The events that occur shortly after fertilization are crucial to ensuring the proper execution of developmental programs in embryogenesis and, thus, to the survival of the embryo. These events include establishment of the anterior-posterior axis and construction of a bipolar mitotic spindle. Using mutations in the
spd-2 gene, we have shown that these two events are dependent on the presence of a functional centrosome. b -tubulin immunofluorescence shows that
spd-2 embryos lack the organized arrays of microtubules normally associated with the sperm pronucleus after fertilization; foci of g -tubulin are also absent from early
spd-2 embryos. As a result,
spd-2 embryos show a disruption of all aspects of embryonic polarity that have been investigated. Furthermore, the lack of a centrosome prevents the establishment of a bipolar mitotic spindle, causing the majority of
spd-2 embryos to fail in the first division. (see abstract by O'Connell et al). In order to further understand the function of the
spd-2 gene product in early development, we have initiated cloning of the
spd-2 gene. The
spd-2 gene is defined by two temperature-sensitive alleles,
oj29 and
oj42 . Most likely the stronger allele,
oj29 shows 100% embryonic lethality when L4 animals are shifted to the restrictive temperature; furthermore,
spd-2(
oj29) shows a completely penetrant sterile, uncoordinated phenotype when animals are exposed to the restrictive temperature during postembryonic development. Standard genetic mapping techniques were used to position
spd-2 between
let-89 and
unc-29 on chromosome I. Transgenic lines were established with six of the cosmids that span this genetic region, and one cosmid, F43G9, gives embryonic rescue. We are currently testing this transgenic line for further indication of both embryonic and post-embryonic rescue. Furthermore, in order to determine which predicted open-reading frame on F43G9 corresponds to the
spd-2 gene, we are injecting double-stranded RNA corresponding to these ORFs into wild-type worms to look for phenocopy of
spd-2 .