Expression study of cadherin7 and cadherin20 in the embryonic and adult rat central nervous system
© Takahashi and Osumi; licensee BioMed Central Ltd. 2008
Received: 27 February 2008
Accepted: 19 September 2008
Published: 19 September 2008
Vertebrate classic cadherins are divided into type I and type II subtypes, which are individually expressed in brain subdivisions (e.g., prosomeres, rhombomeres, and progenitor domains) and in specific neuronal circuits in region-specific manners. We reported previously the expression of cadherin19 (cad19) in Schwann cell precursors. Cad19 is a type II classic cadherin closely clustered on a chromosome with cad7 and cad20. The expression patterns of cad7 and cad20 have been reported previously in chick embryo but not in the developing and adult central nervous system of mammals. In this study, we identified rat cad7 and cad20 and analyzed their expression patterns in embryonic and adult rat brains.
Rat cad7 protein showed 92% similarity to chick cad7, while rat cad20 protein had 76% similarity to Xenopus F-cadherin. Rat cad7 mRNA was initially expressed in the anterior neural plate including presumptive forebrain and midbrain regions, and then accumulated in cells of the dorsal neural tube and in rhombomere boundary cells of the hindbrain. Expression of rat cad20 mRNA was specifically localized in the anterior neural region and rhombomere 2 in the early neural plate, and later in longitudinally defined ventral cells of the hindbrain. The expression boundaries of cad7 and cad20 corresponded to those of region-specific transcription factors such as Six3, Irx3 and Otx2 in the neural plate, and Dbx2 and Gsh1 in the hindbrain. At later stages, the expression of cad7 and cad20 disappeared from neuroepithelial cells in the hindbrain, and was almost restricted to postmitotic cells, e.g. somatic motor neurons and precerebellar neurons. These results emphasized the diversity of cad7 and cad20 expression patterns in different vertebrate species, i.e. birds and rodents.
Taken together, our findings suggest that the expression of cad7 and cad20 demarcates the compartments, boundaries, progenitor domains, specific nuclei and specific neural circuits during mammalian brain development.
In the early neural plate, the brain primodium is subdivided into several domains, i.e., neuromeres, to generate regional differences and units . The hindbrain primordium is morphologically divided into lineage-restricted functional metameric units called rhombomeres . Neuroepithelial cells are initially scattered in the hindbrain neuroepithelium, but are sorted gradually into each compartment . In the forebrain, that consists of the diencephalon and telencephalon, the primordium is longitudinally divided into prosomeres, which are defined by the expression border of various transcription factors such as homeodomain (HD) proteins [4, 5], and a lineage restriction of neuroepithelial cells between each subdivision has been reported in the caudal diencephalon of the chick embryo . In each region of the hindbrain and spinal cord, the neuroepithelium is also regionalized into basal and alar plates, which are separated at a groove called the sulcus limitans. Several molecular makers, e.g., HD transcription factors, subdivide the neuroepithelium into several discrete progenitor domains that give rise to different types of neurons along the dorsoventral (D-V) axis .
Previous studies demonstrated the expression of cadherin superfamily genes encoding cell adhesion molecules in the brain and the spinal cord, with distinct expression patterns that correspond with the subdivisions of the brain and the spinal cord [8–12]. These studies proposed that cadherin-mediated differential cell affinity establishes various compartments and regionalizes the neuroepithelium. Vertebrate cadherin superfamily genes are categorized into subfamilies, such as classic cadherins, protocadherins, and desmosomal cadherins . Classic cadherins have cadherin-repeats in an extracellular region called EC (extracellular cadherin) domain and associate with β-catenin and p120-catein in the cytoplasmic domains that connect to the actin cytoskeleton . The EC1 domain of classic cadherin shows adhesive properties that enhance the homophilic binding of cadherin. Classic cadherins are categorized into type I and type II groups with or without conserved amino acids, His-Ala-Val (HAV) within EC1 domain . The adhesive affinities of type I cadherins have been studied extensively. Cells expressing single type I cadherin, such as E-cadherin or N-cadherin, prefer to adhere to those expressing the same cadherin via homophilic interaction rather than heterophilic binding. For example, the neuroectoderm is segregated from the ectoderm by distinct affinity of N-cadherin and E-cadherin during the formation of the neural tube . In contrast, R-cadherin and N-cadherin, which are type I cadherins, interact in a heterophilic manner , and the adhesive interaction between individual subtypes of type II cadherin is not always homophilic in nature . Therefore, the complexity of homophilic and/or heterophilic interactions between each type II cadherin subtype may be involved in several developmental processes beyond tissue segregation during early embryogenesis.
