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Slitrk6 expression profile in the mouse embryo and its relationship to that of Nlrr3
Gene Expression Patterns 3 (2003) 727–733 www.elsevier.com/locate/modgep
Slitrk6 expression profile in the mouse embryo and its relationship to that of Nlrr3 Jun Aruga* Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan Received 26 May 2003; received in revised form 30 June 2003; accepted 30 June 2003
Abstract Slitrk6 is a member of the Slitrk family of proteins, which are integral membrane proteins possessing two leucine-rich repeat (LRR) domains and a carboxy-terminal domain partially similar to that in the trk neurotrophin receptor proteins. Here, I show that Slitrk6 is uniquely expressed in various organs, different from other Slitrk genes which are predominantly expressed in neural tissues. In the developing mouse embryo, Slitrk6 expression was detected in the otic cyst, lateral trunk epidermis and its underlying mesenchymal tissue, limb bud, maxillary process, pharyngeal arches, cochlea, retina, tongue, tooth primordium, central nervous system (CNS), and the visceral organ primordia including of the lung, gastrointestinal tract (particularly in the enteric neurons) and pancreas. The expression in these organs occurred in a spatially restricted manner. In the CNS, the expression was highly compartmentalized in the dorsal thalamus, cerebellum and medulla. The expression compartment in the thalamus in which Slitrk6 was expressed was closely related to the Gbx2-expressing prosomere 2. Interestingly, the Slitrk6 expression in the CNS, cochlea, tongue, tooth primordial, and other organs was partially complementary to the expression of Nlrr3, which belongs to another family of neuronal LRR-containing transmembrane proteins. The complementary expression of the two proteins in the dorsal thalamus persisted from E13.5 to the adult stage. q 2003 Elsevier B.V. All rights reserved. Keywords: Neural fold; Inner ear; Limb bud; Neural crest; External sensory organ; Sensory neuroepithelium; Skin; Vibrissae; Hair root; Transmembrane protein; Neural development; Cerebellar development; Esophagus; Stomach; Intestine; Inner muscular layer; Neural patterning; Cerebellar Compartmentalization; Gene expression; Slit; PGP9.5; Brain tumor; Medulloblastoma
1. Results and discussion The Slitrk family was identified as a family of neuronal transmembrane proteins that control neurite outgrowth (Aruga and Mikoshiba, 2003; Aruga et al., 2003). When overexpressed in neurons, either induction of a single neurite or inhibition of neurite outgrowth is observed, depending on the Slitrk subtype. Structurally, these proteins are characterized by two leucine-rich repeat (LRR) domains located amino-terminally to the transmembrane domain. LRR domains are known to be present in many proteins, and mediate protein – protein interactions (Kobe and Deisenhofer, 1995). The LRR domains in the Slitrk family proteins are highly similar to those in the entire Slit family of proteins, which are known to control axon guidance and Abbreviations: LRR, leucine-rich repeat. * Tel.: þ81-48-467-9745; fax: þ 81-48-467-9744. E-mail address: [email protected] (J. Aruga). 1567-133X/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1567-133X(03)00141-8
branching (Brose and Tessier-Lavigne, 2000). Another characteristic structural feature of the Slitrk proteins is the carboxy-terminally located tyrosine residues flanked by amino acid sequences similar to those in the carboxyterminal domain of trk neurotrophin receptor (Patapoutian and Reichardt, 2001). The carboxy-terminal domain is conserved in all the Slitrk proteins, except Slitrk1. The expression profiles of the mouse Slitrk genes are unique for each gene of the family. In particular, highly compartmentalized expression of Slitrk6 expression is observed in the thalamic nuclei, different from the expression pattern of other Slitrk genes, which are broadly expressed in neural tissues (Aruga and Mikoshiba, 2003). The unique expression pattern of Slitrk6 in the thalamus led me to investigate the comprehensive expression profile of this gene in developing mouse embryos. I therefore carried out a series of in situ hybridization experiments using a Slitrk6 probe.
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Fig. 1. Slitrk6 expression in E8.5–E12.5 embryos. (A) –(K) show Slitrk6 expression as revealed by in situ hybridization. (L) shows positive immunohistochemical staining with anti-PGP9.5 (pan-neural marker). (A– G) Results of whole-mount in situ hybridization. Lateral (A, C, E) and dorsal (B, D, F) view of E8.5 embryo in which embryonic turning had already occurred (A, B), E9.5 embryo (C, D) and E10.5 embryo (E, F). (G) Isolated forelimb buds from E10.5 embryos. Top, dorsal view; bottom, ventral view. (H –K) Results of section in situ hybridization. (H) A thoracic transverse section of E11.5 embryos. Hybridization signals were detected in the epidermis, its underlying mesenchyme (asterisk) of the proximal limb bud, and the lung bud. (I) A transverse section through the abdomen of E12.5 embryos. Expression was detected in the lung buds, esophagus, stomach and midgut, but not in the liver. (J, K, L) Higher magnification view of the lung bud and esophagus (J) and midgut (K, L). (L) The PGP9.5 protein was localized in enteric neurons. (K) and (L) are adjacent sections. The Slitrk6 signals were detected in the lateral part of the lung bud, mesenchyme surrounding the main bronchi, enteric neurons and the subepithelial layer of the midgut. AO, aorta; BA, branchial arch; BR, bronchus; EN, enteric neuron; EP, epithelial layer; ES, esophagus; FL, forelimb; HF, head neural fold (the region indicated by a curved line in A); HL, hindlimb; IM, inner muscular layer; LB, limb bud; LE, trunk lateral epidermis; LU, lung bud; LV, Liver (outlines indicated by broken lines in I); MG, midgut; OC, otic cyst; OM, outer muscular layer; SC, spinal cord (outlines indicated by broken lines in H); ST, stomach. Scale bars in (H) and (I) indicate 1 mm, and those in (J), (K), and (L) indicate 100 mm.
