Developmental Expression Profiles of Celsr (Flamingo) Genes in the Mouse

Mechanisms of Development 112 (2002) 157–160 www.elsevier.com/locate/modo Gene expression pattern Developmental expression proﬁles of Celsr (Flamingo) genes in the mouse

F. Tissir1, O. De-Backer, A.M. Gofﬁnet1, C. Lambert de Rouvroit*

Neurobiology Unit 2853, FUNDP School of Medicine, B5000 Namur, Belgium Received 10 October 2001; received in revised form 8 November 2001; accepted 8 November 2001

Abstract Celsr, also called Flamingo (Fmi) genes encode proteins of the cadherin superfamily. Celsr cadherins are seven-pass transmembrane proteins with nine cadherin repeats in the extracellular domain, and an anonymous intracellular C-terminus. The Drosophila Fmi gene regulates epithelial planar cell polarity and dendritic ﬁeld deployment. The three Flamingo gene orthologs in man and rodents are named, respectively, CELSR1–3 and Celsr1–3. Celsr1 and 2 are expressed during early development, in the brain and epithelia. In this report, we characterized further Celsr genes in the mouse, and examined their developmental pattern of expression. Each Celsr is expressed prominently in the developing brain following a speciﬁc pattern, suggesting that they serve distinct functions. q 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Brain development; Cerebral cortex; Cadherin; In situ hybridization; Genomic structure; Frizzled; Flamingo; G-protein coupled receptor

1. Results and discussion developing brain following a specific pattern, suggesting that they serve distinct functions. Celsr, also called Flamingo (Fmi) genes encode proteins The Celsr3 cDNA was reconstituted by alignment of the of the cadherin superfamily (Nollet et al., 2000; Redies, human CELSR3 cDNA with mouse genomic and expressed 2000; Takeichi et al., 2000; Tepass et al., 2000). Celsr sequence tag (EST) sequences, and verified by reverse tran- proteins have nine N-terminal cadherin repeats, followed scriptase polymerase chain reaction (RT-PCR) and sequen- by six EGF-like domains, seven putative transmembrane cing. Like its two homologs Celsr1 and 2 (Wu and Maniatis, segments and an intracellular C-terminus. The Drosophila 2000), it is encoded by 35 exons (Fig. 1). The first exon is Fmi gene regulates planar cell polarity and dendritic field longer than 4000 nucleotides (nt) and encodes the nine deployment (Chae et al., 1999; Usui et al., 1999; Gao et al., cadherin repeats. Other exons range in size from 79 to 2000). The three Flamingo gene orthologs in man and 1660 nt and are separated by small introns (range: 94– rodents are named, respectively, CELSR1–3 and Celsr1–3 1831). The three Celsr protein sequences are more than (Table 1; Hadjantonakis et al., 1997; Nakayama et al., 1998; 50% identical in their extracellular and transmembrane Usui et al., 1999; Vincent et al., 2000; Wu and Maniatis, segments but differ in their cytoplasmic portions. 2000). Celsr1 and 2 are expressed during early develop- At embryonic day 10 (E10, Fig. 2A–C), Celsr genes were ment, in the brain and epithelia (Hadjantonakis et al., expressed in the embryonic central nervous system (CNS), 1997; Formstone and Little, 2001). However, brain expres- in the emerging dorsal root ganglia (DRG) and cranial gang- sion patterns have not been studied in detail (Shima et al., lia, and to a lower level in epithelia. In the CNS, expression 2001). In this report, we characterized further Celsr genes in of the three genes was uniform along the rostrocaudal axis, the mouse, and examined their developmental pattern of but different in the radial dimension. Celsr1 mRNA was expression. Each Celsr is expressed prominently in the detected in ventricular zones (VZ) but not in marginal zones (MZ), and weakly in other structures. Celsr2 expression was strong in the VZ in all sectors of the brain, and lower in the MZ. As also shown by Formstone and Little * Corresponding author. Tel.: 132-81-72-42-78; fax: 132-81-72-42-80. E-mail address: [email protected] (C. Lambert de Rouv- (2001), Celsr3 expression was almost complementary to roit). that of Celsr1, low in VZ but strong in MZ. At E12 (Fig. 1 Present address: Developmental Genetics Unit, UCL Medical School, 2D–F), Celsr1 was detected at high levels in the VZ in all B1200 Brussels, Belgium.

