Morphogenesis of the optic tectum in the medaka (Oryzias latipes): A morphological and molecular study, with special emphasis on cell proliferation

Morphogenesis of the Optic Tectumin the Medaka (Oryzias latipes):

A Morphological and Molecular Study,With Special Emphasis on Cell

Proliferation

VAN NGUYEN,1 KARINE DESCHET,1 TORSTEN HENRICH,2 ESTELLE GODET,1

JEAN-STEPHANE JOLY,1 JOCHEN WITTBRODT,2 DANIEL CHOURROUT,3

AND FRANCK BOURRAT1*1Laboratoire de Genetique des Poissons, INRA Domaine de Vilvert,

78350 Jouy-en-Josas, France2Developmental Biology Program, EMBL, D-69012 Heidelberg, Germany

3Molecular Genetics Group, SARS International Center for Molecular Marine Biology,N-5008 Bergen, Norway

ABSTRACTWe analyzed the medaka optic tectum (OT) morphogenesis by using 5-bromo-2’-

deoxyuridine (BrdU) immunohistochemistry (with a new method we developed for pulse-labeling embryos) and in situ hybridization with three probes, two for recently clonedhomeobox genes (Ol-Prx3 [Paired-Related-Homeobox3] and Ol-Gsh1 [Genetic-Screen-Homeobox1]) and one for Ol-tailless. The tectal anlage first appears as a sheet of proliferatingcells expressing Ol-Gsh1 and Ol-tailless but not Ol-Prx3. Cells subsequently cease toproliferate in a superficial and rostral zone and begin to express Ol-Prx3. When tectallamination begins, the proliferative zone (mpz) becomes restricted to a crescent at the OTmedial, caudal, and lateral margin. This mpz functions throughout the fish’s entire life. Itproduces cells that are added at the OT’s edge as radial rows, spanning every layer of the OT.The cells of the mpz continue to express Ol-tailless in the adult, whereas Ol-Gsh1 expression isturned off. When superficial layers form, Ol-Prx3 expression becomes restricted to theunderlying deep layer, where it persists in the adult. Ol-Prx3 seems to be a marker for thedifferentiation of a subset of deep cells and allows analysis of tectal lamination, whereasOl-tailless and Ol-Gsh1 could be involved in the control of tectal cell proliferation. This studyconstitutes a first step toward molecular approach to OT development in anamniotes. Wecompare and discuss the expression patterns of the homologs of the genes studied, and moregenerally the morphogenetic patterns of the medaka tectum, with those encountered in othercortical structures and in other vertebrate groups. J. Comp. Neurol. 413:385–404,1999. r 1999 Wiley-Liss, Inc.

Indexing terms: development; BrdU; in situ hybridization; tailless; Prx3; Gsh-1

In vertebrate development, after the phase of territoryspecification (patterning), the various structures of thecentral nervous system (CNS) are built by morphogeneticprocesses, such as cell proliferation, death (apoptosis), andmigration (Raff, 1996). Our study focuses on neuronalproliferation and more specifically on the search for genesinvolved in its regulation. We have chosen to address thisquestion by using the optic tectum of a small aquariumfish, the Japanese medaka (Oryzias latipes).

The medaka, along with its more successful ‘‘sistermodel,’’ the zebrafish (Danio rerio), is becoming an increas-ingly popular animal among developmental biologists.Small, fertile, easy to breed and nurture, with wholly

transparent eggs and embroyos, it is an ideal subject.Moreover, the number of developmental genes cloned inthis species is rapidly growing, and the development of

Grant sponsor: Institut National de la Recherche Agronomique; Grantsponsor: Institut National de la Sante et de la Recherche Medicale; Grantsponsor: European Commission Biotechnology Program; Grant number:BI02-CT93-0430.

*Correspondence to: F. Bourrat, Laboratoire de Genetique des Poissons,INRA Domaine de Vilvert, 78350 Jouy-en-Josas, France.E-mail: [email protected]

Received 22 December 1998; Revised 21 April 1999; Accepted 2 July 1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 413:385–404 (1999)

r 1999 WILEY-LISS, INC.

genetic tools for manipulating their expression can beexpected in the near future, including embronic stem(ES)-like cells (Hong et al., 1998) and an apparently intacttransposable element (Koga et al., 1996).

The tectum mesencephali, or optic tectum (OT), is aprominent dorsal structure in the CNS of anamniotevertebrates and an important center in the processing ofsensory (mainly, but not exclusively, visual) inputs. Histo-logically, it is also one of the most differentiated areas ofthe brain in most fishes and amphibians, with a clearcortical (i.e., laminated) organization of its neuronal ele-ments (Northcutt, 1983; Vanegas and Ito, 1983).

Not surprisingly, its ontogenesis has been the subject ofnumerous detailed studies, the vast majority of whichhave dealt with the interplay between retinal and tectalgrowth. Indeed, the retinotectal system of amphibians andfishes has long been, and largely remains, the model ofchoice for the analysis of projectional specificity (establish-ment of topography) in vertebrates (see, for example, thereviews of Fraser and Hunt, 1980; Easter et al., 1985;Fraser, 1992; Holt and Harris, 1993; Flanagan and Vander-haeghen, 1998).

By comparison, the morphogenesis of the tectum itself infishes and amphibians, aside from the arrival, distribu-tion, and shift of its retinal inputs, has received lessattention. The late phases of its development (in juvenilefish), and its continuous growth in the adult are docu-mented in goldfish (Raymond and Easter, 1983), trout(Mansour-Robaey and Pinganaud, 1990), and Xenopus(Straznicky and Gaze, 1972). As for the embryonic develop-ment of the tectum, there seem to be some discrepanciesbetween the results obtained in goldfish (Raymond, 1986)and Xenopus (Gaze and Grant, 1992) and those obtained inzebrafish (Rahmann, 1968; Schmatolla and Ehrmann,1973; see also Huang and Sato, 1998). To the best of ourknowledge, no data are available on tectal development inthe medaka.

The initial aim of this study was therefore to understandhow and when proliferative and migratory phenomenonstake place in the medaka OT; from this point we sought toanalyze the expression pattern of several recently clonedgenes in the context of OT morphogenesis.

We have performed a morphological analysis of tectalmorphogenesis, coupled with an extensive study of neuro-nal proliferation with the thymidine analog 5-bromo-28-deoxyuridine (BrdU). In the course of this work we devel-oped a new method for pulse-labeling large numbers of fishembryos at any stage with BrdU. We have also tracedtectal lamination with a newly cloned gene, Ol-Prx3 -themedaka homolog of mammalian Prx3, a member of thePaired-related class of homeobox genes, which specificallylabels neurons in the deepest layer of the medaka tectum(Joly et al., 1997). We have identified a correlation betweenthe expression of two developmental genes, Ol-Gsh1 (themedaka homolog of the mouse Gsh-1 [Genetic ScreenHomeobox-1; Deschet et al., 1998]) and Ol-tailless (themedaka homolog of the tailless orphan nuclear receptor ofDrosophila [Pignoni et al., 1990] and mice [Monaghan etal., 1995]) with proliferative events in the tectum. Webelieve that these genes could participate in the control oftectal proliferation, since one is expressed transiently(Ol-Gsh1) and the other permanently (Ol-tailless) in thetectal proliferative zones.

