A Morpholino Phenocopy of the
 colourless
 Mutant
K. Dutton,
*
 J. R. Dutton, A. Pauliny, and R. N. Kelsh
Department of Biology and Biochemistry, University of Bath, Bath, United Kingdom
Received 12 April 2001; Accepted 29 May 2001
Publishedonline23July2001;DOI10 1002 gene 1062
Key words:
 neural crest,
 sox10
, Waardenburg-Shah syn-drome, pigmentation, neurocristopathy
 We have utilized the modified antisense RNA morpho-lino technology to effectively phenocopy zebrafis
colourless
 / 
 sox10
 
cls
 ) mutations. The
 cls
 locus wasidentified in mutagenesis screens (Kelsh 
 et al.
, 1996;Malicki
 et al.
, 1996). Homozygous mutants are char-acterized by extensive defects in nonectomesenchy-mal fates (neurons, glia, and pigment cells) derivedfrom the neural crest (Kelsh and Eisen 2000; Kelsh 
 et al.
, 2000a, 2000b). Additionally,
 cls
 mutants havesmall otic vesicles (Whitfield
 et al.
, 1996). Zebrafish 
cls
 mutants are models for two human neurocristopa-thies, Hirschsprung’s disease, characterized by few or no enteric ganglia, and Waardenburg-Shah syndrome, which combines Hirschsprung’s disease with pigmentdefects. We have recently shown that the
 cls
 mutantphenotype results from disruptions in the zebrafish 
 sox10
 homologue and that
 sox10
 expression is firstseen at approximately 11 h postfertilization (hpf) inpremigratory neural crest cells (A. Pauliny and R. N.Kelsh, unpublished). The
 cls
 mutant phenotype canfirst be detected at 21 hpf in dopachrome tautomerase(dct) in situ or scored visibly at 27 hpf (Kelsh 
 et al.
,2000a). Thus, we demonstrate that morpholino oligoscan effectively phenocopy late embryonic phenotypesin zebrafish. We have characterized morphants gener-ated using morpholino oligos designed to target thezebrafish 
 sox10
 homologue. The phenotypes resultingfrom injection of these morpholinos are consistent with the
 cls
 mutant phenotype affecting both neuralcrest derivatives and otic vesicles with an optimalresponse achieved with a dose of 16.5 ng (Fig 1 andTable 1). In strong mutant phenotypes, no normalmelanophores are seen. However, even with higdoses of morpholino, some normal melanophores arepresent, implying that the characterized strong mu-tant alleles are likely nulls. At lower doses, all mor-phant embryos are reminiscent of the single reported weak 
 cls
 allele, which is not presently available (Mal-icki
 et al.
, 1996). As most known
 cls
 alleles display strong phenotypes, the ability to produce a gradedseries of 
 cls
 hypomorphs with morpholinos will beinvaluable in examining the role of 
 sox10
 in neuralcrest development.
LITERATURE CITED
Kelsh RN, Eisen JS. 2000. The zebrafish 
 colourless
 gene regulatesdevelopment of non-ectomesenchymal neural crest derivatives.Development 127:515–525.Kelsh RN, Brand M, Jiang Y-J, Heisenberg CP, Lin S, Haffter P, Odenthal J, Mullins MC, van Eeden FJ, Furutani-Seiki M, Granato M, Ham-merschmidt M, Kane DA, Warga RM, Beuchle D, Vogelsang L,Nusslein-Volhard C. 1996. Zebrafish pigmentation mutations andthe processes of neural crest development. Development 123:369–389.Kelsh RN, Dutton K, Medlin J, Eisen JS. 2000a. Expression of zebrafish 
 fkd6 
 in neural crest-derived glia. Mech Dev 93:161–164.Kelsh RN, Schmid B, Eisen JS. 2000b. Genetic analysis of melanophoredevelopment in zebrafish embryos. Dev Biol 225:277–293.Malicki J, Schier AF, Solnica-Krezel L, Stemple DL, Neuhauss SCF,Stainier DYR, Abdelilah S, Rangini Z, Zwartkruis F, Driever W.1996. Mutations affecting development of the zebrafish ear. De- velopment 123:275–283.Marusich MF, Furneaux HM, Henion PD, Weston JA. 1994. Hu neuronalproteins are expressed in proliferating neurogenic cells. J Neuro-biol 25:143–155. Whitfield TT, Granato M, van Eeden FJ, Schach U, Brand M, Furutani-Seiki M, Haffter P, Hammerschmidt M, Heisenberg CP, Jiang YJ,Kane DA, Kelsh RN, Mullins MC, Odenthal J, Nusslein-Volhard C.1996. Mutations affecting development of the zebrafish inner ear and lateral line. Development 123:241–254.
