DEVELOPMENTAL BIOLOGY 183, 183±194 (1997) ARTICLE NO. DB968501 View metadata,Cadherin-6 citation and similar Expressionpapers at core.ac.uk Transiently Delineates brought to you by CORE Speci®c Rhombomeres, Other Neural Tube provided by Elsevier - Publisher Connector Subdivisions, and Neural Crest Subpopulations in Mouse Embryos Takayoshi Inoue, Osamu Chisaka, Hiroaki Matsunami, and Masatoshi Takeichi Department of Biophysics, Faculty of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-01, Japan Mammalian cadherin-6 (K-cadherin, cad6) was originally identi®ed by means of the polymerase chain reaction, but its biological functions have not yet been determined. We analyzed the expression pattern of the mouse homologue of this cadherin during development and found that it was transiently expressed in restricted rhombomeres and in other subdivi- sions of the neural plate and tube. In the midbrain and anterior hindbrain of E8.0-8.5 embryos, cad6 was expressed only in neural crest-generating regions. In contrast, in the posterior hindbrain and contiguous spinal cord of these embryos, cad6 occurred throughout the neural plate, forming a sharp anterior limit at the future rhombomere 4 and 5 boundary. Subsequently, this neural plate expression became con®ned to rhombomere 6, although most of the neural crest-generating areas remained positive throughout the body. Neural crest cells expressing cad6 migrated out of the neural tube, and subsequently accumulated mainly along peripheral nerves. We then studied the effect of Hoxa-1 mutation on the expression of cad6, as their expressions spatiotemporally overlapped with each other in the early posterior hindbrain. In E8.0-8.5 Hoxa-1 mutants, cad6 expression was suppressed in the region of rhombomeres 4 to 6, although that in the other regions was not essentially affected. At later stages, however, cad6-positive crest cells appeared and migrated out of rhombomeres 4 to 6, indicating that the suppression of cad6 expression was transient and restricted to early stages. Importantly, this effect of the Hoxa-1 mutation concurred with the timing of the expression of this gene. We also studied Hoxa-3 mutants, but found no effect of this mutation on the cad6 expression pattern. These ®ndings suggest that cad6 may contribute to the formation of the segmental structure of the early brain through its ability to confer speci®c adhesiveness on cells and that Hoxa-1 may be required for early cad6 expression in the posterior hindbrain. q 1997 Academic Press INTRODUCTION well as differentiated epithelial layers (Hirai et al., 1989; Hermiston et al., 1995), and that of N-cadherin impairs neu- Cadherins represent a superfamily of transmembrane or ral morphogenesis (Matsunaga et al., 1988; Barami et al., surface-anchored proteins characterized by the presence of 1994). With respect to homophilic interactions between the unique repeated motifs on their extracellular domain classic cadherins, like subtypes prefer to bind together (Ta- (Ranscht, 1994). Members of one subfamily, called classic keichi, 1995), although heterotypic interactions were ob- cadherins, have been identi®ed as homophilic cell±cell ad- served between certain cadherin subtypes (Murphy-Erdosh hesion molecules (Takeichi, 1988). They are subdivided into et al., 1995). As a result, cells expressing a particular cad- two groups, types I and II (Suzuki et al., 1991; Takeichi, herin selectively adhere to those expressing the same cad- 1995). The type-I group includes E-, N-, and R-cadherin, and herin. It was thus proposed that cadherins are essential not the type-II group includes cadherin-5 to -12. Each of these only for physical linking of cells but also for selective associ- molecules is expressed in a unique pattern in the body, ation of different cell types. and dysfunction of them causes defective morphogenesis of Two kinds of morphogenetic events, likely relevant to tissues, as demonstrated by a number of studies. For exam- the above functions of cadherins in selective cell associa- ple, blocking of E-cadherin disorganizes preimplantation tion, were recently reported. First, we found that two type- embryos (Larue et al., 1994; Riethmacher et al., 1995) as II cadherins, cadherin-6B and cadherin-7, were expressed 0012-1606/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved. 183 AID DB 8501 / 6x1b$$$$41 02-19-97 11:55:05 dbal 184 Inoue et al. during speci®c steps of neural crest development in the cause the cad6 expression pattern in the neural plate was chicken embryo (Nakagawa and Takeichi, 1995). The reminiscent of that of Hox genes, we tested the possibility chicken cadherin-6B (cad6B) is exclusively expressed in the that the former may be regulated by these genes and found future neural crest regions of the neural fold. When neural that in Hoxa-1 mutants, the cad6 expression in the posterior crest cells begin migration, they cease the expression of hindbrain was temporarily suppressed. cad6B and instead begin to express cadherin-7 (cad7). This cad7 expression occurs