Our final conclusion is that the medaka tectum, albeit atypical cortical structure, is built by morphogenetic pro-cesses that are radically different from those at work inmammalian or avian cortices. We discuss the possibledevelopmental and evolutionary consequences of thesedifferences and the interest of the medaka model for anunderstanding of the genetic control of morphogenesis oflaminated structures.

MATERIALS AND METHODS

Fish strains and breeding conditions

Medaka embryos and adults of an Orange-Red strain(generously provided by Dr. A. Shima, Tokyo University)were used in all experiments. Fish were raised in 20-litertanks at 25°C, with a 12-hour day/12-hour night cycle(standard regime), or 14-hour light/10-hour darkness (re-production regime). Eggs were collected daily and incu-bated in Petri dishes in Yamamoto’s embryo rearing me-dium (Yamamoto, 1975) at 28°C. Embryos were stagedaccording to Iwamatsu (1994). Animal care and experi-ments were conducted in accordance with national regula-tions.

External morphogenesis andhistological methods

Embryos were fixed for 24 hours at 4°C in PFA (4%paraformaldehyde in 0.12 M phosphate buffer, pH 7.4).Some were subsequently cleared with graded glycerol/phosphate-buffered saline (PBS; 0.1 M, pH 7.4) baths,embedded in 1.5% low melting point agarose, photo-graphed with a Leica M 10 stereomicroscope, and drawnwith a camera lucida. Others were embedded in paraffinand cut at 8 µm in the transverse, sagittal, or horizontalplane. Sections were stained with Cresyl violet-thionineand photographed with a Leica DMRD photomicroscope.

Proliferation and migration analysis

Embryos were dechorionated with hatching enzyme(Yasumasu et al., 1994) following the procedure of Waka-matsu et al. (1993). They were then bathed in a solution of1 g/liter BrdU (Sigma) in balanced salt solution (BSS; 1.3%NaCl; 0.08% KCl, 4% MgSO4 7H2O, 0.001% phenol red) for2 hours. Embryos were permeabilized by the addition of2% Tween-20 or 0.0006–0.001% saponin to the BrdUsolution, a protocol not described previously, which provednecessary for effective labeling of the embryos. Alevins andadults were bathed for 3–5 hours in a solution of 1 g/literBrdU in aquarium water. Embryos or fishes were eitherfixed immediately (for localization of proliferative zones)or kept for various periods (to follow the fate of labeledcells). They were anesthetized by immersion in ice-coldwater and fixed for 1 hour in Clarke’s solution (75%ethanol, 25% acetic acid). Whole embryos or adult brainswere wax-embedded and processed immunocytochemi-cally to reveal neurons that incorporated the marker.

Immunohistochemistry

Embryos and adult brains were sectioned at 8 µm with aLeica rotary microtome. Sections were treated according toBourrat and Sotelo (1991). Briefly, they were dewaxed,treated with 2 N HCl in PBS-Triton (PBST; Triton X-100,Sigma) for 30 minute at room temperature, rinsed severaltimes in PBST, and incubated in primary antibody anti-BrdU (Sigma, diluted to 1:500 to 1:1,000 in PBS-gelatine-

386 V. NGUYEN ET AL.

Triton-azide) overnight at room temperature. Sectionswere rinsed 4 times in PBST and incubated for 1 hour inthe secondary antibody (goat anti-mouse IgG biotinylated[Amersham], diluted to 1:200 in PBS-gelatine-Triton).After several rinses in PBST, sections were incubated instreptavidin-biotinylated horseradish peroxidase (HRP)complex (Amersham, diluted to 1:250 in PBS-gelatine-Triton). HRP activity was revealed with a standard DABprocedure. Sections were rehydrated, counterstained withCresyl violet, and mounted in Entellan (Merck).

In situ hybridization

Sense and antisense probes for Ol-Prx3 and Ol-Gsh1were prepared as described elsewhere (Joly et al., 1997,and Deschet et al., 1998, respectively). The tailless cDNAwas isolated as follows: a 530-bp fragment encoding medakatailless was amplified by using reverse transcriptase poly-merase chain reaction (RT-PCR) from total RNA isolatedfrom early neurula stage embryos (stage 17, Iwamatsu,1994) by using degenerate PCR primers specific for tailless(up: 58 AAR CAY TAY GGI GTI TAY GCI TG; low: 58 TTIACY TCR TGI GGR TAY TTI GG). PCR conditions were asfollows: 5 cycles at 94°C for 1 minute, 48°C for 2 minutes,72°C for 4 minutes, followed by 30 cycles with annealing at53°C. The resulting PCR product was cloned into theTopoTA vector (Invitrogen), sequenced, and subsequentlyused to isolate a 2.2-kb full-length tailless cDNA. Senseand antisense digoxigenin-UTP probes (SHOM, 38HOM)were then prepared following the procedure described inJoly et al. (1997). For in situ hybridization, embryos weredechorionated with hatching enzyme and fixed overnightat 4°C or 1 hour at room temperature in PFA. Followingrehydration, embryos were processed according to Joly etal. (1997). Whole adult brains were processed the sameway, except that proteinase K treatment was extended upto 45 minutes and detection to up to 3 days. Sense probes,used as controls, did not give any detectable signal. Somebackground was observed, especially around ventricularspaces, in the experiments with long detection times.

RESULTS

Morphological studies

The optic tectum becomes recognizable as a distinctstructure at stage 22 (1 day and 14 hours of developmentat 26°C; corresponding to the appearance of the heartanlage, just after the formation of the otic placodes). Atthis stage, a bilateral fold separates the mesencephalicneural tube into dorsal and ventral halves. In transversesections, the tectal anlage appears as the dorsal domain,relatively thick laterally and thinning at the midline (Fig.1A). Histologically, it appears as a typical pseudo-stratifiedneuroepithelium (Fig. 1A).

This anlage rapidly spreads laterally and caudally,assuming the shape of a sheet of cells, thicker at its center,and very thin medially and laterally where it joins theventral mesencephalic domain (Fig. 1B). Until stages29–30, the tectum shows no sign of differentiation in theradial dimension (no layers are visible); its expansion ismerely planar, but not uniform: specifically, it appears tobe maximal caudally and ventrolaterally.

At stages 29–30 (3 days and 2–10 hours of developmentat 26°C; corresponding to the onset of blood circulation inthe somites), an external layer becomes apparent (Fig. 1C).

Its texture is at first only fibrous, but quickly (by stage 31,i.e., about 12 hours later) some perikarya are seen in itsrostral aspect. From this stage onward, the radial differen-tiation proceeds quickly (Fig. 1D); it follows a clear rostralto caudal gradient and also a less prominent lateral tomedial one. Throughout this process the most differenti-ated area (i.e., the area where the layers are thicker andmost visible) is located at the rostral and slightly lateralpole of the tectum. At hatching (stage 39; occurring at 9–10days in the medaka) at least some of the layers classicallydescribed in the adult tectum (Northcutt, 1983; Vanegasand Ito, 1983) are distinguishable (Fig. 1E).