Table 1 Nomenclature of Celsr genes

Name Others, man Human map Mouse Mouse map Rat

CELSR1 FMI2 Chr 22 Celsr1, Fmi2 Chr 15 CELSR2 EGFL2 Chr 1 Celsr2, Fmi1 Chr 3 MEGF3 CELSR3 MEGF2, EGFL1, FMI1 Chr 3 Celsr3 Chr 9 MEGF2 brain areas, and to some extent in epithelia. The signal was and incipient dentate gyrus, cerebellar cortex and pontine negligible in other organs. Celsr2 expression was strong in nuclei. Celsr3 was expressed at highest levels in differen- VZ and lower in MZ, except in the telencephalic MZ where tiating neuronal fields, particularly in the olfactory bulb, the it was prominent. The DRG and cranial ganglia expressed cerebral and hippocampal cortical plate, the external layer low amounts of Celsr2 mRNA, and there was also some of the tectum, pontine nuclei (not shown) and the external weak signal in epithelia, particularly in nasal cavities. granular layer of the cerebellum. At postnatal day 10 (P10, Celsr3 mRNA was expressed in a pattern almost comple- Fig. 2M–O), Celsr1 mRNA expression was weak and mentary to that of Celsr1, with highest levels in MZ, such as confined to germinative zones, namely the residual VZ in the cortical primordium (mainly the preplate) and weaker telencephalon and midbrain, and the external tiers of the signal in the VZ. Celsr3 was expressed in DRG and cranial cerebellar external granule cell layer. Celsr2 mRNA was ganglia. At E15 (Fig. 2G–I), Celsr1 was highly expressed in no longer detected in the VZ, but was found in high concen- the VZ in all brain areas, particularly in the telencephalon. tration in postmigratory fields. In the olfactory bulb, heavy No Celsr1 mRNA was detected in differentiating neuronal expression was associated with the granule cell layer. In the fields. Celsr1 signal was also present in epithelia, including cerebral cortex, high expression at the external and inter- the olfactory epithelium, in the developing lungs and kidney mediate levels was superposed to a diffuse moderate signal. cortex. Celsr2 expression remained prominent in the brain. A high signal was detected in the pyramidal cell layer of the The intensity was highest in all VZ. The signal was less hippocampus and in granule cells of the dentate gyrus. Inner intense in differentiating fields than in VZ, except in the and outer cerebellar granule cells were strongly positive. cerebral hemispheres, and to a lesser extent in the tectum Celsr3 mRNA was expressed in differentiating neuronal and cerebellum. In addition, there was strong Celsr2 expres- fields in all brain areas. There was strong signal in the sion in the kidney cortex, moderate signal in the skin, nasal olfactory bulb, in the cortical plate – with highest intensity pits and developing lungs. The Celsr3-related signal was in its superficial tiers –, in hippocampal pyramidal cells and restricted to the brain and to the olfactory epithelium. In in the dentate gyrus. Expression was high in the external and the brain, it was low in VZ but strong in external fields, inner granule cell layers of the cerebellum; in the external particularly those with ongoing migration, such as the tele- layer, it was associated with postmitotic cells of the inner ncephalic cortical plate, the olfactory bulb, the cerebellum tiers. At P21 (Fig. 2P–R), Celsr1 expression was detected in and the tectum. the remnants of the telencephalic VZ. Celsr2 expression In the newborn and postnatal stages, Celsr-related signals remained strong in pyramidal hippocampal neurons, granule were studied on brain sections. In the newborn (Fig. 2J–L), cells of the dentate gyrus, in the cerebral cortex, olfactory the expression of Celsr1 remained restricted to the VZ. bulb and basal forebrain. Celsr3 mRNA levels were much Celsr2 remained expressed in the VZ as well as in migrating lower than at previous stages, except in cerebellar granule and postmigratory cells throughout the brain. Expression cells and, to a lesser extent, in the dentate gyrus and olfac- was particularly high in the cortical plate, hippocampus tory bulb.

