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
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alized by calcein staining following a slightly modified procedure
(23). Embryos at 5 days postfertilization (dpf) were immersed in
0.2% calcein for 7 min, rinsed three times with fresh egg water,
and mounted on glass slides with 4% methyl-cellulose after a
30-min period. Calcified bone was analyzed by fluorescence
microscopy by using a green fluorescence filter.
Histology and Methylene-Blue Staining. Embryos at 24 hours post-
fertilization (hpf) were fixed in 4% paraformaldehyde (Sigma)
overnight at 4°C, then gradually dehydrated with an ethanol
series. For embedding, dehydrated embryos were immersed in
JB-4 plastic resin (Polyscience) overnight and hardened by
a Leica (Deerfield, IL) RM2165 microtome. Samples were
stained in 1% methylene blue (Sigma) dissolved in 1% sodium
tetraborate, followed by rinsing in water.
Isolation and Identification of the kto Mutation. Two alleles of a
recessive lethal mutation were obtained in an ethylnitrosourea-
based mutagenesis screen. Bulk segregant and further mapping
placed the mutations on chromosome 14 in the critical interval
shown in Fig. 1A. Bacterial artificial chromosome clones derived
from this area of the genome provided candidate genes, includ-
ing a gene encoding the zebrafish homolog of TRAP230. The
partial ORF that had been available for kto?trap230 was ex-
panded by the RACE procedure, and wild-type and mutant
cDNAs were sequenced. The zebrafish trap230 sequence is
deposited in GenBank, accession no. DQ133567. Allele ktoy81
contains a stop codon at amino acid 399, whereas ktoy82has a
G3A transition in a splice donor site (Fig. 1B). Sequencing of
genomic DNA confirmed that ktoy82mutant mRNA is spliced
from a cryptic GT donor site four nucleotides downstream of the
normal site, leading to a frame shift at amino acid 683, followed
by chain termination. Thus, both alleles lead to early truncations
of the protein, likely representing null mutations.
Kto?trap230 mRNA is present in maternal RNA and is widely
expressed in the early embryo but concentrated in the head by
48 hpf (Fig. 1C).
kto Function Is Required for Brain Morphogenesis. Phenotypic ab-
normalities are first visible in kto mutant embryos during
midsomitogenesis. Although different brain regions are formed,
morphogenesis is abnormal with reduced extension along the
anterior–posterior axis leading to a wavy appearance and failure
to inflate the ventricles in fore- and midbrain, although the
fourth ventricle is largely normal (Fig. 2 A–F). Development of
the trunk and tail is not obviously affected. Heart edema is
apparent at 24 hpf and increases subsequently, but the initial
morphogenesis of the heart is normal. Injection of a trap230 MO
generated a phenocopy of the mutation (Fig. 2 G–I). The
identification of the mutant gene was confirmed further by
rescue of brain development by injection of trap230 mRNA into
mutant embryos (Fig. 2 J–L).
Deficits in brain morphogenesis were studied further with the
aid of marker genes. The floating head (flh) gene is expressed in
two regions of the forebrain, the epiphysis, and telencephalon,
which are separated by a clear gap in wild-type embryos (Fig.
3A). In kto embryos both regions express flh as the wild type, but
the domains are fused into a continuous region due to the
reduced expansion of the brain in the anterior–posterior direc-
tion (Fig. 3B). This phenotype is closely matched in embryos
injected with trap230 MO (Fig. 3D). Reduced expansion of the
brain was also visualized by Wnt1 expression. Wild-type and
ary and along the dorsal midline of the midbrain, but the midline
domain is shortened in kto (Fig. 3 E and F).
