Bürglin TR, Finney M, Coulson A, Ruvkun G (1989) Caenorhab- ditis elegans has ... Mangalam HJ, Albert VR, Ingraham HA, Kapiloff M, Wilson L,. Nelson C ...
Dev Genes Evol (1998) 208:235–244
© Springer-Verlag 1998
O R I G I NA L A RT I C L E
&roles:Karine Deschet · Franck Bourrat · Daniel Chourrout Jean-Stéphane Joly
Expression domains of the medaka (Oryzias latipes) Ol-Gsh 1 gene are reminiscent of those of clustered and orphan homeobox genes
&misc:Received: 5 January 1998 / Accepted: 23 March 1998
&p.1:Abstract Screening of a medaka (Oryzias latipes) adult brain cDNA library, with a degenerated probe corresponding to the most conserved region of helix III of the homeodomain, led to the isolation of a gene homologous to a murine orphan Hox gene, named Gsh-1. We have called this gene Ol-Gsh 1 (Oryzias latipes-Gsh 1). Molecular analysis of the Ol-Gsh 1 putative protein points to potential functional domains which are highly conserved between fish and mouse genes. Whole-mount in situ hybridization shows that Ol-Gsh 1 is expressed in several waves during embryonic development. Transcripts are found in many regions of the central nervous system: the spinal cord, dorsal rhombencephalon, optic tectum, dorsal diencephalon, hypothalamus anlagen and rostral telencephalon. This multimodal expression pattern, strikingly conserved between fish and mammals, is reminiscent of both clustered and orphan homeobox genes. In addition, each expression wave is initiated in the fish embryo earlier than in the mammalian embryo, relative to the time scale defined by somitogenesis. We propose that Ol-Gsh 1 may be involved in conserved developmental pathways and in particular may be linked to proliferation events. Mouse Gsh-1 was shown to participate in neuro-endocrine functions of the hypothalamus. From late developmental stages onwards, Ol-Gsh 1 expression is also restricted to the hypothalamus. The expression pattern in this structure raises interesting Edited by J. Campos-Ortega K. Deschet (✉) · F. Bourrat · J.-S. Joly Laboratoire de Génétique des Poissons, Domaine de Vilvert, Institut National de la Recherche Agronomique, F-78 352 Jouy-En-Josas Cedex, France D. Chourrout1 Laboratoire de Génétique des Poissons, Domaine de Vilvert, Institut National de la Recherche Agronomique, F-78 352 Jouy-En-Josas Cedex, France Present address: 1 SARS International Centre for Molecular Marine Biology, High Technology Center, Thormohlengst 55, N-5020 Bergen, Norway&/fn-block:
questions concerning a fully or partially conserved function for these genes. &kwd:Key words Homeobox · CNS development · Hypothalamus · Medaka · In situ hybridization&bdy:
Introduction The medaka, Oryzias latipes, is a daily spawning freshwater fish which produces numerous transparent embryos and has a short life cycle. Classical genetic work in the medaka has given rise to highly inbred lines and mutant strains (Hyodo-Taguchi and Sakaizumi 1993). Studies of medaka gene function benefit from a method of gene transfer by nuclear injection (Ozato et al. 1986). Recent progress on the culture and reimplantation of embryonic stem cells (Hong et al. 1996) and the identification of an apparently intact transposable element (Koga et al. 1996) are two other advantages of this fish species. Our interest in the developmental genes of the central nervous system (CNS) led us to undergo a search for homeobox genes expressed in the medaka. Many vertebrate homeobox genes belong to the Hox subfamily. Hox genes are grouped in complexes and are prominently expressed along the antero-posterior axis of the embryo in colinearity with their cluster position (MacGinnis and Krumlauf 1992; Keynes and Krumlauf 1994). A specific combinatorial spatio-temporal expression of Hox genes was shown to participate in the specification of functional units of the hindbrain, the rhombomeres (Kessel and Gruss 1991). Nevertheless, numerous other homeobox genes are dispersed in the genome and display expression patterns distinct from those of clustered Hox genes, such as transcriptional domains rostral to the hindbrain (Lu et al. 1992; Roberts et al. 1995). These “orphan” genes generally encode a homeodomain quite divergent from that of the Drosophila Antennapedia gene and phenotypes of mutant mice generated by homologous recombination demonstrate that they fulfil new functions
