Alvarez-Bolado, G.

Ashery-Padan, R.

Gessler, M.

Gruss, P.

Klamt, B.

English

MPG.PuRe

Gene expression patternFjx1, the murine homologue of the Drosophila four-jointed gene, codes fora putative secreted protein expressed in restricted domainsof the developing and adult brainRuth Ashery-Padana, Gonzalo Alvarez-Boladoa, Barbara Klamtb, Manfred Gesslerb, Peter Grussa,*aMax-Planck Institute of Biophysical Chemistry, Goettingen, D37077, GermanybBiocenter of the University of Wuerzburg, Lehrstuhl Physiol. Chemie I, Am Hubland, D-97074 Wuerzburg, GermanyReceived 27 August 1998; revised version received 28 October 1998; accepted 6 November 1998AbstractThe Drosophila gene four jointed (fj) codes for a secreted or cell surface protein important for growth and differentiation of legs andwings and for proper development of the eyes. Here we report the cloning of the mouse four-jointed gene (fjx1) and its pattern of expressionin the brain during embryogenesis and in the adult. In the neural plate, fjx1 is expressed in the presumptive forebrain and midbrain, and inrhombomere 4, however a small rostral/medial area of the forebrain primordium is devoid of expression. Expression of fjx1 in the neuraltube can be divided into three phases. (1) In the embryonic brain fjx1 is expressed in two patches of neuroepithelium: in the midbraintectum and the telencephalic vesicles. (2) In fetal and early postnatal brain fjx1 is expressed mainly by the primordia of layered tele-ncephalic structures: cortex (ventricular layer and cortical plate), olfactory bulb (subependymal layer and in the mitral cell layer). Inaddition expression is observed in the superior colliculus. (3) In the adult, fjx1 is expressed by neurons evenly distributed in thetelencephalon (isocortex, striatum, hippocampus, olfactory bulb, piriform cortex), in the Purkinje cell layer of the cerebellum, andnumerous medullary nuclei. In the embryo, strong expression can further be seen in the apical ectodermal ridge of fore- and hindlimbsand in the ectoderm of the branchial arches. Ó 1999 Elsevier Science Ireland Ltd. All rights reserved.Keywords: Apical ectodermal ridge; Cortex; four-jointed; Forebrain development; Olfactory bulb; Regionalization; Telencephalon1. IntroductionDrosophila four-jointed (fj) is expressed in the eye, leg,wing imaginal disks and in the optic lobe (Villano and Katz,1995; Brodsky and Steller, 1996). The null-mutation of theDrosophila fj gene causes reduced and altered growth of theleg and wing along the proximal distal axis (Villano andKatz, 1995; Brodsky and Steller, 1996). Mosaic analysisreveals that this protein works in a non-cell-autonomousmanner (Tokunaga and Gerhart, 1976). In accordance, invitro biochemical studies show that Fj is either a cell surfaceor a secreted protein (Villano and Katz, 1995). Fj has, there-fore, been suggested to be an intercellular signal involved inregulating proximo-distal growth of parts of the legs andwings and in the differentiation of the eyes (Brodsky andSteller, 1996).2. ResultsWe have identified a novel mouse gene with highsequence similarity to the Drosophila fj gene (Fig. 1A)and we have followed its expression pattern through devel-opment. Based on the sequence similarity and the resem-blance in expression pattern, we suggest that this cDNA isencoding the homologue of the Drosophila fj gene. Wetherefore, termed this new murine gene four-jointed x1(fjx1). Partial fjx1 cDNA clones were isolated from anembryonic day (E) 8.5 mouse embryo cDNA library usinga human genomic probe from chromosome 11p13 (Thate etal., 1995). Since the human gene does not contain an intronMechanisms of Development 80 (1999) 213–2170925-4773/99/$ - see front matter Ó 1999 Elsevier Science Ireland Ltd. All rights reserved.PII : S0925-4773(98)00218-4* Corresponding author. Tel.: +49-551-2011361; fax: +49-551-2011504;e-mail: pgruss@gwdg.de(M. Gessler et al., in preparation), complete mouse genomicclones were isolated in parallel and sequenced. The murinesequence has an open reading frame encoding a polypeptideof 437 amino acids. The calculated relative molecular massof the conceptual translation product is 48 912 Da, and itsisoelectric point would be 10.55. Comparison of the pre-dicted amino acids sequence with the currently availabledatabase entries revealed significant homology to the Dro-sophila Fj protein (50% similarity, 42% identity, see Fig. 1).In addition to sequence similarity, Fj and Fjx1 share struc-tural motives (Fig. 1A); a putative transmembrane domain,and a signal sequence cleavage site (von Heijne, 1986) arelocated in the N-terminal end of both proteins. In the C-terminal region of both proteins consensus sites for as-paragine linked glycosylation are present. In addition, aconsensus sequence for alpha