Multiple Deficits in the Formation of Neural Crest Derivatives in kto
Embryos. Neural crest cells arise in the dorsal aspect of the neural
tube, migrate to multiple locations in the embryo, and give rise
to a large variety of derivatives (24–27). Given the apparent
requirement for kto function in morphogenesis, we tested neural
crest formation in mutant embryos. Already at the six-somite
stage, there is a pronounced reduction in the expression of the
crest-specific genes snail2 (28) and sox9b (19) (Fig. 4 A–D). A
similar reduction was seen for other early neural crest markers
such as crestin, foxd3, sox10, and ap2a and was further exacer-
bated at later stages (not shown). These observations suggest
that cells that normally become neural crest progenitors either
the kto locus on chromosome 14, not precisely to scale. Below the cM scale,
markers that are polymorphic in a map cross with the TL strain are shown in
black, and markers polymorphic in the WIK map cross are shown in red.
Recombinants per number of meioses tested are listed below. Note that we
have repositioned simple sequence length polymorphism marker z22128
based on these results. Bacterial artificial chromosome (BAC) clones from this
region are shown below. A portion of the kto?trap230 gene is located at the
end of BAC clone zC119P14. (B) TRAP230 protein domain structure and point
mutations in two kto alleles. L, leucine-rich domain; LS, leucine-serine rich
domain; PQL, proline–glutamine–leucine-rich domain; OPA, opposite-paired
domain. The ktoy81allele carries a stop codon. A point mutation in a splice
donor site in ktoy82leads to splicing from a cryptic site four nucleotides
downstream, which results in a frame shift and premature termination. (C)
Expression of trap230 as seen by in situ hybridization. Lateral views except
widespread expression at 90% epiboly; (c) in kto embryo at 90% epiboly,
arrowheads point to presumptive cartilage primordia; (e) at 48 hpf, trap230
expression is limited to the head (arrow). di, diencephalon; e, eye; h, hind-
brain; m, midbrain; mhb, mid-hindbrain boundary; ov, otic vesicle; te, telen-
Positional cloning of the zebrafish kto locus. (A) Genetic mapping of
www.pnas.org?cgi?doi?10.1073?pnas.0509457102Hong et al.
fail to differentiate or are lost early in development. To test the
latter possibility, we carried out TUNEL staining for cells
undergoing apoptosis. The moderate increase in TUNEL-
positive cells that was observed does not appear to account for
the phenotypes that arise in the kto mutant (Fig. 4 E and F).
Pigment cells are major derivatives of the neural crest, and
kto embryos showed a reduction but not elimination of pig-
mentation at 48 hpf (Fig. 5 A and B). Iridophores, a subset of
pigment cells, could not be detected in mutant embryos by 5
dpf (not shown). In the formation of pigment cells, neural crest
cells emerge from their dorsal origin and migrate in a ventral
direction before differentiation (29, 30). This behavior de-
pends on the function of multiple genes in chromatophore
precursors. In 24-hpf embryos, the mitfa?nacre and c-kit genes
mark cells in the dorsal aspect of the neural tube and in streams
that extend across the trunk to the ventral side (31–33). These
streams of migratory cells are absent in kto embryos even
though mitfa and c-kit-positive cells arise in the mutant (Fig.
5 C and D and data not shown). These observations suggest
that melanocyte precursors are generated in kto embryos but
fail to migrate in a ventral direction, although it is also possible
that the cells do migrate but quickly lose their differentiation
blue. Lateral views (A, D, and G) and dorsal views (B, E, H, J–L) of the brain. Wild type (A–C), kto mutant (D–F), and embryos (G–I) injected with 2 ng of
trap230 MO. Rescue of mutant (J–L), wild type (J), and kto embryo (K and L) injected with 100 pg of trap230 mRNA; the embryo was genotyped to ascertain
that it was mutant. Arrowheads point to regions malformed in mutants and morphants. di, diencephalon; e, eye; m, midbrain; mhb, mid-hindbrain
boundary; te, telencephalon.
Brain phenotype of kto mutants. Embryos at 24 hpf. Live embryos (A, B, D, E, G, H, J–L) and sagittal sections (C, F, and I) stained with methylene
C–E) and kto mutant (B and F). (A–D) In situ hybridization with flh; a gap
(arrowhead) is seen in expression between telencephalon and diencephalon
of trap230 MO (D). (E and F) wnt1 expression in dorsal midbrain and mid-
hindbrain boundary. Arrow shows reduced midbrain size in kto mutants. di,
diencephalon; m, midbrain; mhb, mid-hindbrain boundary; te, telencephalon.
