O-GlcNAc modifications regulate cell survival and epiboly during zebrafish development.
BACKGROUND
The post-translational addition of the monosaccharide O-linked beta-N-acetylglucosamine (O-GlcNAc) regulates the activity of a wide variety of nuclear and cytoplasmic proteins. The enzymes O-GlcNAc Transferase (Ogt) and O-GlcNAcase (Oga) catalyze, respectively, the attachment and removal of O-GlcNAc to target proteins. In adult mice, Ogt and Oga attenuate the response to insulin by modifying several components of the signal transduction pathway. Complete loss of ogt function, however, is lethal to mouse embryonic stem cells, suggesting that the enzyme has additional, unstudied roles in development. We have utilized zebrafish as a model to determine role of O-GlcNAc modifications in development. Zebrafish has two ogt genes, encoding six different enzymatic isoforms that are expressed maternally and zygotically.


RESULTS
We manipulated O-GlcNAc levels in zebrafish embryos by overexpressing zebrafish ogt, human oga or by injecting morpholinos against ogt transcripts. Each of these treatments results in embryos with shortened body axes and reduced brains at 24 hpf. The embryos had 23% fewer cells than controls, and displayed increased rates of cell death as early as the mid-gastrula stages. An extensive marker analysis indicates that derivatives of three germ layers are reduced to variable extents, and the embryos are severely disorganized after gastrulation. Overexpression of Ogt and Oga delayed epiboly and caused a severe disorganization of the microtubule and actin based cytoskeleton in the extra-embryonic yolk syncytial layer (YSL). The cytoskeletal defects resemble those previously reported for embryos lacking function of the Pou5f1/Oct4 transcription factor spiel ohne grenzen. Consistent with this, Pou5f1/Oct4 is modified by O-GlcNAc in human embryonic stem cells.


CONCLUSIONS
We conclude that O-GlcNAc modifications control the activity of proteins that regulate apoptosis and epiboly movements, but do not seem to regulate germ layer specification. O-GlcNAc modifies the transcription factor Spiel ohne grenzen/Pou5f1 and may regulate its activity.
Webster
Danielle M
DM
Department of Cellular Biology, The University of Georgia, Athens, GA 30602, USA. dwebster@hodges.edu
Teo
Chin Fen
CF
Sun
Yuhua
Y
Wloga
Dorota
D
Gay
Steven
S
Klonowski
Kimberly D
KD
Wells
Lance
L
Dougan
Scott T
ST
D000117:Acetylglucosamine / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D019521:Body Patterning; D001921:Brain / Q000196:embryology / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival; D003599:Cytoskeleton / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D020440:Gene Duplication; D018507:Gene Expression Regulation, Developmental; D005855:Germ Layers / Q000378:metabolism; D006801:Humans; D017403:In Situ Hybridization; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D017351:N-Acetylglucosaminyltransferases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D010802:Phylogeny; D011499:Protein Processing, Post-Translational; D015017:Yolk Sac / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics / Q000378:metabolism; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism*; D001619:beta-N-Acetylhexosaminidases / Q000235:genetics / Q000378:metabolism
D007527:Isoenzymes; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D029961:Zebrafish Proteins; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase; C110628:hexosaminidase C; D001619:beta-N-Acetylhexosaminidases; D000117:Acetylglucosamine