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in regional specification of the mid- and forebrain (Wurst et al. 1994 ; Ang et al. 1996). The ontogenesis and function of the hypothalamo-hypophyseal axis is dependant upon several key genes to development, such as the related class III POU domain factors, Brn-1, -2 and -4 (Rosenfeld et al. 1996) and the Pit-1 homeobox gene (Mangalam et al. 1989). The targeted disruption of the murine orphan Hox gene Gsh-1 (Genetic screen homeobox-1) has brought new data to the determination of the genetic hierarchy involved in this axis patterning (Li et al. 1996). Gsh-1-deficient mice exhibit a pleiotropic phenotype characterized by dwarfism, sexual infantilism and perinatal mortality correlating with a hypocellular pituitary and reduced level of several adenohypophyseal hormones. The growthhormone-releasing factor (GHRH) is not expressed in the hypothalamic arcuate nucleus of mutants. Moreover, binding of the Gsh-1 protein on the GHRH gene promoter has been demonstrated. These data suggest that the murine Gsh-1 gene could be involved in the early specification of the hypothalamus and in the regulation of hypothalamic-releasing factor expression. The severe phenotype of Gsh-1-deficient mice, compared to those of the dwarf and little mouse (Li et al. 1990; Lin et al. 1993), may reflect, globally, a crucial regulatory role upstream of one or more molecular cascade(s) leading to the normal development of the hypothalamic-pituitary axis. In this paper, we report the characterization of a fulllength cDNA of the medaka Gsh-1 gene isolated by screening an adult brain library and the study of its expression pattern by whole-mount in situ hybridization. It shows several waves of expression in both the anterior and posterior parts of the developing central nervous system and transcriptional domains in specific hypothalamic nuclei during adult stage. These data lead us to discuss hypothetical roles played by Ol-Gsh 1 during the phylotypic period of medaka development, which are both relevant to clustered Hox genes and dispersed homeobox genes. Our results allow us to also question the level of conservation of hypothalamic functions of the GSH-1 genes between fish and mammals.
Materials and methods Fish strains and breeding conditions Embryos and adults from an orange-red strain (kindly provided by Prof. Shima, Tokyo University) were used. Fish were raised in 20l tanks, at 25°C, under a standard light regime (12 h light/12 h dark). Adults were placed under a reproduction regime (14 h light/10 h dark). Embryos were incubated in petri dishes, in Yamamoto embryo rearing medium (Yamamoto 1975), at 27°C. Embryonic stages were determined according to Iwamatsu (1994). cDNA library screening An amplified λZAPII library (Stratagene, USA) of putatively fulllength medaka brain cDNAs (Joly et al. 1997) was constructed. A 1024-times degenerated oligonucleotide coding for the 8 amino
acids in the most conserved region of helix III of the homeodomain, KIWFQNRR (named HB1 in Bürglin et al. 1989), was used to screen 6×105 clones, according to conditions given by the authors (Bürglin et al. 1989). cDNA sequencing Sequencing was performed directly on double-stranded DNA inserts in Bluescript SK- plasmids (Stratagene, USA) rescued from λZAPII and purified using a commercial kit (Qiagen, USA). An automatic sequencer (ABI 310, Perkin Elmer) was used. Extension products were synthesised on both strands, after initiation with specific primers. The dT-Rhodamine sequencing kit (Perkin Elmer) was used according to the instructions of the manufacturer. The Wisconsin Sequence Analysis package (Genetic Computer Group, Madison, Wis.) was used for DNA fragment assembly. Whole-mount in situ hybridization and histological methods After plasmid linearisation with Hind III, digoxigenin-UTP labelled riboprobes were transcribed from the full-length cDNA using T7 and T3 phage polymerases respectively (Promega, USA) in order to obtain sense and antisense probes. Embryos were fixed overnight at 4°C, with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) and dechorionated with fine forceps. Specimens were dehydrated and stored in methanol at –20°C. Following rehydration in graded methanol baths, embryos were processed as described by Joly et al. (1993). An overnight reaction was performed, using the DIG nucleic acid detection kit (Boehringer Mannhein). After in situ hybridization, embryos were briefly post-fixed in 4% paraformaldehyde in PBS. Some specimens were cleared with graded PBS/glycerol baths and photographed with a Leica M 10 stereomicroscope. Following dehydration, others were wax-embedded and sectioned at 8 µm using a Leica Jung Biocut microtome either in transverse, sagittal or horizontal planes. Sections were mounted on slides and were counterstained with Nuclear Fast Red (Gabe 1968). Photographs were taken with a Leica DMRD photomicroscope. Detection of transcripts in the adult brain CNS was performed using 2-month-old medakas killed into ice-cold water and fixed for 24 h in paraformaldehyde at 4°C. Whole brains were dissected out and processed as per whole embryos, except that proteinase K treatment was extended to 30 min and detection to 3 days.