The brain does not extend properly in kto mutants. Wild-type (A and
kto (B, D, and F) at the six-somite stage. snail2 expression (A and B) and sox9b
expression (C and D) are reduced in the mutant. (E and F) Cell death as
in dying cells can be seen in the mutant. e, eye; h, hindbrain; ov, otic vesicle;
sp, spinal cord; tb, tail bud.
Neural crest development in kto embryos. Wild type (A, C, and E) and
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kto Is Required for Pharyngeal Arch Formation. Neural crest cells
originating from the dorsal aspect of the hindbrain migrate in
three streams in a ventral direction to generate a large portion
of the pharyngeal arches (34). We observed severe disruption in
arch formation in embryos that lack kto gene function. This
deficit can be seen at 24 hpf by the analysis of the expression of
genes such as dlx2a, which marks the forming arch structures
(Fig. 6 A and B). In kto embryos, these structures are reduced
and malformed, as seen by the fusion of anterior elements. At a
slightly later stage of development (31 hpf), arch structures can
be visualized in transgenic animals by the expression of GFP
under the control of the fli promoter (35); in kto mutant embryos
that carry the transgene, branchial arches are largely absent,
whereas arches 1 and 2 seem to be fused (Fig. 6 C and D). Arches
form by the contribution and interaction of tissues derived from
the neural crest, mesoderm, and endoderm (34). To evaluate the
requirement for kto function in different derivatives, we com-
pared the expression of nkx2.3, which acts in the endoderm (36),
with that of dHAND, which labels crest-derived cells (37).
Examination of dHAND confirms the drastic reduction and
disorganization of the crest-derived arch components in the
mutant embryo (Fig. 6 E and F). The endodermal lining of the
arches, as assayed by nkx2.3, still forms, but pouches that
normally separate the body of each arch do not develop (Fig. 6
G and H). Loss of endodermal pouches was confirmed by using
the antibody zn5 (Fig. 6 I and J). Given the deficits in neural
crest-derived structures, it is not surprising that pharyngeal
cartilage elements could not be detected in the mutant embryo
by 5 dpf (Fig. 6 K and L). Pharyngeal muscles (Fig. 6 M and N)
and bones of the head likewise fail to form in the mutant
embryos (Fig. 6 O–R). Thus, deficits in neural crest formation
starting in the early embryo are followed by extensive failure of
pharyngeal arch formation in the kto mutant embryo.
Pronephric Cells Are Specified, But Kidney Morphogenesis Fails in kto
Mutants. The vertebrate kidney is derived from the interme-
diate mesoderm as a bilaterally symmetric primordium. Al-
though the pronephric ducts and tubules remain as bilaterally
paired structures, cells at the rostral end of the nephric field
coalesce and migrate medially to form glomeruli fused at the
midline (38). This process is marked by wt1 expression, which
is first observed in the anterior nephrogenic mesenchyme and
subsequently becomes restricted to the podocyte cells of the
glomeruli (38, 39). In early somitogenesis stages, wt1 marks
two bilaterally paired symmetrical regions with indistinguish-
able appearance in wild-type and kto mutant embryos (Fig. 7
A and B). By 24 hpf, however, when the anterior pronephric
cells have begun to coalesce in wild-type embryos, two distinct
regions of wt1-positive cells remain in kto embryos (Fig. 7 C
and D), and they persist as distinct regions through the next day
of development (not shown). Injection of trap230 MO pro-
duces a closely similar phenotype (Fig. 7 E and F). As a result,
B) Pigment patterning of wild-type and kto embryos at 48 hpf. Pigmentation
is reduced in the mutant embryo, especially in ventral regions. (C and D)
nacre?mitfa expression at 24 hpf. Arrows and arrowheads point to dorsal
neural tube and migrating neural crest cells, respectively. n, notochord.