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1.1. Expression in embryonic day (E) 8.5 – 10.5 embryos In the developing mouse embryo, weak expression of Slitrk6 was first detected in E8.5 embryos after embryonic turning had fully occurred (Fig. 1A,B). At this stage, the signal was limited to the head folds of the midbrain region, otocyst-forming ectoderm and the lateral end of the epidermal ectoderm. The expression in the otocyst and the lateral epidermis became stronger one day later (Fig. 1C,D). The expression in the otocyst at E9.5 was especially strong in the rostro-lateral part of the cysts and was diminished one day later. On the other hand, the lateral epidermal expression continued into expression in the proximal limb bud at E10.5. The expression in the limb bud was stronger in the dorsal aspect than the ventral aspect (Fig. 1G), and was limited to the epidermis and the underlying mesenchymal cells located at the junction of limb bud and the trunk as seen in transverse sections (Fig. 1H). In addition to the limb buds, the branchial arches also expressed Slitrk6, strongly in the first branchial arches. There was a faint signal in the form of a thin line along the AP axis, corresponding to the dorsal branches of the spinal nerves (asterisk in Fig. 1F, data not shown). 1.2. Slitrk6 expression in visceral organs Transverse sections through the thoracic region in E11.5 embryos revealed a hybridization signal in the lung bud (Fig. 1H). Thereafter, in E12.5 and E14.5 embryos, Slitrk6 expression was detected strongly in the visceral organs (Fig. 1I,J,K; data not shown). In the lung bud, the signals were detected in the distal end of the parenchyma and mesenchymal tissue around the major bronchi (Fig. 1J). In the gastrointestinal tract, Slitrk6 was expressed almost throughout, including the esophagus, stomach, midgut, and the oral cavity (Fig. 1I,J,K, and 4K). A pair of adjacent transverse sections of the midgut revealed Slitrk6 signals located on a layer of cells expressing the pan-neuronal marker PGP9.5 (Krammer et al., 1993; Fig. 1K,L). Colocalization of Slitrk6 and PGP9.5 was also observed in the esophagus and stomach (data not shown). Thus, Slitrk6 was widely expressed in enteric neurons. In addition to the enteric neuron layer, Slitrk6 expression was also noted in the subepithelial region. On the whole, Slitrk6 expression in the gastrointestinal tract was seen in two layers (Fig. 1I,K). Among the organs related to the gastrointestinal tract, the pancreas expressed Slitrk6 at E14 (data not shown), but the liver showed no significant expression. 1.3. Slitrk6 expression in the CNS is highly compartmentalized and closely related to the expression of Nlrr3 To identify a gene whose expression is correlated with that of Slitrk6, I hypothesized that a gene with an LRR motif may be expressed in a complementary pattern, as observed
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for members of other neuronal transmembrane proteins, such as the cadherin family (Yagi and Takeichi, 2000). To test this hypothesis, a group of LRR motif-containing genes deposited in a current database was examined in terms of the expression patterns in the brain (data not shown). As a result, I found that the expression pattern of Nlrr3 (new LRR protein3) is closely related to that of Slitrk6. Nlrr3 has been characterized as a single LRR domain possessing transmembrane protein belonging to the vertebrate Nlrr family (Taniguchi et al., 1996). Nlrr3 is expressed in both developing and mature neural tissue, and is known to be up-regulated during neural regeneration (Taniguchi et al., 1996; Borman et al., 1999; Fukamachi et al., 2001). In a coronal section through the diencephalon at E13.5, Slitrk6 expression was detected in a part of the dorsal thalamus. There was a roughly complementary expression of Nlrr3 expression in the same tissue; while the Nlrr3 signal was distributed in the ventrolateral part of the dorsal thalamus, no Slitrk6 signal was observed in this region (Fig. 2C,D). Besides the dorsal thalamus, strong Nlrr3 expression was also detected in the thalamic eminence, and weak expression was noted in the cerebral cortex, pretectum and pyriform cortex. At E17.5, the complementary expression patterns of Nlrr3 and Slitrk6 became even clearer. Slitrk6 expression was confined to the posterior thalamic nucleus, ventral lateral geniculate nucleus and ventral posterolateral thalamic nucleus, which constituted its expression compartment (Fig. 2G,I). In contrast, Nlrr3 was expressed more broadly in the thalamus, but the signals were absent or weak in the Slitrk6-expressing region (Fig. 2H,J). This tendency for complementary expression of the two genes was also recognizable in the adult stage. In a coronal section through the habenular nuclei, Nlrr3 expression was detected in the medial and lateral habenular nuclei, while Slitrk6 expression was detected in the surrounding nuclei (intermediodorsal thalamic nuclei and lateral geniculate nuclei) (Fig. 2K,L). Thus, Slitrk6 and Nlrr3 are expressed in a nearly complementary manner to each other from the midgestational stage to the adult stage. I next characterized the nature of Slitrk expression in the diencephalon. It is known that many genes are expressed in a compartmentalized fashion in the developing diencephalon (Rubenstein et al., 1998). When regions expressing Slitrk6 were compared with those expressing Gbx2 in coronal sections, it became clear that the expression regions of Slitrk6 and Gbx2 overlapped at E15.5 (Fig. 2E,F), but not at E12.5 (Fig. 2A,B). It has been reported that Gbx2 is expressed in a compartment corresponding to the dorsal thalamus (p2 in the prosomere model, Bulfone et al., 1993). Therefore, it was considered that the region expressing in the diencephalon was almost identical to the dorsal thalamic (p2) compartment. The difference in the onset of expression between Gbx2 and Slitrk6 suggests that Slitrk6 expression occurs after the establishment of the compartment demarcated by Gbx2 and other genes.
