Author Correction: Insulin resistance in cavefish as an adaptation to a nutrient-limited environment.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Insulin resistance in cavefish as an adaptation to a nutrient-limited environment.

Periodic food shortages are a major challenge faced by organisms in natural habitats. Cave-dwelling animals must withstand long periods of nutrient deprivation, as-in the absence of photosynthesis-caves depend on external energy sources such as seasonal floods. Here we show that cave-adapted populations of the Mexican tetra, Astyanax mexicanus, have dysregulated blood glucose homeostasis and are insulin-resistant compared to river-adapted populations. We found that multiple cave populations carry a mutation in the insulin receptor that leads to decreased insulin binding in vitro and contributes to hyperglycaemia. Hybrid fish from surface-cave crosses carrying this mutation weigh more than non-carriers, and zebrafish genetically engineered to carry the mutation have increased body weight and insulin resistance. Higher body weight may be advantageous in caves as a strategy to cope with an infrequent food supply. In humans, the identical mutation in the insulin receptor leads to a severe form of insulin resistance and reduced lifespan. However, cavefish have a similar lifespan to surface fish and do not accumulate the advanced glycation end-products in the blood that are typically associated with the progression of diabetes-associated pathologies. Our findings suggest that diminished insulin signalling is beneficial in a nutrient-limited environment and that cavefish may have acquired compensatory mechanisms that enable them to circumvent the typical negative effects associated with failure to regulate blood glucose levels.

Stable transgenesis in Astyanax mexicanus using the Tol2 transposase system.

BACKGROUND: Astyanax mexicanus is a well-established fish model system for evolutionary and developmental biology research. These fish exist as surface forms that inhabit rivers and 30 different populations of cavefish. Despite important progress in the deployment of new technologies, deep mechanistic insights into the genetic basis of evolution, development, and behavior have been limited by a lack of transgenic lines commonly used in genetic model systems. RESULTS: Here, we expand the toolkit of transgenesis by characterizing two novel stable transgenic lines that were generated using the highly efficient Tol2 system, commonly used to generate transgenic zebrafish. A stable transgenic line consisting of the zebrafish ubiquitin promoter expresses enhanced green fluorescent protein ubiquitously throughout development in a surface population of Astyanax. To define specific cell-types, a Cntnap2-mCherry construct labels lateral line mechanosensory neurons in zebrafish. Strikingly, both constructs appear to label the predicted cell types, suggesting many genetic tools and defined promoter regions in zebrafish are directly transferrable to cavefish. CONCLUSION: The lines provide proof-of-principle for the application of Tol2 transgenic technology in A. mexicanus. Expansion on these initial transgenic lines will provide a platform to address broadly important problems in the quest to bridge the genotype-phenotype gap.

Analysis of dynamic changes in retinoid-induced transcription and epigenetic profiles of murine Hox clusters in ES cells.

The clustered Hox genes, which are highly conserved across metazoans, encode homeodomain-containing transcription factors that provide a blueprint for segmental identity along the body axis. Recent studies have underscored that in addition to encoding Hox genes, the homeotic clusters contain key noncoding RNA genes that play a central role in development. In this study, we have taken advantage of genome-wide approaches to provide a detailed analysis of retinoic acid (RA)-induced transcriptional and epigenetic changes within the homeotic clusters of mouse embryonic stem cells. Although there is a general colinear response, our analyses suggest a lack of strict colinearity for several genes in the HoxA and HoxB clusters. We have identified transcribed novel noncoding RNAs (ncRNAs) and their cis-regulatory elements that function in response to RA and demonstrated that the expression of these ncRNAs from both strands represent some of the most rapidly induced transcripts in ES cells. Finally, we have provided dynamic analyses of chromatin modifications for the coding and noncoding genes expressed upon activation and suggest that active transcription can occur in the presence of chromatin modifications and machineries associated with repressed transcription state over the clusters. Overall, our data provide a resource for a better understanding of the dynamic nature of the coding and noncoding transcripts and their associated chromatin marks in the regulation of homeotic gene transcription during development.

Improved transcription and translation with L-leucine stimulation of mTORC1 in Roberts syndrome.

