The Lhx1-Ldb1 complex interacts with Furry to regulate microRNA expression during pronephric kidney development

The molecular events driving specification of the kidney have been well characterized. However, how the initial kidney field size is established, patterned, and proportioned is not well characterized. Lhx1 is a transcription factor expressed in pronephric progenitors and is required for specification of the kidney, but few Lhx1 interacting proteins or downstream targets have been identified. By tandem-affinity purification, we isolated FRY like transcriptional coactivator (Fryl), one of two paralogous genes, fryl and furry (fry), have been described in vertebrates. Both proteins were found to interact with the Ldb1-Lhx1 complex, but our studies focused on Lhx1/Fry functional roles, as they are expressed in overlapping domains. We found that Xenopus embryos depleted of fry exhibit loss of pronephric mesoderm, phenocopying the Lhx1-depleted animals. In addition, we demonstrated a synergism between Fry and Lhx1, identified candidate microRNAs regulated by the pair, and confirmed these microRNA clusters influence specification of the kidney. Therefore, our data shows that a constitutively-active Ldb1-Lhx1 complex interacts with a broadly expressed microRNA repressor, Fry, to establish the kidney field.Fil: Espiritu, Eugenel B.. University of Pittsburgh; Estados UnidosFil: Crunk, Amanda E.. University of Pittsburgh; Estados UnidosFil: Bais, Abha. University of Pittsburgh; Estados UnidosFil: Hochbaum, Daniel. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cervino, Ailen Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Phua, Yu Leng. University Of Pittsburgh Medical Center; Estados UnidosFil: Butterworth, Michael B.. University of Pittsburgh; Estados UnidosFil: Goto, Toshiyasu. Tokyo Medical And Dental University; JapónFil: Ho, Jacqueline. University Of Pittsburgh Medical Center; Estados UnidosFil: Hukriede, Neil A.. University of Pittsburgh; Estados UnidosFil: Cirio, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Deep small RNA sequencing from the nematode Ascaris reveals conservation, functional diversification, and novel developmental profiles

Eukaryotic cells express several classes of small RNAs that regulate gene expression and ensure genome maintenance. Endogenous siRNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs) mainly control gene and transposon expression in the germline, while microRNAs (miRNAs) generally function in post-transcriptional gene silencing in both somatic and germline cells. To provide an evolutionary and developmental perspective on small RNA pathways in nematodes, we identified and characterized known and novel small RNA classes through gametogenesis and embryo development in the parasitic nematode Ascaris suum and compared them with known small RNAs of Caenorhabditis elegans. piRNAs, Piwi-clade Argonautes, and other proteins associated with the piRNA pathway have been lost in Ascaris. miRNAs are synthesized immediately after fertilization in utero, before pronuclear fusion, and before the first cleavage of the zygote. This is the earliest expression of small RNAs ever described at a developmental stage long thought to be transcriptionally quiescent. A comparison of the two classes of Ascaris endo-siRNAs, 22G-RNAs and 26G-RNAs, to those in C. elegans, suggests great diversification and plasticity in the use of small RNA pathways during spermatogenesis in different nematodes. Our data reveal conserved characteristics of nematode small RNAs as well as features unique to Ascaris that illustrate significant flexibility in the use of small RNAs pathways, some of which are likely an adaptation to Ascaris' life cycle and parasitism

Dynamic Regulation of Hepatic Lipid Droplet Properties by Diet

<div><p>Cytoplasmic lipid droplets (CLD) are organelle-like structures that function in neutral lipid storage, transport and metabolism through the actions of specific surface-associated proteins. Although diet and metabolism influence hepatic CLD levels, how they affect CLD protein composition is largely unknown. We used non-biased, shotgun, proteomics in combination with metabolic analysis, quantitative immunoblotting, electron microscopy and confocal imaging to define the effects of low- and high-fat diets on CLD properties in fasted-refed mice. We found that the hepatic CLD proteome is distinct from that of CLD from other mammalian tissues, containing enzymes from multiple metabolic pathways. The hepatic CLD proteome is also differentially affected by dietary fat content and hepatic metabolic status. High fat feeding markedly increased the CLD surface density of perilipin-2, a critical regulator of hepatic neutral lipid storage, whereas it reduced CLD levels of betaine-homocysteine S-methyltransferase, an enzyme regulator of homocysteine levels linked to fatty liver disease and hepatocellular carcinoma. Collectively our data demonstrate that the hepatic CLD proteome is enriched in metabolic enzymes, and that it is qualitatively and quantitatively regulated by diet and metabolism. These findings implicate CLD in the regulation of hepatic metabolic processes, and suggest that their properties undergo reorganization in response to hepatic metabolic demands.</p></div

Methionine-Cysteine pathway proteins associated with CLD.