For the sake of simplicity, and also because the tech-niques we used only allow the visualization of neuronalcell bodies, we have, in the following descriptions, groupedthese layers into: 1) a deep zone, which corresponds to theperiventricular gray layer (pgz); this zone contains thevast majority of cell bodies; 2) an intermediate or centralzone (cz), corresponding to the stratum griseum centrale,or central zone of Northcutt (1983); and 3) a superficialzone (sz) corresponding to the superficial white and grayzone of Northcutt (1983). The term external zone (ez) isused for both the cz and sz, when it is not possible todistinguish between these layers (i.e., at early stages).This external zone contains very few cell bodies in embry-onic stages (Fig. 1D,E). An enormous growth of the tectumtakes place after hatching. It is characterized by a relativethinning of the pgz and a corresponding relative thicken-ing of the cz and sz, which becomes less parvocellular (Fig.1F,H). We did not attempt to quantify this growth, butqualitatively the sz and cz seems to contain fewer cellbodies at hatching than in the adult, suggesting animportant post-hatching maturation. Nevertheless, it isobvious even without quantitative measurements, thatthe main growth of the tectum occurs in the planardimension (see the scales of Fig. 1E–H), i.e., it becomeslonger and broader much more quickly than thicker.Whereas the tectal sheet is only slightly curved in embry-onic and early post-hatching stages (Fig. 1A–E), it iscurved by tangential growth until it covers the adultmesencephalon (Fig. 1H).

Special attention has been paid throughout the develop-ment process to the histological structure of the tectalmargin. From stages 27–28 (2 days and 10 hours; appear-ance of the pectoral fin buds) onward, this area appears asmorphologically distinct: the periphery of the tectum (me-dially, caudally, and laterally) is occupied by an epitheliumof closely packed, tightly adherent cells (Fig. 2A–D). Thiszone spans the entire thickness of the tectal sheet at earlystages; later, when lamination begins, it is restricted to themargin of the pgz. At the tectal margin, and especially atits central aspect (close to the torus longitudinalis), the pgzappears to be curved almost up to the dorsal aspect of thetectal plate (Fig. 2A,C,D).

Proliferation studies with BrdU

In preliminary studies, we found the medaka chorion tobe almost impervious to BrdU: exposure of chorionatedmedaka embryos overnight in BrdU solution (1g/liter inaquarium water) led to no labeling whatsoever (results notshown). The same held true when we bathed dechorion-ated embryos in BrdU diluted in BSS, suggesting that theimpermeability might be due to the embryonic skin as wellas to the chorion.

FISH OPTIC TECTUM MORPHOGENESIS 387

Figure 1


We therefore submitted dechorionated embryos to avariety of treatments: mechanical wounds, painstakinglyperformed with sharpened needles or broken glass, provedto be either inefficient (if small) or deleterious to theembryos; enzymatic treatments (including trypsin, pro-nase, proteinase K, collagenase, hyaluronidase, and li-pase, in standard or calcium-free buffers) were also ineffi-cient.

Only detergent treatment led to reproducible labeling ofproliferating cells, as indicated in Materials and Methods.This treatment is not innocuous: a variable proportion(decreasing with age) of embryos exhibited general malfor-mations and were therefore systematically discarded. Un-der these conditions it was never possible to label all theembryos in a given experiment (around 50% in mostcases).

Experiments with survival time 0. A pulse labeling of2 hours followed by immediate fixation and treatment ofthe embryos, fry, or adults (survival time 0) allowed us todetermine the location of the tectal germinative epithe-lium (Figs. 3, 5A, 6A,B).

Between stages 22 (appearance of the tectal anlagen)and 26, labeled nuclei were found in all three dimensionsof the tectal sheet. We did not detect regional differences inthe mitotic activity of the tectal cells at these stages (Fig.3A,B).

At stages 26–27 (2 days and 6 hours; appearance ofmelanin in the dorsal part of the retina; beginning ofnotochord vacuolization) a transition occurred: a superfi-cial, rostral, and lateral zone of the tectal plate was devoidof labeling, whereas the rest of the structure was filled

with BrdU-positive nuclei (Fig. 3C). Very quickly, thelabeled zone became restricted to the lateral, caudal, andmedial margins of the tectum, and by stages 29–30 BrdUpositive nuclei were only observed in this peripheral zone(Fig. 3D). This situation persisted during embryonic andadult life. Exposure of embryos older than stage 30, of fry,and of adult fishes systematically produced the samelabeling pattern. The difference between these stages wasmerely quantitative: the area of BrdU positive nucleiprogressively declined from a width of about 10–12 cells intransverse sections at stage 30 (Fig. 3D) to a width of 5–6cells at hatching (Fig. 3E,F) and to 1–3 rows of cells inadults (2–3 months, i.e., at sexual maturity; Fig. 6A,B). Ageneral picture of the location of the proliferative crescentis provided by three-dimensional (3D) reconstruction ofthe developing OT at stage 30 (Fig. 5A).

A closer examination of the position of the labeled arearevealed that it corresponded to the marginal epithelialzone described above in the morphological study (Fig. 2): atearly stages, when no lamination was visible in thetectum, it spanned the entire depth of the tectal plate.Later, it was restricted to the pgz.

Experiments in which embryos and fishes were al-

lowed to survive for various durations

Exposures performed before stages 26–27. With shortsurvival times (for example, embryos bathed at stage 24and sacrificed at stage 27), labeled nuclei were found in theentire tectal plate (not shown). With longer survival times(we usually killed the embryos at stage 39, i.e., hatchingstage), BrdU pulse labeling given between stages 20 and

Fig. 1. (Continued) Histological development of the medaka optictectum. Transverse paraffin sections (8 µm) of medaka embryo, fry,and adult optic tectum, stained with cresyl-violet-thionine. A: Stage22. The OT (arrowhead) becomes recognizable at this stage; it is madeof a typical pseudostratified neuroepithelium. B: Stage 24. The OT(arrowhead) has thickened and widened but otherwise remains apseudostratified epithelium; no layers are visible. C: Stage 30. Firstsign of lamination: a superficial, fibrous, and parvocellular layerbecomes visible (open arrow). The rest of the tectum (deep layer) ismade of small packed cells (stars). D: Stage 36. The tectum is made ofa superficial parvocellular zone (ez, open arrow) and of a deepperiventricular zone (pgz, stars). E: Stage 39 (hatching stage). Samesituation as in D, but the external zone has thickened and can besubdivided into a superficial zone (sz) and a central zone (cz). F: Stage

41 (young fry). The same three zones as in E are now fully developed.The general shape of the optic plate is no longer straight, but curveslaterally to cover the mesencephale. G: Drawing of an adult medakaCNS, in lateral view, with rostral to the right. The bar indicates thelevel of the section in H. H: Adult stage. Caudal aspect of the optictectum in transverse section. The curved, almost hemispheric tectumis a typical cortical structure, with well-delineated layers of cells. Thesz and cz have proportionally increased compared with the pgz (stars).In C–H, the periphery of the pgz (the margin of the tectum) has adistinctive histological appearance (small arrows). It is made of small,tightly packed cells. See also Figure 2. Ce, cerebellum; Ey, eye; Ht,hypothalamus; OT, optic tectum; Te, tegmentum (ventral mesen-cephale); Tel, telencephale; Ts, torus semicircularis. Scale bars 5 50µm in A–F; 1 mm in G; 100 µm in H.


26–27 led essentially to the same result: labeled nucleiwere found only at the centrorostral pole of the tectum(Fig. 4A,B). These BrdU-positive nuclei were found in alltectal layers; however, as at stage 39 (hatching) the sz andcz are still parvocellular and the positive nuclei are mostconspicuous in the pgz (Fig. 4A,B).