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Fig. 2.
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the pontine neuroepithelium (ventricular layer) (Fig. 3A,B). The strong expression of Slitrk6 in this region had a sharp ventral border, and strong Nlrr3 expression was observed ventrally to this border. Thus, I observed complementary patterns of expression of Slitrk6/Nlrr3 in three different regions of the CNS. 1.4. Complementary expression of Slitrk6/ Nlrr3 in other head structures
Fig. 3. Slitrk6 and Nlrr3 expression in E13–15 cerebellum and medulla. Parasagittal sections through the cerebellar primordia at E13.5 (A, B) and E15.5 (C, D). (A) and (B), and (C) and (D) are adjacent sections in the same magnification. Sections were hybridized with Slitrk6 (A, C), and Nlrr3 (B, D). 4V, fourth ventricle; CNE, cerebellar ventricular layer (neurospithelium); CP, cerebellar primordium; EGL, external germinal layer; dPNE, deep layer of pontine ventricular layer (neuroepithelium); sPNE, superficial layer of pontine ventricular layer (neuroepithelium); RL, rhombic lip; TE, tectum. Scale bars indicate 1 mm.
In the CNS, Slitrk6 expression was also observed in the cerebellum and pontine area. Sagittal sections at E13.5 of the cerebellar primordia revealed that Slitrk6 is expressed in a part of the ventricular layer in the cerebellum and medulla (Fig. 3A). Later, at E15.5, the Slitrk6 expression became confined to the caudal compartment, while strong Nlrr3 expression was noted in the adjacent rostral and caudal regions, again representing a complementary expression pattern with Slitrk6 (Fig. 3C,D). Both Slitrk6 and Nlrr3 signals were absent in the external germinal layer, where the granule neuron precursors are clustered. Both signals were, instead, densely distributed in a deeper layer where Purkinje cell precursors are known to be located (Altman and Bayer, 1997). In addition to being bound caudally by Slitrk6, Nlrr3 expression in the rostral compartment had another boundary on the rostral side. Thus, four domains exist in the cerebellar primordia along the rostrocaudal direction. These patterns of expression are intriguing and may be related to regionalization of the developing cerebellum. In addition to the cerebellum, I observed a region showing a complementary pattern of expression of Slitrk6 and Nlrr3:
In sections obtained at E13.5 and E15.5, strong Slitrk6 hybridization signals were detected in the developing cochlear organs (Fig. 4A,C,D,E). The signals were evident in the hair-cell generation region (primitive sensory neuroepithelium) of the cochlear duct in E13.5 and E15.5 embryos. Interestingly, the spiral ganglion located adjacently, which contains neurons forming synapses with the hair cells, expressed Nlrr3. It is noteworthy that the lateral edge of the spiral ganglion showed a Slitrk6 hybridization signal, that is, the neurons in this region expressed Slitrk6 (Fig. 4E), apparently forming a complementary pattern with Nlrr3 expression (Fig. 4F). However, it is also possible that there are cell processes from developing hair cell at this stage. The validity of these interpretations should be further tested by additional examination of localization of both these proteins. In addition to the cochlear organs, Slitrk6 was also significantly expressed in other head structures. In the eyes, expression of Slitrk6 was observed in the retinal outer neuroblastic layer, eyelid, extraocular orbital mesenchymal tissue whereas Nlrr3 was very weakly expressed in the retinal ganglion cell layer (Fig. 4G,H). The expression in the eyelids continued into that in the epidermal and dermal layers of the head surrounding the eyelids. Strong epidermal and dermal expression was also observed in the maxillary and mandibular regions (Fig. 4A,C,K,M). In the maxillary region, Slitrk6 expression was recognized in the dermal layer surrounding the outer hair root sheath of the vibrissae (Fig. 4C,I). In addition, Nlrr3 expression was also detected weakly in the layer surrounding the area expressing Slitrk6 (Fig. 4J). Complementary Slitrk6/Nlrr3 expression was also observed in the tongue, teeth primordia and the nasal epithelium. In E13.5 embryos, Slitrk6 was expressed in the upper surface of the tongue (Fig. 4M), while Nlrr3 expression was detected in the lateral surface of the tongue adjacent to the region showing Slitrk6 expression (Fig. 4N). At the same stage, Slitrk6 was also detected in the invaginating oral epithelium forming the upper molar teeth promordia (Fig. 4M),
R Fig. 2. Highly compartmentalized expression of Slitrk6 and Nlrr3 in the developing thalamus. Sections were hybridized with Slitrk6 (A, C, E, G, I, K), Gbx2 (B, F) and Nlrr3 (D, H, J, L). (A –H, K, L) Coronal sections through the thalamic nuclei at E12.5 (A, B), E13.5 (C, D), E15.5 (E, F), E17.5 (G, H) and two months after birth (K, L). (I, J) Parasagittal sections at E17.5. (B), (D), (F), (H), (J), and (L) are adjacent sections in the same magnification of (A), (C), (E), (G), (I), and (K), respectively. Note that Slitrk6 and Nlrr3 are expressed in a partially complementary pattern throughout development and that Slitrk6 and Gbx2 are expressed in the same compartment at E15.5. 3V, third ventricle; AB, anterobasal nucleus; CC, cerebral cortex; DT (p2), dorsal thalamus (prosomere 2); EL, eyelid; FR, fasciculus retroflexus; HP, hippocampus; IDT, intermediodorsal thalamic nucleus; LGN, lateral geniculate nucleus; LH, lateral habenular nucleus; LV, lateral ventricle; MH, medial habenular nucleus; OB, olfactory bulb; PC, pyriform cortex; PO, posterior thalamic nucleus; PT, pretectum (prosomere 1); TE, thalamic eminence; VLG, ventrolateral geniculate nucleus; VPT, ventral posterolateral thalamic nucleus; VT, ventral thalamus. Scale bars indicate 1 mm.
