Embryonic expression and activity ofdoughnut, a secondRYK homologin Drosophila

Andrew C. Oatesa,*, Joshua L. Bonkovskyb,d, Danielle V. Irvinea,1,Leonard E. Kellyc, John B. Thomasb, Andrew F. Wilksa,2

aLudwig Institute for Cancer Research, Melbourne Tumour Biology Branch, P.O. Box 2008, Royal Melbourne Hospital, Melbourne, Victoria 3050, AustraliabMolecular Neurobiology Laboratory, The Salk Institute for Biological Studies, P.O. Box 85800, San Diego, CA 92186, USA

cDepartment of Genetics, University of Melbourne, Parkville, Victoria 3052, AustraliadDepartment of Biomedical Sciences, University of California, San Diego, CA 92186, USA

Received 15 June 1998; revised version received 14 September 1998; accepted 17 September 1998

Abstract

In theDrosophilaembryo, a subset of muscles require expression and function of theRYKsubfamily RTK genederailed(drl) for correctattachment. We have isolated a secondRYKhomolog,doughnut(dnt), from Drosophila. The DNT protein exhibits 60% amino acid identityto DRL, and is structurally as similar to the mammalian RYK proteins as is DRL, indicating an ancient duplication event.dnt is expressedin dynamic patterns in the embryonic epidermis, being found at high level in epithelia adjacent to cells that are invaginating into the interiorof the embryo, including ventral furrow, cephalic furrow, fore- and hindgut, optic lobe and tracheal pits.dnt is capable of a partial rescue ofthe muscle attachment defect ofdrl − /− embryos, indicating that it encodes a receptor with a related and significantly overlappingbiochemical function. 1998 Elsevier Science Ireland Ltd. All rights reserved

Keywords: Drosophila melanogaster; Receptor tyrosine kinase; Development; Embryogenesis; P-element mediated transgenesis;RYKgenefamily; derailed; doughnut; Gene duplication

1. Introduction

Three members of the RYK subfamily of tyrosine kinaseshave been identified, one each in mouse (Hovens et al.,1992), human (Stacker et al., 1993), andDrosophila(Call-ahan et al., 1995). RYK proteins are characterized by theirrelatively short extracellular domains, and an intracellularkinase domain that contains a significant degree of diver-gence to other PTKs within highly conserved amino acidmotifs (Hanks and Quinn, 1991; Hovens et al., 1992). In

Drosophilamutants of theRYK homologderailed (drl), asubset of somatic muscles fails to select the correct attach-ment points on the epidermis, instead migrating beyond theappropriate site before attaching (Callahan et al., 1996). Thedrl phenotype is a starting point for investigation of thefunction of the RYK gene family, suggesting a role forthese receptors in the control of cell migration.

2. Results and discussion

2.1. Cloning and structure of a second RYK homolog inDrosophila

We isolated a cDNA fromDrosophila encoding a full-lengthRYK family member that we have nameddoughnut(dnt,protein DNT, accession number AJ224361). An align-ment of DNT with DRL and the mammalian RYK genes(Fig. 1) revealed the presence of conserved features

Mechanisms of Development 78 (1998) 165–169

0925-4773/98/$ - see front matter 1998 Elsevier Science Ireland Ltd. All rights reservedPII S0925-4773(98)00167-1

* Corresponding author. Present address: Department of Molecular Biol-ogy, Princeton University, Washington Road, Princeton, NJ 08544, USA.Tel.: +1 609 2583983; fax: +1 609 2581035; e-mail: aoates@molbio.prin-ceton.edu

1 Present address: The Murdoch Institute for Research into Birth Defects,Royal Children’s Hospital, Flemington Road, Victoria 3050, Australia.

2 Present address: Mutation Research Centre, St. Vincent’s Hospital, 41Victoria Parade, Victoria 3065, Australia.

166 A.C. Oates et al. / Mechanisms of Development 78 (1998) 165–169

expected in a member of theRYKRTK subfamily: seven ofthe 11 Hanks kinase motifs (Hanks and Quinn, 1991) inDNT show RYK-specific variations in sequence. DNT andDRL form a natural clade in a phylogenetic tree of theRYKfamily (data not shown), hencednt anddrl were likely gen-erated by gene duplication within the lineage leading toDrosophila. We have localized thednt gene to band 37D2on salivary gland interphase polytene chromosomes byhybridization of a cDNA probe (data not shown).

2.2. Analysis of dnt transcript in the Drosophila embryo byin situ hybridization

dnt transcript was detected during embryogenesis in ser-ies of dynamic domains in the ectodermally derived epider-mis, many of which bordered on sites of epithelialinvagination, including ventral furrow, cephalic furrow,fore- and hindgut, optic lobe and tracheal pits (see legendto Fig. 2). This expression pattern is distinct from that ofdrl(Callahan et al., 1995).

