The zebrafish kohtalo?trap230 gene is required
for the development of the brain, neural crest,
and pronephric kidney
Sung-Kook Hong*, Caroline E. Haldin†, Nathan D. Lawson*, Brant M. Weinstein*, Igor B. Dawid*‡,
and Neil A. Hukriede*†‡
*Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892;
and†Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, W1152 BSTWR, Pittsburgh, PA 15261
Contributed by Igor B. Dawid, October 31, 2005
Mutation of the gene encoding the Mediator component thyroid
hormone receptor-associated protein (TRAP)230?MED12 affects
the development of multiple systems in zebrafish embryogenesis.
We isolated two ethylnitrosourea-induced alleles in the gene
encoding this protein and named the locus kohtalo (kto) after the
homologous locus in Drosophila. Homozygous kto mutant ze-
brafish embryos show defects in brain, neural crest, and kidney
development and die at ?6 days postfertilization. In the affected
tissues, differentiation is initiated and many cell type-specific
genes are expressed, but there is a failure of morphogenesis and
failure to complete differentiation. These results suggest that
for cell mobility, cell sorting, and tissue assembly.
branchial arches ? Danio rerio ? Mediator ? morphogenesis ? TRAP230
all organisms. Transcriptional regulation requires a protein
complex, named Mediator, that facilitates the interaction of
sequence-specific transcription factors and RNA polymerase II.
In metazoans, at least two types of Mediator complexes are
recognized, larger forms known as thyroid hormone receptor-
associated protein (TRAP)?SMCC, DRIP, and ARC and
smaller forms known as PC2 and CRSP. The large Mediator
complex includes four polypeptides not found in smaller forms
of the complex (1–5). These polypeptides, TRAP230?MED12,
TRAP240?MED13, CDK8, and Cyclin C, or Srb8-11 in yeast,
constitute a distinct functional module that may act as a repres-
sor (6–8). Genetic studies of the kohtalo (kto) gene (9) that
encodes TRAP230 in Drosophila melanogaster have focused on
its function in the wing and eye disks (10, 11). Mutant cells
proliferate, survive, and initiate but do not complete differen-
tiation; most notably, these cells do not respect compartment
boundaries, leading to disorganized tissue architecture (10, 11).
In Caenorhabditis elegans, the TRAP230 homolog is an essential
gene (12, 13) and is required for asymmetric cell division in the
T blast cell lineage (14). Vertebrate mutations in the gene
encoding TRAP230 have not been reported.
In a zebrafish mutagenesis screen, we isolated two alleles of an
embryonic lethal mutation that results in abnormal development
of the brain, neural crest, and kidney. The mutated gene encodes
the zebrafish homolog of TRAP230?MED12; we refer to the
genetic locus as kto (9–11) and to the protein product as
TRAP230 (15). We show that kto mutant embryos are not
grossly compromised in cell proliferation and survival and are
capable of initiating differentiation of multiple cell types. A
common feature of the malformations that ensue is the failure
of organogenesis, with deficits in proper tissue extension, cell
movements, and generation of tissue architecture.
he orderly expression of batteries of genes in a temporally
and spatially regulated way is critical to the development of
Materials and Methods
Identification of kto Mutations by Genetic Mapping. Mutations were
recovered after ethylnitrosourea mutagenesis. Mapping lines
were generated by mating kto heterozygotes to the EK and WIK
strains, and the location of the mutation relative to polymorphic
markers was determined as described (16, 17).
Knockdown Experiments. A morpholino (MO) antisense oligonu-
cleotide for trap230, 5?-TCAGAACGCCGAAGGCAGCCAT-
CAT-3?, was designed and purchased from Gene Tools (Philo-
math, OR); the bold sequence CAT corresponds to the start
Cloning. We found genomic sequence of a portion of the kto?
trap230 gene in scaffold 1226 (Zv4) from the Sanger Institute
(Cambridge, U.K.). The sequence was amplified and subcloned.
The RACE-PCR kit (Clontech) was used according to the
manufacturer’s protocol with primers 5?-CTCAAAGCACTC-
CAGCACCCAGGTGA-3? for 5?-RACE and 5?-ATCCAC-
CCAAACATGAGGCCCAATCAG-3? for 3?-RACE. Full-
length cDNA was amplified with the Expand high-fidelity PCR
system (Roche Applied Science, Indianapolis) and cloned into
pCS2?or pBluescript SK?. To generate RNA probe, a 1.4-kb
fragment spanning the region from 1.8 to 3.3 kb of the ORF was
used. Sequences were analyzed with DNASIS MAX, Version 2.0
(MiraiBio, Hitachi, Tokyo).
TUNEL Assay, in Situ Hybridization, and Immunohistochemistry. For
TUNEL assay, terminal deoxynucleotidyl transferase and buffer
were purchased from Invitrogen, and digoxigenin-11-dUTP and
BM purple for detection were from Roche. Digoxigenin- or
fluorescein-labeled antisense RNA probes were prepared from
linearized template DNAs using an RNA labeling kit (Roche).
Whole-mount in situ hybridization (18, 19) and two-color in situ
hybridization (20) were performed as described, and immuno-
staining was carried out as described (21). F-59 slow muscle
antibody (Developmental Studies Hybridoma Bank) was used at
1:100. Monoclonal zn-5 antibody (DM-GRASP) was obtained
from Zebrafish International Resource Center, Eugene, OR.
Horseradish peroxidase-conjugated anti-mouse IgG was used as
secondary antibody (1:1,000, Sigma), and staining was per-
formed with Fast diaminobenzidine (Sigma) in 1? PBS.
Alcian Blue and Calcein Staining. Cartilage staining was carried out
by using Alcian blue (Sigma) (22). Bone structures were visu-
Conflict of interest statement: No conflicts declared.
Freely available online through the PNAS open access option.
Abbreviations: dpf, days postfertilization; hpf, hours postfertilization; MO, morpholino;
kto, kohtalo; TRAP, thyroid hormone receptor-associated protein.
Data deposition: The sequence reported in this paper has been deposited in the GenBank
database (accession no. DQ133567).
‡To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or hukriede@
© 2005 by The National Academy of Sciences of the USA
www.pnas.org?cgi?doi?10.1073?pnas.0509457102 PNAS ?
December 20, 2005 ?
vol. 102 ?
no. 51 ?
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