The optic vesicle comprises a pool of bi-potential progenitor cells from which the retinal pigment epithelium (RPE) and neural retina fates segregate during ocular morphogenesis. Several ...transcription factors and signaling pathways have been shown to be important for RPE maintenance and differentiation, but an understanding of the initial fate specification and determination of this ocular cell type is lacking. We show that Yap/Taz-Tead activity is necessary and sufficient for optic vesicle progenitors to adopt RPE identity in zebrafish. A Tead-responsive transgene is expressed within the domain of the optic cup from which RPE arises, and Yap immunoreactivity localizes to the nuclei of prospective RPE cells. yap (yap1) mutants lack a subset of RPE cells and/or exhibit coloboma. Loss of RPE in yap mutants is exacerbated in combination with taz (wwtr1) mutant alleles such that, when Yap and Taz are both absent, optic vesicle progenitor cells completely lose their ability to form RPE. The mechanism of Yap-dependent RPE cell type determination is reliant on both nuclear localization of Yap and interaction with a Tead co-factor. In contrast to loss of Yap and Taz, overexpression of either protein within optic vesicle progenitors leads to ectopic pigmentation in a dosage-dependent manner. Overall, this study identifies Yap and Taz as key early regulators of RPE genesis and provides a mechanistic framework for understanding the congenital ocular defects of Sveinsson's chorioretinal atrophy and congenital retinal coloboma.
Hedgehog signaling drives oncogenesis in several cancers, and strategies targeting this pathway have been developed, most notably through inhibition of Smoothened (SMO). However, resistance to ...Smoothened inhibitors occurs by genetic changes of Smoothened or other downstream Hedgehog components. Here we overcome these resistance mechanisms by modulating GLI transcription through inhibition of bromo and extra C-terminal (BET) bromodomain proteins. We show that BRD4 and other BET bromodomain proteins regulate GLI transcription downstream of SMO and suppressor of fused (SUFU), and chromatin immunoprecipitation studies reveal that BRD4 directly occupies GLI1 and GLI2 promoters, with a substantial decrease in engagement of these sites after treatment with JQ1, a small-molecule inhibitor targeting BRD4. Globally, genes associated with medulloblastoma-specific GLI1 binding sites are downregulated in response to JQ1 treatment, supporting direct regulation of GLI activity by BRD4. Notably, patient- and GEMM (genetically engineered mouse model)-derived Hedgehog-driven tumors (basal cell carcinoma, medulloblastoma and atypical teratoid rhabdoid tumor) respond to JQ1 even when harboring genetic lesions rendering them resistant to Smoothened antagonists. Altogether, our results reveal BET proteins as critical regulators of Hedgehog pathway transcriptional output and nominate BET bromodomain inhibitors as a strategy for treating Hedgehog-driven tumors with emerged or a priori resistance to Smoothened antagonists.
Refractive errors in vision can be caused by aberrant axial length of the eye, irregular corneal shape, or lens abnormalities. Causes of eye length overgrowth include multiple genetic loci, and ...visual parameters. We evaluate zebrafish as a potential animal model for studies of the genetic, cellular, and signaling basis of emmetropization and myopia. Axial length and other eye dimensions of zebrafish were measured using spectral domain-optical coherence tomography (SD-OCT). We used ocular lens and body metrics to normalize and compare eye size and relative refractive error (difference between observed retinal radial length and controls) in wild-type and lrp2 zebrafish. Zebrafish were dark-reared to assess effects of visual deprivation on eye size. Two relative measurements, ocular axial length to body length and axial length to lens diameter, were found to accurately normalize comparisons of eye sizes between different sized fish (R2=0.9548, R2=0.9921). Ray-traced focal lengths of wild-type zebrafish lenses were equal to their retinal radii, while lrp2 eyes had longer retinal radii than focal lengths. Both genetic mutation (lrp2) and environmental manipulation (dark-rearing) caused elongated eye axes. lrp2 mutants had relative refractive errors of -0.327 compared to wild-types, and dark-reared wild-type fish had relative refractive errors of -0.132 compared to light-reared siblings. Therefore, zebrafish eye anatomy (axial length, lens radius, retinal radius) can be rapidly and accurately measured by SD-OCT, facilitating longitudinal studies of regulated eye growth and emmetropization. Specifically, genes homologous to human myopia candidates may be modified, inactivated or overexpressed in zebrafish, and myopia-sensitizing conditions used to probe gene-environment interactions. Our studies provide foundation for such investigations into genetic contributions that control eye size and impact refractive errors.
