Chuan Shen et al. (2004) Mid-west Worm Meeting "C. elegans hypoxia response: Elucidating the function and regulation of the HIF-1 transcription factor"

Jo Anne Powell-Coffman, Chuan Shen, Kelly S Gillette, Sara Belliveau

[Mid-west Worm Meeting, 2004]

The C. elegans hif-1 gene is orthologous to the mammalian hypoxia inducible factor alpha units, which have been described as master regulators of transcriptional response to hypoxia (low oxygen). Mutants lacking hif-1 function are viable in normoxic conditions, but they cannot adapt to hypoxia (Jiang et al. 2001, PNAS 98: 7916). The stability of the HIF-1 protein is regulated by the evolutionarily conserved EGL-9/ VHL-1 pathway. In normoxia, a proline in the oxygen-dependent degradation domain of HIF-1 is hydroxylated by EGL-9, enabling HIF-1 to bind VHL-1. VHL-1 targets HIF-1 for degradation (Epstein et al. 2001, Cell 107: 43). To further understand the role of HIF-1 in hypoxia response, we compared the mRNA expression patterns of wild-type, hif-1-deficient, and vhl-1-deficient animals in normoxic and in hypoxic conditions using microarray technology. We identified over 100 genes that exhibit a >2-fold difference in mRNA expression in hypoxic conditions (p < 0.05). The majority of these genes are regulated in a hif-1-dependent manner. We hypothesized that some of the transcriptional targets of HIF-1 might also be required for survival of hypoxia. To test this, we used mutation or RNAi to deplete gene function and assayed viability in normoxia and hypoxia. We report that several individual HIF-1 target genes have essential roles in adaptation to hypoxia. We aim to use genetic methods to identify VHL-independent regulators of HIF-1 and to isolate additional alleles of HIF-1. In pilot screens, we have identified several mutations that alter the expression of HIF-1 target genes. One of these mutations results in a constitutively expressed, truncated HIF-1 protein. The other mutations map to other regions of the genome. We are working towards identification and characterization of these potential HIF-1 regulators. This project is supported by the American Heart Association.

Sara Vassalli et al. (2006) European Worm Meeting "Identification of Genes required for Anchor Cell Attachment and Invasion during Vulval Development"

ALEX HAJNAL, Sara Vassalli

[European Worm Meeting, 2006]

Sara Vassalli and Alex Hajnal. During vulval development in the hermaphrodite, three out of six equivalent vulval precursor cells (the VPCs P3.p to P8.p) are induced by a signal from the gonadal anchor cell (AC). The LIN-3 EGF growth factor produced by the AC activates the EGFR/RAS/MAPK pathway in the VPCs to specify the primary cell fate. Prior to vulval induction, the gonad is separated from the VPCs by two basal laminas covering each tissue such that the position of the AC relative to the VPCs is variable when the animal moves. At the time of vulval induction in the early L3 stage, however, the AC attaches to the basal side of the future primary cell P6.p. After the first round of vulval cell divisions (Pn.px stage), the basal laminas separating the gonad from the vulval cells start to dissolve precisely under the AC. After the second round of divisions (Pn.pxx stage), the basal laminas between the AC and the four primary vulval cells are interrupted, and the AC invades the vulval tissue.. While the mechanism of vulval induction is well studied, less is known about genes regulating the attachment of the AC to P6.p and the following invasion step. To study these two processes, we performed a forward genetic screen for mutants with defects in AC positioning and/or invasion. Since worms with an abnormal or missing connection between the gonad and the vulva usually display a protruding vulva (Pvl) phenotype, we first screened for animals with a Pvl phenotype and then examined them for defects in the positioning or invasion of the AC. Among 5300 F1 clones, we identified 8 sterile mutants with a misplaced AC and 2 mutants, in which the AC is normally positioned but fails to dissolve the basal laminas and invade the vulval tissue. Details about the cloning and analysis of the genes identified in this screen will be presented at the meeting.