Exposures performed after stage 28. In these experi-ments (exposure at stages 28–34; BrdU detection per-formed at stages 34–39) the pattern of labeled nuclei wasquite different: it appeared as a crescent of labeled nucleilocated laterally, caudally, and medially in the tectum.Only the rostral pole of the tectal plate did not containBrdU-positive cells. The differences between the variousexperiments of this group were merely quantitative: theyounger the exposure stage (for example, stages 28–30versus 34), the more numerous the labeled nuclei observedin this peripheral crescent; and the longer the survivaltime after exposure (for example, survival until stage 39versus stage 34 after exposure performed at stage 30), thefurther from the margin of the tectum the crescent ofBrdU-positive nuclei. This tectal growth is illustrated inFigure 5B by 3D reconstruction of medaka OT at stage 39(BrdU exposure at stage 32).

On transverse sections, the labeled cells were arrayed ina column of BrdU-positive cells spanning the whole thick-ness of the tectal plate (Fig. 4C,D). This held true in thepost-hatching fry and in the adult (Fig. 6C–F). In this latercase, it should be noted that, for all that the proliferationzone was restricted to the marginal pgz, BrdU-positivecells were later found in all tectal layers (Fig. 6C–E). Therewas strictly no tangential dispersion: no positive cells werefound at any time, after any survival time, in any otherplace in the developing tectum but in this radial column(Fig. 6C).

Figure 2

Fig. 2. Histology of the developing tectum’s marginal zone. Paraf-fin sections (8 µm) stained with cresyl violet-thionin. A, C: Transversesections taken at the dorsal midline. B: Horizontal section. D: Sagittalsection, taken at the caudal pole of the developing OT. A: Stage 36. B:Stage 39 (hatching stage). C, D: Stage 41 (fry). The marginal zone ofthe tectum (arrowheads) exhibits a histologically distinct aspect. Itbelongs to the pgz but is composed of more adherent, tightly packedcells. Scale bars 5 20 µm in A, C, D; 50 µm in B.

Fig. 3. Proliferation in the developing medaka optic tectum. BrdUpulse labeling, survival time 0. Paraffin sections (8 µm) of medakaembryos treated for BrdU immunohistochemistry and counterstainedwith Cresyl violet-thionin. Embryos were killed and processed immedi-ately after administration of a pulse of BrdU. A: Schematic drawingsof medaka embryos at stage 24 (1, sagittal view), stage 27, and stage39 (2 and 3, dorsal views), anterior to the right. The approximate levelsof sections in B, C, E, and F are indicated. The level of the section for Dis the same as that in C, in a slightly older embryo. The tectalproliferative zone is indicated in gray. B: Stage 24, horizontal section.Cells proliferate over the whole extent of the tectal plate (arrowheads).C: Stage 27, transverse section. A superficial and central zone of thetectal plate has ceased to proliferate (stars). Cells in the rest of thestructure are still mitotically active (arrowheads). D: Stage 32,transverse section. The proliferative zone is restricted to the edges ofthe tectal plate (arrowheads). The whole depth of the central area is nolonger mitotically active (stars). E,F: Stage 39 (hatching stage),transverse section (E), and sagittal section (F). The proliferative zoneis restricted to the very margin of the tectum (arrowheads), correspond-ing to the mpz (see text). This is the only proliferative area in the nowdifferentiated tectum, where layers (sz, cz, pgz) are recognizable.Melanophores are present in the skin at that stage (open triangles in Eand F). Ce, cerebellum anlage; Rh, rhombencephale; Te, tegmentum(ventral mesencephale); sz, superficial zone; cz, central zone; pgz,periventricular gray zone; ez, external zone. Scale bars 5 50 µm.


Figure 3


At higher magnification, this radial column of BrdU-positive cells (Fig. 4E and F, corresponding to a exposure atstage 32, and survival to stage 39) appeared to be quiteordered. The labeled nuclei appeared to be arrayed in a

graded fashion, the most heavily marked ones beingfurther from the tectal margin. Again, there was littledispersion or mixing of the heavily and lightly labelednuclei.

Figure 4


Gene expression patterns

The in situ hybridization (ISH) method performed onwhole embryos or adult brains revealed the expressionpattern of three recently cloned genes in the developingmedaka tectum.

Ol-Prx3 and the development of tectal lamination.

We have recently isolated a medaka gene, Ol-Prx3, whichbelongs to a new class of homeobox genes and is alsorelated to the paired class of transcription factors (Joly etal., 1997). Its expression pattern is complex, both withinand outside the CNS.

Ol-Prx3 expression was first detected at stage 26 in themost superficial and rostral part of the tectum (Fig.7A,B,E). It was limited to a flat disc of cells, 1) which didnot extend to the central and lateral edges of the tectalplate; and 2) where all the cells appeared to express thegene (i.e., the expression pattern was neither in patchesnor in a salt-and-pepper fashion). At stages 28–29 (justbefore the first signs of lamination appeared; see above),the Ol-Prx3-expressing area expanded, both radially andtangentially: radially, it covered roughly the superficialtwo-thirds of the tectum, and tangentially it spread close,but not up to, the margin of the tectal plate (Fig. 7C,D).This left the peripheral zone of the tectum devoid ofOl-Prx3 expression (Fig. 7C). The labeling no longerappeared homogenous, but rather distributed in patches ofvarying intensity, although no cell or group of cells totallydevoid of labeling was observed (Fig. 7C). Starting fromstage 30, and the distinctive appearance of upper tectal

Fig. 4. Building of the medaka optic tectum: BrdU pulse labelingexperiments in embryos, survival until hatching. Paraffin sections (8µm) of medaka embryos or fry treated for BrdU immunohistochemis-try and counterstained with Cresyl violet-thionin. A,B: Sagittal (A)and transverse (B) sections of stage 39 (hatching stage) medakaembryos having received a pulse of BrdU at stage 26. Only thecentrorostral zone of the tectum is labeled (stars). At this stage, thetectum consists of an external zone (ez; open arrow) and a deep pgz.C,D: Sagittal (C) and transverse (D) sections of stage 39 (hatchingstage) medaka embryos having received a pulse of BrdU at stage 32. Amarginal column of cells is labeled (stars). This column spans thewhole thickness of the developing tectum, including the superficiallayers (small arrows). At this stage, most tectal cells are located in thepgz, where the labeling is thereforemost conspicuous. The dashedlines delineate the ventricular aspect of the optic tectum. E,F: Detailof the zone labeled in the developing medaka tectum, following thesame experiments as in C and D. E: Sagittal section of the OT caudalpole. F: Horizontal section, caudal aspect of the OT. A gradient oflabeling is visible (open arrow), the cells further away from the tectalmargin being the most heavily labeled. G: An interpretation of thegradient seen in E and F. A population (P) of proliferating andrenewing stem cells is proposed to exist at the very edge (margin) ofthe tectum. After BrdU pulse labeling (open arrow), the marker isdiluted in the following rounds of mitosis, and the rows of cells aredisplaced centrally by the constant addition of new cells at the margin.In this hypothesis, the BrdU labeling cannot be distinguished afterthree rounds of mitosis. It is proposed (see text) that a phase of radialmigration leads to the building of the external layers (small arrows inC, D, F). Ce, cerebellum; Te, tegmentum (ventral mesencephale); sz,superficial zone; cz, central zone; mpz, marginal proliferative zone.Scale bars 5 50 µm.

