Worm Breeder's Gazette 12(2): 62 (January 1, 1992)
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.
ctk(L5b) is the first of a tandem pair of transcription units that encode novel transmembrane protein-tyrosine kinases (WBG 11(2):44). To investigate its potential role in development, we have examined its developmental expression pattern using a lacZ fusion gene construct. An appropriate reporter gene plasmid was constructed by inserting a nuclearly localized ß-galactosidase protein-coding region (Fire et. al. (1990) Gene 93: 189) in-frame at codon 10 of the ctk(L5b) gene (see Figure). Stable lines containing this construct were established by cotransformation with a rol-6 ( su1006 )plasmid (Mello et al., WBG 11(1):18) or a lin-12 (+)plasmid (H. Wikinson, pers. comm.). In general, a higher percentage of ß-galactosidase staining animals was observed with the lin-12 (+)cotransformants.
All nuclear staining, with a single exception, is consistent with expression being restricted to hyp7 .(The single exception is observed in the adult head where the six embryonically-derived nuclei of hyp6 stain.) The fusion gene is specifically expressed in all nuclei of the hyp7 syncytium beginning in L1 and continuing into adulthood. ß-galactosidase activity is first detected in 23 hypodermal nuclei (identified by their characteristic morphology and position) in young larvae. The observed pattern is identical to the reported positions of the 23 embryonic nuclei which comprise the hyp7 syncytium of newly hatched animals. Consistent with expression in hyp7 ,which increases in size by fusing with post-embryonically derived cells, additional nuclear staining is observed as development continues. Particularly striking is the coincidence of the developmental staining pattern with the reported pattern of cell fusions between hyp7 and Pn.p-derived cells. Nuclear staining coincident with P1 .pand P2 .pis detected first (fusion at ~10 hours post-hatching), closely followed by P9 .pand P10 .p(fusion at ~11 h), and then P11 .p(fusion at ~12 h). In L3 larvae, staining coincident with P3 .pa, P3 .pp, P4 .pa, P4 .pp, P8 .pa,and P8 .pp(fusion at ~30 h) is also detected. As expected, no expression is detected coincident with the P(5-7).p or P12 .pdescendants, which become the vulva or preanal hypodermis ( hyp12 ),respectively. Altogether 110 monocucleate cells are recruited so that the adult hyp7 syncytium contains a total of 133 nuclei; a similar number of staining nuclei is observed in transformed animals.
Similar N-terminal fusion constructs with the downstream transcription unit ctk(L5a) failed to exhibit any ß-galactosidase activity (possibly because translation initiates downstream of the two insertion sites chosen). However, preliminary results with C-terminal fusion constructs exhibit similar, diffuse staining patterns for both genes. An additional fusion construct, with the modified lacZ gene inserted after the third ATG codon of ctk(L5a) is currently being microinjected in an attempt to corroborate this result.
[See Figure 1]