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Fig. 4. Slitrk6 and Nlrr3 expression in head structures. Slitrk6 (A, C, D, E, G, I, K, M) and Nlrr3 (B, F, H, J, L, N) expression in parasagittal (A–C, E, F, I –L) and coronal (D, G, H, M, N) of the head at E13.5 (A– C, I– N) and E15.5 (D –H). (A)–(B), (E)– (F), (G)–(H), (I)– (J), (K)–(L), and (M)–(N) are the pairs of adjacent sections in the same magnification. (D –F) Higher magnification of inner ear region. (G, H) Higher magnification of eye. (I, J) Higher magnification of vibrissae region. (E), (F), (I), and (J) are the differential interference contrast views. In the developing inner ear, Slitrk6 is primarily expressed in the primitive sensory neuroepithelium that later develops into cochlear hair cells, and Nlrr3 is expressed in the cochlear spiral ganglion cells which form synapses with the hair cells. Slitrk6/Nlrr3 complementary expression was also observed in vibrissae, nasal epithelium, tongue, and teeth primordia. CD, cochlear duct; EL, eyelid; FB, forebrain; GCL, retinal ganglion cell layer; HN, hypoglossal nerve; IE, inner ear; IRS, inner root sheath; LE, lens; MN, mandibular process; MNE, mesenchymal tissue surrounding nasal olfactory epithelium; MTE, epithelial part of the molar tooth primordium; MTM, mesenchymal tissue surrounding the molar tooth primordium; MX, maxillary process; OM, orbital mesenchymal tissue; ONE, nasal olfactory epithelium; ONL, retinal outer nuclear layer; ORS, outer root sheath; PO, pons; PSN, primitive sensory neuroepithelium; SG, cochlear spiral ganglion; TB, petrosus part of temporal bone (primordial cartilage); TN, trigeminal ganglion; TO, tongue; VE, vestibular part of the inner ear; VB, vibrissae. Scale bars indicate 1 mm in (A)– (D), (G), (H), (K)–(N), and 100 mm in (E), (F), (I), (J).
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and Nlrr3 expression was detected in the surrounding mesenchymal tissue (Fig. 4N). In contrast, this epithelium– mesenchyme relationship was reversed in the nasal epithelium, since Nlrr3 was densely expressed in the posterior part of the nasal epithelium (Fig. 4L), whereas Slitrk6 was expressed in the surrounding mesenchymal cells (Fig. 4K). Besides at sites related to the Nlrr3 expression sites, Slitrk6 was also broadly expressed in mesenchymal tissues constituting the maxillary and mandibular structures (Fig. 4A,K,M).
2. Materials and methods 2.1. Animals CD-1 mice were purchased from Nihon SLC and Nihon CLEA. Embryonic day (E) 0.5 was defined as noon of the day on which a vaginal plug was observed. All animal experiments were conducted according to the guidelines for animal experiments in RIKEN. 2.2. In situ hybridization and immunohistochemisty In situ hybridization was performed on sections of the embryos as described (Aruga and Mikoshiba, 2003). Wholemount hybridization was carried out as described (Wilkinson et al., 1992). The probes for Slitrk6 were as described (Aruga and Mikoshiba, 2003). For the Nlrr3 probe, a cDNA fragment was generated by PCR using the following primers: 50 -TGAACTTTCAGCCTCTTG-30 and 50 -CTGTCAGCTTCTACATCC-30 . The probe for Gbx2 was provided by T. Inoue (T. Inoue, unpublished). Anti PGP9.5 antibody was purchased from Neuromics (Minnesota). The immuhistochemical staining was carried out on paraformadehyde-fixed frozen sections (10 mm) using the Vectastain ABC kit (Vector Laboratories) with 3,30 -diaminobenzidine as the chromogenic substance. The immunostained sections were couterstained with methyl green.
Acknowledgements I thank ATDC RIKEN BSI for their technical assistance, and T. Inoue for his helpful advice. This work was supported
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by grants from the Nakabayashi Trust For ALS Research and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology.