Results Identification of the Ol-Gsh 1 cDNA A screening of a medaka brain cDNA library with a 1024-fold degenerated oligonucleotide corresponding to the most conserved region of helix III of the homeodomain led to the isolation of one clone named 1.1. The 1.1 cDNA sequence was found to be most similar to the Gsh-1 cDNA sequence, a recently isolated murine orphan Hox gene (Singh et al. 1991; Valerius et al. 1995). In reference to this high degree of homology, we decided to name the 1.1 cDNA, Oryzias latipes-Genetic screen homeobox 1 (Ol-Gsh 1). Its 1.377-kb-long sequence seems to be complete at the 3’ end, with a polyadenylation signal and a poly-A tail. At the 5’ end, a putative initiator codon is found at base 105. In fact, the encoded methionine preceeds a sequence of 21 amino acids (Fig. 1) which is well conserved between the medaka and mouse GSH-1 proteins (see below). One stop codon
237 Fig. 1 Alignment between putative Oryzias latipes-Gsh1 (Ol-Gsh 1) protein sequence and mouse Gsh-1. Amino acids conserved between the mouse and medaka proteins are indicated by two points, and conserved amino acids substitutions by one point. Conserved functional domains between both proteins are represented by boldface type [Aa 1–21 21 amino acids corresponding to the amino-terminal conserved region, identified as a SNAG related domain; Aa 24–28 SH3binding related domain (PPLFP); Aa 137–196 homeodomain including a nuclear localization signal (KHKK) at the carboxy-terminal region]. The murine specific polyalanine stretch is underlined&ig.c:/f
10 20 30 40 50 MPRSFLVDSLILREANEKGSENN--PPLFPYAMHSPHHAHGL-PGSCHSRKAGMLCFCPL ::::::::::.::::..: . .. :::::::. :: ::: ::.::.::::.:: ::: MPRSFLVDSLVLREASDKKAPEGSPPPLFPYAVPPPHALHGLSPGACHARKAGLLCVCPL 10 20 30 40 50 60 60 70 80 90 100 110 CMAASQLH--PSPPTLPLLKASFPPFSSQYCHSALSRQHASSNSIS-----LSQGAGIYQ :..::::: :.::.:::::::::::.:::::. :.:::. : ... . .:..:: CVTASQLHGPPGPPALPLLKASFPPFGSQYCHAPLGRQHSVSPGVAHGPAAAAAAAALYQ 70 80 90 100 110 120 120 130 140 150 160 170 AAYSVPDPRQFHCISLENSNGKLQSSKRMRTAFTSTQLLELEREFTSNMYLSRLRRIEIA ..: .::::::::::...:...: :::::::::::::::::::::.:::::::::::::: TSYPLPDPRQFHCISVDSSSNQLPSSKRMRTAFTSTQLLELEREFASNMYLSRLRRIEIA 130 140 150 160 170 180 180 190 200 210 220 TYLNLSEKQVKIWFQNRRVKHKKEGKSSGQRTGS---------HNCKCSSLSAARCSEEE ::::::::::::::::::::::::::.:..: :. ..:::::::.:.:::. TYLNLSEKQVKIWFQNRRVKHKKEGKGSNHRGGAGAGAGGGAPQGCKCSSLSSAKCSED190 200 210 220 230 230 240 DDDIPISPSSSEKEDVDLSVSP ::..:.::::: :.: ::.:.: DDELPMSPSSSGKDDRDLTVTP 240 250 260
Ol-Gsh 1 m-Gsh 1
Ol-Gsh 1 m-Gsh 1
Ol-Gsh 1 m-Gsh 1
Ol-Gsh 1 m-Gsh 1
Ol-Gsh 1 m-Gsh 1
Medaka GSH-1 Mouse GSH-1 Mouse GSH-2
SKRMRTAFTSTQLLELEREFTSNMYLSRLRRIEIATYLNLSEKQVKIWFQNRRVKHKKEG --------------------A--------------------------------------G-------------------S---------------------------------------
98% 97%
Hydra
CNOX2
---I---Y--I------K--QN-R--------Q--VI-D-T--------------W--DK
75%
Mouse Mouse Mouse
HOXA2 -R-L---Y-N-------K—-HF-K--C-P--V---AL-D-P-R---V------M---RQHOXB3 ---A---Y--A--V---K--HF-R--C-P--V-M-NL-----R-I--------M-Y--DQ HOXA11 TRKK-CPY-KY-IR------FFSV-INKEK-LQLSRM---TDR----------M-E--IN
67% 67% 45%
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ZEN ZEN-1
G--A---Q--A--V------HHGK----P---Q--EN-----R-I--------M-----Q L--S------V--V---N--K-----Y-T------QR-S-C-R----------M-F--NI
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R--G-QTY-RY-T----K--HF-R--T-R------HA-C-T-R-I--------M-W---N
62%
Fig. 2 Comparison between Ol-Gsh 1 (medaka) and other homeodomains. Dashes correspond to amino acids that are common to the Ol-Gsh 1-deduced homeodomain and other Hox gene encoded homeodomains. Species are indicated on the left followed by gene names. Diagnostic residues of the third paralogous group HOX homeodomains are indicated in boldface. On the right are listed the percentage identity when comparing each homeodomain to the Ol-Gsh 1 protein. The Ol-Gsh 1 homeodomain could be related to genes of the second and third paralogous groups (murine Hox A2 and Hox B3, and Hydra Cnox-2) whereas it exihibits little identity with the mouse Hox A11, an Abd-B class gene. (Dm Drosophila melanogaster, Tc Tribolium castaneum)&ig.c:/f