F-cadherin (F-cad), a member of type II classic cadherins, is expressed in the area adjacent to the sulcus limitans, the boundary between the basal and alar plates, which restricts positioning of neuroepithelial cells in Xenopus embryos [18, 19]. In the chick neural tube, cadherin7 (cad7) is expressed not only in migratory neural crest cells but also in the domain ventral to the sulcus limitans of the neural tube [20–22]. R-cadherin (R-cad) and cadherin6 (cad6) are expressed in the primodia of the mouse cerebral cortex and striatum, respectively, and their differential cell affinities mediate segregation at the corticostriatal boundary . Other subtypes of classic cadherins also have multiple functions in the developing and adult brain, such as involvement in neuronal migration and connectivity of specific neuronal circuits, and synaptic formation in the brain. For example, cad6, cad8 and cad11 are observed in specific neural circuits [24, 25], and α N-catenin and N-cadherin, which are localized in the pre- and postsynaptic regions, modulate dendritic spine formation in an activity-dependent manner . So far, more than 20 members of type II classic cadherin have been identified. In Schwann cell precursors, we reported previously the expression of rat cad19 gene , which is a type II subtype clustered with cad7 and cad20 on chromosome 13. In mice, cad7 is expressed only in adult tissues , whereas cad20, a F-cad homologue, is expressed in early neural tissues [28, 29]. However, little is known about the expression patterns of cad7 and cad20 in the developing and adult rodent brains. In this study, we focused on cad7 and cad20 genes and analyzed their expression patterns in the developing and adult rat brains. The results showed that the expression borders of cad7 and cad20 corresponded with those of regional compartments and boundaries, which were marked with the expression of region-specific transcription factors. Cad7 and cad20 were also expressed in neurons of several nuclei that form the cerebellar/precerebellar circuitry in the late embryonic and adult hindbrain. The results suggest the contribution of cad7 and cad20 in the formation of compartment/boundary and specific neuronal circuitry in the rat hindbrain.
Isolation of rat cad7 and cad20
Characterization of cad20/cad19/cad7 gene cluster in different species.
Ensemble Gene model
In this study
In this study
 Takahashi and Osumi, 2005
 Moore et al., 2004
 Takahashi and Osumi, 2005
 Kools et al., 2000
 Kools et al., 2000
Human CDH7, CDH19 and CDH20 genes are clustered on chromosome18q22-p23 . Accordingly, we examined whether similar gene clusters are conserved in the rat, mouse and chick genomes. Cad7 and cad20 genes were closely localized in the same chromosome with cad19 (Fig. 1B). It is noteworthy that in the chick genome, the position of cad7, MN-cad and cad19  gene cluster on chromosome 2 is different from that of cad7 and cad20 on human, rat, and mouse genomes (Fig. 1C, Table 1).
Region-specific expression of cad7in the developing rat embryo
Expression borders of cad7 and cad20are adjacent to those of transcription factors in the early neural plate
Next, we compared the expression domains of cad7 and cad20 with those of transcription factors at E10.5 stage (10–12 somite stage). Otx2 and Gbx2 are HD transcription factors that establish the MHB with mutual repression . Remarkably, the posterior border of cad7 expression corresponded with the MHB (Fig. 3I, M). The second domain of cad20 was identified in the hindbrain in r2 region at areas positive for Gbx2 (Fig. 3K, L and 3N) whilst the expression of Otx2 was excluded (Fig. 3J, N). At E10.5, the posterior border of cad20 in the forebrain became sharper (Fig. 3O). These results indicate that the expression borders of cad7 and cad20 correspond to the prosomere and rhombomere boundaries in the early neural plate.