References Altman, J., Bayer, S.A., 1997. The ascent and settling of Purkinje cells in relation to their time and site of origin, In: Development of the Cerebellar System, CRC Press, New York, pp. 206 –229. Aruga, J., Mikoshiba, K., 2003. Identification and characterization of Slitrk, a novel neuronal transmembrane protein family controlling neurite outgrowth. Mol. Cell. Neurosci., in press. Aruga, J., Yokota, N., Mikoshiba, K., 2003. Human SLITRK family genes, genomic organization and expression profiling in normal brain and brain tumor tissue. Gene, in press. Bormann, P., Roth, L.W.A., Andel, D., Ackerman, M., Reinhard, E., 1999. zfNLRR, a novel leucine-rich repeat proteins is preferentially expressed during regeneration in zebrafish. Mol. Cell. Neurosci. 13, 167–179. Brose, K., Tessier-Lavigne, M., 2000. Slit proteins: key regulators of axon guidance, axonal branching, and cell migration. Curr. Opin. Neurobiol. 10, 95–102. Bulfone, A., Puelles, L., Porteus, M.H., Frohman, M.A., Martin, G.R., Rubenstein, J.L.R., 1993. Spatially restricted expression of Dlx-1, Dlx2 (Tes-1), Gbx-2, and Wnt3 in the embryonic day 12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries. J. Neurosci. 13, 3155–3172. Fukamachi, K., Matsuoka, Y., Kitanaka, C., Kuchino, Y., Tsuda, H., 2001. Rat neuronal leucine-rich repeat protein-3, cloning and regulation of the gene expression. Biochem. Biophys. Res. Com. 287, 257–263. Kobe, B., Deisenhofer, J., 1995. Proteins with LRRs. Curr. Opin. Struct. Biol. 5, 409– 416. Krammer, H.J., Karahan, S.T., Rumpel, E., Klinger, M., Kuhnel, W., 1993. Immunohistochemical visualization of the enteric nervous system using antibodies against protein gene product (PGP) 9.5. Anat. Anz. 175, 321– 325. Patapoutian, A., Reichardt, L.F., 2001. Trk receptors: mediators of neurotrophin action. Curr. Opin. Neurobiol. 11, 272–280. Rubenstein, J.L., Shimamura, K., Martinez, S., Puelles, L., 1998. Regionalization of the prosencephalic neural plate. Annu. Rev. Neurosci. 21, 445 –477. Taniguchi, H., Tohyama, M., Takagi, T., 1996. Cloning and expression of a novel gene for a protein with lecine-rich repeats in the developing mouse nervous system. Mol. Brain Res. 36, 45 –52. Wilkinson, D.G., 1992. Whole mount in situ hybridization of vertebrate embryos. In: Wilkinson, D.G., (Ed.), In situ Hybridization, A Practical Approach, IRL Press, Oxford, pp. 75–84. Yagi, T., Takeichi, M., 2000. Cadherin superfamily genes: functions, genomic organization, and neurologic diversity. Genes Dev. 14, 1169–1180.
Slitrk6 expression profile in the mouse embryo and its relationship to that of Nlrr3 Jun Aruga* Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan Received 26 May 2003; received in revised form 30 June 2003; accepted 30 June 2003
Abstract Slitrk6 is a member of the Slitrk family of proteins, which are integral membrane proteins possessing two leucine-rich repeat (LRR) domains and a carboxy-terminal domain partially similar to that in the trk neurotrophin receptor proteins. Here, I show that Slitrk6 is uniquely expressed in various organs, different from other Slitrk genes which are predominantly expressed in neural tissues. In the developing mouse embryo, Slitrk6 expression was detected in the otic cyst, lateral trunk epidermis and its underlying mesenchymal tissue, limb bud, maxillary process, pharyngeal arches, cochlea, retina, tongue, tooth primordium, central nervous system (CNS), and the visceral organ primordia including of the lung, gastrointestinal tract (particularly in the enteric neurons) and pancreas. The expression in these organs occurred in a spatially restricted manner. In the CNS, the expression was highly compartmentalized in the dorsal thalamus, cerebellum and medulla. The expression compartment in the thalamus in which Slitrk6 was expressed was closely related to the Gbx2-expressing prosomere 2. Interestingly, the Slitrk6 expression in the CNS, cochlea, tongue, tooth primordial, and other organs was partially complementary to the expression of Nlrr3, which belongs to another family of neuronal LRR-containing transmembrane proteins. The complementary expression of the two proteins in the dorsal thalamus persisted from E13.5 to the adult stage. q 2003 Elsevier B.V. All rights reserved. Keywords: Neural fold; Inner ear; Limb bud; Neural crest; External sensory organ; Sensory neuroepithelium; Skin; Vibrissae; Hair root; Transmembrane protein; Neural development; Cerebellar development; Esophagus; Stomach; Intestine; Inner muscular layer; Neural patterning; Cerebellar Compartmentalization; Gene expression; Slit; PGP9.5; Brain tumor; Medulloblastoma
1. Results and discussion The Slitrk family was identified as a family of neuronal transmembrane proteins that control neurite outgrowth (Aruga and Mikoshiba, 2003; Aruga et al., 2003). When overexpressed in neurons, either induction of a single neurite or inhibition of neurite outgrowth is observed, depending on the Slitrk subtype. Structurally, these proteins are characterized by two leucine-rich repeat (LRR) domains located amino-terminally to the transmembrane domain. LRR domains are known to be present in many proteins, and mediate protein – protein interactions (Kobe and Deisenhofer, 1995). The LRR domains in the Slitrk family proteins are highly similar to those in the entire Slit family of proteins, which are known to control axon guidance and Abbreviations: LRR, leucine-rich repeat. * Tel.: þ81-48-467-9745; fax: þ 81-48-467-9744. E-mail address: [email protected] (J. Aruga). 1567-133X/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1567-133X(03)00141-8
branching (Brose and Tessier-Lavigne, 2000). Another characteristic structural feature of the Slitrk proteins is the carboxy-terminally located tyrosine residues flanked by amino acid sequences similar to those in the carboxyterminal domain of trk neurotrophin receptor (Patapoutian and Reichardt, 2001). The carboxy-terminal domain is conserved in all the Slitrk proteins, except Slitrk1. The expression profiles of the mouse Slitrk genes are unique for each gene of the family. In particular, highly compartmentalized expression of Slitrk6 expression is observed in the thalamic nuclei, different from the expression pattern of other Slitrk genes, which are broadly expressed in neural tissues (Aruga and Mikoshiba, 2003). The unique expression pattern of Slitrk6 in the thalamus led me to investigate the comprehensive expression profile of this gene in developing mouse embryos. I therefore carried out a series of in situ hybridization experiments using a Slitrk6 probe.