is found in frame at position 834. Thus, Ol-Gsh 1 predicted protein possess 243 amino acids. The Ol-Gsh 1 encoded homeodomain has 59 and 58 amino acids out of 60 in common with the mouse Gsh-1 and Gsh-2 homeodomains respectively. The full-length mouse and medaka GSH-1 proteins share 67% of their
amino acids, the amino and the carboxy-terminal regions of both proteins being particularly well-conserved (Fig. 1). In contrast, the Ol-Gsh 1 protein exhibits very little homology with the mouse Gsh-2-encoded protein (Hsieh-Li et al. 1995) outside the homeodomain. From these data we propose that Ol-Gsh 1 and m-Gsh-1 (mouse-Gsh-1) are true orthologs. Features of the GSH 1 family As previously established for the murine Gsh-1 protein, the Ol-Gsh 1 homeodomain is related to those defined by the Antennapedia class genes. It only shows 45% identity with the Hox A11 homeodomain encoded by an Abdominal-B type clustered Hox gene (Fig. 2). More precisely, additional comparisons (Fig. 2) demonstrate its high degree of homology with homeodomains encoded
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by the second and third paralogous groups of clustered Hox genes. Finally, it is interesting to note that Ol-Gsh 1 exhibits a significant homology (72%), restricted to the homeodomain, with the Zerknüllt proteins, Zen-1 and Tc Zen, the genes of which map to the Drosophila melanogaster and the Tribolium castaneum homeotic complexes respectively. Putative functional domains other than the homeodomain of the Ol-Gsh 1 protein Outside the homeodomain, several motifs are conserved between the mouse and medaka GSH-1 proteins. Both proteins have a high content of proline residues at the Nterminal region (Fig. 1), a feature which has been shown to mediate transcriptional activation (Mermod et al. 1989; Han and Manley 1993). Based upon the consensus sequence of Barfold et al. (1993), a potential SH3-binding domain might be present in this region (Fig. 1). In contrast, Ol-Gsh 1 does not exhibit the polyalanine repeat observed in its murine homolog (Fig. 1). Such a stretch, commonly found in developmental genes (Poole et al. 1985; MacDonald et al. 1986), has been proposed to confer transcriptional repressor function (Han and Maley 1993). Moreover, the Ol-Gsh 1 protein possess the first 7 of 21 amino acids of the SNAG consensus (Fig. 1), first identified in the Gfi-1 rat, mouse and human protooncoprotein families (Grimes et al. 1996). This transcriptional repressor domain is also related to sequences located at the N termini of the vertebrate SnailSlug protein family which are involved in the early ontogenesis of the mesoderm and neural crest (Angela-Nieto et al. 1994). In addition, a potential nuclear localization signal is detected at the carboxy-terminal region of the homeodomain (Fig. 1). Ol-Gsh 1 expression pattern during embryonic development and adult stage In order to determine the RNA spatio-temporal distribution, whole-mount in situ hybridizations using a digoxigenin riboprobe were performed. Some specimens were further processed for an histological analysis. Onset of expression in the hind and forebrain A narrow stripe of transcripts is detected in the hindbrain, at the neurula stage (from stage 18 to 20, beginning of somitogenesis; Fig. 3B). This restricted domain might delineate the presumptive territory of a rhombomere, although morphological boundaries could not be clearly observed in the rhombencephalon at this early stage. Shortly after, at stage 21, expression expands as transversal patches in the rostral rhombencephalon (Fig. 3C). A novel band of transcripts also appears in the dorsal diencephalon, between the two optic vesicles (Fig. 3C).
