Glucosamine 6-phosphate N-acetyltransferase (GNA-2) is essential for the biosynthesis of the amino sugar UDP-N-acetylglucosamine (UDP-GlcNAc), which is necessary to synthesise a wide variety of glycoconjugates, including N- and O-glycan-modified glycoproteins, proteoglycans, GPI linkers, O-GlcNAc-modified proteins and glycopolymers including chitin and hyaluronan. We have shown previously that a
gna-2 allele deleted for the entire coding sequence (
qa705) is Mel, with most embryos arrested as multinucleated single cells. Embryos are also defective in meiosis and polar body extrusion, and display reduced or mislocalised microtubules, polarity and osmotic defects (Pod), decreased eggshell WGA binding and decreased embryonic O-GlcNAc residues. In addition,
gna-2(
qa705) embryos lack the zone (extraembryonic matrix, EEM) between the embryo and the eggshell and are permeable to the lipophilic dye, FM 4-64. Chitin, a linear polymer of GlcNAc, is deposited in the eggshell very shortly after fertilisation. It was detected in N2 as a bright ring surrounding the embryo using a fluorescently labeled chitin-binding reagent (kindly donated by Y. Zhang, NEB). Conversely, in
gna-2(
qa705) , chitin was undetectable. To test the hypothesis that chitin deficiency contributes to the
gna-2(
qa705) Mel phenotype, we performed RNAi with
chi-1 and determined that eggshell chitin was missing and the embryonic lethal phenotype resembled
gna-2(
qa705) . In the hermaphrodite germline,
gna-2 RNA is bound and translationally repressed by GLD-1 1 . Other GLD-1 targets include the chitin-binding domain motif containing
cej-1 and B0280.5 , and
cej-1/B0280.5 coRNAi resulted in an embryonic lethality that resembled that of
gna-2 RNAi 1 . We performed
cej-1/B0280.5 coRNAi and found that embryos were multinucleated, polarity-defective and inappropriately permeable to FM 4-64. These results suggest a model where
gna-2 is made available for UDP-GlcNAc synthesis following relief of GLD-1 translational repression in the proximal germline. After fertilisation, chitin is synthesised from UDP-GlcNAc and extruded from the plasma membrane. CEJ-1 and B0280.5, synthesised following relief of GLD-1 RNA translational repression, are secreted and bind chitin, where, together with chitin, they support the production of a hydrophilic EEM. This EEM is required to complete meiosis and for further development, including polarisation. Using fluorescently labeled WGA, we previously reported the appearance of embryonic cortical patches in wildtype embryos that arise transiently at meiosis and disappear by the first cell division. These patches do not bind the chitin-binding reagent and they persist when a robust chitinous eggshell and an EEM have already formed. This suggests that they are not intermediates in the production of eggshell chitin, but rather, are non-chitin GlcNAc-containing structures that are dependent on GNA-2. Recently, Pellettieri et al. 2 demonstrated that the kinase MBK-2, which coordinates the degradation of a number of maternally supplied proteins (MEI-1, MEI-2, OMA-1, PIE-1), is present at meiosis in cortical patches. Using a GFP antibody in embryos carrying an MBK-2::GFP fusion, we determined that the WGA patches are coincident with MBK-2 patches during meiosis. We are currently testing the hypothesis that deficiency of GNA-2/chitin/CEJ-1/B0280.5 results in decreased MBK-2 activation, perhaps due to defects in the EEM. Decreased MBK-2 activation would then result in inappropriate perdurance of target maternal proteins, resulting in defects in development and polarisation. In summary, these results demonstrate that GNA-2 is necessary for eggshell chitin synthesis, and that chitin and the functionally redundant chitin-binding domain containing proteins, CEJ-1/B0280.5, are absolutely required for development and polarity at the one-cell stage. Our results also demonstrate that MBK-2 cortical patches colocalise with WGA and experiments are currently underway to investigate the development and composition of these patches. 1 Lee, M.H. and Schedl, T. Genes Dev. 15, 2408-2420 (2001) 2 Pellettieri, J., Reinke, V., Kim, S.K. and Seydoux, G. Dev. Cell 5, 451-462 (2003)