Browsing by Author "Bejsovec, Amy M"

Developmental physiology is very sensitive to nutrient availability. For instance, in the nematode C. elegans, newly hatched L1-stage larvae require food to initiate postembryonic development. Despite the essential role of food in C. elegans development, the contribution of food perception versus ingestion on physiology has not been delineated. We used a pharmacological approach to uncouple the effects of food (bacteria) perception and ingestion in C. elegans. Perception was not sufficient to promote postembryonic development in L1-stage larvae. However, L1 larvae exposed to food without ingestion failed to develop upon return to normal culture conditions, instead displaying an irreversible arrest phenotype. Inhibition of gene expression during perception rescued subsequent development, demonstrating that the response to perception without feeding is deleterious. Perception altered DAF-16/FOXO subcellular localization, reflecting activation of insulin/IGF signaling (IIS). However, genetic manipulation of IIS did not modify the irreversible arrest phenotype caused by food perception, revealing that wild-type function of the IIS pathway is not required to produce this phenotype and that other pathways affected by perception of food in the absence of its ingestion are likely to be involved. Gene expression and Nile red staining showed that food perception could alter lipid metabolism and storage. We found that starved larvae sense environmental polypeptides, with similar molecular and developmental effects as perception of bacteria. We conclude that actual ingestion of food is required to initiate postembryonic development in C. elegans. We also conclude that polypeptides are perceived as a food-associated cue in this and likely other animals, initiating a signaling and gene regulatory cascade that alters metabolism in anticipation of feeding and development, but that this response is detrimental if feeding does not occur.

The C. elegans insulin-like signaling network supports homeostasis and developmental plasticity. The genome encodes 40 insulin-like peptides and one known receptor. Feedback regulation has been reported, but the extent of feedback and its effect on signaling dynamics in response to changes in nutrient availability has not been determined. We measured mRNA expression for each insulin-like peptide, the receptor daf-2, components of the PI3K pathway, and its transcriptional effectors daf-16/FOXO and skn-1/Nrf at high temporal resolution during transition from a starved, quiescent state to a fed, growing state in wild type and mutants affecting daf-2/InsR and daf-16/FOXO. We also analyzed the effect of temperature on insulin-like gene expression. We found that most PI3K pathway components and insulin-like peptides are affected by signaling activity, revealing pervasive positive and negative feedback regulation at intra- and inter-cellular levels. Reporter gene analysis demonstrated that the daf-2/InsR agonist daf-28 positively regulates its own transcription and that the putative agonist ins-6 cross-regulates DAF-28 protein expression through feedback. Our results show that positive and negative feedback regulation of insulin-like signaling is widespread, giving rise to an organismal FOXO-to-FOXO signaling network that supports homeostasis during fluctuations in nutrient availability.

L1 arrest (or "L1 diapause") is associated with increased stress resistance, supporting starvation survival. Loss of the transcription factor daf-16/FOXO results in arrest-defective and starvation-sensitive phenotypes. We show that daf-16/FOXO regulates L1 arrest cell-nonautonomously, suggesting that insulin/IGF signaling regulates at least one additional signaling pathway. We used mRNA-seq to identify candidate signaling molecules affected by daf-16/FOXO during L1 arrest. dbl-1/TGF-β, a ligand for the Sma/Mab pathway, daf-12/NHR, and daf-36/oxygenase, an upstream component of the daf-12 steroid hormone signaling pathway, were up-regulated during L1 arrest in a daf-16/FOXO mutant. Using genetic epistasis analysis, we show that dbl-1/TGF-β and daf-12/NHR steroid hormone signaling pathways are required for the daf-16/FOXO arrest-defective phenotype, suggesting that daf-16/FOXO represses dbl-1/TGF-β, daf-12/NHR and daf-36/oxygenase. The dbl-1/TGF-β and daf-12/NHR pathways have not previously been shown to affect L1 development, but we found that disruption of these pathways delayed L1 development in fed larvae, consistent with these pathways promoting development in starved daf-16/FOXO mutants. Though the dbl-1/TGF-β and daf-12/NHR pathways are epistatic to daf-16/FOXO for the arrest-defective phenotype, disruption of these pathways does not suppress starvation sensitivity of daf-16/FOXO mutants. This observation uncouples starvation survival from developmental arrest, indicating that DAF-16/FOXO targets distinct effectors for each phenotype and revealing that inappropriate development during starvation does not cause the early demise of daf-16/FOXO mutants. We show that daf-16/FOXO promotes developmental arrest cell-nonautonomously by repressing pathways that promote larval development.

