The
daf-2 gene codes for the singular insulin-like growth factor receptor in C. elegans, acting as a control point in multiple developmental and physiological pathways that depend on the integrated response to at least forty different insulin-like peptides (Murphy and Hu, 2013). The functions of
daf-2 in dauer formation, longevity, germ-line, and early larval arrest have been extensively studied. A strong
daf-2 allele,
e979, is homozygous viable at 15oC, despite a partially penetrant embryonic lethal phenotype at 25oC (Gems et al., 1998). The presumed null allele,
m65, does not produce fertile homozygous adults due to 100% dauer arrest, and does not display embryonic lethality due to maternal rescue from heterozygous mothers. While several
daf-2 alleles display low penetrance embryonic lethality at 25oC, the
e979 allele displays the most penetrant embryonic arrest phenotype (Gems et al., 1998; Patel et al., 2008), making it the best choice to investigate the embryonic requirement for insulin signaling. The
e979 mutation causes a C146Y substitution that is thought to interrupt an existing disulfide bond interaction with C181, destabilizing the typical fold of the insulin receptor, and impairing the L1 domains function in ligand binding (Patel et al., 2008).To investigate the role of insulin-like signaling in embryonic development, we examined
daf-2(
e979) and parallel N2 wild-type and
daf-2(
e1370) controls at 25oC. The canonical
e1370 allele causes a strong dauer arrest phenotype, but a very low penetrance embryonic arrest phenotype. While 98.9% of
daf-2(
e979) embryos reached early elongation midway through embryogenesis (comma stage), 10.6% of embryos arrested without hatching (Fig. 1A). Similar to previous reports, 23.5% of
daf-2(
e979) animals arrested development as fully-elongated L1 animals at or after hatching (Gems et al., 1998; Baugh, 2013). Examination of arrested
daf-2(
e979) embryos by DIC microscopy revealed that they had some of the features of late embryogenesis, such as an apparently normal pharynx, but had failed to elongate properly (Fig. 1B). We followed five control wild-type embryos and six
daf-2(
e979) embryos by time-lapse microscopy over an 8 hour period at 25oC beginning at the mid-embryogenesis comma stage. While all of the wild-type and five of the
daf-2(
e979) embryos exhibited normal elongation and morphogenesis, one
daf-2(
e979) embryo failed to elongate, beginning just before the two-fold stage (Fig. 1C). The failing embryo twitched normally at this stage, indicating that it had functional muscles, but was unable to elongate past the two-fold length and retracted somewhat over a one hour period. After several hours, blebs appeared at the anterior end of the embryo and it eventually ruptured. These observations suggest that insulin-like signaling plays a role in embryonic elongation in C. elegans. A role for
daf-2 in embryo elongation was previously suggested based on synthetic genetic interactions of other
daf-2 alleles with the
let-502 Rho-binding kinase (Piekny, et al., 2000), but has not been described for
daf-2 mutations alone.The embryonic elongation process is driven by the migration, fusion, and contraction of the hypodermal epithelium (Priess and Hirsh, 1986; Costa et al., 1998; Chisholm and Hardin, 2005). Circumferential actin microfilaments connect the longitudinal margins of the belt desmosomes surrounding each hypodermal cell and contractile activity leads to the change in hypodermal cell shape seen in elongation. Therefore, elongation depends on both the mechanistic contraction of actin microfilaments and the arrangement and differentiation of the hypodermal cells themselves.