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Identification and expression of the lamprey Pax6 gene: evolutionary origin of the segmented brain of vertebrates

Yasunori Murakami1,2,*,{ddagger}, Michio Ogasawara3,*, Fumiaki Sugahara1, Shigeki Hirano4, Nori Satoh3 and Shigeru Kuratani1,2

1 Department of Biology, Okayama University, Okayama 700-8530, Japan
2 Evolutionary Morphology Research Team, Center for Developmental Biology (CDB), RIKEN, Kobe, Japan
3 Department of Zoology, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan
4 Department of Medical Technology School of Health Sciences Faculty of Medicine, Niigata University, Niigata 951-8518, Japan
* These authors contributed equally to this work



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  		Fig. 1. Comparison of amino acid sequences of Pax6 genes. Predicted amino acid sequences of L. japonica Pax6 cDNA clone (LjPax6) and various Pax6 proteins of vertebrates including Oryzias (Loosli et al., 1998), Danio (Nornes et al., 1998), Xenopus (Jaworski et al., 1997), Fugu (Miles et al., 1998), Gallus (Kawakami et al., 1997), Mus (Walter and Gruss, 1991), Homo (Ton et al., 1991), and the amphioxus Branchiostoma floridae (Glardon et al., 1998), are compared. LjPax6 amino acids are shown in blue, the paired domains in yellow and the homeodomains in red. Conserved amino acids are boxed.

 


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  		Fig. 2. Phylogenetic position of LjPax6. A neighbor-joining tree based on a comparison of the deduced amino acid sequences of full-length clones of Pax6. The sequence of mouse Pax2 was used as an outgroup. Branch lengths are proportional to the number of amino acid substitutions. The numbers indicate the ratio (%) of relative robustness of each node as assessed by bootstrap analysis.

 


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  		Fig. 3. LjPax6 expression in developing embryos of L. japonica using whole-mount in situ hybridization. Lateral (A-E,G) and ventral (F,H) views. Anterior is towards the left in all the embryos. (A) LjPax6 expression at stage 19. The weak expression in the anterior neural tube is indicated by a white arrowhead. (B) Stage 20. Transcripts are detected in the anterior neural tube and hindbrain (black arrowhead). (C) Stage 21. Expression is restricted to the rostral hindbrain (black arrowhead) and no transcripts are seen in the posterior hindbrain (black arrow). (D) Stage 22. LjPax6 expression is detected in the forebrain and the spinal cord. The part of the hindbrain that corresponds to r4 is devoid of LjPax6 expression (black arrow). (E,F) Stage 23. Forebrain expression is intensified. Transcripts are also detected in the dorsal oral ectoderm (arrow in F). (G,H) Stage 24. LjPax6 transcripts appear in the optic vesicle (white arrow), the ectoderm behind the optic vesicle (arrowhead) and the dorsal oral ectoderm (arrow in H). LjPax6 is weakly expressed in r4 (arrow in G).

 


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  		Fig. 4. Histological analysis of LjPax6 expression. (A-G) Transverse sections of a stage 26 embryo except for the whole-mount embryo in A, in which the levels of the sections (B-G) are shown by arrows. Box indicates the enlarged portion in H. It is clear from A, that LjPax6 is expressed in the nasohypophysial plate (arrow). (B-G) High levels of LjPax6 transcripts are found in the dorsal telencephalon (arrow in B), the diencephalon (arrows in C,D), the optic stalk (arrowheads in C-E), the eye (arrowhead in F) and the dorsal oral ectoderm (arrow in G), whereas no transcripts are seen in the midbrain (arrow in F). (H) LjPax6 expression in the oral ectoderm. LjPax6 expression is seen in the dorsal oral ectoderm (DOE), the anterior velum ectoderm (AVE) and the posteriodorsal velum ectoderm (PDVE).