The Wingless (Wg)/Wnt signal transduction pathway directs a variety of cell fate decisions in developing animal embryos. Despite the identification of many Wg pathway components to date, it is still not clear how these proteins work together to generate cellular identities. We have carried out a series of genetic screens in Drosophila to identify new components involved in Wg signaling. Two mutant lines that modify wg-mediated epidermal patterning carry mutations in the RacGap50C gene. These mutations on their own cause cuticle pattern disruptions that include replacement of ventral denticles with naked cuticle, which indicate that the Wg pathway is ectopically activated in mutant embryos. In addition, RacGap50C mutations also interact genetically with naked cuticle and Axin, known negative regulators of the Wg pathway. These phenotypes suggest that the RacGap50C gene product participates in the negative regulation of Wg pathway activity.

In addition to the role of RacGap50C in regulating Wg signaling, RacGap50C, with the kinesin like protein Pavarotti (Pav), is essential for cytokinesis in dividing cells. Surprisingly, Pav, like RacGap50C is able to repress Wnt stimulated reporter gene activity in mammalian tissue culture. Expression of RacGap50C or Pav does not alter the levels of endogenous BetaCatenin, nor does it prevent BetaCatenin from entering the nucleus and binding the transcription factor TCF. However, reporter gene activity is significantly diminished in cells expressing RacGap50C or Pav, indicating an inability of this nuclear BetaCatenin to activate transcription. RacGap50C and Pav are also found in the nucleus of interphase cells and this subcellular localization may be relevant to their role in regulation of the Wnt pathway. Although RacGap50C and Pav colocalize, our data indicate distinct roles for these two proteins in Wnt signaling, as they show different genetic interactions with nkd in Drosophila embryos and with TCF in mammalian cells.

In addition, I have identified regions of the RacGap50C proteins that are dispensable for Wnt signaling repression. Pav-binding region, and the cysteine rich region of RacGap50C are not required for the activity of RacGap50C in Drosophila embryos, nor is GAP activity. Conversely, the middle region of the RacGap50C, which contains a nuclear localization signal, and the RacGap domain, which contains regions required for cofactor binding, are required for RacGap50C regulation of Wnt signaling. In summary, these results indicate a unique role for RacGap50C and Pav in regulating Wnt signaling at the level of BetaCatenin target gene activation.

During development, morphogenetic processes require the integration of numerous signals to properly shape cells and tissues. These signals are interpreted by cells to induce the precise transcriptional circuitry that controls morphogenesis. In the fly embryonic epidermis, the fly Wnt, wingless (wg), generates naked cuticle zones that separate the ventral denticle belts by repressing expression of shavenbaby (svb), which encodes a transcription factor required for denticle formation. What is not known is how Wg and Svb interact to produce the stereotyped diversity of denticle shapes within each belt. One possibility is that graded levels of Svb, responding to graded levels of Wg signaling, determine denticle shape. Indeed, we found that the svb promoter responds differentially to altered Wg activity levels. However, artificially increasing the levels of ectopic svb does not produce morphologically distinct denticles, suggesting that additional factors are involved. We have discovered that a second Wg-responsive transcription factor, encoded by SoxNeuro (SoxN), cooperates with Svb to shape the denticles. Co-expressing SoxN with svb is sufficient to rescue the morphology of denticles in an ectopic location. Furthermore, embryos that lack Svb activity retain the ability to produce a small number of rounded, reduced ventral denticles, due to SoxN activity. We found that svb;SoxN double mutant embryos secrete cuticles that completely lack ventral denticles and dorsal hairs. We also found that a group of known Svb target genes belonging to the zona pellucida family of proteins are differentially activated by SoxN. Finally, we discovered two novel target genes of SoxN, CG16885 and CG30101, which are expressed in denticle-producing cells and which are regulated independently of Svb. SoxN was shown previously to down-regulate Wg signaling and to promote expression of svb. Here, we propose that SoxN acts with Svb, in an additional, direct role, to promote denticle morphogenesis.