Worm Breeder's Gazette 13(2): 95 (February 1, 1994)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

lin-32 ,a gene specifying neuroblast cell fate, encodes a bHLH protein

Connie Zhao, Scott W. Emmons

Department of Molecular Genetics,
Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461

In C. elegans males, rays are derived postembryonically from lateral hypodermal cells V5 , V6 ,and T. The gene lin-32 is required for the expression of the ray sublineage leading to the generation of rays. lin-32 is also necessary for the generation of certain touch neurons, as well as the Q and postdeirid neurons. lin-32 functions in specifying neuroblast cell fate: lin-32 loss-of-function mutations cause the transformation of neuroblast cells into hypodermal cells. We are carrying out a genetic and molecular analysis of lin-32 in order to understand its role in the generation of the nervous system.

We identified a 5kb genomic fragment that rescued lin-32 mutations. The 5kb fragment was used as a probe to screen the Chris-Martin size-selected cDNA library After screening about two million phage plaques, we isolated two positive cDNA clones, both with the size of 1.4 kb. Restriction mapping and sequencing analysis showed that the two cDNA are identical, and therefore may be due to the amplification of the cDNA library. The cDNA has a poly(A) tail of 130 A residues and surprisingly, it seems that the cDNA also contains a 300 bp intron. We are using RT-PCR to confirm this. Expression of the lin-32 cDNA under a heat shock promoter rescued lin-32 mutations. Furthermore, heat shock experiments showed that the ectopic expression of lin-32 caused the generation of gaps in alae, presumably due to the generation of ectopic ray neurons at the expense of body seam cells.

We have completely sequenced the genomic region and the cDNA of lin-32 .Sequencing data showed that lin-32 encodes a protein with a basic-helix-loop-helix (bHLH) domain, which defines a class of transcription factors that control cell type determination. At the protein level, lin-32 has 46% identity and 79% similarity in the bHLH domain to the achaete-scute homolog in rats. Extensive studies in the development of peripheral sense organs in Drosophila have established that achaete-scute complex (AS-C) genes play a crucial role in specifying the neuroblast fate of sensory mother cells, which are the precursors of sense organs. It has also been shown that the mammalian achaete-scute homolog functions in controlling the development of neuronal progenitors. The similarity in both biological function and molecular structure of all these genes suggests a central and ubiquitous role of this family of proteins in specifying the neuronal cell fate.

We are in the process of determining the expression pattern of lin-32 .By expressing a fusion construct of the lin-32 promoter with the lac-Z gene, we have so far observed that lin-32 is expressed in the ventral nerve cord in L1 and L2 ,and in the tail region of adult males. We are trying to generate an integrated line for further study of the lin-32 expression pattern in wild type and in a variety of mutant backgrounds.