amidation, a common mod-ification in proteolytically processed neuropeptides (Eipperet al., 1992), is predicted in Fj and Fjx1. Finally, the pre-dicted topology of both proteins implies that the C-terminalregion is processed in the endoplasmic reticulum andsecreted (Hartmann et al., 1989). This is in agreementwith in vitro experimental evidence showing that the Dro-sophila Fj is glycosylated and secreted (Villano and Katz,1995).In order to determine the expression pattern of fjx1,polyA RNA from embryos of E7–17 was examined byNorthern blot analysis (Fig. 1B). A single fjx1 transcriptof 2.8 kb was detected from E11 onward. A transcript ofthe same size was also detected in the adult brain (Fig. 1B).A weaker signal corresponding to 1.7 kb is detected at E7,but not during the later stages of development.2.1. Embryonic pattern of expressionIn the neural plate (E8.5; Fig. 1C,D) fjx is expressed in alarge rostral area which corresponds to the presumptiveforebrain and midbrain (Fig. 1C), although excluding a ros-tral/medial region (asterisk in Fig. 1D). In addition, fjx1 ismost probably expressed in rhombomere 4 (Fig. 1C; identi-fied following morphological criteria). At E9.5 (Fig. 1E,F),fjx1 expression is observed in the limb buds and in theectoderm of the first branchial arches (arrow head in Fig.1F). In the brain, expression is detected in two distinctregions, the dorsal mesencephalon (tectum) and prosence-phalon (presumptive isocortex). The expression in the tec-tum and the cortex persists at E10.5. (Fig. 1G,H) and E11.5(Fig. 1I,J). In the cortex, fjx1 is expressed specifically by arestricted dorsolateral patch of the neuroepithelium. Strongexpression is observed in the ectoderm of the branchial archand the oral ectoderm (Fig. 1K). In the limbs fjx1 isexpressed in the AER (Fig. 1I,J).2.2. Fetal and early postnatal pattern of expression (Fig.2A–H)During the histogenetic period of brain development fjx1is highly expressed by a number of layered structures, par-ticularly in the telencephalon (Fig. 2A–D), but also in thetectum/superior colliculus and the cerebellum (Fig. 2E–H).2.3. Adult pattern of expressionIn the adult (postnatal day 30), fjx1 is strongly expressedby numerous cells evenly distributed throughout the tele-ncephalon (Fig. 2I). Expression seems particularly intensein the piriform cortex, the hippocampus and the olfactorybulb. In the diencephalon, only the dorsal thalamusexpresses fjx1, although weakly. The Purkinje layer of thecerebellum, as well as numerous medullary nuclei alsoexpress our gene.3. MethodsAn E8.5 day mouse embryo cDNA library (a gift of B.Hogan) and a mouse genomic library (strain 129SVJ) werescreened with a 1.7 kb NotI–BamHI fragment from thehuman NE3 locus (D11S3892) using standard conditionsexcept for a reduced hybridization and washing temperatureof 55 ° C (Sambrook et al., 1989). The isolation of the humangenomic probe was described in Thate et al., 1995. Theregion homologous to fjx1 was sequenced and a 2088 bpFig. 1. (A) Comparison between the predicted amino acid sequences of the four jointed genes in mouse and Drosophila. The sequence of mouse Fjx1 (black)as compared with Drosophila Fj (green) reveals high sequence homology (50% similarity, 42% identity; comparison done by the Bestfit GCG program). Apredicted (Kyte and Doolittle, 1982) hydrophobic cluster is located between AA 7–27 in Fjx and between AA 77–103 in Fj (underlined). A potential signalsequence cleavage site (von Heijne, 1986) is predicted in both proteins (red). In both proteins putative glycosylation sites (blue) are present. Alpha amidationconsensus sequences (XGR/K,R/K) are predicted between residues 1–4 and 431–433 in Fjx1 and between residues 535 and 538 in Fj. (B) Expression of fjx1mRNA during embryogenesis and in the adult brain. PolyA RNA from embryos of E7,11,15,17 (left) and 20 mg of total RNA from adult brain (right) wereexamined by Northern blot analysis using a 1 kb probe encompassing most of the transcribed region. The size of the marker and the labeled bands is indicatedin kb. (C–K) Pattern of expression of fjx1 during early development shown by whole mount in situ hybridization. (C) Dorsal and (D) head-on views of labeledneural plates (E8.5). The asterisk in (D) shows the non-labeled medial/rostral area in the presumptive forebrain. The dotted line shows the profile of therostral end of the neural plate. (E) E9.5 embryo treated with antisense probe. (F) Sagittal section of E9.5 embryo, showing a detail of the labeledneuroepithelial regions. Arrowhead points at the epibranchial ectoderm. (G) Expression of fjx1 at E10.5. The thick line in the tectum shows the approximateplane of section of (H). (H) Transverse section of the embryo in (G) showing the labeled tectal neuroepithelium. (I) Expression