Cad7 and cad20-expressing domains correspond to progenitor domain boundary along the D-V axis
Even at E12.5, the expression domain of cad7 was clearly maintained in neuroepithelial cells in the dorsal hindbrain (Fig. 4E). The ventral border of cad7 coincided with the dorsal border of Dbx2, and the cad7 domain partially overlapped with the progenitor domains of the dorsal Lbx1-expressing interneurons  (Fig. 4F–H). Remarkably, a narrow longitudinal stripe of cad20 expression appeared in the ventral region of the hindbrain (Fig. 4I, J). This pattern was similar to the ventral expression of cad20 in the mouse embryo . To elucidate the relationship between the expression domain of cad20 and the neural progenitor domain, we compared the expression of cad20 with several progenitor domain markers. The results showed similar expression patterns for transcription factor Gata2 in cad20 domain and the V2 interneuron progenitor domain (Fig. 4K, L) . Cad20 expression was also detected in BrdU-incorporated S-phase cells (Fig. 4L). Taken together, these results suggest that cad7 and cad20 are expressed in different progenitor cells in the developing hindbrain.
Cad7expression delineates rhombomere boundaries
Cad7 and cad20expression in motor neurons and precerebellar neurons at later developmental stages
Cad7expression in the external germinal layer of the cerebellum
Next, we examined the expression of cad7 in the developing cerebellum. At E20.5, cad7 expression overlapped with that of Pax6 in the external germinal layer (EGL) containing progenitors of granule cells that later migrate inwards from EGL (Fig. 7A, B, E, F, M, N). On the other hand, cad7 expression was not detected in the upper rhombic lip (URL) (Fig. 7F), but was identified in the deep cerebellar nucleus and the epithelium of the anterior hindbrain (Fig. 7J and 7L), which differed from the expression of Pax6 (Fig. 7I and 7K). Previous studies in the chick cerebellum at later embryonic stages have shown that cad7 transcripts are restricted to the Purkinje cell layer and internal granular layer (IGL) and are not in the EGL , and that the immunoreactivity of cad7 protein is absent in the ventricular zone of the developing cerebellum . Considered together, the results suggest diversity of cad7 expression patterns in the chick and rat cerebellum.
It is has been reported that Pax6 regulates cell adhesion in the cerebral cortex [55, 56] and cerebellar granule cell precursors . R-cadherin is a downstream gene of Pax6 in the ventricular zone of the developing cerebral cortex . Cad7 expression was still detected in EGL cells of the Pax6 homozygous mutant rat (Fig. 7D and 7H). These results suggest that cad7 expression in EGL is regulated by a Pax6-independent pathway.
Cad7 and cad20expression in the adult brainstem and cerebellum
Expression of cad7 and cad20in early brain subdivision
Wizenmann and Lumsden analyzed rhombomere cells of the chick embryo by re-aggregation assay and were the first to report that segregation of rhombomere neuroepithelial cells between even and odd rhombomeres was probably mediated by calcium-dependent molecules such as cadherins . However, the candidate cadherin molecules expressed in specific rhombomeres or rhombomere boundary cells have not yet been identified in the chick embryo. On the other hand, other studies reported R-cad mRNA expression in the midbrain and odd number of rhombomeres, as well as cad6 expression in even number of rhombomeres in the mouse neural plate [36, 37]. Interestingly, in our analysis, we found specific expression of cad20 in the r2 of the rat embryo at E10.5 (Fig. 3) and that the posterior limit of cad7 expression was consistent with that of Otx2 at E10.5 (Fig. 3). Taking into consideration the transient expression of R-cad and cad6 at early stages and distinct cell adhesiveness of different cadherins, it is conceivable that classic cadherin subtypes including cad20 are involved in segregation of cells at the interface between rhombomeres in rodent embryos. Taken together, it is likely that distinct cadherin subtypes mediate compartmentalization, although further studies are needed to confirm this conclusion.