2.3. Targeted expression of dnt to muscles partially rescuesthe drl mutant muscle phenotype

To explore the degree of functional conservation betweendntanddrl, we tested whetherdntcould substitute fordrl inmuscle attachment site selection. IndrlR343 null mutantembryos, muscles 21–23 elongate but fail to properly attachto their ventral attachment sites, ‘bypassing’ their propersite in approximately 20% of hemisegments (Callahan etal., 1996).dnt expression in adrlR343 null using theME4enhancer, which drives high level expression in muscles21–23 (Table 1; Callahan et al., 1996), gave partial rescueof the mutant phenotype, reducing the incidence of the‘bypass’ to 10% and 7% in the independent linesME4-dnt1 and ME4-dnt3, respectively (Table 1). We introducedboth ME4-dnt1 and ME4-dnt3 into drlR343 mutants and the

incidence of ‘bypass’ did not further decrease (9%), indicat-ing that the system was saturated with respect todnt func-tion. Thus, DNT expression indrl − /− muscles can partiallysubstitute for DRL function.

3. Experimental procedures

dnt was amplified fromDrosophilaembryonic cDNA byPCR using two primer pairs targeted to conserved regions ofthe RYK genes. External primer pair: (GCIGA(A/G)(C/T)TITA(C/T)TA(C/T)GT)–(AAIA(A/G)(C/T)TC(A/G)T-CIGG(A/G)CA); internal primer pair: (GCIGCI(A/C)GIAA(C/T)TG(C/T)GTIATIGA(C/T)GA)–(TAICC(A/G)TC(C/T)TTIA(A/G)(A/G)TA). Standard techniques wereused for subcloning, PCR cycle sequencing (PE-AppliedBiosystems, Foster City, CA), screening of embryoniccDNA libraries (Sambrook et al., 1989), chromosomal loca-lization (Phillips et al., 1994) and in situ hybridization (Leh-mann and Tautz, 1994). Embryos were mounted in glyceroland photographed under dark-field illumination with aNikon Microphot-FX microscope using Kodak 160T Eckta-chrome film (Eastman-Kodak, Rochester, NY).

P[ME4-dnt] was constructed with thednt cDNA down-stream of thehsp70promoter in P[ME4] (Callahan et al.,1996; J. Botas, unpublished data). P[ME4-dnt] was intro-duced into the fly germ line by standard methods (Rubin andSpradling, 1982) to create theME4-dnt1 andME4-dnt3 lineswhich carry insertions on the second and third chromo-somes, respectively. Analysis of the ‘bypass’ phenotypewas as previously described (Callahan et al., 1996).

Acknowledgements

We thank Guo-Fen Tu for DNA sequencing, AndrewRunting for assistance with computing and Steve Stacker,

Fig. 1. Amino acid sequence alignment between RYK family proteins. DNT, DRL (Callahan et al., 1995), mouse (Mm) (Simoneaux et al., 1995), and human(Hs) RYK (Stacker et al., 1993). Sequence identities are boxed. Domain organization is indicated by labeled square brackets: RH1, RYK homology domain1; RH2, RYK homology domain 2; TM, transmembrane domain; KIN, kinase domain. Conserved serine residues are labeled with an asterisk. Hankssubdomains are indicated with the appropriate roman numerals.

Fig. 2. Dynamic expression of thednt transcript during embryonic development. Whole-mount in situ hybridization with a DIG-labeled probe was carried outon stagedDrosophilaembryos. All embryos are displayed in the same orientation unless otherwise indicated: anterior (A) to the left and dorsal (D) to the topof the figure. (a) Precellular (syncytial) blastoderm showing ubiquitous distribution of maternal transcript. (b) Cellular blastoderm. Note the absence oftranscript from the termini of the embryo. (c) Cellular blastoderm. ep, anlage of the epipharynx. (d) Cellular blastoderm (stage 5). hy, anlage of thehypopharynx; ma, anlage of the anterior midgut; p, pole cells (out of focal plane). Large arrowheads indicate Gap gene-like bands of expression; the arrowindicates the anteriormost interstripe at the future site of the cephalic furrow formation. (e) Ventrolateral view of gastrulating embryo (stage 6). cf, cephalicburrow; vf, ventral furrow. Note absence of transcript within the cephalic and ventral furrows. (f) Onset of germband extension. pr, proctoderm. (g)Mid-germband extension. Arrows indicate segment polarity gene-like stripes. (h) Germband extended embryo (stage 9). Arrows indicate the position of 13segmental stripes of equal width along the dorsoventral axis. (i) Early stage 10 embryos. The lower embryo displays a ventral aspect. Note the ventralthinning of the stripes, and the concomitant lateral expansion to form circular patch domains. Arrow indicates a cluster of cells cleared of transcript. Nomorphological signs of invagination of the tracheal pits are visible in these embryos. (j) Stage 11 embryo with a full array of invaginated tracheal pits. es,esophagus; ol, site of future optic lobe invagination; tp1 and tp10, tracheal pits 1 and 10. (k) Higher magnification view of the embryo in 4J, showingdoughnut-shaped expression domains surrounding each tracheal pit. T2 and T3, thoracic segments 2 and 3; A1, abdominal segment 1. Note the absence oftranscript in the interior of the tracheal pit. (l) Germband retracted embryo, late stage 12. ps, posterior spiracle. Small arrows indicate a restricted patch oftranscript overlying trachea 1 and 10.