Fig. 5. Reconstructions and whole-mount of medaka developingtecta. A,B: 3D reconstructions of medaka OT after a BrdU pulselabeling at stage 30, and survival time 0 (A), or BrdU pulse labeling atstage 32 and survival until stage 39 (hatching stage; B). Every othersection of technically good cases was drawn with a camera lucida, andthe position of labeled nuclei was plotted on drawings of ‘‘flattened’’optic tecta. Heavily labeled nuclei are denoted by a black dot andlightly labeled ones by an open circle. In each case, one of the drawingused for the reconstruction is represented on the left. A: At stage 30,proliferation has ceased at the rostral pole (star) and in the centralzone of the OT. The proliferative zone is crescent shaped, at themedial, caudal, and lateral edge of the tectum. This corresponds to the

situation in D. B: The tectal zone built between stages 32 and 39 isindicated by arrowheads. The tectum therefore grows by addition ofrows of cells at its medial, caudal, and lateral margin. This corre-sponds to the situation illustrated in Figure C and D. See text, andFigure 4G, for further details on the gradient of lightly and heavilylabeled nuclei. C: Whole-mount in situ hybridization of a medakaembryo at stage 31 with an Ol-tailless antisense probe. Dorsal view,with rostral to the top. The labeling (small arrows) corresponds to thetectal mpz. The star points to the rostral pole of the tectum, the firstzone to become post-mitotic. The horizontal line indicates a levelcorresponding to the one shown in Figure 8C and D. Ce, cerebellum;E, epiphysis; Ey, eye; Tel, telencephale. Scale bar 5 100 µm in C.


Figure 6


layers, the labeling appeared to ‘‘sink’’ in the tectum, i.e.,the sz and cz were totally devoid of Ol-Prx3-positive cells(Fig. 7F). Only the underlying pgz contained labeled cells,and they were distributed in patches (Fig. 7F). Betweenthese groups of Ol-Prx3-expressing cells were areas devoidof any labeling. It should be noted that, as at previousstages, the most lateral, most central, and most caudalarea of the pgz did not contain labeled cells (Fig. 7F); aperipheral crescent of Ol-Prx3-negative cells was thereforepresent during all developmental stages in the medakatectum.

This pattern remained essentially the same up to stage39 (hatching stage, Fig. 7G,H); the most conspicuousdifference was that the patchy appearance became moreand more clearly recognizable (Fig. 7G). At fry stages(defined as stages 40–43 in Iwamatsu’s developmentaltable) and in the adult, this Ol-Prx3 expression pattern(patches in pgz) persisted, as described in Joly et al. (1997).In addition, a few cells became positive in the cz. Theywere not detected at hatching (Fig. 7G,H) but were con-spicuous at the adult (31 months; sexual maturity) stage(see Fig. 3D and H in Joly et al., 1997).

Ol-tailless, Ol-Gsh1, and the tectal proliferative zone.

Ol-tailless is the medaka homolog of the tailless gene ofDrosophila (Pignoni et al., 1990) and mice (Monaghan etal., 1995). tailless belongs to the nuclear receptor genefamily but has presently no known ligand. In the medakadeveloping tectum, it exhibited a simple and straightfor-ward expression pattern: it paralleled at all embryonic andadult stages the zone labeled in BrdU experiments withsurvival time 0. Thus, between stages 22 and 26, Ol-tailless was expressed in all cells of the whole tectal plate(Fig. 8A,B). From stage 26, the expression began to berestricted to a peripheral crescent. A central and rostral

area of the tectal plate was devoid of Ol-tailless positivecells. Starting at stages 29–30 (Fig. 8C,D), and continuingafter hatching in the fry, and also in adult fish (Fig. 8E,F),Ol-tailless expression was restricted to an horseshoe ofcells located at the lateral, caudal, and medial margin ofthe developing tectum. This crescent was clearly visible inwhole-mount preparations of embryos (Fig. 5C). Essen-tially all the cells in this area appeared to express theOl-tailless gene (Fig. 8C–F). This expression pattern thusclosely paralleled, qualitatively and quantitatively, theBrdU labeling with survival time 0 (i.e., the tectal prolifera-tive zone; compare Fig. 5A and C), as it progressivelydecreased in width as the embryos, then the fry, andultimately the juveniles grew in size.

Ol-Gsh1 is an homeobox gene recently isolated andcharacterized in our laboratory (Deschet et al., 1998). Itsexpression pattern is quite complex and dynamic in theCNS. In the developing tectum, it was expressed in muchthe same way as Ol-tailless, with the following differences:it began to be expressed at stage 23, shortly after the tectalanlagen became recognizable (Fig. 9A,D). At that stage, alltectal cells were Ol-Gsh1 positive. This situation persistedup to stages 27–28, when Ol-Gsh1 expression paralleledthe restriction of the proliferation zone to the tectalmargin: a central, rostral, and superficial zone of the tectalplate ceased to express Ol-Gsh1. At stages 29–30, itsexpression was limited to a crescent of cells at the lateral,caudal, and medial margin of the developing tectum (Fig.9B,D). Within this zone, all cells appeared to be Ol-Gsh1positive. The level of expression of Ol-Gsh1 then began todecrease: it was still rather intensely expressed at stage33, but at stage 39 (hatching) its expression became weak(Fig. 9C). In fry and adult fishes, Ol-Gsh1 appeared turnedoff in the tectum, or was expressed at a very low level(corresponding to the limit of sensitivity of our in situmethod; data not shown).

DISCUSSION

Methodological considerations

We wanted to take advantage of the medaka peculiari-ties (above all, availability of large numbers of transparentembryos) to label embryos with BrdU en masse. Exposureseemed to us the most obvious method, particularly as wehad validated it on fry and adults. To our surprise, medakaembryos, even dechorionated ones, were almost totallyimpervious to BrdU, despite the small size of this mol-ecule. Moreover, the embryonic skin proved to be resistantto a variety of proteolytic enzymes, and only mild deter-gent treatments allowed BrdU to penetrate the embryos,at stages where neither the mouth nor the gill covers wereopen. This might indicate that some of the moleculesresponsible for the embryonic skin imperviousness arelipidic or proteolipidic in nature; however, lipase treat-ments gave no results whatsoever.

We believe that these results should interest the grow-ing number of people using medaka or zebrafish embryosas model systems, as drug treatments of embryos could berather difficult to carry out in these animals.

Radial growth of the medakaoptic tectum

The vast majority of studies dealing with tectal develop-ment in anamniotes have treated it only as an aspect of, orin close relation with, the more general problem of the