Lineage restriction of neuroepithelial cells in diencephalic subdivisions has been elucidated in the chick embryo [6, 59]. The chick diencephalon is firstly subdivided into three regions; the presumptive pretectum, dorsal thalamus, and ventral thalamus. Previous studies suggested that differential expression of types I and II cadherins demarcates diencephalic subdivisions in the chick and mouse embryos [11, 12, 34, 60]. In the present study, the expression of cad7 overlapped with that of cad20 in the rat neural plate in the forebrain region. Furthermore, a previous cell lineage analysis suggested that the midbrain/diencephalon boundary restricts cell mixing [61, 62], and that cad6 delineates the midbrain/diencephalon boundary in the mouse neural plate . In fact, the posterior border of cad7 became clearer at E11.5 compared with at E10.5 and the sharp expression boundary was maintained at E12.5 as in the case of Otx2 expression (Fig. 2E, I). Otx2 regulates the cell adhesion property of neuroepithelial cells in mice , and overexpression of Otx can induce cell aggregation in zebrafish embryos . ZLI is not a cell population derived from specialized cells at p2/3 boundary, but is itself a compartment that originates from the area that does not express Lunatic fringe (L-fng) . The p2/3 border is demarcated by the expression of Six3 and Irx3 in the early neural plate , where ZLI is established. However, in the early neural plate, whether the p2/3 boundary restricts cell mixing has not been elucidated in both avian and rodent embryos. Interestingly, we found that the posterior border of cad20 expression was consistent with the p2/3 border, which is mediated by mutual repression of Six3/Fez1/Fez like1 and Irx3/Irx1 (Fig. 3) [40, 65]. Our finding suggests the involvement of cad20 restricted expression in establishment of ZLI-signalling centre formed at the p2/3 boundary.
Expression of cad7 and cad20in the hindbrain progenitor domains and rhombomere boundary
Although the hindbrain and spinal cord are subdivided into the basal and alar plates at the sulcus limitans defined by a morphological groove in the vertebrates, a recent study using molecular markers has shown that the basal/alar boundary corresponds to the dorsal border of cad7 expression in the chick neural tube . Our study showed that longitudinal expression of cad20 resembles those of chick cad7 and Xenopus F-cad at the hindbrain level (Fig. 4) [18, 20] and that the sulcus limitans in the rat hindbrain is somewhat related to the expression of cad20. Remarkably, along the anterior-posterior (A-P) axis, cad20 mRNA expression was gradually downregulated in the basal plate from r7 to the spinal cord, and restricted in V2 interneuron progenitor cells (Fig. 5). Moreover, the cad7-expressing domain continued through the caudal hindbrain to the spinal cord (Figs. 2, 4 and 5), suggesting that the basal/alar boundary could be established or maintained by distinct cadherin subtypes in the rostral hindbrain and spinal cord. It was noteworthy that both cad7 and cad20 were expressed at r6 in E11.5 along the D-V axis (Fig. 4), and double staining for Dbx2/cad7 and Gsh1/cad20 showed partial overlap of cad7 and cad20 expression at r6 level in the E11.5 hindbrain (Fig. 4). Since heterophilic adhesion has been reported within distinct type II cadherins , and EC1, which is a critical sequence for calcium-dependent adhesion, is highly conserved between cad7 and cad20 proteins (Fig. 1), determination of the adhesion properties of cad7 and cad20 proteins should be the next research priority.
In the ventral spinal cord and hindbrain, transcription factors such as Pax, Nkx and Dbx families regulate the formation of progenitor domains by HD codes and specification of neuronal subtypes by mutual repression activities [7, 43]. In the chick spinal cord, ectopically expressed Pax7 can repress cad7 in the ventral region and Shh can induce cad7 in the dorsal region . Therefore, it is possible that rat cad7 and cad20 are regulated by Shh signalling pathway.
A previous study indicated that segregation of boundary cells is mediated by radical fringe-mediated activation of the Notch signalling pathway in the zebrafish hindbrain . In the mouse, although three fringe genes are not expressed at rhombomere boundaries , expression of Hes1, a target gene of Notch signalling, persists at high levels in boundary cells in the hindbrain . Rhombomere boundary cells exhibit a static feature contrary to rhombomere centre cells, i.e., a slow rate of proliferation and interkinetic nuclear migration, and their nuclei are located on the ventricular surface . Since cad7 expression in rhombomere boundaries actually starts after the formation of boundaries, such expression at rhombomere boundaries implicates differential cell adhesiveness between boundary and non-boundary cells in maintenance of boundary cells. Considering the expression of cad7 in ZLI, a boundary in the chick diencephalon , our findings could be interpreted to mean that cadherin-mediated cell-to-cell contact serves to restrict intermingling of boundary cells and compartment cells, thereby maintaining boundary regions.