BACKGROUND: Roberts syndrome (RBS) is a human developmental disorder caused by mutations in the cohesin acetyltransferase ESCO2. We previously reported that mTORC1 signaling was depressed and overall translation was reduced in RBS cells and zebrafish models for RBS. Treatment of RBS cells and zebrafish RBS models with L-leucine partially rescued mTOR function and protein synthesis, correlating with increased cell division and improved development. RESULTS: In this study, we use RBS cells to model mTORC1 repression and analyze transcription and translation with ribosome profiling to determine gene-level effects of L-leucine. L-leucine treatment partially rescued translational efficiency of ribosomal subunits, translation initiation factors, snoRNA production, and mitochondrial function in RBS cells, consistent with these processes being mTORC1 controlled. In contrast, other genes are differentially expressed independent of L-leucine treatment, including imprinted genes such as H19 and GTL2, miRNAs regulated by GTL2, HOX genes, and genes in nucleolar associated domains. CONCLUSIONS: Our study distinguishes between gene expression changes in RBS cells that are TOR dependent and those that are independent. Some of the TOR independent gene expression changes likely reflect the architectural role of cohesin in chromatin looping and gene expression. This study reveals the dramatic rescue effects of L-leucine stimulation of mTORC1 in RBS cells and supports that normal gene expression and translation requires ESCO2 function.

Notch2 regulates BMP signaling and epithelial morphogenesis in the ciliary body of the mouse eye.

The ciliary body (CB) of the mammalian eye is responsible for secreting aqueous humor to maintain intraocular pressure, which is elevated in the eyes of glaucoma patients. It contains a folded two-layered epithelial structure comprising the nonpigmented inner ciliary epithelium (ICE), the pigmented outer ciliary epithelium (OCE), and the underlying stroma. Although the CB has an important function in the eye, its morphogenesis remains poorly studied. In this study, we show that conditional inactivation of the Jagged 1 (Jag1)-Notch2 signaling pathway in the developing CB abolishes its morphogenesis. Notch2 is expressed in the OCE of the CB, whereas Jag1 is expressed in the ICE. Conditional inactivation of Jag1 in the ICE or Notch2 in the OCE disrupts CB morphogenesis, but neither affects the specification of the CB region. Notch2 signaling in the OCE is required for promoting cell proliferation and maintaining bone morphogenetic protein (BMP) signaling, both of which have been suggested to be important for CB morphogenesis. Although Notch and BMP signaling pathways are known to cross-talk via the interaction between their downstream transcriptional factors, this study suggests that Notch2 maintains BMP signaling in the OCE possibly by repressing expression of secreted BMP inhibitors. Based on our findings, we propose that Jag1-Notch2 signaling controls CB morphogenesis at least in part by regulating cell proliferation and BMP signaling.

Delayed correlation of mRNA and protein expression in rapamycin-treated cells and a role for Ggc1 in cellular sensitivity to rapamycin.

To identify new molecular targets of rapamycin, an anticancer and immunosuppressive drug, we analyzed temporal changes in yeast over 6 h in response to rapamycin at the transcriptome and proteome levels and integrated the expression patterns with functional profiling. We show that the integration of transcriptomics, proteomics, and functional data sets provides novel insights into the molecular mechanisms of rapamycin action. We first observed a temporal delay in the correlation of mRNA and protein expression where mRNA expression at 1 and 2 h correlated best with protein expression changes after 6 h of rapamycin treatment. This was especially the case for the inhibition of ribosome biogenesis and induction of heat shock and autophagy essential to promote the cellular sensitivity to rapamycin. However, increased levels of vacuolar protease could enhance resistance to rapamycin. Of the 85 proteins identified as statistically significantly changing in abundance, most of the proteins that decreased in abundance were correlated with a decrease in mRNA expression. However, of the 56 proteins increasing in abundance, 26 were not correlated with an increase in mRNA expression. These protein changes were correlated with unchanged or down-regulated mRNA expression. These proteins, involved in mitochondrial genome maintenance, endocytosis, or drug export, represent new candidates effecting rapamycin action whose expression might be post-transcriptionally or post-translationally regulated. We identified GGC1, a mitochondrial GTP/GDP carrier, as a new component of the rapamycin/target of rapamycin (TOR) signaling pathway. We determined that the protein product of GGC1 was stabilized in the presence of rapamycin, and the deletion of the GGC1 enhanced growth fitness in the presence of rapamycin. A dynamic mRNA expression analysis of Deltaggc1 and wild-type cells treated with rapamycin revealed a key role for Ggc1p in the regulation of ribosome biogenesis and cell cycle progression under TOR control.

Piwi is required in multiple cell types to control germline stem cell lineage development in the Drosophila ovary.