Expression in the spinal cord From stage 23 (12 somite stage) onwards, a strong expression expands to encompass most of the rhombencephalon (Fig. 3D). It forms two continuous bilaterally symmetrical bands (Figs. 3D–G, 4C). Analysis of histological sections of stage 26 embryos shows that transcripts extend up to the caudal metencephalon, corresponding to the ventro-caudal part of the cerebellum anlagen (Fig. 4B). Furthermore, at stage 23, a weaker expression is present in the spinal cord, where its intensity decreases caudally (Fig. 3 D). The maximal level of this latter signal is observed at stages 26–27 (compare signals in Fig. 3D and E). Transverse serial sections in this region unravel two contiguous expression domains at the dorsal midline of the embryo. Organization of the expression in the mesencephalic tectum A novel domain of transcription can be detected in the optic tectum from stage 24 onwards (beginning of heart beating; Fig. 3E). Globally, Ol-Gsh 1 expression extends along the whole thickness of this structure. By these stages, transcripts also appear in the telencephalon (Figs. 3E–G, 4B). Then, until stage 29 (34 somite stage), transcripts are present in the forebrain, optic tectum, rhombencephalon and spinal cord (Fig. 4E). The anterior region of the cerebellum remains negative (Fig. 4B, E). Decrease in intensity and extent of signals in the distinct expression domains From stage 29 to 33 (completion of eyes pigmentation and notochord cavitation), Ol-Gsh 1 expression progressively decreases in the spinal cord (compare Fig. 3E and F, representing whole-mount in situ hybridizations performed at stages 26 and 31 respectively). Furthermore, by stage 29, the signal becomes progressively restricted to the most anterior part of the dorsal rhombencephalon (Fig. 3G). On histological sections, transcripts are detected in restricted regions of the mesencephalon that are the lateral margins of the optic tectum and the torus semi-circularis (Fig. 4F). Thus, because the signal is no longer present in the central zone of the tectum, two symmetrical signals can be seen in the ventral diencephalon when the embryo is observed dorsally (Fig. 3F, G) : histological analysis shows that expression is confined to a structure which we identify as the anlagen of the hypothalamus. Although not visible by whole-mount in situ hybridization at earlier stages, sections show that transcription is effective from stage 26 in this region (Fig. 4-B, E). Late expression in the hypothalamus During the last stages of organogenesis (spleen, blood vessel and heart development), Ol-Gsh 1-expressing
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Fig. 3A–H Whole-mount in situ hybridization using a specific Ol-Gsh 1 RNA probe. Signal appears as a blue-purple precipitate. A–D Anterior is to the left; E–H anterior is to the top. A–G Dorsal views; H ventral view. A Schematic drawing of a stage 20 medaka embryo. B Stage 20. A strong signal is observed in a restricted domain of the hindbrain (white open arrow). C Stage 21. Bands of labelling can be seen in the anterior hindbrain (white open arrow), whilst a new transcription domain appears in the dorsal diencephalon (white arrowhead). D Stage 23. The hindbrain domain of expression (white open arrow) is formed from two symmetrical bands, whilst transcription extends in the spinal cord (black arrow). E Stage 26. From stage 24 onwards, a strong ex-
pression is detected in the mesencephalic optic tectum (white star). A more discrete signal is seen in the telencephalon (black arrowhead). Transcription now stretches the length of the spinal cord (black arrow). F Stage 31. Expression becomes restricted to the lateral margins of the optic tectum (white star), and progressively decreases in the spinal cord (black arrow). G Higher magnification of the head of a stage 33 embryo. Two deep symmetrical signals localized in the prospective region of the hypothalamus (white arrow) are clearly detected below the mesencephalic tectum (white star). H Higher magnification of the head of a stage 38 embyo. The most prominent signal is now restricted to the hypothalamus (white arrow; scale bars A–D 100 µm, E–H 200 µm)&ig.c:/f