Expression of cad7in precerebellar neurons and cerebellum during late embryonic and adult periods
Interestingly, the expression of cad7 and cad20 mRNAs in the neuroepithelium disappeared by E14.5, and their expression was detected in postmitotic neurons in the pons and medulla oblongata (Figs. 6 and 7). Previous studies reported that cadherins also contribute to cell migration in the brain . In the cerebellum, Purkinje cells are generated from epithelial progenitor cells and migrate to the deep layer . Experimentally, cad7 and cad6b-overexpressed progenitor cells preferentially migrated into Purkinje cell domain, which endogenously expresses these cadherins in the chick cerebellum . The lower RL cells generate four types of precerebellar neurons to form the pontine, reticulotegmental, lateral reticular and external cuneate nuclei [73, 74]. These precerebellar neurons project to the granule cells in the cerebellum . The dorsal cells of the caudal hindbrain generate the inferior olive neurons, and express cadherin subtypes, e.g., cad6, cad8 and cad11 [23, 24]. Interestingly, at later stages, we observed switching of cad7 expression from epithelial cells to migrating postmitotic neurons. Although the expression of various cadherin subtypes in precerebellar neurons had been reported previously , the expression of cad7 was more specifically detected than that of other cadherins in all precerebellar nuclei. Neurons expressing cad7 (X/XII cranial motor neurons, pontine and external cuneate neurons) aggregated at late embryonic stages (Figs. 6 and 7), suggesting the involvement of cad7 in cell sorting mechanisms, in agreement with the functions of type II classic cadherin subtypes in the chick spinal cord .
We found sparse cad7 expression in precerebellar neurons in the adult hindbrain (Fig. 8). The cadherin-catenin complex including N-cadherin/αN-catenin is involved in the formation of synaptic contact [26, 75]. Furthermore, recent studies have shown that type II subtypes, cad11 and cad13, also have specific roles in synaptic function including modulation of long-term potential and neurotransmission [76, 77], and that cad8 has an important role in transmission of sensory information from sensory neurons to the dorsal horn neurons in the spinal cord . Therefore, the expression pattern of cad7 in the hindbrain and cerebellum suggests that cad7 may physiologically modulate the cerebellar/precerebellar neural circuitry.
Genomic organization of cad20/cad19/cad7cluster and expression among different species
Cadherin family genes evolutionally duplicated  and formed as several clusters on chromosomes [80, 81]. In the human genome, CDH5 (VE-cad)/CDH1 (E-cad)/CDH3 (P-cad) and CDH8/CDH11/CDH13/CDH15 are located on chromosome 16q21/22 , and CDH20/CDH19/CDH7 are clustered on 18q22-23 . Comparison of the expression of such clustered cadherin genes among distinct species is important in order to identify common control element. However, our results showed that the distribution of rat cad7 and cad19 to that of cad20 was inconsistent with the localization of their homologues in the chick (Fig. 1, Table 1). Chick cad7 is also expressed in neural crest-derived cells as well as in the ventral neural tube [20, 21]. Our previous and present studies showed that cad19 but not cad7 was expressed in the neural crest cell lineage at the trunk level in the rat , suggesting that the distribution of cad7 and cad19 genes is associated with their expression patterns. However, the expression of chick cad19 was detected in part of neural crest-derived cells and neural tube but not in the dorsal domain and rhombomere boundary cells in the hindbrain (our unpublished observation). Taken together, our analysis suggests that the expression patterns of clustered cad20/cad19/cad7 genes are regulated by species-specific gene regulatory elements in avian and rodent embryos, which is in contrast to the highly conserved expression patterns of HD protein genes in the central nervous system.
The present study demonstrated that cell populations that express cad7 and cad20 are diversified in the rat and chick. The expression of these cadherin subtypes demarcates compartments, boundaries, progenitor domains, specific nuclei and circuits during mammalian hindbrain development.