Fig. 6. Sustained growth of the adult medaka optic tectum.Paraffin sections (8 µm) of adult medaka optic tectum, treated forBrdU immunohistochemistry. No counterstain. A,B: Transverse (A)and sagittal (B) sections of the central (A) and posterior (B) tecta ofadult (2 months old) medaka killed and processed immediately after 5hours of BrdU pulse labeling. A small population of cells continues toproliferate at the tectal margin (mpz, arrowheads). It is located in thedeepest (pgz) layer. C,D: Transverse (A) and sagittal (B) sections of thecentral (A) and posterior (B) tecta of adult (2 months old) medakakilled and processed 2 months (C) or 2 weeks (D) after 5 hours of BrdUpulse labeling. The labeling is restricted to a column of cells (stars)spanning the whole thickness of the tectum, including the central zone(cz) and superficial zone (szl small arrows). There is no tangentialdispersion of the labeled cells. E: Transverse section of adult medakaoptic tectum at low magnification. Same experiments as in C and D,survival time 2 months. Most of the tectum is built after hatching, in aslow and continued process. The area between the columns of labeledcells (stars in pgz, and small arrows in cz and sz) represents the extentof the tectum at the time of the BrdU pulse labeling (2-month-oldmedaka). The area between the columns of labeled cells and the tectalmargins represents the part of the tectum built during the survivalperiod. F: Schematic drawing of a medaka adult brain, dorsal view,with anterior to the right. The levels of sections represented in A,B,and E are indicated. The top part of the drawing (left OT) representsthe situation in E, with the proliferative zone (gray crescent), and thetectal area built during the 2 months between BrdU pulse labeling andprocessing of the brain (hatched area). The bottom part (right OT)depicts the situation in A and B (survival time 0), with the tectal mpzin gray. Rh, rhombencephale; ce, cerebellum; OT, optic tectum; Tel,telecephale; pgz, periventricular gray zone; Cge, granular layer ofcerebellum; Te, tegmentum (ventral mesencephale); Tl, torus longitu-dinalis; Ts, torus semicircularis; Vc, valvula cerebelli. Scale bars 5 50µm in A and C; 100 µm in B, D, and E.


Figure 7


development of the retinotectal system in these animals.The building of the tectum per se has received much lessattention.

To our knowledge, the most precise study on this matteris the one of Raymond and Easter (1983; with additionalresults in Raymond, 1986), carried out on the goldfish. Wehave used it as a guideline in the following discussion.

The medaka’s optic tectum is built in essentially twosteps; in the first one, extending from stage 22 to stage26–27 (roughly 1 day at 27°C) the tectal anlage becomesrecognizable and all its cells are mitotically active. As seenin experiments with long survival times, the cells gener-ated during this first step will ultimately form the rostralpole of the optic tectum.

A transition takes place at stages 27–28: for the firsttime a well-defined zone of the developing tectum containscells that do not incorporate BrdU. This zone is located atthe superficial and rostral pole of the tectal plate.

The second step corresponds to the situation docu-mented in detail by Raymond and Easter (1983) in thegoldfish, as well as by other authors in other species(Mansour-Robaey and Pinganaud, 1990 in the trout; Straz-niky and Gaze, 1972 in Xenopus). Essentially, the tectumis built by the activity of a proliferative zone located at itslateral, posterior, and medial edge. This mitotic zone istherefore shaped like a crescent, or horseshoe, the aper-ture of which lies at the rostromedial pole of the tectum.

As pointed out by Raymond and Easter (1983), and alsoby previous authors, this neuroepithelium is histologicallydistinct from the surrounding structures; it can be de-

scribed as ‘‘ventricular’’ since it always faces ventricularspaces. However, our BrdU results clearly demonstratethat when the tectal anlagen is not morphologically lami-nated (up to stages 29–30) this neuroepithelium spans thewhole depth of the tectal plate (up to its dorsal aspect); atlater stages it spans the whole depth of the innermost (pgz)layer of this structure. Moreover, the major part of thetectal pgz, which overlies the third ventricle, stops toproliferate early in embryonic development. We agree withRaymond and Easter (1983) that the term ‘‘ventricular’’cannot be used here in exactly the same way as it is used todescribe the proliferative zone of the cerebral cortex orcerebellum of mammals (see also the discussion below). Inthe following discussion, we refer to this proliferativeepithelium as the marginal proliferative zone (mpz).

It should be noted that the mpz continues to functionduring the fish’s whole life, although its activity constantlydecreases, as already shown in the goldfish by Raymondand Easter (1983).

Analysis of animals allowed to survive for variousperiods after BrdU pulse-labeling shows that cells gener-ated in the mpz at a given time do not disperse tangen-tially in the plane of the tectum. This was already pointedout by Raymond and Easter (1983) in juvenile goldfish,and we have confirmed and extended their conclusions byshowing that it holds true at any stage (embryo, fry, adult).After a pulse of BrdU, labeled cells are later found to bedisposed in a radial row (column of cells) spanning all thetectal layers (see, for example, Fig. 5C).

Fig. 7. (Continued) Expression pattern of the homeobox geneOl-Prx3 in medaka tectal development. Paraffin sections (8 µm) ofmedaka embryos and fry processed in toto for in situ hybridizationwith an antisense Ol-Prx3 probe. Counterstain: Nuclear Fast red.A,B: Transverse (A) and sagittal (B) sections of stage 26 medakaembryos. Ol-Prx3 expression in the tectum (arrowheads) starts in asuperficial and rostral zone, corresponding to the zone that first ceasesto proliferate (compare with Fig. C). C,D: Transverse (C) and sagittal(D) sections of stage 29 medaka embryos. Ol-Prx3 expression zone(arrowheads) has spread and occupies the superficial part of thedeveloping tectum, with the exception of the marginal areas (stars),which are the mitotically active zones (see Fig. 3D). Ol-Prx3 isexpressed in other parts of the developing CNS, as well as outside thebrain (for example, in branchial arches, triangles in Fig. 7D).E: Schematic drawings of medaka embryos at stage 26 (1) and 36 (2),

dorsal views, with anterior to the right. The levels of the sectionsrepresented in A, B, and F are indicated. The shaded zone in 1represents the Ol-Prx3-positive area. F: Ol-Prx3 expression at stage36 (transverse section). When the superficial layers (open arrow)differentiate, Ol-Prx3 is not expressed in them, but in the upper part ofthe underlying pgz (arrowheads). The expression does not reach themargin (mpz) of the developing tectum (stars). G,H: Transverse (G)and sagittal (H) sections of stage 39 (hatching stage) medaka embryos.Ol-Prx3 expression (arrowheads) is restricted to patches of cells in theupper part of the pgz. The marginal (proliferative) zone is devoid oflabeling (stars), as is the external zone (ez). Ce, cerebellum; Ey, eye;Ht, hypothalamus; L, lens; R, retina; Rf, reticular formation (of therhombencephale) Rh, rhombencephale; Te, tegmentum (ventral mesen-cephale); Tel, telencephale; Ts, torus semicircularis; Th, thalamus.Scale bars 5 50 µm.


Fig. 8. Expression pattern of the orphan nuclear receptor Ol-tailless in medaka tectal development. Paraffin sections (8 µm) ofmedaka embryos and fry processed in toto for in situ hybridizationwith an antisense Ol-tailless probe. Counterstain: Nuclear Fast red.A,B: Transverse sections of stage 22 (A) and stage 24 (B) medakaembryos. All tectal cells express Ol-tailless (arrowheads).C,D: Transverse sections of stage 31 medaka embryos. When the ezbegins to differentiate (open arrows) and the proliferation becomesrestricted to the tectal margin, Ol-tailless expression is restricted tothis marginal zone (arrowheads). The rest of the pgz (stars) does notexpress Ol-tailless. These sections are taken at a level corresponding

approximately to that indicated in Figure 5C. E,F: Sagittal (E) andtransverse (F) sections of adult medaka optic tecta. Ol-tailless expres-sion persists in the adult and is restricted to the marginal proliferativezone (mpz, arrowheads; compare with Figs. 2 and 6A and B). Smallarrows point to nonspecific (background) labeling present in theventricular spaces. Ce, cerebellum; Ey, eye; Ht, hypothalamus; L, lens;R, retina; Te, tegmentum (ventral mesencephale); sz, superficial zone;cz, central zone; pgz, periventricular gray zone; Vc, valvula of thecerebellum; Tl, torus longitudinalis. Scale bars 5 25 µm in A–E; 50 µmin F.

