Kazumasa Hada et al. (2010) East Asia Worm Meeting "The nuclear receptor gene nhr-25 plays multiple roles in the C. elegans heterochronic gene network to control the larva-to-adult transition"

Kazumasa Hada, Frank J. Slack, Hiroshi Hasegawa, Yasunori Kanaho, Ryusuke Niwa, Masako ASAHINA

[East Asia Worm Meeting, 2010]

Developmental timing in C. elegans is controlled by heterochronic genes, mutations in which cause changes in the relative timing of developmental events. One of the heterochronic genes, let-7, encodes a microRNA (miRNA) that is highly evolutionarily conserved, suggesting that similar genetic pathways control developmental timing across phyla. Recently we have reported that the expression in seam cells of apl-1, a homolog of Alzheimer's amyloid precursor protein gene, is regulated by the let-7 family miRNAs and their target heterochronic genes, such as hbl-1 and lin-41 (Niwa et al. Dev. Biol. 315: 418-425, 2008). We proposed that the expression analysis of apl-1 in seam cells is a new approach to elucidate the let-7-dependent terminal differentiation pathway in C. elegans. Here we report that the nuclear hormone receptor gene nhr-25 is identified as the apl-1 regulator and controls the larva-to-adult transition in C. elegans. During the course of an RNAi screen, we found that nhr-25 positively regulated apl-1 expression in seam cells. Similar to hbl-1 and lin-41, nhr-25 controlled not only the temporal apl-1 expression but also other let-7-dependent developmental timing functions. In addition to the let-7- and nhr-25-directing the molting process (Hayes et al. Development 133: 4631-4641, 2006), we showed that nhr-25 also controlled adult-specific collagen gene col-19 expression, seam cell division, seam cell fusion and alae formation. Loss of nhr-25 function leaded to the precocious expression of the adult-specific collagen gene col-19 in the larval stage, while loss-of-nhr-25 function resulted in retarded phenotypes in seam cell division, seam cell fusion and alae formation. These results suggest that nhr-25 has multiple roles in both promoting and inhibiting C. elegans adult programs.

Eugeni V Entchev et al. (2005) International Worm Meeting "Role of sterols during dauer formation in C.elegans"

Arndt W Schmidt, Christoph Thiele, Teymuras V Kurzchalia, Samuel Ward, Fanny Mende, Eugeni V Entchev, Hans-joachim Knolker, Vitali Matyash, Michaela Wilsch-Brauninger

[International Worm Meeting, 2005]

C.elegans can not synthesize cholesterol de novo but sterols are absolutely required for worm development. Cholesterol auxotrophy makes the worm a valuable system for studying sterol metabolism and functions by replacing dietary cholesterol with different derivatives and analogs. As a starting point for our investigation on the role of sterols in C.elegans, we developed a protocol for complete elimination of sterols in the medium and food. After one generation, strict sterols depletion caused uniform arrest into a dauer-like larvae with incomplete molting. Studying the metabolism of cholesterol under the conditions of cholesterol depletion indicated reduced levels of nonmethylated sterols and accumulation of 4-alpha methylated sterols. This prompted us to test how the methylated sterols influence dauer formation. Substitution of cholesterol by the 4-alpha methylated sterol lophenol was sufficient to complete dauer larva formation in the second generation. However, our data indicate that methylated sterols do not actively induce the dauer formation but rather that the reproductive growth requires a cholesterol derived hormone that can not be produced from methylated sterols. Using the effect of lophenol on growth, we have partially purified activity, named gamravali, which promotes the reproduction. In addition, effect of lophenol allowed us to identify the step at which gamravali is required during the reproductive development. In the absence of gamravali, the nuclear hormone receptor DAF-12 is activated and triggers dauer formation program by allowing in neurons the nuclear import of DAF-16, a forkhead domain (FOXO) transcription factor required for dauer formation. Our results reveal a novel function of DAF-16 downstream of DAF-12 in order to complete dauer differentiation. Reference: Matyash V, Entchev EV, Mende F, Wilsch-Brauninger M, Thiele C, Schmidt AW, Knolker, HJ, Ward, S, Kurzchalia, TV. Sterol-Derived Hormone(s) Controls Entry into Diapause in Caenorhabditis elegans by Consecutive Activation of DAF-12 and DAF-16. PLoS Biol, 2004. 2(10): p. e280.

Axel Bethke et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "LIN-29/ZnF co-activates DAF-12/NHR to drive miR-84 expression, placing miR-84 towards the end of the heterochronic pathway"

Nicole Fielenbach, Axel Bethke, Adam Antebi, Linyan Meng

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

The nuclear hormone receptor DAF-12 acts as the switch between continuous development or arrest at the dauer diapause during the L2 stage. It also works in the heterochronic circuit, promoting L3-stage specific developmental programs. Another heterochronic factor, the zinc finger protein LIN-29 acts during L4 to promote terminal differentiation of various tissues like gonad or hypodermis, a function referred to as the larval-to-adult switch. Despite this terminal position, here we present evidence that LIN-29 is involved in transcriptional activation of the let-7 sister mir-84, which was thought to act at an earlier step. Previously, we had shown DAF-12 and ligand dependent activation of mir-84 promoter constructs in human cell culture and in worms. We wondered whether any of the heterochronic factors could serve as DAF-12 co-activators to augment transcription. Remarkably, we found that co-transfection of LIN-29 increased DAF-12 dependent transcription two-fold, but had no such activity alone, suggesting LIN-29 may be a DAF-12 co-activator. Accordingly, point mutation of DAF-12 response elements strongly decreased DAF-12 and ligand dependent activation as well as the co-activator effect, The DNA-motive that LIN-29 binds to is unknown, but EMSA with an oligo containing two functional DAF-12 sites show no LIN-29 binding, making it unlikely that LIN-29 can bind directly to DAF-12 response elements. Taken together, the experiments suggest that LIN-29 works through DAF-12 to exert these transcriptional effects. The mir-84 promoter also shows DAF-12 and LIN-29 dependence in vivo. In particular, the lin-29 null mutants exhibited decreased pharyngeal mir-84p::GFP expression, even below the level observed in a daf-12 null. Moreover, a lin-29, daf-12 double null nearly lost all GFP expression. These in vivo data are consistent with the notion that LIN-29 acts through DAF-12 and its response elements, but also hint at a DAF-12 independent function of LIN-29 on the mir-84 promoter. The placement of LIN-29 upstream of mir-84 inverts its conventional placement as the terminal factor in the heterochronic pathway. Interestingly, Hayes et al showed that mir-84 over expression can suppress lin-29 for supernumerary molting heterochronic phenotypes. This, together with the data presented here, suggests that LIN-29 could drive terminal differentiation in the hypodermis through activating mir-84 expression, placing miR-84 downstream of LIN-29 to the very end of the heterochronic signaling cascade. We are currently testing this hypothesis.