Animal experiments were carried out in accordance with National Institute of Health guidelines for care and use of laboratory animals. The Committee for Animal Experiments of Tohoku University Graduate School of Medicine approved the experimental procedures described in this study. The midday of the vaginal plug was designated as embryonic day 0.5 (E0.5). Pregnant Sprague-Dawley (SD) rats were purchased from Charles River Japan. Pax6 homozygous mutant rat embryos were obtained by crossing of male and female Small eye rat heterozygotes (rSey2/+) , which were maintained at Tohoku University Graduate School of Medicine.
Identification of rat cad7 and cad20
Rat cad7 and cad20 genomic sequences were identified through BLAST genome search of NCBI and BLAT database at UCSC Genome Bioinformatics. cDNA fragments encoding ORF of rat cad7 and cad20 were amplified by RT-PCR using oligonucleotide primers designed based on the identified rat genomic sequences. Total RNA taken from the head including the hindbrain of E12.5 rat embryos was purified with RNeasy column (Qiagen, Hilden, Germany), and cDNA was synthesized using oligo dT primer and reverse transcriptase (Superscript II, Invitrogen, San Diego, CA). The primer sets were as follow: 5' fragment of cad7 (1–1467: 5'-ATGAAGCTGGGCAAAGTGGAG-3' and 5'-AGTGGTTTCATACTCCATGGC-3'), 3' fragment of cad7 (1447–2361: 5'-GCCATGGACTATGAAACCACT-3' and 5'-AGGCTATGAGTACAAACTCTC-3'), 5' fragment of cad20 (1–1071: 5'-ATGTGGACTACAGGTAGAATG-3' and 5'-ATTGGATCCTTCCACCTTCAG-3'), and 3' fragment of cad20 (1051–2450: 5'-CTGAAGGTGGAAGGATCCAAT-3' and 5'-TGAGAACGTCTGGATTTGGGT-3'). Amplification was performed using a thermal cycler (Mastercycler gradient; Eppendorf, Hamburg, Germany) using Taq DNA polymerase (Promega, Madison, WI) under the following conditions: denaturation for 5 min at 96°C, annealing for 1 min at 63.5°C (cad7), 60.8°C (cad20), extension for 1 min at 72°C, 35 cycles. The amplified products were blunted using T4 DNA polymerase (Invitrogen) and inserted into EcoR V site of pBluescript SKII (-). DDBJ accession numbers are AB121031 (rat cad7) and AB121033 (rat cad20).
Alignments and phylogenetic analysis of cadherin family
Multiple alignment of amino acid sequences of Cad7 and Cad20 and phylogenetic analysis for full length of amino acid sequences of type II classic cadherins were performed by Clustal W program and gene database. The gene accession numbers of cadherins used to characterize protein sequences are AB121032 (rat cad19), AJ007607 (human cad19), X95600 (mouse cad8), D21253 (mouse cad11), D252990 (rat K-cad/cad6), D82029 (mouse cad6), D42149 (chick cad6b), AB035301 (human cad7), AK034096 (mouse cad7), D42150 (chick cad7), AF217289 (human cad20), AF007116 (mouse cad20), AF459439 (chick MN-cad, the same sequence is identified as the chick F-cadherin homolog, AF465257), X85330 (Xenopus F-cad), L33477 (human cad12/Br-cad), XP_226899 (rat cad18), XP_001054792 (rat cad24/EY-cad), AY260900 (human cad24) and NM_019161 (rat cad22). Phylogenetic tree was drawn by Tree View software (Taxonomy and Systematics at Glasgow). The scale bar was set to represent 10% differences.
