Evolution of New cis-Regulatory Motifs Required for Cell-Specific Gene Expression in Caenorhabditis

Patterning of C. elegans vulval cell fates relies on inductive signaling. In this induction event, a single cell, the gonadal anchor cell, secretes LIN-3/EGF and induces three out of six competent precursor cells to acquire a vulval fate. We previously showed that this developmental system is robust to a four-fold variation in lin-3/EGF genetic dose. Here using single-molecule FISH, we find that the mean level of expression of lin-3 in the anchor cell is remarkably conserved. No change in lin-3 expression level could be detected among C. elegans wild isolates and only a low level of change-less than 30%-in the Caenorhabditis genus and in Oscheius tipulae. In C. elegans, lin-3 expression in the anchor cell is known to require three transcription factor binding sites, specifically two E-boxes and a nuclear-hormone-receptor (NHR) binding site. Mutation of any of these three elements in C. elegans results in a dramatic decrease in lin-3 expression. Yet only a single E-box is found in the Drosophilae supergroup of Caenorhabditis species, including C. angaria, while the NHR-binding site likely only evolved at the base of the Elegans group. We find that a transgene from C. angaria bearing a single E-box is sufficient for normal expression in C. elegans. Even a short 58 bp cis-regulatory fragment from C. angaria with this single E-box is able to replace the three transcription factor binding sites at the endogenous C. elegans lin-3 locus, resulting in the wild-type expression level. Thus, regulatory evolution occurring in cis within a 58 bp lin-3 fragment, results in a strict requirement for the NHR binding site and a second E-box in C. elegans. This single-cell, single-molecule, quantitative and functional evo-devo study demonstrates that conserved expression levels can hide extensive change in cis-regulatory site requirements and highlights the evolution of new cis-regulatory elements required for cell-specific gene expression

Evolutionary Conservation of the Heterochronic Pathway in C. elegans and C. briggsae

Heterochronic genes control the sequence and timing of developmental events during four larval stages of Caenorhabitis nematodes. Mutations in these genes may cause skipping or reiteration of developmental events. C. briggsae is a close relative of C. elegans. These species have similar morphology and share the same ecological niche. C. briggsae undergoes the same developmental pathway consisting of four larval stages before reaching adulthood. It also has the same set of heterochronic genes. Lin-28 is one of the heterochronic genes that also exists in other animals from flies to humans. It conservatively blocks the maturation of let-7 miRNA, the process is generally associated with the stem cell state. lin-28 is silenced as cells differentiate. C. elegans mutants of lin-28 have a reduced number of seam cells and precocious alae. Despite the highly conserved protein sequence, C. briggsae develop a distinct phenotype when its lin 28 is disrupted. Worms did not have a characteristic vulval development defect, they also became lethargic and had a reduced fertility. This observation led to a question of how conserved the heterochronic pathway is in close species

Centro-affine normal flows on curves: Harnack estimates and Ancient solutions

We prove that the only compact, origin-symmetric, strictly convex ancient solutions of the planar $p$ centro-affine normal flows are contracting origin-centered ellipses.Comment: I changed the title and fixed some typos. To appear in Annales de l'Institut Henri Poincar\'e (C) Analyse Non Lin\'eair

C. elegans LIN-28 controls temporal cell fate progression by regulating LIN-46 expression via the 5\u27 UTR of lin-46 mRNA

Lin28/LIN-28 is a conserved RNA-binding protein that promotes proliferation and pluripotency and can be oncogenic in mammals. Mammalian Lin28 and C. elegans LIN-28 have been shown to inhibit biogenesis of the conserved cellular differentiation-promoting microRNA let-7 by directly binding to unprocessed let-7 transcripts. Lin28/LIN-28 also bind and regulate many mRNAs in diverse cell types. However, the determinants and consequences of LIN-28-mRNA interactions are not well understood. Here, we report that C. elegans LIN-28 represses the expression of LIN-46, a downstream protein in the heterochronic pathway. We find that lin-28 and sequences within the lin-46 5\u27 UTR are required to prevent LIN-46 expression at early larval stages. Moreover, we find that precocious LIN-46 expression caused by mutations in the lin-46 5\u27 UTR is sufficient to cause precocious heterochronic defects similar to those of lin-28(lf) animals. Thus, our findings demonstrate the biological importance of the regulation of individual target mRNAs by LIN-28