in a subpopulation-speci®c manner. For example, in the trunk, cad7 is expressed only in crest MATERIALS AND METHODS cells populating the dorsal and ventral roots and spinal nerves, whereas it is never intensely expressed in the cranial Animals and dorsal root ganglia or in the cranial mesenchyme, where ICR mice were mated, and embryos at various stages were col- neural crest cells are known to populate. Based on these lected. The precise staging of collected embryos was made ac- and other observations, we hypothesized that neural crest cording to Theiler (1972). For Hoxa-1 or Hoxa-3 mutants, mice cells migrate while maintaining their cadherin-mediated as- heterozygous for targeted disruptions of the Hoxa-1 or Hoxa-3 gene sociations and that cad7 serves for the sorting of particular (Chisaka et al., 1991, 1992) were intercrossed to produce embryos, subsets of neural crest cells from the other subpopulations. and their genotypes were determined by polymerase chain reaction As for the second morphogenetic event indicating cadherin- (PCR) methods as described previously (Carpenter et al., 1993; dependent selective cell association, R-cadherin was found Manley et al., 1995). The Splotch heterozygous mice were provided to be expressed in a restricted fashion in speci®c rhombo- by Dr. S. Aizawa (Kumamoto University), and mated to produce meres of the embryonic chicken or mouse brain (Ganzler embryos. Their genotypes were determined by their characteristic phenotype of the neural tube (Douglas et al., 1991). and Redies, 1995; Redies, 1995; Matsunami and Takeichi, 1995), some of which corresponded to certain ``neuro- meres.'' Cell aggregation assays conducted in vitro showed Cloning of Mouse cad6 cDNA that cells derived from R-cadherin-positive and -negative To obtain cadherin cDNAs, we carried out PCR using mixed regions segregated from each other in a cadherin-dependent primers which can amplify multiple cadherin subtypes (Suzuki et manner. These ®ndings led us to propose that R-cadherin al., 1991). As templates, cDNAs were prepared from mRNAs iso- confers speci®c aggregating ability on cells belonging to re- lated from newborn mouse brains. By this PCR, about 160-bp frag- stricted subdivisions of the fetal brain, so as to con®ne them ments were ampli®ed, and these fragments were subcloned into the to the subdivisions. We argued that this mechanism may pUC18 EcoRI site. To obtain full-length cDNAs, we constructed contribute to the formation or maintenance of the seg- random primed lgt10 cDNA libraries of postnatal brains derived mented structure of the fetal brain (Matsunami and Takei- from ICR mice by use of the cDNA Synthesis kit (Amersham). We chi, 1995). It was also reported that a novel cadherin, F- screened these libraries with the above PCR fragments radiolabeled. cadherin, is expressed along several boundary regions in the Many overlapping clones were obtained, and among them one open Xenopus embryonic brain (Espeseth et al., 1995). reading frame (ORF) was found. The above observations on the developing brains sug- gested that other cadherins may also be expressed in a seg- Probes for in Situ Hybridization ment-speci®c manner in the embryonic brain; this sugges- tion prompted us to search for such cadherins. It is known Sense or antisense cad6 RNA probes labeled with digoxigenin (Dig)-11-UTP (Boehringer-Mannheim) were prepared from that neuroepithelial cells do not cross the boundaries be- pBSII1.0B, which contains the 202±1229 BamHI fragment of cad6 tween rhombomeres subdividing the hindbrain (Fraser et cDNA. For the antisense probe, the EcoRI linearized vector was al., 1990), although a small percentage of them can (Birg- transcribed with T3 RNA polymerase (Gibco BRL); for the sense bauer and Fraser, 1994), and that odd and even rhombomeres probe, the XbaI linearized vector was transcribed with T7 RNA do not randomly intermix with each other (Clarke and polymerase (Gibco BRL). Hoxa-1 antisense RNA probes labeled Lumsden, 1993; Guthrie et al., 1993; Graham et al., 1993). with Dig-11-UTP were prepared from a newly constructed pBlue- These phenomena can be explained by assuming segment- script vector containing the 1233±1950 EcoRI±BamHI fragment of speci®c adhesiveness of the cells comprising the hindbrain Hoxa-1 cDNA (LaRosa and Gudas, 1988). For its antisense probe, (Lumsden, 1990), and cadherins could be involved in this the EcoRI-linearized vector was transcribed with T7 RNA polymer- hypothetical mechanism. In the present study, we analyzed ase. R-cadherin antisense RNA probes labeled with Dig-11-UTP and Krox-20 antisense RNA probes labeled with ¯uorescein (FITC)- the expression pattern of mouse cadherin-6 (cad6) during 12-UTP (Boehringer Mannheim) were prepared, as described pre- embryogenesis, which is the mouse homolog of the pre- viously (Matsunami and Takeichi, 1995; Carpenter et al., 1993). viously cloned rat K-cadherin (Xiang et al., 1994) or human All probes prepared were puri®ed by use of Quick Spin Columns cadherin-6 (Shimoyama et al., 1995).