Cloning of rat cDNAs encoding transcription factors
To obtain rat Gsh1, Krox20 and Gbx2 cDNAs, the corresponding genomic sequence was amplified by genomic PCR using oligonucleotide primers. Rat Dbx2 and Lbx1 cDNAs were amplified by RT-PCR using mRNA prepared from E12.5 SD rat embryos. Primer sets were designed based on the sequences, which are homologous to mouse Gsh1, rat Krox20, rat Gbx2, mouse Dbx2 and mouse Lbx1 mRNAs. Used oligonucleotide primers were as follow, Gsh1 (5'-CAGCAGCAGCCAAGGTGATT-3' and 5'-CCACGGAGATGCAGTGAAAC-3'), Krox20 (5'-TCAACATTGACATGACCGGAG-3' and 5'-GAATGAGACCTGGGTCCATAG-3'), Gbx2 (5'-ACGAGTCAAAGGTGGAAGATG-3' and 5'-TGACTTCGAATAGCGAACCTG-3'), Dbx2 (5'-TGCTGACCCAGGACTCAAATT-3' and 5'-GGATACCAAAGAAGCCAGAAG-3') and Lbx1 (5'-GAGATGACTTCCAAGGAGGAC-3' and 5'-ATCAGGCTGTAGTGGAAGGAA-3'). Amplification was performed under following conditions: denaturation for 5 minutes at 96°C, annealing for 1 min at 68.1°C (Gsh1), 55.5°C (Krox20), 60.8°C (Gbx2), 60.8°C (Dbx2) and 60.8°C (Lbx1), extension for 1 min at 72°C, 35 cycles. To clone the amplified products, these fragments were blunted using T4 DNA polymerase (Invitrogen) and inserted into EcoR V site of pBluescript SKII (-) (Stratagene, La Jolla, CA). Cloned cDNAs were confirmed by sequencing. DDBJ accession numbers are AB197922 (rat Gsh1), AB264614 (rat Krox20), AB266843 (rat Gbx2), AB121147 (rat Dbx2) and AB197923 (rat Lbx1).
Whole-mount in situhybridization
Whole-mount in situ hybridization was performed as described previously [27, 52]. Dissected embryos were fixed in 4% paraformaldehyde (PFA)/phosphate buffered saline (PBS) overnight at 4°C. Digoxigenin (DIG)-labelled riboprobes were synthesized by in vitro transcription with DIG RNA labelling mix (Roche, Mannheim, Germany) and T3 or T7 RNA polymerase (Promega). All synthesized probes were purified with Quick Spin Columns G-25 (RNA) (Roche) to remove unincorporated nucleotides. For detection of cad7 mRNA, three kinds of probes transcribed from different cad7 cDNA fragments (1–460, 567–1460 and 1992–2361) were used in hybridization. To detect cad20 mRNA, four kinds of probes transcribed from different cad20 cDNA fragments (1–546, 1051–1497, 1639–1956 and 2014–2403) were mixed in hybridization. Two-colour whole-mount in situ hybridization was performed using the protocol described on the internet http://www.anat.ucl.ac.uk/research/sternlab/INSITU.htm. For double colour detection, fluorescein-labelled riboprobes for Dbx2, Gsh1, Otx2 and Gbx2 were generated with fluorescein RNA labelling mix (Roche), and simultaneously hybridized with DIG-labelled cad7 or cad20 riboprobes, respectively. After the first colour reaction with alkaline phosphatase (AP)-conjugated anti-DIG antibody (1:5000, Roche) and NBT/BCIP (Wako Pure Chemical Industries, Osaka, Japan), embryos were fixed in 4% PFA/PBS at room temperature overnight and incubated in TBST containing 0.1% Tween20 at 65°C for 1 hour to inactivate AP completely. The second colour signal was detected with AP-conjugated anti-fluorescein antibody (1:5000, Roche) and INT/BCIP (Roche). Drs. I. Matsuo, A. Mansouri and P. Gruss kindly provided mouse Otx2, Six3 and Irx3 cDNAs for synthesis of riboprobes, respectively. Images were recorded by cooled colour CCD camera (Penguin 600CL, Pixera, San Jose, CA).