The general picture that emerges is that the tectum isbuilt by an initial period of widespread proliferation thatgenerates the rostromedial pole of the OT. This structure isused as a kind of nucleation center on which the OT is builtby continuous addition of rows, or radial columns, of cellsgenerated by the mpz at the lateral, caudal, and medialedge of the OT.

Building tectal layers in the medaka

In medaka’s OT morphogenesis, does radial neuronalmigration take place as it does in other cortical structures?An indirect, and purely morphological, argument can bederived from the parvocellular character of the externallayers at hatching, compared with the situation in juve-niles and adults. Since no proliferation was ever detected

in the sz and cz, it seems that the external layers arepopulated by cells coming from the underlying pgz. Asecond argument comes from the BrdU results in juvenileand adult fish: as the mpz is located exclusively in the pgz,whereas cells labeled later are found in a row spanning alltectal layers, it follows that cells must be displaced radi-ally to built the cz and sz. Another argument can be drawnfrom the Ol-Prx3 in situ experiments: Ol-Prx3 is initiallyturned on in a superficial domain and the labeled cellsthen appear to sink within the developing tectal plate,finally settling down in the upper third of the pgz (deepest)layer.

The simplest interpretation of this pattern is that Ol-Prx3 expression is monophasic, as it appears to be every-where else in the medaka CNS (Joly et al., 1997): the

Fig. 9. Expression pattern of the homeobox gene Ol-Gsh1 inmedaka tectal development. Paraffin sections (8 µm) of medakaembryos and fry processed in toto for in situ hybridization with anantisense Ol-Gsh1 probe. Counterstain: Nuclear Fast red. A: Sagittalsection of a stage 24 embryo. Ol-Gsh1 expression encompasses thewhole tectal plate (arrowheads). B: Transverse section of a stage 30embryo. Ol-Gsh1 is restricted to the marginal (proliferative) zone(arrowheads). The rest of the pgz (stars) does not express Ol-Gsh1.C: Transverse sections of a stage 39 embryo (hatching stage). Expres-

sion is restricted to the tectal margin (arrowheads), which correspondsto the proliferative zone (mpz). The intensity of the labeling hasalready decreased. The open triangle points to a skin melanophore.D: Schematic drawings of medaka embryos at stage 24 (1) and 30 (2),dorsal view, with anterior to the right. The levels of the sectionsrepresented in A and B are indicated. The shaded zones represent thearea where tectal cells are Ol-Gsh1 positive. Ce, cerebellum; Rh,rhombencephale; Te, tegmentum (ventral mesencephale); ez, externalzone; pgz, periventricular gray zone. Scale bars 5 25 µm.


superficially located, Ol-Prx3-positive cells seen at earlystages are the same as those located in the adult pgz. Inother words, the Ol-Prx3-negative sz and cz differentiatelater and above the pgz. There is therefore an inside-outgradient in the building of the OT. Since Ol-Prx3 expres-sion is limited to the upper part of the pgz, it could even beproposed that the Ol-Prx3-negative cells of the sz (cz andez) are in fact coming from the deepest part of the pgz(which also remains Ol-Prx3 negative at all stages).

A last and indirect argument comes from the fact thatthe OT of teleostean fish has been shown to contain radialglia, even in the adult (Manso et al., 1995; Stevenson andYoon, 1982). The structure classically recognized as anessential guide for radial migration is therefore present atall stages in the tectum.

Figure 10 recapitulates our results and shows the modelwe propose for tectal morphogenesis in the medaka.

As a final word of caution, we would like to stress thatthe scenario we propose is still hypothetical. If little doubtremains concerning the general picture of marginal prolif-eration and growth of the OT in the medaka, the exactextent of migration and movement of post-mitotic cells isstill dubious. For example, even if we believe it to beunlikely, the change of Ol-Prx3 expression pattern fromsuperficial to deep could be the result of a switch-off in thesuperficial layers followed by a switch-on in the deep one.The final answer to this question must await the character-ization of specific molecular markers for the differentiatedtectal superficial cells, just as Ol-Prx3 is a marker of pgzdifferentiated cells. It will then be possible to follow theirfate and movements after they have left the mpz and toexamine whether they indeed come from the deepest partof the pgz, as suggested above.

Comparison with the situationin other teleosts

We have discussed our results in light of those ofRaymond and Easter (1983) in the goldfish Carassiusauratus. Our data match theirs perfectly, and indeedsomewhat extend their conclusions since we have consid-ered a greater number of developmental stages. The sameholds true for the results of Mansour-Robaey and Pingan-aud (1990), who studied retinal and tectal growth at threestages in the trout Oncorhynchus mykiss. In this teleost,the OT appears to be built by the same morphogeneticmechanism.

Nonetheless, this may not be a feature common to allteleosts. In the zebrafish Danio rerio the OT does not seemto grow continuously in juvenile and adults (Rahmann,1968; Schmatolla and Erdmann, 1973), rather, cell prolif-eration in the tectum of this species appears to be limitedto the first 10 days of development (Schmatolla andErdmann, 1973). This phenomenon appears rather surpris-ing, since the zebrafish is phylogenetically a close relativeof the goldfish. It is unclear why this species seems tobehave differently, in this respect, from what seems to be awidespread feature in teleosts (the medaka, goldfish, andtrout are representative of divergent and unrelated groupsof teleosts).

In a recent report (Huang and Sato, 1998) tai-ji, a POUdomain gene that appears to be expressed in progenitorcells of embryonic and adult CNS, was characterized in thezebrafish. Interestingly, tai-ji expression in the tectum isnot wholly turned off in the adult but persists in cellsscattered all over the tectal pgz. No restriction of tai-ji

expression to a marginal zone (mpz) was reported. Further-more, Huang and Sato have shown that ‘‘Subsets oftai-ji-expressing cells within these germinal zones incorpo-rate BrdU.’’ These data appear to be in conflict both withthe earlier results of Rahmann (1968) and Schmatolla andErdmann (1973) in zebrafish, and with those of Raymondand Easter (1983) in goldfish and our own in medaka.Indeed, they indicate that neurogenesis does persists inadulthood in Danio’s optic tectum, but not according to an‘‘mpz pattern.’’ More work is needed to elucidate thisproblem, but it seems clear that the zebrafish and medaka,two increasingly popular models in developmental biology,might be more different than is usually assumed.

Genetic regulation of tectal proliferation

Further understanding of the tectal pattern of morpho-genesis could be gained by dissecting its genetic mecha-nisms, in a developmental and evolutionary perspective.

In this respect, the tailless gene provides an interestingcandidate. tailless is a gene encoding a member of theorphan nuclear steroid receptor superfamily. It was firstisolated in Drosophila (Pignoni et al., 1990); a homolog wasthen cloned in mice, where its expression pattern wasdescribed (Monaghan et al., 1995). The tailless gene isexpressed in proliferative neuroepithelia of the forebrainand, tardily and at rather low level, in the dorsal midbrain.Mice carrying a null allele of tailless have been recentlygenerated by homologous recombination (Monaghan et al.,1997). They exhibit limbic defects (entorhinal and olfac-tory cortices, amygdala, and hippocampus reduced in size),the authors explicitly stating that ‘‘anatomical defectsoutside the limbic system were not found in the miceexamined.’’ These abnormalities led the authors to suggesta role for tailless in ventricular zone proliferation anddifferentiation.