167A.C. Oates et al. / Mechanisms of Development 78 (1998) 165–169

168 A.C. Oates et al. / Mechanisms of Development 78 (1998) 165–169

Gary Hime, Ashley Bruce, Marie Phillips and Tony Burgessfor discussions and constructive criticism on the manuscript.A.C.O. is the recipient of an Anti Cancer Council of Vic-toria Postgraduate Research Scholarship. J.L.B. is supportedby NIH/NIGMS Training Grant 5T32GM07198 for theMedical Scientist Training Program. D.V.I. was the holderof an Anti Cancer Council of Victoria Summer VacationScholarship. J.B.T. is supported by grants from the NIH.

References

Callahan, C.A., Muralidhar, M.G., Lundgren, S.E., Scully, A.L., Thomas,

J.B., 1995. Control of neuronal pathway selection by aDrosophilareceptor protein-tyrosine kinase family member. Nature 376, 171–174.

Callahan, C.A., Bonkovsky, J.L., Scully, A.L., Thomas, J.B., 1996.derailed is required for muscle attachment site selection inDrosophila. Development 122, 2761–2767.

Hanks, S.K., Quinn, A.M., 1991. Protein kinase catalytic domain sequencedatabase: identification of conserved features of primary structure andclassification of family members. Methods Enzymol. 200, 38–62.

Hovens, C.M., Stacker, S.A., Andres, A.C., Harpur, A.G., Ziemiecki, A.,Wilks, A.F., 1992. RYK, a receptor tyrosine kinase-related moleculewith unusual kinase domain motifs. Proc. Natl. Acad. Sci. USA 89,11818–11822.

Lehmann, R., Tautz, D., 1994. In situ hybridization to RNA. Methods CellBiol. 44, 575–597.

Phillips, A.M., Martin, J., Bedo, D.G., 1994. In situ hybridization to poly-tene chromosomes ofDrosophila melanogasterand other dipteranspecies. Methods Mol. Biol. 33, 193–209.

Rubin, G.M., Spradling, A.C., 1982. Genetic transformation ofDrosophilawith transposable element vectors. Science 218, 348–353.

Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY.

Simoneaux, D.K., Fletcher, F.A., Jurecic, R., Shilling, H.G., Van, N.T.,Patel, P., Belmont, J.W., 1995. The receptor tyrosine kinase-related gene(ryk) demonstrates lineage and stage-specific expression in hematopoie-tic cells. J. Immunol. 154, 1157–1165.

Stacker, S.A., Hovens, C.M., Vitali, A., Pritchard, M.A., Baker, E.,Sutherland, G.R., Wilks, A.F., 1993. Molecular cloning and chromoso-mal localisation of the human homologue of a receptor related to tyr-osine kinases (RYK). Oncogene 8, 1347–1356.

Table 1

Partial rescue ofdrl muscle defects by targeteddnt expression

Genotypea No. ofembryos

No. ofhemisegments

% ‘Bypass’b

drlR343 10 115 24± 2.8drlR343, ME4-drl 10 111 0drlR343, ME4-dnt1 13 143 10± 2.1*drlR343; ME4-dnt3 12 136 7± 1.6*drlR343, ME4-dnt1; ME4-dnt3 12 113 9± 2.3*

aAll genotypes were in aw1118 background. Hr. 13–15 embryos were dis-sected and stained with RITC-conjugated phalloidin (Callahan et al.,1996).bDefined as hemisegments/embryo± SEM. A hemisegment was classifiedas a ‘bypass’ if at least one muscle of the 21–23 group projected ventrallybeyond muscle 13, attaching at least two longitudinal muscle widths moreventrally than normal (Callahan et al., 1996).*Significantly less thandrlR343 (P , 0.001 by Student’st-test).

169A.C. Oates et al. / Mechanisms of Development 78 (1998) 165–169