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Fig. 1. Slitrk6 expression in E8.5–E12.5 embryos. (A) –(K) show Slitrk6 expression as revealed by in situ hybridization. (L) shows positive immunohistochemical staining with anti-PGP9.5 (pan-neural marker). (A– G) Results of whole-mount in situ hybridization. Lateral (A, C, E) and dorsal (B, D, F) view of E8.5 embryo in which embryonic turning had already occurred (A, B), E9.5 embryo (C, D) and E10.5 embryo (E, F). (G) Isolated forelimb buds from E10.5 embryos. Top, dorsal view; bottom, ventral view. (H –K) Results of section in situ hybridization. (H) A thoracic transverse section of E11.5 embryos. Hybridization signals were detected in the epidermis, its underlying mesenchyme (asterisk) of the proximal limb bud, and the lung bud. (I) A transverse section through the abdomen of E12.5 embryos. Expression was detected in the lung buds, esophagus, stomach and midgut, but not in the liver. (J, K, L) Higher magnification view of the lung bud and esophagus (J) and midgut (K, L). (L) The PGP9.5 protein was localized in enteric neurons. (K) and (L) are adjacent sections. The Slitrk6 signals were detected in the lateral part of the lung bud, mesenchyme surrounding the main bronchi, enteric neurons and the subepithelial layer of the midgut. AO, aorta; BA, branchial arch; BR, bronchus; EN, enteric neuron; EP, epithelial layer; ES, esophagus; FL, forelimb; HF, head neural fold (the region indicated by a curved line in A); HL, hindlimb; IM, inner muscular layer; LB, limb bud; LE, trunk lateral epidermis; LU, lung bud; LV, Liver (outlines indicated by broken lines in I); MG, midgut; OC, otic cyst; OM, outer muscular layer; SC, spinal cord (outlines indicated by broken lines in H); ST, stomach. Scale bars in (H) and (I) indicate 1 mm, and those in (J), (K), and (L) indicate 100 mm.
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1.1. Expression in embryonic day (E) 8.5 – 10.5 embryos In the developing mouse embryo, weak expression of Slitrk6 was first detected in E8.5 embryos after embryonic turning had fully occurred (Fig. 1A,B). At this stage, the signal was limited to the head folds of the midbrain region, otocyst-forming ectoderm and the lateral end of the epidermal ectoderm. The expression in the otocyst and the lateral epidermis became stronger one day later (Fig. 1C,D). The expression in the otocyst at E9.5 was especially strong in the rostro-lateral part of the cysts and was diminished one day later. On the other hand, the lateral epidermal expression continued into expression in the proximal limb bud at E10.5. The expression in the limb bud was stronger in the dorsal aspect than the ventral aspect (Fig. 1G), and was limited to the epidermis and the underlying mesenchymal cells located at the junction of limb bud and the trunk as seen in transverse sections (Fig. 1H). In addition to the limb buds, the branchial arches also expressed Slitrk6, strongly in the first branchial arches. There was a faint signal in the form of a thin line along the AP axis, corresponding to the dorsal branches of the spinal nerves (asterisk in Fig. 1F, data not shown). 1.2. Slitrk6 expression in visceral organs Transverse sections through the thoracic region in E11.5 embryos revealed a hybridization signal in the lung bud (Fig. 1H). Thereafter, in E12.5 and E14.5 embryos, Slitrk6 expression was detected strongly in the visceral organs (Fig. 1I,J,K; data not shown). In the lung bud, the signals were detected in the distal end of the parenchyma and mesenchymal tissue around the major bronchi (Fig. 1J). In the gastrointestinal tract, Slitrk6 was expressed almost throughout, including the esophagus, stomach, midgut, and the oral cavity (Fig. 1I,J,K, and 4K). A pair of adjacent transverse sections of the midgut revealed Slitrk6 signals located on a layer of cells expressing the pan-neuronal marker PGP9.5 (Krammer et al., 1993; Fig. 1K,L). Colocalization of Slitrk6 and PGP9.5 was also observed in the esophagus and stomach (data not shown). Thus, Slitrk6 was widely expressed in enteric neurons. In addition to the enteric neuron layer, Slitrk6 expression was also noted in the subepithelial region. On the whole, Slitrk6 expression in the gastrointestinal tract was seen in two layers (Fig. 1I,K). Among the organs related to the gastrointestinal tract, the pancreas expressed Slitrk6 at E14 (data not shown), but the liver showed no significant expression. 1.3. Slitrk6 expression in the CNS is highly compartmentalized and closely related to the expression of Nlrr3 To identify a gene whose expression is correlated with that of Slitrk6, I hypothesized that a gene with an LRR motif may be expressed in a complementary pattern, as observed
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for members of other neuronal transmembrane proteins, such as the cadherin family (Yagi and Takeichi, 2000). To test this hypothesis, a group of LRR motif-containing genes deposited in a current database was examined in terms of the expression patterns in the brain (data not shown). As a result, I found that the expression pattern of Nlrr3 (new LRR protein3) is closely related to that of Slitrk6. Nlrr3 has been characterized as a single LRR domain possessing transmembrane protein belonging to the vertebrate Nlrr family (Taniguchi et al., 1996). Nlrr3 is expressed in both developing and mature neural tissue, and is known to be up-regulated during neural regeneration (Taniguchi et al., 1996; Borman et al., 1999; Fukamachi et al., 2001). In a coronal section through the diencephalon at E13.5, Slitrk6 expression was detected in a part of the dorsal thalamus. There was a roughly complementary expression of Nlrr3 expression in the same tissue; while the Nlrr3 signal was distributed in the ventrolateral part of the dorsal thalamus, no Slitrk6 signal was observed in this region (Fig. 2C,D). Besides the dorsal thalamus, strong Nlrr3 expression was also detected in the thalamic eminence, and weak expression was noted in the cerebral cortex, pretectum and pyriform cortex. At E17.5, the complementary expression patterns of Nlrr3 and Slitrk6 became even clearer. Slitrk6 expression was confined to the posterior thalamic nucleus, ventral lateral geniculate nucleus and ventral posterolateral thalamic nucleus, which constituted its expression compartment (Fig. 2G,I). In contrast, Nlrr3 was expressed more broadly in the thalamus, but the signals were absent or weak in the Slitrk6-expressing region (Fig. 2H,J). This tendency for complementary expression of the two genes was also recognizable in the adult stage. In a coronal section through the habenular nuclei, Nlrr3 expression was detected in the medial and lateral habenular nuclei, while Slitrk6 expression was detected in the surrounding nuclei (intermediodorsal thalamic nuclei and lateral geniculate nuclei) (Fig. 2K,L). Thus, Slitrk6 and Nlrr3 are expressed in a nearly complementary manner to each other from the midgestational stage to the adult stage. I next characterized the nature of Slitrk expression in the diencephalon. It is known that many genes are expressed in a compartmentalized fashion in the developing diencephalon (Rubenstein et al., 1998). When regions expressing Slitrk6 were compared with those expressing Gbx2 in coronal sections, it became clear that the expression regions of Slitrk6 and Gbx2 overlapped at E15.5 (Fig. 2E,F), but not at E12.5 (Fig. 2A,B). It has been reported that Gbx2 is expressed in a compartment corresponding to the dorsal thalamus (p2 in the prosomere model, Bulfone et al., 1993). Therefore, it was considered that the region expressing in the diencephalon was almost identical to the dorsal thalamic (p2) compartment. The difference in the onset of expression between Gbx2 and Slitrk6 suggests that Slitrk6 expression occurs after the establishment of the compartment demarcated by Gbx2 and other genes.