The piRNA pathway plays an important role in maintaining genome stability in the germ line by silencing transposable elements (TEs) from fly to mammals. As a highly conserved piRNA pathway component, Piwi is widely expressed in both germ cells and somatic cells in the Drosophila ovary and is required for piRNA production in both cell types. In addition to its known role in somatic cap cells to maintain germline stem cells (GSCs), this study has demonstrated that Piwi has novel functions in somatic cells and germ cells of the Drosophila ovary to promote germ cell differentiation. Piwi knockdown in escort cells causes a reduction in escort cell (EC) number and accumulation of undifferentiated germ cells, some of which show active BMP signaling, indicating that Piwi is required to maintain ECs and promote germ cell differentiation. Simultaneous knockdown of dpp, encoding a BMP, in ECs can partially rescue the germ cell differentiation defect, indicating that Piwi is required in ECs to repress dpp. Consistent with its key role in piRNA production, TE transcripts increase significantly and DNA damage is also elevated in the piwi knockdown somatic cells. Germ cell-specific knockdown of piwi surprisingly causes depletion of germ cells before adulthood, suggesting that Piwi might control primordial germ cell maintenance or GSC establishment. Finally, Piwi inactivation in the germ line of the adult ovary leads to gradual GSC loss and germ cell differentiation defects, indicating the intrinsic role of Piwi in adult GSC maintenance and differentiation. This study has revealed new germline requirement of Piwi in controlling GSC maintenance and lineage differentiation as well as its new somatic function in promoting germ cell differentiation. Therefore, Piwi is required in multiple cell types to control GSC lineage development in the Drosophila ovary.

Defective FGF signaling causes coloboma formation and disrupts retinal neurogenesis.

The optic fissure (OF) is a transient opening on the ventral side of the developing vertebrate eye that closes before nearly all retinal progenitor cell differentiation has occurred. Failure to close the OF results in coloboma, a congenital disease that is a major cause of childhood blindness. Although human genetic studies and animal models have linked a number of genes to coloboma, the cellular and molecular mechanisms driving the closure of the OF are still largely unclear. In this study, we used Cre-LoxP-mediated conditional removal of fibroblast growth factor (FGF) receptors, Fgfr1 and Fgfr2, from the developing optic cup (OC) to show that FGF signaling regulates the closing of the OF. Our molecular, cellular and transcriptome analyses of Fgfr1 and Fgfr2 double conditional knockout OCs suggest that FGF signaling controls the OF closure through modulation of retinal progenitor cell proliferation, fate specification and morphological changes. Furthermore, Fgfr1 and Fgfr2 double conditional mutant retinal progenitor cells fail to initiate retinal ganglion cell (RGC) genesis. Taken together, our mouse genetic studies reveal that FGF signaling is essential for OF morphogenesis and RGC development.

Poised RNA polymerase II changes over developmental time and prepares genes for future expression.

Poised RNA polymerase II (Pol II) is predominantly found at developmental control genes and is thought to allow their rapid and synchronous induction in response to extracellular signals. How the recruitment of poised RNA Pol II is regulated during development is not known. By isolating muscle tissue from Drosophila embryos at five stages of differentiation, we show that the recruitment of poised Pol II occurs at many genes de novo and this makes them permissive for future gene expression. A comparison with other tissues shows that these changes are stage specific and not tissue specific. In contrast, Polycomb group repression is tissue specific, and in combination with Pol II (the balanced state) marks genes with highly dynamic expression. This suggests that poised Pol II is temporally regulated and is held in check in a tissue-specific fashion. We compare our data with findings in mammalian embryonic stem cells and discuss a framework for predicting developmental programs on the basis of the chromatin state.

Prevention of the neurocristopathy Treacher Collins syndrome through inhibition of p53 function.

Treacher Collins syndrome (TCS) is a congenital disorder of craniofacial development arising from mutations in TCOF1, which encodes the nucleolar phosphoprotein Treacle. Haploinsufficiency of Tcof1 perturbs mature ribosome biogenesis, resulting in stabilization of p53 and the cyclin G1-mediated cell-cycle arrest that underpins the specificity of neuroepithelial apoptosis and neural crest cell hypoplasia characteristic of TCS. Here we show that inhibition of p53 prevents cyclin G1-driven apoptotic elimination of neural crest cells while rescuing the craniofacial abnormalities associated with mutations in Tcof1 and extending life span. These improvements, however, occur independently of the effects on ribosome biogenesis; thus suggesting that it is p53-dependent neuroepithelial apoptosis that is the primary mechanism underlying the pathogenesis of TCS. Our work further implies that neuroepithelial and neural crest cells are particularly sensitive to cellular stress during embryogenesis and that suppression of p53 function provides an attractive avenue for possible clinical prevention of TCS craniofacial birth defects and possibly those of other neurocristopathies.

