Genetic and biochemical evidence that gastrulation defects in Pofut2 mutants result from defects in ADAMTS9 secretion.
Protein O-fucosyltransferase 2 (POFUT2) adds O-linked fucose to Thrombospondin Type 1 Repeats (TSR) in 49 potential target proteins. Nearly half the POFUT2 targets belong to the A Disintegrin and Metalloprotease with ThromboSpondin type-1 motifs (ADAMTS) or ADAMTS-like family of proteins. Both the mouse Pofut2 RST434 gene trap allele and the Adamts9 knockout were reported to result in early embryonic lethality, suggesting that defects in Pofut2 mutant embryos could result from loss of O-fucosylation on ADAMTS9. To address this question, we compared the Pofut2 and Adamts9 knockout phenotypes and used Cre-mediated deletion of Pofut2 and Adamts9 to dissect the tissue-specific role of O-fucosylated ADAMTS9 during gastrulation. Disruption of Pofut2 using the knockout (LoxP) or gene trap (RST434) allele, as well as deletion of Adamts9, resulted in disorganized epithelia (epiblast, extraembryonic ectoderm, and visceral endoderm) and blocked mesoderm formation during gastrulation. The similarity between Pofut2 and Adamts9 mutants suggested that disruption of ADAMTS9 function could be responsible for the gastrulation defects observed in Pofut2 mutants. Consistent with this prediction, CRISPR/Cas9 knockout of POFUT2 in HEK293T cells blocked secretion of ADAMTS9. We determined that Adamts9 was dynamically expressed during mouse gastrulation by trophoblast giant cells, parietal endoderm, the most proximal visceral endoderm adjacent to the ectoplacental cone, extraembryonic mesoderm, and anterior primitive streak. Conditional deletion of either Pofut2 or Adamts9 in the epiblast rescues the gastrulation defects, and identified a new role for O-fucosylated ADAMTS9 during morphogenesis of the amnion and axial mesendoderm. Combined, these results suggested that loss of ADAMTS9 function in the extra embryonic tissue is responsible for gastrulation defects in the Pofut2 knockout. We hypothesize that loss of ADAMTS9 function in the most proximal visceral endoderm leads to slippage of the visceral endoderm and altered characteristics of the extraembryonic ectoderm. Consequently, loss of input from the extraembryonic ectoderm and/or compression of the epiblast by Reichert's membrane blocks gastrulation. In the future, the Pofut2 and Adamts9 knockouts will be valuable tools for understanding how local changes in the properties of the extracellular matrix influence the organization of tissues during mammalian development.
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, United States.
Brian A
BA
Benz
Department of Biomedical Engineering, Cleveland Clinic Lerner Research Institute, Cleveland, OH, United States.
Sumeda
S
Nandadasa
Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, United States.
Megumi
M
Takeuchi
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, United States.
Richard C
RC
Grady
Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, United States.
Hideyuki
H
Takeuchi
Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, United States.
Rachel K
RK
LoPilato
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, United States.
Shinako
S
Kakuda
Department of Biomedical Engineering, Cleveland Clinic Lerner Research Institute, Cleveland, OH, United States.
Robert P T
RPT
Somerville
Department of Biomedical Engineering, Cleveland Clinic Lerner Research Institute, Cleveland, OH, United States.
Suneel S
SS
Apte
Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, United States. Electronic address: rhalti@uga.edu.
Robert S
RS
Haltiwanger
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, United States. Electronic address: bernadette.holdener@stonybrook.edu.
Bernadette C
BC
Holdener
D000071119:ADAMTS9 Protein / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000650:Amnion / Q000196:embryology; D000818:Animals; D019521:Body Patterning; D002460:Cell Line; D053595:Embryonic Stem Cells; D005260:Female; D005647:Fucosyltransferases / Q000235:genetics*; D054262:Gastrulation / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008648:Mesoderm / Q000196:embryology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009154:Mutation
D005647:Fucosyltransferases; C000619307:PoFut2 protein, mouse; D000071119:ADAMTS9 Protein; C545434:Adamts9 protein, mouse
Adamts9; Gastrulation; O-Fucosylation; Pofut2; Thrombospondin type I repeats