Identification of Carnitine Transporter CT1 Binding Protein Lin-7 in Nervous System

_L-Carnitine is an essential component of mitochondrial fatty acid b-oxidation in the muscle and may control the acetyl moiety levels in the brain for acetylcholine synthesis. Carnitine transporter 1(CT1)is the high affinity _L-carnitine transporter whose localization was observed in the kidney, testis, liver, skeletal muscle and brain. To clarify the molecular mechanism of carnitine transport, we sought to find the interacting protein that may be related to the transport function of CT1. Using the intracellular C-terminal region of rat CT1 containing PDZ(PSD95/DLG/ZO-1)motif as bait, we performed the yeast two-hybrid screening against rat brain cDNA library. Thirty two positive clones were obtained from the 2.7×10^7 clones screened. One of them was PDZ domain-containing protein Lin-7. We found that Lin-7 interacts specifically with C-termini of CT1:deletion and mutation of the CT1 C-terminal PDZ-motif abolished the interaction with Lin-7 in the yeast two-hybrid assay. In addition, a PDZ domain within Lin-7 associates with the CT1 C-terminal. The association of CT1 with Lin-7 enhanced _L-carnitine transport activities in HEK293 cells although there is no statistical significance. Coexpression of Lin-7 and CT1 is identified in motor neurons of the spinal cord ventral horn together with Lin-2, a binding partner of Lin-7 known to assemble proteins involved in synaptic vesicle exocytosis and synaptic junctions. Therefore, Lin-7 interacts with CT1 and may regulate their subcellular distribution or function in central nervous system

LIN-42, the Caenorhabditis elegans PERIOD homolog, Negatively Regulates MicroRNA Transcription

During C. elegans development, microRNAs (miRNAs) function as molecular switches that define temporal gene expression and cell lineage patterns in a dosage-dependent manner. It is critical, therefore, that the expression of miRNAs be tightly regulated so that target mRNA expression is properly controlled. The molecular mechanisms that function to optimize or control miRNA levels during development are unknown. Here we find that mutations in lin-42, the C. elegans homolog of the circadian-related period gene, suppress multiple dosage-dependent miRNA phenotypes including those involved in developmental timing and neuronal cell fate determination. Analysis of mature miRNA levels in lin-42 mutants indicates that lin-42 functions to attenuate miRNA expression. Through the analysis of transcriptional reporters, we show that the upstream cis-acting regulatory regions of several miRNA genes are sufficient to promote highly dynamic transcription that is coupled to the molting cycles of post-embryonic development. Immunoprecipitation of LIN-42 complexes indicates that LIN-42 binds the putative cis-regulatory regions of both non-coding and protein-coding genes and likely plays a role in regulating their transcription. Consistent with this hypothesis, analysis of miRNA transcriptional reporters in lin-42 mutants indicates that lin-42 regulates miRNA transcription. Surprisingly, strong loss-of-function mutations in lin-42 do not abolish the oscillatory expression patterns of lin-4 and let-7 transcription but lead to increased expression of these genes. We propose that lin-42 functions to negatively regulate the transcriptional output of multiple miRNAs and mRNAs and therefore coordinates the expression levels of genes that dictate temporal cell fate with other regulatory programs that promote rhythmic gene expression

The \u3cem\u3elet-7\u3c/em\u3e MicroRNA Family Members \u3cem\u3emir\u3c/em\u3e-48, \u3cem\u3emir\u3c/em\u3e-84, and mir-241 Function Together to Regulate Developmental Timing in \u3cem\u3eCaenorhabditis elegans\u3c/em\u3e