N-cadherin expression level distinguishes reserved versus primed states of hematopoietic stem cells.

Osteoblasts expressing the homophilic adhesion molecule N-cadherin form a hematopoietic stem cell (HSC) niche. Therefore, we examined how N-cadherin expression in HSCs relates to their function. We found that bone marrow (BM) cells highly expressing N-cadherin (N-cadherin(hi)) are not stem cells, being largely devoid of a Lineage(-)Sca1(+)cKit(+) population and unable to reconstitute hematopoietic lineages in irradiated recipient mice. Instead, long-term HSCs form distinct populations expressing N-cadherin at intermediate (N-cadherin(int)) or low (N-cadherin(lo)) levels. The minority N-cadherin(lo) population can robustly reconstitute the hematopoietic system, express genes that may prime them to mobilize, and predominate among HSCs mobilized from BM to spleen. The larger N-cadherin(int) population performs poorly in reconstitution assays when freshly isolated but improves in response to overnight in vitro culture. Their expression profile and lower cell-cycle entry rate suggest N-cadherin(int) cells are being held in reserve. Thus, differential N-cadherin expression reflects functional distinctions between two HSC subpopulations.

Transcription alters chromosomal locations of cohesin in Saccharomyces cerevisiae.

In eukaryotic cells, cohesion between sister chromatids allows chromosomes to biorient on the metaphase plate and holds them together until they separate into daughter cells during mitosis. Cohesion is mediated by the cohesin protein complex. Although the association of this complex with particular regions of the genome is highly reproducible, it is unclear what distinguishes a chromosomal region for cohesin association. Since one of the primary locations of cohesin is intergenic regions between converging transcription units, we explored the relationship between transcription and cohesin localization. Chromatin immunoprecipitation followed by hybridization to a microarray (ChIP chip) indicated that transcript elongation into cohesin association sites results in the local disassociation of cohesin. Once transcription is halted, cohesin can reassociate with its original sites, independent of DNA replication and the cohesin loading factor Scc2, although cohesin association with chromosomes in G2/M is not functional for cohesion. A computer program was developed to systematically identify differences between two ChIP chip data sets. Our results are consistent with a model for cohesin association in which (i) a portion of cohesin can be dynamically loaded and unloaded to accommodate transcription and (ii) the cohesin complex has preferences for features of chromatin that are a reflection of the local transcriptional status. Taken together, our results suggest that cohesion may be degraded by transcription.

Wingless directly represses DPP morphogen expression via an armadillo/TCF/Brinker complex.

BACKGROUND: Spatially restricted morphogen expression drives many patterning and regeneration processes, but how is the pattern of morphogen expression established and maintained? Patterning of Drosophila leg imaginal discs requires expression of the DPP morphogen dorsally and the wingless (WG) morphogen ventrally. We have shown that these mutually exclusive patterns of expression are controlled by a self-organizing system of feedback loops that involve WG and DPP, but whether the feedback is direct or indirect is not known. METHODS/FINDINGS: By analyzing expression patterns of regulatory DNA driving reporter genes in different genetic backgrounds, we identify a key component of this system by showing that WG directly represses transcription of the dpp gene in the ventral leg disc. Repression of dpp requires a tri-partite complex of the WG mediators armadillo (ARM) and dTCF, and the co-repressor Brinker, (BRK), wherein ARM.dTCF and BRK bind to independent sites within the dpp locus. CONCLUSIONS/SIGNIFICANCE: Many examples of dTCF repression in the absence of WNT signaling have been described, but few examples of signal-driven repression requiring both ARM and dTCF binding have been reported. Thus, our findings represent a new mode of WG mediated repression and demonstrate that direct regulation between morphogen signaling pathways can contribute to a robust self-organizing system capable of dynamically maintaining territories of morphogen expression.

A complex oscillating network of signaling genes underlies the mouse segmentation clock.

The segmental pattern of the spine is established early in development, when the vertebral precursors, the somites, are rhythmically produced from the presomitic mesoderm. Microarray studies of the mouse presomitic mesoderm transcriptome reveal that the oscillator associated with this process, the segmentation clock, drives the periodic expression of a large network of cyclic genes involved in cell signaling. Mutually exclusive activation of the notch-fibroblast growth factor and Wnt pathways during each cycle suggests that coordinated regulation of these three pathways underlies the clock oscillator.