Genome-wide loss-of-function analysis of deubiquitylating enzymes for zebrafish development.
BACKGROUND
Deconjugation of ubiquitin and/or ubiquitin-like modified protein substrates is essential to modulate protein-protein interactions and, thus, signaling processes in cells. Although deubiquitylating (deubiquitinating) enzymes (DUBs) play a key role in this process, however, their function and regulation remain insufficiently understood. The "loss-of-function" phenotype studies can provide important information to elucidate the gene function, and zebrafish is an excellent model for this goal.


RESULTS
From an in silico genome-wide search, we found more than 90 putative DUBs encoded in the zebrafish genome belonging to six different subclasses. Out of them, 85 from five classical subclasses have been tested with morpholino (MO) knockdown experiments and 57 of them were found to be important in early development of zebrafish. These DUB morphants resulted in a complex and pleiotropic phenotype that, regardless of gene target, always affected the notochord. Based on the huC neuronal marker expression, we grouped them into five sets (groups I to V). Group I DUBs (otud7b, uchl3 and bap1) appear to be involved in the Notch signaling pathway based on the neuronal hyperplasia, while group IV DUBs (otud4, usp5, usp15 and usp25) play a critical role in dorsoventral patterning through the BMP pathway.


CONCLUSIONS
We have identified an exhaustive list of genes in the zebrafish genome belonging to the five established classes of DUBs. Additionally, we performed the corresponding MO knockdown experiments in zebrafish as well as functional studies for a subset of the predicted DUB genes. The screen results in this work will stimulate functional follow-up studies of potential DUB genes using the zebrafish model system.
Tse
William K F
WK
Laboratory of Developmental Signalling and Patterning, Genes and Development Division, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore. kftse@imcb.a-star.edu.sg
Eisenhaber
Birgit
B
Ho
Steven H K
SH
Ng
Qimei
Q
Eisenhaber
Frank
F
Jiang
Yun-Jin
YJ
D000818:Animals; D019521:Body Patterning; D019485:Bone Morphogenetic Proteins / Q000235:genetics; D004625:Embryo, Nonmammalian; D047108:Embryonic Development; D010450:Endopeptidases / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques; D051880:Receptors, Notch / Q000235:genetics; D017422:Sequence Analysis, DNA; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics*
D019485:Bone Morphogenetic Proteins; D051880:Receptors, Notch; D029961:Zebrafish Proteins; D010450:Endopeptidases