A morpholino phenocopy of the colourless mutant

We have utilized the modified antisense RNA morpholino technology to effectively phenocopy zebrafish colourless/sox10 (cls) mutations. The cls locus was identified in mutagenesis screens (Kelsh et al., 1996; Malicki et al., 1996). Homozygous mutants
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   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 zebrafish  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firstseen at approximately 11 h postfertilization (hpf) inpremigratory neural crest cells (A. Pauliny and R. N.Kelsh, unpublished).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 high doses 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:  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
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