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Fig. 2.
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the pontine neuroepithelium (ventricular layer) (Fig. 3A,B). The strong expression of Slitrk6 in this region had a sharp ventral border, and strong Nlrr3 expression was observed ventrally to this border. Thus, I observed complementary patterns of expression of Slitrk6/Nlrr3 in three different regions of the CNS. 1.4. Complementary expression of Slitrk6/ Nlrr3 in other head structures
Fig. 3. Slitrk6 and Nlrr3 expression in E13–15 cerebellum and medulla. Parasagittal sections through the cerebellar primordia at E13.5 (A, B) and E15.5 (C, D). (A) and (B), and (C) and (D) are adjacent sections in the same magnification. Sections were hybridized with Slitrk6 (A, C), and Nlrr3 (B, D). 4V, fourth ventricle; CNE, cerebellar ventricular layer (neurospithelium); CP, cerebellar primordium; EGL, external germinal layer; dPNE, deep layer of pontine ventricular layer (neuroepithelium); sPNE, superficial layer of pontine ventricular layer (neuroepithelium); RL, rhombic lip; TE, tectum. Scale bars indicate 1 mm.
In the CNS, Slitrk6 expression was also observed in the cerebellum and pontine area. Sagittal sections at E13.5 of the cerebellar primordia revealed that Slitrk6 is expressed in a part of the ventricular layer in the cerebellum and medulla (Fig. 3A). Later, at E15.5, the Slitrk6 expression became confined to the caudal compartment, while strong Nlrr3 expression was noted in the adjacent rostral and caudal regions, again representing a complementary expression pattern with Slitrk6 (Fig. 3C,D). Both Slitrk6 and Nlrr3 signals were absent in the external germinal layer, where the granule neuron precursors are clustered. Both signals were, instead, densely distributed in a deeper layer where Purkinje cell precursors are known to be located (Altman and Bayer, 1997). In addition to being bound caudally by Slitrk6, Nlrr3 expression in the rostral compartment had another boundary on the rostral side. Thus, four domains exist in the cerebellar primordia along the rostrocaudal direction. These patterns of expression are intriguing and may be related to regionalization of the developing cerebellum. In addition to the cerebellum, I observed a region showing a complementary pattern of expression of Slitrk6 and Nlrr3:
In sections obtained at E13.5 and E15.5, strong Slitrk6 hybridization signals were detected in the developing cochlear organs (Fig. 4A,C,D,E). The signals were evident in the hair-cell generation region (primitive sensory neuroepithelium) of the cochlear duct in E13.5 and E15.5 embryos. Interestingly, the spiral ganglion located adjacently, which contains neurons forming synapses with the hair cells, expressed Nlrr3. It is noteworthy that the lateral edge of the spiral ganglion showed a Slitrk6 hybridization signal, that is, the neurons in this region expressed Slitrk6 (Fig. 4E), apparently forming a complementary pattern with Nlrr3 expression (Fig. 4F). However, it is also possible that there are cell processes from developing hair cell at this stage. The validity of these interpretations should be further tested by additional examination of localization of both these proteins. In addition to the cochlear organs, Slitrk6 was also significantly expressed in other head structures. In the eyes, expression of Slitrk6 was observed in the retinal outer neuroblastic layer, eyelid, extraocular orbital mesenchymal tissue whereas Nlrr3 was very weakly expressed in the retinal ganglion cell layer (Fig. 4G,H). The expression in the eyelids continued into that in the epidermal and dermal layers of the head surrounding the eyelids. Strong epidermal and dermal expression was also observed in the maxillary and mandibular regions (Fig. 4A,C,K,M). In the maxillary region, Slitrk6 expression was recognized in the dermal layer surrounding the outer hair root sheath of the vibrissae (Fig. 4C,I). In addition, Nlrr3 expression was also detected weakly in the layer surrounding the area expressing Slitrk6 (Fig. 4J). Complementary Slitrk6/Nlrr3 expression was also observed in the tongue, teeth primordia and the nasal epithelium. In E13.5 embryos, Slitrk6 was expressed in the upper surface of the tongue (Fig. 4M), while Nlrr3 expression was detected in the lateral surface of the tongue adjacent to the region showing Slitrk6 expression (Fig. 4N). At the same stage, Slitrk6 was also detected in the invaginating oral epithelium forming the upper molar teeth promordia (Fig. 4M),