<p>(A) Schematic diagram representing the methionine/cysteine pathway. Boxes represent proteins, circles represent metabolites. Green colored boxes indicate proteins identified on hepatic CLD. (B) Quantitative immunoblot analysis of BHMT levels in liver extracts from non-fasted male mice (Control) and refed male mice on LF- or HF-diets. Insets show immunoblots of 50 μg of total liver homogenate protein from 3 mice probed with antibodies to BHMT or β-actin. The graph shows the average (± SD) BHMT levels normalized to β-actin from Control (3), LFD (N = 3) and HFD (N = 3) refed mice. Asterisks indicate LFD and HFD values differ from Control values (p<0.005). (C) Quantitative immunoblot analysis of BHMT levels in CLD protein extracts from refed male mice on LF- or HF-diets. Insets show immunoblots of 25 μg of CLD protein from 3 mice. The graph shows the average (± SD) BHMT levels normalized to 25 μg of CLD protein from LFD (N = 3) and HFD (N = 3) mice. (D) CLD BHMT levels normalized to CLD TG content. Values are means (± SD) for LFD (N = 3) and HFD (N = 3) refed animals. Asterisks in C and D indicate HFD values differ from LFD values (p<0.001).</p

Specific HFD CLD Protein KEGG Pathways.

<p>Specific HFD CLD Protein KEGG Pathways.</p

Unique protein patterns of isolated hepatic CLD.

<p>A) Coomassie blue staining profiles of proteins in liver homogenates and isolated CLD. B) Immunoblot analyses of equal amounts (25μg) of protein from liver homogenate, PNS, initial CLD, final CLD wash, and final enriched CLD fractions reacted with antibodies to GRP78, Plin2, PEX3, and VDAC.</p

Diet effects Plin2 on CLD.

<p>(A) Representative confocal Plin2 immunofluorescence (green) images of liver sections from fasted male mice refed with LF (LFD) – and HF (HFD)-diets and stained with antibodies to Plin2. Nuclei (blue) were stained with DAPI. Asterisks indicate central veins. (B) Quantitative immunoblot analysis of Plin2 levels in enriched CLD protein extracts from fast-fed male mice on LF (LFD)- or HF(HFD)-diets. Insets show immunoblots of 25 μg of CLD protein from 3 mice. The graph shows the average (± SD) Plin2 levels normalized to 25 μg of total CLD protein from LF (N = 3) and HF (N = 3) mice. (C) CLD Plin2 levels normalized to CLD TG content. Values are means (± SD) for LF (N = 3) and HF (N = 3) refed animals. Asterisks in B and C indicate HFD values differ from LFD values (p<0.0001). (D) Transcript levels of PLIN family members in livers of fasted and refed mice on LFD and HFD quantified by qRT-PCR using primers listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067631#pone.0067631.s001" target="_blank">Table S1</a>. Values are means± SD normalized to 18S RNA. Asterisks indicate Plin2 transcript levels are significantly elevated over transcript levels for Plins 1,3,4, and 5. (E) Effects of HF feeding on Plin2 surface density in HEK293 cells stably expressing Plin2-VSV. Images are representative electron micrographs of anti-PLIN2-gold particle labeled cells that were cultured in oleic acid-supplemented media for 0h, 4h or 24h. An enlarged micrograph of a CLD at 24h is shown. Average (± SD) Plin2 surface densities on CLD at each time point are shown for 50–75 CLD from triplicate cultures. The experiment was repeated twice with similar results. Asterisks indicate statistically significant differences from T = 0 time point, double dagger indicates statistically significant differences between 4h and 24h time points.</p

Hepatic CLD differs from other core CLD proteomes.

<p>(A) Functional categories of common- and liver-specific proteins categorized according to gene ontology (GO) annotations. (B) Association networks of common- and liver-specific CLD associated proteins predicted by the STRING 9.0 program with a confidence level of 0.8. Network edges represent predicted functional associations with different line colors standing for various types of evidence used in establishing the level of confidence. Red, fusion evidence; green, neighborhood evidence; blue, co-occurrence evidence; purple, experimental evidence; yellow, text-mining evidence; black, co-expression evidence. Non-network proteins are not shown.</p

HFD induces expression of proteins from different pathways.

<p>(A) Functional categories of LF- and HF-specific CLD associated proteins categorized according to gene ontology (GO) annotations. (B) Association networks of LF- and HF-specific CLD associated proteins predicted by the STRING 9.0 program with a confidence level of 0.8. Color codes are as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067631#pone-0067631-g003" target="_blank">Figure 3</a>.</p

Diet effects on metabolism and hepatic lipid storage.

<p>(A) Effects of LF- and HF-refeeding on energy intake in fasted male mice. Values are means (± SD) for 4 animals in each group. Asterisk indicates HFD values differ from LFD values (p<0.01). (B) Energy usage in non-fasted, fasted and LF- and HF-refed male mice as determined by RER. Non-fasted and fasted values correspond to averages (± SD) of 8 animals obtained prior to refeeding. LF- and HF-refeeding values correspond to averages (± SD) for 4 animals in each group. Asterisk indicates values differ from non-fasted controls (p<0.001); dagger indicates values differ from non-fasted and fasted values (p<0.001); double dagger indicates values differ from LF refed and non-fasted values (p<0.001). (C) Representative images of frozen liver sections from LF- and HF-refed male stained with BODIPY and imaged by laser confocal (BODIPY) and CARS microscopy (200X magnification). Asterisks indicate central veins. (D) Representative surface-view of 3D projection images of single cells within liver sections from LF (LFD)- and HF (HFD) -refed mice obtained at 600X magnification. Nuclei are shown in blue.</p