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Fig. 4A–I Ol-Gsh 1 RNA spatio-temporal distributions during embryonic development viewed by serial sections performed after whole-mount in situ hybridization. A, D, G Drawings of 26, 29, and 38 stage medaka embryos respectively. A, G Dorsal view is to the right, lateral view is to the left. D Lateral view is to the top, dorsal view is to the bottom. The planes of corresponding sections are indicated. B Detail of a sagittal section of a stage 26 embryo ; anterior side is to the left. At that stage, a signal is present in the telencephalon (Tel), in the dorsal diencephalon (Di), in the hypothalamus anlagen (HA), in the mesencephalic tectum (OT), and in the rostral rhombencephalon (Rh). Ventral side of the cerebellum (Ce) is also labelled. C Transverse section of a stage 26 embryo ; dorsal side is to the top. The signal appears as two symmetrical contiguous bands in the most caudal part of the rhombencephalon (Rh). E Sagittal section of a stage 29 embryo ; anterior side is to
38 the left. The signal is detected in the optic tectum (OT), in the dorsal diencephalon (Di), in the prospective hypothalamus (HA), and all along the spinal cord (SC). F Transverse section of a stage 29 embryo. The dorsal side is to the top. The signal can be observed in the torus semi-circularis and in the lateral margins of the optic tectum (OT). This latter domain has been identified as mitotically active at this stage. H, I Sagittal and horizontal sections respectively of a stage 38 embryo. At that stage, the labelling is restricted to two rostro-ventral symmetrical regions of the central nervous system belonging to the hypothalamus anlagen (HA, Ce cerebellum, Di diencephalon, HA hypothalamus anlagen, NGp nucleus glomerulosus poterioris, OT optic tectum, Rh rhombencephalon, Tel telencephalon, TSC torus semi-circularis, SC spinal cord; scale bars A, D, G 500 µm; B, C, E, F 100 µm; H, I 150 µm)&ig.c:/f
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Fig. 5A–G Ol-Gsh 1 RNA distribution in 2-month-old medaka viewed by serial sections performed after whole-mount in situ hybridization. A, B A lateral and a ventral view of a medaka brain drawing respectively ; the anterior side is always to the left. The planes of corresponding sections are indicated. C—D Anterior side is to the left; E–G dorsal side is to the top. C, D Sagittal and horizontal sections respectively. Expression is seen in the hypothalamus dorsalis. E–G Transverse sections. E A general view of a brain section through the posterior hypothalamus. F, G Detailed views of the rostral and caudal hypothalamus respectively. E–G
Two different expression domains can be recognized : one is parietal (black triangle) and probably included in the nucleus lateralis tuberis, the analog of the arcuate nucleus. The other looks like a ring in the posterior part of the hypothalamus (black arrow); such an evolving shape is reminiscent of the hypothalamus dorsalis. (Ce cerebellum, Di diencephalon, Hyp hypothalamus, Mes mesencephalon, Met metencephalon, OB olfactory bulb, ON optic nerves, Pit pituitary, Rh rhombencephalon, Tel telencephalon, TSC torus semi-circularis; scale bars A–D 1 mm; E 500 µm; F, G 150 µm)&ig.c:/f
cells become restricted to the hypothalamus presumptive territory (Figs. 3H, 4 H, I). A very slight expression is still detectable in the optic tectum (data not shown). Serial sections performed in the adult brain show that Ol-Gsh 1-expressing cells are found in two distinct hypothalamic nuclei. One of the Ol-Gsh 1 transcription domains is localized dorsally in the hypothalamus (arrows in Fig. 5C–G): each half of the hypothalamus exhibits a population of stained cells having the shape of an anteroposterior tube (arrows in Fig. 5C, D). On caudal sections, staining appears as a ring (Fig. 5E, G). According to a recent medaka brain atlas (Anken and Bourrat 1998), a nucleus, the hypothalamus dorsalis (Hd; also called the recessus lateralis), has a coincident shape and
localization in the hypothalamus. The expression expands all along this nucleus in the adult brain, most if not all cells of which are labelled. Another domain of expression is found close to the ventral region of the third ventricle (triangles in Fig. 5E–G). Rostrally, this domain is parietal (triangle in Fig. 5-F). More caudally it becomes more lateral (triangles in Fig. 5E, G). Contrary to the labelling in Hd, stained cells are scattered in this domain, which corresponds to the Tv/Hv (nucleus ventralis tuberis / hypothalamus periventricularis ventralis) or the Hc (hypothalamus caudalis), or both.