of fjx1 at E11.5. (J) Sagittalsection through the embryo shown in (I). (K) Transverse section through the face of a littermate of the embryo shown in (I and J), demonstrating the labelingin the epibranchial and the oral ectoderm. Abbreviations: AER, apical ectodermal ridge; AQ, aqueduct; CTRL, control; CTX, cortex; FB, forebrain; MB,midbrain; pos, preotic sulcus; rh4, fourth rhombomere; TC, tectum.214 R. Ashery-Padan et al. / Mechanisms of Development 80 (1999) 213–217Fig. 1.215R. Ashery-Padan et al. / Mechanisms of Development 80 (1999) 213–217Fig. 2. Expression of fjx1 in fetal and adult brains demonstrated by in situ hybridization. (A,B) Sagittal sections through a mouse P0 brain (A, dark field; B,bright field) showing fjx1 expression in the cortex and olfactory bulb. (C,D) High-magnification photograph of the P0 cortex (C, dark field; D, bright field)showing fjx1 expression in the ventricular layer and in the cortical plate of the developing isocortex. (E,F) Transverse section (E, bright field; F, dark field)through the P0 brainstem showing expression in the superior colliculus and Purkinje cell layer of the cerebellum. (G,H) Sagittal section through the P0brainstem (G, bright field; H, dark field) showing expression in the ependymal layer of aqueduct and fourth ventricle, as well as in the inferior olive and thecerebellum. (I) Sagittal section through a P30 brain. Notice the strong expression in the telencephalon especially in the dentate gyrus, in layer 2 of thePiriform cortex and in the olfactory bulb. Fjx1 expression in main olfactory bulb is observed in the subependymal zone, the mitral cell layer and in theglomerular layer but not in the granular layer or the outer plexiform layer. Abbreviations: 1, isocortical layer 1; 4V, fourth ventricle; AQ, aqueduct; CA3,region 3 of Ammon’s horn (hippocampus); CTX, cortex; CBXp, Purkinje cell layer of cerebellar cortex; CP, caudate putamen; cpl, cortical plate; DG, dentategyrus (hippocampus); DOR, dorsal thalamus; GB, globus pallidus; HIP, hippocampus; IC, inferior colliculus; il, intermediate layer; IO, inferior olivarynucleus; gl, glomerular layer; gr, granular layer; mi, mitral cell layer; opl, outer plexiform layer; MOB, main olfactory bulb; MY, medulla (myelencephalon);PIR, Piriform cortex; SC, superior colliculus; SEZ, subependymal zone; sub, subventricular layer; ven, ventricular layer (neuroepithelium).216 R. Ashery-Padan et al. / Mechanisms of Development 80 (1999) 213–217segment spanning the entire coding region and the flankinguntranslated regions (EMBL accession number AJ009634).A 32P-labeled probe (SacII–PflMI, 1 kb fragment) washybridized to a commercially available mouse embryoNorthern blot membrane (Clontech 7763-1) and to a mem-brane on which 20 mg total RNA from adult brain has beenblotted. In situ hybridization experiments on whole-mountsor tissue sections were performed as previously described(Simmons et al., 1989; Nieto et al., 1996) using a 1.6 kbprobe (SacII–EcoRI fragment). Identification and namingof brain structures was done after Alvarez-Bolado andSwanson (1996).AcknowledgementsWe thank Tim Thomas for technical advice and DanielMaurus for technical assistance. R. Ashery-Padan was sup-ported by an EMBO postdoctoral fellowship. The DeutscheForschungsgemeinschaft (DFG Ge 539/6) and EC Biomedsupported the work of M. Gessler.ReferencesAlvarez-Bolado, G., Swanson, L.W., 1996. Developmental Brain Maps:Structure of the Embryonic Rat Brain, Elsevier, Amsterdam.Brodsky, M.H., Steller, H., 1996. Positional information along the dorsal–ventral axis of the Drosophila eye: graded expression of the four-jointedgene. Dev. Biol. 173, 428–446.Eipper, B.A., Stoffers, D.A., Mains, R.E., 1992. The biosynthesis of neu-ropeptides: peptide alpha-amidation. Annu. Rev. Neurosci. 15, 57–85.Hartmann, E., Rapoport, T.A., Lodish, H.F., 1989. Predicting the orienta-tion of eukaryotic membrane-spanning proteins. Proc. Natl. Acad. Sci.USA 86, 5786–5790.Kyte, J., Doolittle, R.F., 1982. A simple method for displaying the hydro-pathic character of a protein. J. Mol. Biol. 157, 105–132.Nieto, M.A., Patel, K., Wilkinson, D.G., 1996. In situ hybridization ana-lysis of chick embryos in whole mount and tissue sections. Methods CellBiol. 51, 219–235.Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: ALaboratory Manual. 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Fjx1, the murine homologue of the Drosophila four-jointed gene, codes for a putative secreted protein expressed in restricted domains of the developing and adult brain.

Fjx1, the murine homologue of the Drosophila four-jointed gene, codes for a putative secreted protein expressed in restricted domains of the developing and adult brain.

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