R Fig. 2. Highly compartmentalized expression of Slitrk6 and Nlrr3 in the developing thalamus. Sections were hybridized with Slitrk6 (A, C, E, G, I, K), Gbx2 (B, F) and Nlrr3 (D, H, J, L). (A –H, K, L) Coronal sections through the thalamic nuclei at E12.5 (A, B), E13.5 (C, D), E15.5 (E, F), E17.5 (G, H) and two months after birth (K, L). (I, J) Parasagittal sections at E17.5. (B), (D), (F), (H), (J), and (L) are adjacent sections in the same magnification of (A), (C), (E), (G), (I), and (K), respectively. Note that Slitrk6 and Nlrr3 are expressed in a partially complementary pattern throughout development and that Slitrk6 and Gbx2 are expressed in the same compartment at E15.5. 3V, third ventricle; AB, anterobasal nucleus; CC, cerebral cortex; DT (p2), dorsal thalamus (prosomere 2); EL, eyelid; FR, fasciculus retroflexus; HP, hippocampus; IDT, intermediodorsal thalamic nucleus; LGN, lateral geniculate nucleus; LH, lateral habenular nucleus; LV, lateral ventricle; MH, medial habenular nucleus; OB, olfactory bulb; PC, pyriform cortex; PO, posterior thalamic nucleus; PT, pretectum (prosomere 1); TE, thalamic eminence; VLG, ventrolateral geniculate nucleus; VPT, ventral posterolateral thalamic nucleus; VT, ventral thalamus. Scale bars indicate 1 mm.
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Fig. 4. Slitrk6 and Nlrr3 expression in head structures. Slitrk6 (A, C, D, E, G, I, K, M) and Nlrr3 (B, F, H, J, L, N) expression in parasagittal (A–C, E, F, I –L) and coronal (D, G, H, M, N) of the head at E13.5 (A– C, I– N) and E15.5 (D –H). (A)–(B), (E)– (F), (G)–(H), (I)– (J), (K)–(L), and (M)–(N) are the pairs of adjacent sections in the same magnification. (D –F) Higher magnification of inner ear region. (G, H) Higher magnification of eye. (I, J) Higher magnification of vibrissae region. (E), (F), (I), and (J) are the differential interference contrast views. In the developing inner ear, Slitrk6 is primarily expressed in the primitive sensory neuroepithelium that later develops into cochlear hair cells, and Nlrr3 is expressed in the cochlear spiral ganglion cells which form synapses with the hair cells. Slitrk6/Nlrr3 complementary expression was also observed in vibrissae, nasal epithelium, tongue, and teeth primordia. CD, cochlear duct; EL, eyelid; FB, forebrain; GCL, retinal ganglion cell layer; HN, hypoglossal nerve; IE, inner ear; IRS, inner root sheath; LE, lens; MN, mandibular process; MNE, mesenchymal tissue surrounding nasal olfactory epithelium; MTE, epithelial part of the molar tooth primordium; MTM, mesenchymal tissue surrounding the molar tooth primordium; MX, maxillary process; OM, orbital mesenchymal tissue; ONE, nasal olfactory epithelium; ONL, retinal outer nuclear layer; ORS, outer root sheath; PO, pons; PSN, primitive sensory neuroepithelium; SG, cochlear spiral ganglion; TB, petrosus part of temporal bone (primordial cartilage); TN, trigeminal ganglion; TO, tongue; VE, vestibular part of the inner ear; VB, vibrissae. Scale bars indicate 1 mm in (A)– (D), (G), (H), (K)–(N), and 100 mm in (E), (F), (I), (J).
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and Nlrr3 expression was detected in the surrounding mesenchymal tissue (Fig. 4N). In contrast, this epithelium– mesenchyme relationship was reversed in the nasal epithelium, since Nlrr3 was densely expressed in the posterior part of the nasal epithelium (Fig. 4L), whereas Slitrk6 was expressed in the surrounding mesenchymal cells (Fig. 4K). Besides at sites related to the Nlrr3 expression sites, Slitrk6 was also broadly expressed in mesenchymal tissues constituting the maxillary and mandibular structures (Fig. 4A,K,M).
2. Materials and methods 2.1. Animals CD-1 mice were purchased from Nihon SLC and Nihon CLEA. Embryonic day (E) 0.5 was defined as noon of the day on which a vaginal plug was observed. All animal experiments were conducted according to the guidelines for animal experiments in RIKEN. 2.2. In situ hybridization and immunohistochemisty In situ hybridization was performed on sections of the embryos as described (Aruga and Mikoshiba, 2003). Wholemount hybridization was carried out as described (Wilkinson et al., 1992). The probes for Slitrk6 were as described (Aruga and Mikoshiba, 2003). For the Nlrr3 probe, a cDNA fragment was generated by PCR using the following primers: 50 -TGAACTTTCAGCCTCTTG-30 and 50 -CTGTCAGCTTCTACATCC-30 . The probe for Gbx2 was provided by T. Inoue (T. Inoue, unpublished). Anti PGP9.5 antibody was purchased from Neuromics (Minnesota). The immuhistochemical staining was carried out on paraformadehyde-fixed frozen sections (10 mm) using the Vectastain ABC kit (Vector Laboratories) with 3,30 -diaminobenzidine as the chromogenic substance. The immunostained sections were couterstained with methyl green.
Acknowledgements I thank ATDC RIKEN BSI for their technical assistance, and T. Inoue for his helpful advice. This work was supported
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by grants from the Nakabayashi Trust For ALS Research and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology.
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