In situ hybridization using frozen sections of embryonic tissues was performed as described previously . E12.5 whole rat embryos and heads of E14.5 and E18.5 embryos were fixed in 4% PFA/PBS overnight at °C. For fixation of E20.5 rat foetuses, the brains were dissected and fixed without the pia mater in 4% PFA/PBS overnight. Embryos and foetuses were embedded with optimal cutting temperature (OCT) compound (Sakura, Tokyo) and cut into 12 μm sections with a cryostat (Leica, Nussloch, Germany). DIG-labelled riboprobes were synthesized with T3 or T7 (Promega) or SP6 (Takara Shuzo, Ohtsu, Japan) RNA polymerase and hybridized to sections. To detect the expression of cad7 mRNA, two kinds of riboprobes of cad7 were generated from different cDNA fragments (1–1467 and 1447–2361), and mixed in hybridization. Riboprobes of cad20 were synthesized by from two cDNA fragments (1–1071 and 1051–2450), and used simultaneously in hybridization. Mouse Gata2, rat cad6/K-cad  and rat Sox10 cDNAs were kindly provided by Drs. M. Yamamoto, M. Tanaka and M. Wegner, respectively. For in situ hybridization of the adult brain, 8-week-old male adult rats (postnatal day 60) were deeply anesthetized and decapitated. Brains were immediately dissected out and frozen on powder dry ice. The brains were cut into 14 μm sections, and were post-fixed in 4% PFA/PBS for 15 minutes at room temperature. Sections were acetylated with tri-ethanoamine, HCl and acetic acid for 10 min. The following strategies of hybridization and staining were performed according to the protocol described for embryonic tissues except for changing the concentration of AP-conjugated anti-DIG antibody (1:2000). Images were recorded with colour CCD camera (HC-25000 3CCD, Fuji, Tokyo).
Immunohistochemistry using frozen sections was performed as described previously . Embryos and foetuses were fixed as described above for in situ hybridization. Antigen-enhanced sections, which were boiled in 0.1 M citric acid solution in a microwave, were incubated with anti-Pax6 rabbit polyclonal antibody  (1:500) and anti-Islet1/2 mouse monoclonal antibody (D.S.H.B, 40.2D6, 1:100), which were diluted with 2% goat serum/TBST overnight at 4°C. As secondary antibodies, biotin-conjugated affinity purified anti-rabbit IgG (dilution, 1:200, Jackson Immunoresearch Laboratories, West Grove, PA) and anti-mouse IgG donkey antibodies (1:200, Chemicon International, Inc., Temecula, CA) were used. Signal was enhanced by combination of ABC kit (Vector Laboratories, Burlingame, CA) and enhanced DAB kit (Pierce, Rockford, IL). After in situ hybridization for Sox10 and cad7, sections were incubated with anti-neuron-specific class III β-tubulin antibody (1:2000, Tuj1, MMS-435P, Covance, Madison, WI), and the signal was detected by using Cy3-conjugated affinity purified anti-mouse IgG donkey antibody (1:400, Jackson Immunoresearch Laboratories). Images were recorded with AxioPlanII and AxioCamMRm (Carl Zeiss, Jena, Germany).
BrdU labelling using whole embryo cultures
Short pulse labelling of bromodeoxyuridine (BrdU, Sigma Chemical Co., St. Louis, MO) for cultured rat embryos was performed as described previously . E12.5 rat embryos were precultured for 1 hour and BrdU solution was directly added to the culture medium. Embryos were exposed to BrdU for 20 min and fixed in 4% PFA/PBS. After detection of Gata2 mRNA by in situ hybridization, sections were treated with 2N HCl solution for 15 min at 37°C and neutralized in TBST. Sections were incubated with anti-BrdU mouse monoclonal antibody (Becton-Dickinson, Mountain View, CA, 1:50), and the signal was detected with ABC kit and enhanced DAB kit.
We thank Ms. Sayaka Makino, Ms. Yumi Watanabe and Dr. Yoko Arai for technical support, and Dr. Takayoshi Inoue for critical reading and valuable comments. We also thank Drs. Isao Matsuo, Ahmed Mansouri, Peter Gruss, Masamitsu Tanaka and Masayuki Yamamoto for providing reagents used in this study. Islet1/2 antibody was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. We also thank all other members of Prof. Osumi laboratory for valuable comments and discussion. This work was supported by KAKENHI on Priority Areas-A nuclear system to DECODE (#17054003 to M.T), Molecular Brain Science (#17024001 to N.O.) and on Young Scientist Research B (#17700300 and #20700281 to M.T.) from MEXT of Japan, The Core Research for Evolutional Science and Technology from Japan Science and Technology Corporation (JST) (to N.O), Global COE Program "Basic and Translational Research Center for Global Brain Science" of MEXT and GONRYO Foundation for the promotion of medical Science (to. M.T.).
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