Reduced pigmentation in kto mutant embryos. Lateral views. (A and
in pharyngeal arches at 26 hpf. No distinct pharyngeal arches form (arrow), and expression in posterior arches is essentially extinguished in kto mutants
(arrowhead). (C and D) TG(fli1:EGFP)y1transgenic embryos at 31 hpf. In kto mutant embryos, p1 and p2 seem malformed and fused, whereas the more posterior
separation of p1 and p2 failed, and p3–p7 are largely absent. dHAND expression in the heart is present (white asterisk), but the pectoral fins are lost in kto. (G
and H) Expression of nkx2.3 at 36 hpf visualized endodermal pouches; cells separating individual arches are lost in the mutant (arrowhead). (I and J) zn5 stains
the endodermal pouches, trigeminal ganglia, and sensory neurons. (K and L) Alcian blue staining at 5 dpf. No cartilage elements are visible in the mutant. (M
and N) Detection of larval muscles with F59 antibody at 4 dpf; muscles in the head are lost in the mutant. (O–R) Calcein-stained fluorescent image shows loss
of bones in the head of kto embryos. b, branchyal arch; bh, basihyal; cb, ceratobranchial; ch, ceratohyal; e, eye; fb, forebrain; h, hyoid arch; hh, hyohyoideus;
hs, hyosymplectic; ht, heart; ih, interhyoideus; imp, intermandibularis posterior; m, mandibular arch; mc, Meckel’s cartilage; n, notochord; ov, otic vesicle; p,
pharyngeal arch; pf, pectoral fin, r, rhombomere; sh, sternohyoideus; te, teeth; tg, trigeminal ganglia; tv, transversus ventralis.
www.pnas.org?cgi?doi?10.1073?pnas.0509457102Hong et al.
discernable glomeruli do not form in the mutant embryos. To
confirm the lack of glomerular morphogenesis in kto embryos,
we looked at expression of podocyte-specific genes. Nephrin
and podocin expression in the kidney is restricted to podocyte
cells and initiated by 24 hpf (40). Wild-type and kto embryos
from 24 to 48 hpf were tested for the expression of these two
genes, and neither was detectable in kto embryos (not shown).
This failure of morphogenesis is specific to the pronephros,
because the heart, which also forms from a bilateral population
of mesodermal cells (41), forms and beats in kto embryos
before edema becomes apparent.
kto?trap230, an Essential Gene in Zebrafish, Is Involved in Cell
Behavior and Morphogenesis. The gene that encodes the Medi-
ator component TRAP230, also known as MED12, TRCC11,
HOPA, and in yeast as Srb8 (2–5, 42) is an essential gene in
zebrafish. Database searches indicate there is only a single
trap230 gene in zebrafish, possibly explaining why this gene is
essential for development. The homologous genes are also
essential in Drosophila and C. elegans (10–14), but the phe-
notypes described in these species are not obviously similar. A
common feature, however, is an effect on cell arrangements
and morphogenesis. In Drosophila, the infringement of com-
partment boundaries by kto mutant clones indicates that
TRAP230 function is required for the synthesis of molecules
that regulate cell behavior and interactions (10, 11). The
nature of these molecules remains largely unknown, but they
might include cell adhesion factors, receptors and ligands, and
cytoskeletal components. Similar components might be among
the genes that depend on TRAP230 function in zebrafish,
because in all three tissues studied, major defects in kto
embryos appear to involve cellular behavior such as migration
Abnormal brain morphogenesis is the earliest phenotype
observed in kto embryos. Although all major regions of the
brain form with correct anterior–posterior and dorsal–ventral
polarity, the brain fails to expand normally along the anterior–
posterior axis, giving it a wavy appearance (Figs. 2 and 3).