The microRNA let-7 is a critical regulator of developmental timing events at the larval-to-adult transition in C. elegans. Recently, microRNAs with sequence similarity to let-7 have been identified. We find that doubly mutant animals lacking the let-7 family microRNA genes mir-48 and mir-84 exhibit retarded molting behavior and retarded adult gene expression in the hypodermis. Triply mutant animals lacking mir-48, mir-84, and mir-241 exhibit repetition of L2-stage events in addition to retarded adult-stage events. mir-48, mir-84, and mir-241 function together to control the L2-to-L3 transition, likely by base pairing to complementary sites in the hbl-1 3′ UTR and downregulating hbl-1 activity. Genetic analysis indicates that mir-48, mir-84, and mir-241 specify the timing of the L2-to-L3 transition in parallel to the heterochronic genes lin-28 and lin-46. These results indicate that let-7 family microRNAs function in combination to affect both early and late developmental timing decisions

Genetics of intercellular signalling in C. elegans

Cell-cell interactions play a significant role in controlling cell fate during development of the nematode Caenorhabditis elegans. It has been found that two genes, glp-1 and lin-12, are required for many of these decisions. glp-1 is required for induction of mitotic proliferation in the germline by the somatic distal tip cell and for induction of the anterior pharynx early in embryogenesis. lin-12 is required for the interactions between cells of equivalent developmental potential, which allow them to take on different fates. Comparison of these two genes on a molecular level indicates that they are similar in sequence and organization, suggesting that the mechanisms of these two different sets of cell-cell interactions are similar

Coping with suboptimal water temperature: modifications in blood parameters, body composition, and postingestive-driven diet selection in Nile tilapia fed two vegetable oil blends

The world tilapia production faces seasonal variations. However, very few nutritional studies have addressed suboptimal temperature. We evaluated the effect of two temperatures (20 or 30 °C) and two vegetable oil blends (one rich in corn oil (COR) and one rich linseed oil (LIN)) on tilapia growth, body composition, and blood parameters using a 2 × 2 factorial design with the following treatments: COR-20; LIN-20; COR-30; LIN-30 (Trial 1). In addition, we also evaluated the effect of postingestive signals of dietary oils when the organoleptic properties of diets were isolated (Trial 2). In the Trial 1, 256 fish (15.36 ± 0.14 g) were placed in 16 aquariums and submitted during 30 days to the 2 × 2 factorial designs: COR-20; LIN-20; COR-30; LIN-30. The temperatures were established in two independent water recirculation systems. In the Trial 2, 96 fish (34.02 ± 0.79 g) were placed in 12 aquariums and subjected to the same experimental design of Trial 1, but to evaluate fish feeding behavior. They were allowed to select the encapsulated diets provided in different feeding halls to evaluate if diet preferences are influenced by postingestive signals. As the Trial 1 results show, diets had no significant effects on growth, dietary protein use, and body centesimal composition, but 30 °C induced the best performance and protein deposition (P < 0.05). LIN-20 showed lower very-low-density lipoprotein and cortisol, but higher high-density lipoprotein (HDL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and triglycerides (TG) than COR-20 (P < 0.05). COR-30 presented higher HDL, AST, ALT, TG, and cortisol than LIN-30. The fish fed COR showed lower C20:5n-3 (EPA) and higher n-6 than fish fed LIN (P < 0.05). The fish fed LIN had high n-3 highly unsaturated fatty acid. ∑ polyunsaturated fatty acid was higher at 30 °C. Finally, the tilapia in Trial 2 showed clear diet intake regulation and preference for LIN (P < 0.05), regardless of temperature. In short, lipid sources had no influence on tilapia performance; however, temperature affects carcass lipid deposition as well as fatty acids profile. Notably, the preference for linseed oil can suggest nutritional metabolic issues, contributing to animal behavior knowledge