* Correspondence to: R. N. Kelsh, Department of Biology and Biochem-istry, University of Bath, Bath BA2 7AY, UK.E-mail: bssrnk@bath.ac.uk 
Table 1
Dose–Response Curve
DoseWeakphenocopy
a
Strongphenocopy
b
 n
3 ng 10% 194.5 ng 12% 1056 ng 21% 1057 ng 26% 989 ng 18% 11% 10414 ng 14% 30% 5916.5 ng 44% 24% 21837 ng 49% 29% 43
a
Weak phenocopies had at least 25 melanophores with a mean of67 (SD
25).
b
Strong phenocopies had a mean of 11 (SD
8) but less than 25total melanophores. One to eight cell AB wild-type embryos wereinjected with 4.6 nl of morpholino oligo diluted as recommended byGene Tools, LLC and incubated at 28.5°C. Embryos with more than50% of wild-type pigment at 48 hpf were scored as wild type due tovariations in embryonic development. At doses over 16.5 ng, non-specific deformities became apparent.
© 2001 Wiley-Liss, Inc. genesis 30:188–189 (2001)
 
FIG. 1.
 sox10
 knockdown embryos pheno-copy
 cls
 mutant phenotypes. ( 
a
,
 d
,
 g
,
 j
,
 m
 )Uninjected wild-type embryos shown incomparisonwithembryosinjectedwith16.5ng of a morpholino oligo (5
-GCCACAGGT-GACTTCGGTAGGTTTA-3
 )designedtotar-get the
43 to
19 region of the ( 
b
,
 e
,
 h
,
 k
,
n
 )
 sox10
 sequence and ( 
c
,
 f
,
 i
,
 l
,
 o
 )
 cls
 mu-tant embryos. A morpholino oligo (5
-AT-GCTGTGCTCCTCCGCCGACATCG-3
 ) de-signed to target the
23 to
2 region of the
 sox10
 sequence gave similar phenocopies(data not shown). Panels show lateral viewsof
a
f
 ) live whole-mounts,
g
i
 ) fixed em-bryosprocessedfor
dct 
(Kelsh
etal.
,2000b)in situ hybridization, or ( 
 j
o
 ) anti-Hu mAb16A11 (Marusich
 et al.
, 1994) antibodystaining. ( 
d
f
 ) Lateral views of the otic vesi-cle of a ( 
d
 ) 72 hpf wild-type embryo with thereduced otic vesicle, ( 
e
 ) small otoliths of aninjectedsibling,and
f
 )
cls
mutant.The
dct 
insitu hybridization reveals melanoblasts inthe anterior trunk of a ( 
g
 ) 22 hpf wild-typeembryo; these are absent in an ( 
h
 ) injectedsibling, and ( 
i
 )
 cls
 mutant. Anti-Hu stainingdemonstrates a significant decrease in thenumber of enteric neurons (arrows) in thehindgut of an ( 
k
 ) injected 5-day postfertiliza-tion (dpf) embryo compared with a ( 
 j
 ) wild-type sibling and similar to a ( 
l
 )
 cls
 mutant.Hu-positivedorsalrootganglia(arrow)inthetailofa
m
 )5dpfwild-typeembryoarelack-ing in an ( 
n
 ) injected sibling, and ( 
o
 )
 cls
 mu-tant (e, eye; ov, otic vesicle; s, somite).
189
 A MORPHOLINO PHENOCOPY OF THE
 COLOURLESS
 MUTANT
of 2