Fig. 1. Genomic organization of the mouse Celsr3 gene. Exons are indicated by solid rectangles with the encoded protein shown below; the transcription start site was not mapped. The protein domains were deﬁned used the Pfam database (Bateman et al., 2000). S, signal sequence; E, EGF-like motif; Lam, laminin G domain; LE, laminin-type EGF motif; H, domain present in hormone receptors; GPS, latrophilin-CL1-like G protein coupled receptor proteolytic site; 7TM, seven-pass transmembrane domain (secretin family); P, proline-rich region. Shaded rectangles represent protein domains displaying more than 50% identity with Celsr1 and Celsr2. The Celsr3 sequence has been submitted to Genbank (AF427498). F. Tissir et al. / Mechanisms of Development 112 (2002) 157–160 159

Fig. 2. In situ hybridization studies of Celsr 1–3 mRNA expression during mouse development. [P-33] labeled riboprobes were hybridized to cryostat sections, and the signal was revealed using X-ray films as described (Simmons et al., 1989; Bernier et al., 1999). Primers for probe preparations with RT-PCR were: (i) Celsr1:50-CCACCCCTGAAATCTGACC-30 and 50-CACTCTGTATCCTTGCCATCC-30 (amplifying nucleotides 10162–10608 of sequence GI6753407); (ii) Celsr2:50-TCATCTCTGTAGTGCGCCTG-30 and 50-ATCTCACCGTTCTCTCGTCG-30 (amplifying nucleotides 6506–7121 of sequence GI11995465) ; (iii) Celsr3:50-GCTTGGACACCAGCCTGTCTA-30 and 50 TTAACCCCCACCTGCATAGATGCGA-30 (amplifying nucleotides 8314–9138 of sequence AF427498). Figures are micrographs of autoradiography films. The signals for Celsr1, 2 and 3 are shown, respectively in the left, middle and right series of figures. The following stages are illustrated: E10 (A–C), E12 (D–F), E15 (G–I), P0 (J–L), P10 (M–O) and P21 (P–R). Two examples of the complementarity between Celsr1 and Celsr3 are illustrated at E10 and E15, in which arrowheads point to VZ and arrows show postmigratory fields, in the spinal cord at E10 (see also Formstone and Little, 2001) and in the hemispheres at E15. Bar ¼ 1 mm.

Together with other work in mammals (Hadjantonakis et they play important roles during brain development as they al., 1997, 1998; Nakayama et al., 1998; Wu and Maniatis, do in flies. The three Celsr genes have similar genomic 2000; Shima et al., 2001; Formstone and Little, 2001), our organizations (Fig. 1). The proteins are closely related in analysis of the genomic organization and developmental their extracellular and transmembrane regions, but differ in expression profiles of the mouse Celsr genes suggests that their cytoplasmic domains. Celsr1 and 2 have a cytoplasmic 160 F. Tissir et al. / Mechanisms of Development 112 (2002) 157–160 tail of about 300 amino acids, whereas that of Celsr3 is 590 Formstone, C.J., Little, P.F., 2001. The flamingo-related mouse Celsr residues long. Apart from a proline-rich stretch in Celsr3, family (Celsr1–3) genes exhibit distinct patterns of expression during the cytoplasmic sequences do not contain any conserved embryonic development. Mech. Dev. 109, 91–94. Gao, F.-B., Kohwi, M., Brenman, J.E., Jan, L.Y., Jan, Y.N., 2000. Control motif. All Celsr genes are predominantly expressed in the of dendritic field formation in Drosophila: the roles of Flamingo and developing brain. Whereas Celsr3 expression is strictly competition between homologous neurons. Neuron 28, 91–101. brain specific, Celsr1 and 2 are expressed at low levels in Hadjantonakis, A.-K., Sheward, W.J., Harmar, A.J., de Galan, L., Hoovers, the fetal lungs, kidney and epithelia. Interestingly, the brain J.M.N., Little, P.F.R., 1997. Celsr1, a neural-specific gene encoding expression patterns are different. Celsr1 is mostly confined an unusual seven-pass transmembrane receptor, maps to mouse chromosome 15 and human chromosome 22qter. Genomics 45, 97– to areas of neural cell precursor proliferation, namely VZ 104. and the outer tiers of the external granular layer in the cere- Hadjantonakis, A.-K., Formstone, C.J., Little, P.F.R., 1998. Mcelsr1 is an bellum. 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