16
PLoS One. 2012;7(4):e34671.
Fast homozygosity mapping and identifcation of a
zebrafsh ENU-induced mutation by whole-genome
sequencing.
Voz ML, Coppieters W, Manfroid I, Baudhuin A, Von Berg V, Charlier C, Meyer D, Driever W,
Martial JA, Peers B.
Abstract
Forward genetics using zebrafsh is a powerful tool for studying vertebrate development
through large-scale mutagenesis. Nonetheless, the identifcation of the molecular lesion is still
laborious and involves time-consuming genetic mapping. Here, we show that high-through-
put sequencing of the whole zebrafsh genome can directly locate the interval carrying the
causative mutation and at the same time pinpoint the molecular lesion. The feasibility of this
approach was validated by sequencing the m1045 mutant line that displays a severe hypo-
plasia of the exocrine pancreas. We generated 13 Gb of sequence, equivalent to an eightfold
genomic coverage, from a pool of 50 mutant embryos obtained from a map-cross between
the AB mutant carrier and the WIK polymorphic strain. The chromosomal region carrying the
causal mutation was localized based on its unique property to display high levels of homo-
zygosity among sequence reads as it derives exclusively from the initial AB mutated allele.
We developed an algorithm identifying such a region by calculating a homozygosity score
along all chromosomes. This highlighted an 8-Mb window on chromosome 5 with a score close
to 1 in the m1045 mutants. The sequence analysis of all genes within this interval revealed a
nonsense mutation in the snapc4 gene. Knockdown experiments confrmed the assertion that
snapc4 is the gene whose mutation leads to exocrine pancreas hypoplasia. In conclusion, this
study constitutes a proof-of-concept that whole-genome sequencing is a fast and efective
alternative to the classical positional cloning strategies in zebrafsh.
Dev Biol. 2012 Jun 15;366(2):268-78.
Zebrafsh sox9b is crucial for hepatopancreatic
duct development and pancreatic endocrine cell
regeneration.
Manfroid I, Ghaye A, Naye F, Detry N, Palm S, Pan L, Ma TP, Huang W, Rovira M, Martial JA,
Parsons MJ, Moens CB, Voz ML, Peers B.
Abstract
Recent zebrafsh studies have shown that the late appearing pancreatic endocrine cells are
derived from pancreatic ducts but the regulatory factors involved are still largely unknown.
Here, we show that the zebrafsh sox9b gene is expressed in pancreatic ducts where it labels
the pancreatic Notch-responsive cells previously shown to be progenitors. Inactivation of
sox9b disturbs duct formation and impairs regeneration of beta cells from these ducts in
larvae. sox9b expression in the midtrunk endoderm appears at the junction of the hepatic
and ventral pancreatic buds and, by the end of embryogenesis, labels the hepatopancreatic
ductal system as well as the intrapancreatic and intrahepatic ducts. Ductal morphogenesis and
diferentiation are specifcally disrupted in sox9b mutants, with the dysmorphic hepatopan-
creatic ducts containing misdiferentiated hepatocyte-like and pancreatic-like cells. We also
show that maintenance of sox9b expression in the extrapancreatic and intrapancreatic ducts
requires FGF and Notch activity, respectively, both pathways known to prevent excessive
endocrine diferentiation in these ducts. Furthermore, beta cell recovery after specifc abla-
tion is severely compromised in sox9b mutant larvae. Our data position sox9b as a key player
in the generation of secondary endocrine cells deriving from pancreatic ducts in zebrafsh.