Further, ventricle formation in the forebrain and midbrain
fails. The prominent deficits of kto embryos in pharyngeal arch
development and pigmentation are based on the inability of
the mutant neural crest cells to complete differentiation and
reach their proper positions in the embryo. Analysis of early
molecular markers, including snail2 and sox9b, (24, 43–45),
suggests that mutants initiate neural crest differentiation in
fewer cells and express these genes at lower levels than in
wild-type embryos (Fig. 4 A–D). Among the major neural crest
derivatives, a reduced population of melanocyte precursors
was detected, and these cells failed to migrate ventrally or
extinguished their differentiation markers if they did migrate.
As a result, few melanocytes arise in kto embryos (Fig. 5).
Likewise, the formation of pharyngeal arches is progressively
deficient in the mutant. Reduced populations of cells arise that
express genes involved in arch formation, and those cells that
initiate differentiation show a disorganized pattern (Fig. 6
A–F). The morphogenesis of pharyngeal arches involves
endodermal, mesodermal, and neural crest derivatives (34),
and all of these components show deficits in kto mutant
embryos at later stages (Fig. 6).
Kidney development is initiated in kto embryos with for-
mation of bilateral primordia and nephric ducts, but glomus
patterning fails (Fig. 7), and vegf expression is not seen in the
region where the glomerular capillary tuft forms (data not
shown). It should be considered whether the glomerular
morphogenesis defects seen in kto are due to changes in
circulation. Cardiac defects that affect circulation perturb
glomerulogenesis, and kto embryos have reduced circulation.
However, anterior wt1-positive nephrogenic mesenchyme cells
coalesce in other mutants with compromised circulation such
as island beat (isl), valentine (vtn), and silent heart (sih) (46),
whereas in kto embryos, these cells remain as bilateral mes-
enchymal fields. Thus TRAP230 function appears to be re-
quired not for pronephros specification but rather for glomer-
ular morphogenesis, and this defect does not seem to be
related to impaired circulation.
Complex and Selective Functions of a Mediator Component in Meta-
zoan Development. Although Mediator may affect the transcription
of most or all genes, TRAP230 is part of the distinct kinase module
that appears to be a transient constituent of Mediator (3, 7, 47) and
thus might be involved in the regulation of a subset of genes.
Mutations in C. elegans and Drosophila and now in zebrafish
demonstrate that TRAP230 is essential for development, with
multiple but differential requirements in different cells and tissues.
differentiation, whereas mutant clones in the wing disk cross
compartment boundaries (10, 11). Further, mutant clones have
sharper edges and higher circularity than wild-type clones. These
observations point to a change in cell adhesion properties in kto
mutant cells. In particular, the high circularity suggests that mutant
cells may have stronger adhesive affinity for each other than for
wild-type cells. This interpretation follows the long-standing pro-
posal by Steinberg (48, 49) that attributes cell sorting to adhesive
strength. Increased adhesion to like cells might in part explain the
inhibit the epithelial–mesenchymal transition that is required for
neural crest migration (27, 30). One might even speculate further
genes, for example, genes encoding cell adhesion factors, by the
mutational inactivation of TRAP230, a protein that is part of an
apparent repressor module (6, 7). Although this explanation may
account for prominent aspects of the kto phenotype, it may be
expected that this mutation affects multiple functions in the
We thank Paul Ulanch and Rachel Jackson for technical assistance;
Elizabeth Laver, Brigid Diamond, and Mark Rath for help in fish
husbandry; and Iain Drummond (Massachusetts General Hospital,
Charlestown, MA) for clones. This research was supported by the
Intramural Research Program of the National Institute of Child Health
and Human Development, National Institutes of Health. N.A.H. is
supported by the Carl W. Gottschalk Research Scholar Award from the
American Society of Nephrology.
Wild-type (A, C, E, and F) and kto mutant (B and D); at the five-somite stage,
mutant embryos were identified by genotyping. (A and B) Five-somite stage,
coalesce at the midline in kto (D) and trap230 MO-injected embryos (F).
Anterior nephrogenic mesenchyme migration fails in kto embryos.
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