MOLECULAR CLONING OF A C. elegans LONGEVITY GENE pnc-1 AND INVESTIGATION OF ITS NOVEL FUNCTION IN DEVELOPMENT
Open Access
- Author:
- Huang, Li
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 14, 2007
- Committee Members:
- Wendy Hanna Rose, Committee Chair/Co-Chair
Diana Lynn Cox Foster, Committee Member
Emine Koc, Committee Member
Zhi Chun Lai, Committee Member
Pamela J Mitchell, Committee Member - Keywords:
- nicotiamidase
gonad development
egg-laying defect
EGF signaling
uv1 cells
NAD - Abstract:
- The egg-laying apparatus in C. elegans is a great model system to study morphogenesis and organogenesis in developmental biology. Egg-laying defective (Egl) mutants provide powerful genetic tools to address these issues. ku212 was isolated as an allele with egg laying and associated connection of gonad defective (Cog) phenotypes. The corresponding gene was named cog-3. In this study, phenotypic analyses of ku212 revealed no obvious connection between the vulval and the uterine lumens at the appropriate stage, due to a temporal delay in uterine development. Mild delay in global gonadogenesis relative to development of extra-gonadal tissues results in the lack of temporal synchronization between the vulva and the uterus. ku212 mutants also have a specific uterine fate defect. In wild-type animals, four outer ventral uterine ¦Ð cells express LET-23 EGF receptor and adopt the uterine vulval 1 (uv1) fate in response to a vulval-derived LIN-3 EGF signal. In ku212 mutants, these four uv1 cells undergo necrosis prior to full differentiation. Transformation rescue by pnc-1 and further identification of a nonsense mutation in pnc-1 in ku212 animals confirm that ku212 is an allele of pnc-1, a homolog of a yeast longevity gene Pnc1. pnc-1 encodes a nicotinamidase which converts nicotinamide (NAM) to nicotinic acid (NA) as part of the NAD+ salvage pathway for recycling NAD+. PNC-1(ku212) is predicted to be a truncated protein missing the last 8 amino acids. Although the function of Pnc1 has been extensively studied in longevity in yeast, little is known about its roles in development. C. elegans pnc-1 clearly participates in a variety of developmental processes as demonstrated by multiple developmental defects in the loss-of-function pnc-1(ku212) mutants. The impaired nicotinamidase activity could result in high levels of NAM or low levels of NAD+, leading to the developmental defects in the mutants. Two isoforms of pnc-1 including pnc-1a (with a signal sequence) and pnc-1b (without a signal sequence) are present in C. elegans. Another nicotinamidase Y57G11C.47, or pnc-2, is identified in this study by its ability to rescue pnc-1(ku212) phenotypes. How pnc-1 functions in development and how these three nicotinamidases cooperate in C. elegans are still under investigation. A distinct function of each nicotinamidase confined by their unique localization is postulated. PNC-1a could be acting as a secreted cue functioning non cell-autonomously. PNC-1b and PNC-2 could be restricted in some cell types and mediate biological events cell autonomously. I have detected the distinct expression pattern of pnc-1a in the head and pharyngeal muscle, ASK neuron, distal tip cells, uv2 cells and rectal gland cells. In the process of investigating why uv1 cells undergo necrosis, I found that a gain-of-function mutation in LET-23 EGF receptor and ectopic expression of LIN-3 EGF within the proper temporal constraints can rescue the uv1 death, suggesting a compromised EGF signaling in ku212 mutants. Activation of Ras-MAP kinase pathway by EGFR in the presumptive uv1 cells is required for uv1 cell fate specification. However, a let-60(gain-of-function) Ras cannot prevent uv1 cell death, suggesting the requirement of additional signaling pathways in uv1 cell fate specification.