 


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  		Fig. 5. Comparative expression of regulatory genes in the lamprey brain. Whole-mount in situ hybridization of stage 24 lamprey embryos. (A) Expression of LjPax6. (B) Expression of LjDlx1/6. Transcripts are seen in the anterior forebrain and mandibular arch (MA). (C) Expression of LjTTF1. Transcripts are restricted to the hypothalamus (HPT) and the endostyle (ES). (D) Expression of LjPax2/5/8 at the mid-hindbrain boundary (MHB) and mandibular arch (MA). (E) Expression of LjOtxA is seen in the CNS rostral to the mid-hindbrain boundary (MHB) and in the olfactory placode (OP). (F) Immunohistochemical staining of nerve fibers. Several nerve tracts are observed including the habenular commissure (hc), the medial longitudinal fascicle (mlf), the posterior commissure (pc), the posterior optic tract (pot), the supraoptic tract (sot) and the ventral longitudinal fascicle (vlf). Abbreviations: EP, epiphysis; ES, endostyle; FMB, fore-midbrain boundary; hc, habenular commissure; HPT, hypothalamus; ISN, interstitial nucleus; MA, mandibular arch; MB, midbrain; mlf, medial longitudinal fascicle; MHB, mid-hindbrain boundary; NT, notochord; OE, olfactory epithelium; OP, olfactory placode; OPT, optic stalk; pc, posterior commissure; pot, posterior optic tract; PP, pharyngeal pouch; r4, rhombomere 4; sot, supraoptic tract; vlf, ventral longitudinal fascicle.

 


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  		Fig. 6. Comparison of regulatory gene expression in the lamprey brain. Whole-mount in situ hybridization in stage 26 lamprey embryos. (A) Expression of LjPax6. An arrow indicates the dorsal hindbrain that does not express this gene. (B) Expression pattern of LjDlx1/6. Transcripts are detected in the telencephalon (TE), thalamus (TH), upper lip (UL), lower lip (LL) and velum (VE). (C) Expression of LjTTF1 in the hypothalamus (HPT) and endostyle (ES). (D) LjPax2/5/8. Transcripts are found at the mid-hindbrain boundary (MHB), velar epithelium and endostyle. (E) LjOtxA is expressed in the MHB, the midbrain (MB), the diencephalon (DI), the olfactory placode (OP) and the pharyngeal ectoderm (PE). (F) Immunohistochemical staining of nerve tracts. The habenular commissure (hc), medial longitudinal fascicle (mlf), posterior commissure (pc), posterior optic tract (pot), supraoptic tract (sot) and ventral longitudinal fascicle (vlf) are stained. Abbreviations: ch, optic chiasm; DI, diencephalon; EP, epiphysis; ES, endostyle; FMB, fore-midbrain boundary; hc, habenular commissure; HPT, hypothalamus; ISN, interstitial nucleus; LL, lower lip; MB, midbrain; mlf, medial longitudinal fascicle; MHB, mid-hindbrain boundary; NT, notochord; OE, oral ectoderm; OP, olfactory placode; OPT, optic stalk; pc, posterior commissure; pot, posterior optic tract; PP1 & PP2, pharyngeal pouches; sa, intraencephalic sulcus; sot, supraoptic tract; TE, telencephalon; UL, upper lip; vlf, ventral longitudinal fascicle.

 


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  		Fig. 7. Expression boundaries of regulatory genes in the diencephalon and telencephalon. (A-D) Diencephalic expression domains for LjPax6 (A), LjTTF1 (B), LjDlx1/6 (C) and LjOtxA (D). The outline of the diencephalon is shown by white lines. For each figure, the contours of the gene expression domain are shown by a blue line, which is identified anatomically by the relative positions of the telencephalon (arrowheads), the rostral end of the notochord (white arrows), and the anterior end of the epiphysis (black arrows). Red hatched lines indicate the domains where LjPax6 and LjDlx1/6 are both expressed. White arrowheads in C indicate the expression in the tissue lateral to the brain. (E-G) Regionalized expression patterns of regulatory genes in the stage 26 lamprey telencephalon showing the possible tripartite configuration. In all three embryos, broken white lines indicate the anterior intraencephalic sulcus and arrows indicate the hypothetical boundary between the dorsal and ventral telencephalon assumed in the present study. Expression patterns of LjPax6, LjDlx1/6 and LjEmx are shown in E-G, respectively. (E) LjPax6 is expressed in the dorsal part of the telencephalon (DP+VP). (F) LjDlx1/6 is expressed in the ventral telencephalon. (G) Weak expression of LjEmx is restricted to the dorsal region of the telencephalon, possibly corresponding to the dorsal pallium. Abbreviations: DI, diencephalon; DT, dorsal telencephalon; EP, epiphysis; VT, ventral telencephalon.

