? antagonists, the remainder were inactive. The bona fide PPAR? activators we identified induced adipogenesis in 3T3-L1 cells and mBMSCs. Only 7 of the 17 chemicals predicted to be active by the ToxPi promoted adipogenesis, 1 inhibited adipogenesis, and 2 of the 7 predicted negatives were also adipogenic. Of these 9 adipogenic chemicals, 3 activated PPAR?, and 1 activated RXR?.ToxCast? PPAR? and RXR? assays do not correlate well with laboratory measurements of PPAR? and RXR? activity. The adipogenesis ToxPi performed poorly, perhaps due to the performance of ToxCast? assays. We observed a modest predictive value of ToxCast? for PPAR? and RXR? activation and adipogenesis and it is likely that many obesogenic chemicals remain to be identified.Janesick AS, Dimastrogiovanni G, Vanek L, Boulos C, Chamorro-García R, Tang W, Blumberg B. 2016. On the utility of ToxCast? and ToxPi as methods for identifying new obesogens. Environ Health Perspect 124:1214-1226;?http://dx.doi.org/10.1289/ehp.1510352.
View details for DOI 10.1289/ehp.1510352
View details for PubMedID 26757984
View details for PubMedCentralID PMC4977052
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.
View details for DOI 10.1007/s00018-014-1815-9
View details for PubMedID 25558812
Retinoic acid receptor gamma 2 (RAR?2) 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 RAR?2 is engaged in all stages of axial elongation, not solely as a terminator of axial growth. In the absence of RA, RAR?2 represses transcriptional activity in vivo and maintains the pool of caudal progenitor cells and presomitic mesoderm. In the presence of RA, RAR?2 serves as an activator, facilitating somite differentiation. Treatment with an RAR?-selective inverse agonist (NRX205099) or overexpression of dominant-negative RAR? 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-RAR?2), or by treatment with an RAR?-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 RAR?2-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.
View details for DOI 10.1242/dev.103705
View details for PubMedID 24821986
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.
View details for DOI 10.1242/dev.093716
View details for PubMedID 23824578
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). RAR?2 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.
View details for DOI 10.1242/dev.071456
View details for PubMedID 22354841
View details for PubMedCentralID PMC3283127