Progenitor cell nuclei in the rapidly expanding epithelium of the embryonic vertebrate central nervous system undergo a process called interkinetic nuclear migration (IKNM). Movements of IKNM are ...generally believed to involve smooth migration of nuclei from apical to basal and back during the G1 and G2 phases of the cell cycle, respectively. Yet, this has not been formally demonstrated, nor have the molecular mechanisms that drive IKNM been identified. Using time-lapse confocal microscopy to observe nuclear movements in zebrafish retinal neuroepithelial cells, we show that, except for brief apical nuclear translocations preceding mitosis, IKNM is stochastic rather than smooth and directed. We also show that IKNM is driven largely by actomyosin-dependent forces as it still occurs when the microtubule cytoskeleton is compromised but is blocked when MyosinII activity is inhibited.
Pancreatic cancer (PC) mobilizes myeloid cells from the bone marrow to the tumor where they promote tumor growth and proliferation. Cancer stem cells (CSCs) are a population of tumor cells that are ...responsible for tumor initiation. Aldehyde dehydrogenase-1 activity in PC identifies CSCs, and its activity has been correlated with poor overall prognosis in human PC. Myeloid cells have been shown to impact tumor stemness, but the impact of immunosuppressive tumor-infiltrating granulocytic and monocytic myeloid-derived suppressor cells (Mo-MDSC) on ALDH1
Bright
CSCs and epithelial to mesenchymal transition is not well understood. In this study, we demonstrate that Mo-MDSC (CD11b
+
/Gr1
+
/Ly6G
−
/Ly6C
hi
) significantly increase the frequency of ALDH1
Bright
CSCs in a mouse model of PC. Additionally, there was significant upregulation of genes associated with epithelial to mesenchymal transition. We also found that human PC converts CD14
+
peripheral blood monocytes into Mo-MDSC (CD14
+
/HLA-DR
low/−
) in vitro, and this transformation is dependent on the activation of the STAT3 pathway. In turn, these Mo-MDSC increase the frequency of ALDH1
Bright
CSCs and promote mesenchymal features of tumor cells. Finally, blockade of STAT3 activation reversed the increase in ALDH1
Bright
CSCs. These data suggest that the PC tumor microenvironment transforms monocytes to Mo-MDSC by STAT3 activation, and these cells increase the frequency of ALDH1
Bright
CSCs. Therefore, targeting STAT3 activation may be an effective therapeutic strategy in targeting CSCs in PC.
The Hippo pathway plays major roles in development, regeneration, and cancer. Its activity is tightly regulated by both diffusible chemical ligands and mechanical stimuli. The pathway consists of a ...series of kinases that can control the sub-cellular localization and stability of YAP or TAZ, homologous transcriptional co-factors. Caveolae, small (60–100 nm) bulb-like invaginations of the plasma membrane, are comprised predominantly of caveolin and cavin proteins and can respond to mechanical stimuli. Here, we show that YAP/TAZ, the major transcriptional mediators of the Hippo pathway, are critical for expression of caveolae components and therefore caveolae formation in both mammalian cells and zebrafish. In essence, without YAP/TAZ, the cell loses an entire organelle. CAVEOLIN1 and CAVIN1, the two essential caveolar genes, are direct target genes of YAP/TAZ, regulated via TEA domain (TEAD) transcription factors. Notably, YAP/TAZ become nuclear enriched and facilitate target gene transcription in cells with diminished levels of caveolae. Furthermore, caveolar-mediated shear stress response activates YAP/TAZ. These data link caveolae to Hippo signaling in the context of cellular responses to mechanical stimuli and suggest activity-based feedback regulation between components of caveolae and the outputs of the Hippo pathway.