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Discussion We have cloned and characterized a medaka ortholog of the murine orphan Hox gene named Gsh-1 (for Genetic Screen homeobox-1). Both genes have been isolated using a similar strategy consisting of screening a cDNA library with a degenerated probe coding for a highly conserved eight amino acid sequence found in the recognition helix of most homeodomains.A basic NLS and specific motives outside the homeodomain in the Ol-Gsh 1 open reading frame (ORF) are in agreement with an involvement in the regulation of transcription. In particular, the related SNAG sequences, although not functionally characterized, were proposed to mediate transcriptional repression (Grimes et al. 1996). Ol-Gsh 1 is an Antennapedia-like gene, but its evolutionary origin remains unclear Ol-Gsh 1 homeodomain exhibits a higher degree of identity with the Antp Drosophila homeodomain than with that related to Abd-b, which allows the grouping of both the murine and medaka GSH-1 genes in the Antennapedia class. More precisely, analysis of its homeodomain sequence indicates that Ol-Gsh 1 could be related to the second and third paralogous groups of the clustered Hox genes. A recent work has demonstrated that insect zen genes belong to the third paralogous group, despite the fact that these genes have lost the Hox ancestral function in patterning the antero-posterior axis of the embryo (Falciani et al. 1996). However, although mouse and Oryzias latipes Gsh-1 homeodomains exhibit a high level of identity with these of Zen-1 and Tc Zen, GSH-1 genes lack most of the diagnostic residues of paralogous group 3 (Fig. 2) and display no significant identity with zen outside the homeodomain. Thus, rather than being true orthologs, ZEN and GSH genes could have converged (Falciani et al. 1996). In this respect, it is interesting to note that the HOX C complex in mice lacks both 2 and 3 paralogous genes. This led Bürglin to speculate that the murine Gsh-1 and Gsh-2 genes may directly descend from Hox C2 and Hox C3 (Bürglin 1994). In this simple view, chromosome rearrangements may explain the present location of the murine Gsh-1 gene, as they may have occurred for other genes such as even-skipped, which is conversely dispersed in Drosophila but clustered in the mammalian HOX complex (Dush and Martin 1992). Following this hypothesis, Gsh genes, in their new chromosome location, would have lost diagnostic residues specific of the homeodomains of the third paralogous group genes. Ol-Gsh 1 multimodal expression pattern in the CNS is similar to that of the murine Gsh-1 gene with timing differences Comparison of gene expression in different species at similar developmental stages is difficult because of
changes in developmental sequences during evolution, a phenomenon called heterochrony. The timing of the appearance of features, during the period of development when the Bauplan becomes visible, can be more or less conserved (Richardson 1995). In this respect, the somite count may be one of the most standardized reference scales with which to compare gene expression. Comparison between the expression of Gsh-1 in medaka and mouse embryos demonstrates a similarity of spatio-temporal patterns, but differences in their overall timing. In fact, waves of transcription for both genes affect successively the rhombencephalon, diencephalon, spinal cord, mesencephalic tectum and finally the hypothalamus, but each wave occurs in the medaka slightly earlier than in the mouse. For example, expression in the hindbrain is detected at the start of somitogenesis (stage 18–19) in the medaka and at the six–eight somite stages (E 8,5) in the mouse. Detection of the Gsh-1 transcripts in the diencephalon is obvious from the mid-somite stage (E 9,5:24 somites) onwards in the mouse and from the six somite stage in the medaka. Finally, the hypothalamic signal is observed at E 13,5 in the mouse (when somitogenesis is completed), whereas signal in the hypothalamus anlagen can already be seen from stage 26 in medakas (22 somite stage). Interestingly, the earlier expression of zebrafish developmental genes compared with their murine orthologs has already been observed : the beginning of even-skipped or of T (Brachyury) expression occurs in late blastula stages in the zebrafish (Danio rerio; Schulte-Merker et al. 1992; Joly et al. 1993), whereas it is only observed during gastrulation in the mouse (Bastian and Gruss 1990; Wilkinson et al. 1990). Taken together, these results suggest a conserved function and a conserved general spatio-temporal hierarchy of GSH-1 activation between mouse and medaka. Ol-Gsh 1 expression has features of both clustered and orphan homeobox gene expressions The dynamic Ol-Gsh 1 expression pattern suggests an involvement of this gene in the development of distinct structures of the central nervous system. First, from stage 30 to 33, Ol-Gsh 1 RNA is distributed in mitotically active tissues of the optic tectum and the rostral part of the dorsal rhombencephalon. There is indeed a striking parallelism between Brdu labelling domains (Nguyen and Bourrat, personal communication) and Ol-Gsh 1 expression in these former regions. Thus Ol-Gsh 1 expression could be linked to proliferation events. This would be reminiscent of one function of the Hox genes : for example, the neotenic phenotype generated by Hoxd-13 gene targeting in mice has been interpreted as a disturbed regulation of the timing and extent of local growth rates (Dollé et al. 1993). Moreover, we note that Ol-Gsh 1 is expressed in both rostral and caudal domains of the central nervous system. This gene is indeed characterized, on one hand, by an early restricted expression domain in the hindbrain