 


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  		Fig. 8. The developmental plan of the lamprey brain. (A,B) Comparison of the developmental plans between lamprey and amniote brains. On the basis of the gene expression patterns and nerve tracts described in this study, expected segments such as P1 and P2, as well as the sulcus limitans, are drawn on the stage 26 lamprey brain as unbroken lines (A). Note that the ventral limit of Pax6 expression corresponds to the sulcus limitans both in the lamprey and the mouse (B). Broken lines in A represent the hypothetical boundaries proposed in other studies but not confirmed in this study. Homologies between the domains are based on a comparison with the model proposed by Puelles and Rubenstein (Puelles and Rubenstein, 1993; B). No data were obtained in the present study to show more subdivisions rostral to the P2/P3 boundary. Boundaries of the rhombomeres are based on the reports of Kuratani et al. (Kuratani et al., 1997) and Horigome et al. (Horigome et al., 1999). The region rostral to the sulcus intraencephalicus anterior (sa) has here been tentatively termed the ‘telencephalon’ (T). Note that three gene expression domains are detected in this telencephalon, possibly corresponding to the dorsal and lateral pallium (DP and LP), and the striatum (S). Also note that LjTTF1(Nkx2.1) expression in the lamprey is restricted to the hypothalamus, and that this gene is not expressed in any region rostral to the optic chiasm. In the amniote brain, the rostral expression domain of TTF1 corresponds to the pallidum (Pa in B) which is believed to be absent in the lamprey brain (Nieuwenhuys and Nicholson, 1998). (C) Polygonal configuration of the ammocoete brain postulated by Bergquist, with the segmental boundaries (broken red lines) and the sulcus limitans (longitudinal unbroken red line) proposed in the present study. Note that some of the boundaries were not defined in Bergquist’s model. This polygonal model is not isomorphic with the pattern shown in B. Brain regions are named according to the postulated model of the larval lamprey brain in this study, and those of Bergquist are shown in parentheses. Redrawn from Bergquist and Källén (Bergquist and Källén, 1953). Abbreviations: a.bulb, olfactory bulb; a.c.th., area caudalis thalami of Bergquist and Källén (Bergquist and Källén, 1953); DP, dorsal pallium; Dth, dorsal thalamus; EP, epiphysis; hab., habenula; hc, habenular commissure; HPT, hypothalamus; ISN, interstitial nucleus; LL, lower lip; M, midbrain; MHB, mid-hindbrain boundary; mlf, medial longitudinal fasciculus; NHP, nasohypophysial plate; nt, notochord; oc, optic chiasm; P1-P3, prosomeres; Pa, pallidum; pc, posterior commissure; pcm, prechordal mesoderm; pp1, pharyngeal pouch 1; r1-4, rhombomeres; S, striatum; sa, anterior intraencephalic sulcus; sl, sulcus limitans; soc, supraoptic commissure; tpoc, postoptic tract; UL, upper lip; VEL, velum; VP, ventral pallium.

 


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  		Fig. 9. Evolution of the vertebrate brain – a hypothesis. On the phylogenetic tree of the chordates, hypothetical evolutionary events are positioned according to the present findings in the lamprey. In the common ancestor of chordates, the dorsal nerve chord was patterned anteroposteriorly by some regulatory genes. Segmentation of the brain had not appeared even after the divergence of the amphioxus lineage. The present study suggests that the common ancestor of the vertebrates had already acquired rhombomeric segmentation and at least three longitudinal subdivisions in the forebrain. The most fundamental event in the establishment of the ancestral vertebrate brain is assumed here to have involved cell lineage restriction of neuroepithelial cells, both along the dorsoventral and the anteroposterior axes, to develop compartmentalized polygonal subdivisions. Note that lampreys and hagfishes are considered to form a monophyletic group in this figure, based on recent molecular data from Kuraku et al. (Kuraku et al., 1999) and Mallat and Sullivan (Mallat and Sullivan, 1998). After the divergence of the agnathans and gnathostomes, the evolution of the pallidum may have evolved specifically in the lineage of the latter.

 

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