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•YAP/TAZ are critical for CAVIN1 and CAVEOLIN1 expression and caveolae formation•The essential caveolar genes CAVIN1 and CAVEOLIN1 are direct YAP/TAZ-TEAD target genes•YAP/TAZ are hyperactivated in caveolae-deficient cells•Caveolae facilitate YAP/TAZ-mediated shear stress response
Rausch et al. identifies that the Hippo pathway regulates caveolae abundance via YAP/TAZ-TEAD-mediated transcriptional regulation of the two essential caveolar genes CAVEOLIN1 and CAVIN1. In essence, without YAP/TAZ, the cell loses an entire cellular organelle. In addition, caveolae facilitate shear-stress-mediated signaling via the Hippo pathway.
The goals of this retrospective cohort study were as follows: 1) to describe the long-term prevalence and timing of hearing deterioration following low-dose (12- to 13-Gy marginal dose) stereotactic ...radiosurgery (SRS) for vestibular schwannoma (VS); and 2) to identify clinical variables associated with long-term preservation of useful hearing following treatment.
Patients with serviceable hearing who underwent SRS for VS between 1997 and 2002 were studied. Data including radiosurgery treatment plans, tumor characteristics, pre- and posttreatment pure tone average, speech discrimination scores, and American Academy of Otolaryngology-Head and Neck Surgery hearing class were collected. Time to nonserviceable hearing was estimated using the Kaplan-Meier method. Univariate and multivariate associations with time to nonserviceable hearing were evaluated using Cox proportional hazards regression models.
Forty-four patients met the study criteria and were included. The median duration of audiometric follow-up was 9.3 years. Thirty-six patients developed nonserviceable hearing at a mean of 4.2 years following SRS. The Kaplan-Meier estimated rates of serviceable hearing at 1, 3, 5, 7, and 10 years following SRS were 80%, 55%, 48%, 38%, and 23%, respectively. Multivariate analysis revealed that pretreatment ipsilateral pure tone average (p < 0.001) and tumor size (p = 0.009) were statistically significantly associated with time to nonserviceable hearing.
Durable hearing preservation a decade after low-dose SRS for VS occurs in less than one-fourth of patients. Variables including preoperative hearing capacity and tumor size may be used to predict hearing outcomes following treatment. These findings may assist in pretreatment risk disclosure. Furthermore, these data demonstrate the importance of long-term follow-up when reporting audiometric outcomes following SRS for VS.
During retinal development, neuroepithelial progenitor cells divide in either a symmetric proliferative mode, in which both daughter cells remain mitotic, or in a neurogenic mode, in which at least ...one daughter cell exits the cell cycle and differentiates as a neuron. Although the cellular mechanisms of neurogenesis remain unknown, heterogeneity in cell behaviors has been postulated to influence this cell fate. In this study, we analyze interkinetic nuclear migration, the apical-basal movement of nuclei in phase with the cell cycle, and the relationship of this cell behavior to neurogenesis. Using time-lapse imaging in zebrafish, we show that various parameters of interkinetic nuclear migration are significantly heterogeneous among retinal neuroepithelial cells. We provide direct evidence that neurogenic progenitors have greater basal nuclei migrations during the last cell cycle preceding a terminal mitosis. In addition, we show that atypical protein kinase C (aPKC)-mediated cell polarity is essential for the relationship between nuclear position and neurogenesis. Loss of aPKC also resulted in increased proliferative cell divisions and reduced retinal neurogenesis. Our data support a novel model for neurogenesis, in which interkinetic nuclear migration differentially positions nuclei in neuroepithelial cells and therefore influences selection of progenitors for cell cycle exit based on apical-basal polarized signals.
Coat protein complex II (COPII) factors mediate cargo export from the endoplasmic reticulum (ER), but bulky collagens and lipoproteins are too large for traditional COPII vesicles. Mammalian CTAGE5 ...and TANGO1 have been well characterized individually as specialized cargo receptors at the ER that function with COPII coats to facilitate trafficking of bulky cargoes. Here, we present a genetic interaction study in zebrafish of deletions in
,
, or both to investigate their distinct and complementary potential functions. We found that Ctage5 and Tango1 have different roles related to organogenesis, collagen versus lipoprotein trafficking, stress-pathway activation, and survival. While disruption of both
and
compounded phenotype severity, mutation of either factor alone revealed novel tissue-specific defects in the building of heart, muscle, lens, and intestine, in addition to previously described roles in the development of neural and cartilage tissues. Together, our results demonstrate that Ctage5 and Tango1 have overlapping functions, but also suggest divergent roles in tissue development and homeostasis.
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