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and a widespread expression in the spinal cord and on the other hand, by a transcription in anterior domains, such as the optic tectum, the diencephalon and the telencephalon. Thus, Ol-Gsh 1 combines features of typical clustered Hox genes, never shown to be expressed in regions more rostral than the hindbrain, with those related to the so-called orphan homeobox genes which are expressed rostrally to the met-mesencephalic junction. Indeed, several dispersed genes exhibit multimodal expression, predominantly localized in the rostral brain; in mice, Emx-1 and Emx-2 homeobox genes are expressed in the developing telencephalon (Boncinelli et al. 1993), and Dbx gene exhibits various trasncriptional territories in the telencephalon, diencephalon, hindbrain and spinal cord (Lu et al. 1992). In this respect, distinct developmental mechanisms have been postulated for the determination of rostral domains of the CNS, including mid and forebrain, and for the specification of the posterior regions of the neuraxis (Puelles and Rubenstein 1993; Bally-Cuif and Boncinelli 1997). In conclusion, Ol-Gsh 1 shares the expression pattern of orphan homeobox genes, whilst characterized by unusual features common to the clustered Hox genes. In this context, a further molecular analysis of the Ol-Gsh 1 genomic organisation could complete this initial study. It will be important to know whether the Ol-Gsh 1 gene possesses the molecular features observed in most clustered homeobox genes (Kern et al. 1994) and already identified in its murine ortholog (Li et al. 1996), such as the absence of individualised TATAA and CCAAT boxes and the presence of one single intron upstream from the homeobox. It could also be of interest to identify distinct regulatory elements that direct its expression in embryonic CNS domains characteristic of either clustered Hox genes or orphan homeobox genes. Have GSH-1 genes conserved functions in the hypothalamus between lower and higher vertebrates? From an early stage of embryogenesis onwards (at least from stage 26), Ol-Gsh 1 is expressed in the anlagen of the hypothalamus. Moreover, in the adult CNS, Ol-Gsh 1-expressing cells have been identified to belong to specific nuclei of the medaka hypothalamus. On one hand, the Ol-Gsh 1 transcription domain is detected in a periventricular cell population probably corresponding to the Tv/Hv nuclei and/or the Hc (hypothalamus caudalis), both being included in the nucleus lateralis tuberis (pars anterior and posterior). The teleostean nucleus lateralis tuberis, homologous to the mammalian arcuate nucleus, is known to regulate the endocrine activity of the adenohypophysis of the pituitary gland, via products of hypothalamic-releasing factor genes. The hypothalamic Gsh-1 expression (Valerius et al. 1995) described during embryonic development of the mouse has been shown to be critical for physiological homeostasis of adults (Li et al. 1996). Thus it would be of interest to learn more about
Ol-Gsh 1 function, to determine the evolutionary conservation of the GSH 1 family genes role. On the other hand, Ol-Gsh 1 is expressed in the hypothalamus dorsalis (also called the recessus lateralis). In medaka, and more generally in teleostean fish, this nucleus might represent another source of neuroendocrine peptides. However, neither any specific adenohypophysal releasing factors nor any neurohormones carried to the posterior pituitary have been identified in the dorsal hypothalamus so far (Peter and Fryer 1983). Thus, Ol-Gsh 1 might have acquired a divergent function compared to its murine homolog. Alternatively, we can propose that the Ol-Gsh 1 protein, although expressed in a nucleus apparently having no significant endocrine role, could be involved upstream of an endocrine pathway. &p.2:Acknowledgements We would like to thank Dr C. Cunningham (SARS Centre, Bergen) for careful reading and corrections of this manuscript, Prof. A. Shima and Y. Hyodo-Taguchi for the kind gift of medaka strains, Pascal Lafaux and Marc Vandeputte for skilfull maintenance of fish facility, Estelle Godet for technical assistance and Muriel Mambrini, Elisabeth Perrot and Filomena Ristoratore for help and discussion. This work was supported by the Institut National de la Recherche Agronomique, by the Institut National de la Santé et de la Recherche Médicale, and by the European Commission (Biotechnology Program, Contract BI02CT930430).
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