Such a role is indeed compatible with our results in themedaka OT, where Ol-tailless expression is strictly limitedto proliferative areas. However, the exact mechanisms oftailless action in neural proliferation control remain to beelucidated, as well as the differences in the sites of actionof this gene (apparent lack of abnormalities in the mousedorsal midbrain, where the gene is nonetheless expressed).More information will probably be obtained when theligand of tailless is discovered.

It is more difficult to draw conclusions from our study ofOl-Gsh1 and Ol-Prx3 expression patterns. Homologs ofthese genes have been cloned in mammals. The mouseGsh1 gene seems to be expressed mainly if not exclusivelyin the hypothalamus (Valerius et al., 1995), and Gsh1-/-

knock-out mice exhibit defects related to deficiencies in thehypothalamo-pituitary system (Li et al., 1996). In themedaka Ol-Gsh1 is also strongly expressed in the hypo-thalamus (Deschet et al., 1998), in late embryos and adults(where it remains its only expression site). There is noreport of a possible function of Gsh1 in other domains ofthe CNS.

As for Prx3, its homolog has been cloned in the rat andits expression reported (van Schaick et al., 1997). Itexhibits a complex expression pattern in adult and develop-ing CNS, as well as in extraneural tissues of the head andforelimb. To restrict ourselves to the homolog of the fishoptic tectum (the mammalian superior colliculus), Prx3 isexpressed ‘‘. . . in multiple layers with different levels ofabundance. Highest expression was observed in the super-ficial gray layer.’’ Although in rat and medaka the dorsal


midbrain is a major expression domain for Prx3 (it holdstrue for the tectum, but also for the torus semicircularis inrat and inferior colliculus in medaka), the tectal layers

that express Prx3 seem different anatomically and function-ally, since in the rat they are superficial, corresponding tothe main retino-recipient zone of the tectum in all verte-

Fig. 10. Summary of the results obtained in this study. Semi-schematic drawings of the mesencephalic part of the developingmedaka CNS, as viewed from a caudal and dorsal position. At stages22–24 the whole tectal plate is mitotically active. At stages 26–28 atransition occurs, proliferation occurs in a wide marginal zone (mpz),and Ol-Prx3 is turned on in the area that ceases to proliferate(superficial and rostral pole of the tectum). At stages 30–31 externallayers begin to differentiate, proliferation is restricted to a decreasingmpz, and Ol-Prx3 is expressed in the upper tier of the pgz. Thissituation persists in fry and adult fish, which are characterized by a

decrease of the mpz and a thickening of the external layers (cz and sz)relatively to the pgz. Ol-Prx3 remains turned on in the upper part ofthe pgz, where it is expressed in discontinuous patches of cells. At allstages represented here, the expression domains of Ol-tailless andOl-Gsh1 correspond to the areas where cells are mitotically active(whole tectal plate first, and then the mpz). hy, hypothalamus; mes,ventral mesencephale (tegmentum); t. lat, torus lateralis; cx/sz, central/superficial zones; pgz, periventricular gray zone; mpz, marginalproliferative zone.


brates, and in the medaka they are periventricular, in azone that receives few projections from the retina and ismostly innervated by axons originated in the torus semicir-cularis (Vanegas and Ito, 1983).

There is no report so far of a knockout experiment thatcould give insights into Prx3 function; however, the rat andmouse Prx3 gene has extensive (87% in amino acid se-quence) homology with a human gene, SHOX. This gene ismutated in individuals with idiopathic short stature andin Turner syndrome patients (Rao et al., 1997). However,and even though human SHOX appears to be expressed inthe brain, no neural phenotype has been reported forindividuals bearing such a mutation. It is therefore diffi-cultto speculate at present on Prx3 function in CNS (andespecially tectal) ontogenesis.

Further insights into the regulation of neuronal prolifera-tion and lamination in the developing medaka OT wouldnecessitate manipulation of the expression of the genespotentially involved. Such experiments should becomefeasible as tools for transgenesis in fish are developed inseveral laboratories.

There is more than one way to build a cortex

The medaka tectum (and more generally, the tectum ofseveral teleosts, see above) is built by tangential additionof radial blocks of cells at its central, caudal, and lateralmargin. This remains true whatever the extent of subse-quent radial migration.

We want to stress that this morphogenetic pattern isprofoundly different from that encountered in most amni-ote cortical structures. Indeed, in the latter the generalfigure is an inside-out and/or outside-in pattern of prolifera-tion and migration. The cerebral cortex of mammalsprovides a classical case of inside-out morphogenesis,where the layers are constructed from an underlyingventricular neuroepithelium (Angevine and Sidman, 1961;Rakic, 1972). The building of the cerebellum, in addition tothis process, involves an outside-in step, with the lateappearance of the external granular layer, followed by itsenormous extension, and finally by the inward migrationof the granule neurons (Ramon y Cajal, 1911).

In both cases, cortices are built by successive radialaddition of entire layers over their whole tangential ex-tent. The existence, and extent, of tangential migrationsboth within the neuroepithelium and within establishedlayers of these structures (see, for example, Ryder andCepko, 1994; Rakic, 1995; and references herein) do notalter the general picture summarized above. This patternof morphogenesis is therefore radically at odds with that ofthe medaka OT (and that of other teleosts). The mamma-lian cerebral and cerebellar cortices can be described asgrowing according to a ‘‘tangential morphogenetic pattern’’and the teleostean OT according to a ‘‘radial morphoge-netic pattern’’ (Fig. 11).

This difference in growth mechanisms has several impli-cations; the simplest is that, to the best of our knowledge,the anamniote tecta are never folded, in contrast to thecerebrum and cerebellum of many vertebrates.

The exact mechanism of cerebellar and cerebral corticalfoliation is still a matter of debate, but it probably impliesdifferential proliferation rates within the tangential planeof the structure. For example, in the cerebellum, theformation of fissures and folia has been correlated withunequal rates of mitosis in the external granular layer(EGL; Mares and Lodin, 1970). The abnormal cerebellar

foliation observed in rats after treatment with methylazoxy-methanol acetate (MAM) is a case in point, since that drugintervenes primarily with cell proliferation (Chen andHillman, 1988). More recently, it has been hypothesizedthat the foliation defects in the cerebellum of Engrailed-2-/-

knockout mice could also be due to local abnormalities inEGL proliferation rate (Millen et al., 1994).

In the teleost tectum a proliferative zone extending allover (or under) the surface of the developing structure isnever present. As a consequence, the resulting cortex isnever folded. The same holds true for the retina of anamni-otes. This layered structure grows by addition of rings ofcells at its periphery (Johns, 1977; Straznicky and Gaze,1971). This is clearly a ‘‘radial morphogenetic pattern,’’and the resulting retina is never folded.

Finally, on theoretical grounds, these observations arein general agreement with recent models of morphogenesisin various biological structures (Fleury, 1998). For ex-ample, folded structures as diverse as lung or kidneybranches, or corals, are all built by differential growth (i.e.,differential rates of cell proliferation) in the tangentialplane of the developing structures. In contrast, structuresthat grow peripherally are always smooth, except if con-strained by external factors (for example, a limited spaceor rigid walls around them).

Evolutionary considerations

The ‘‘radial morphogenetic pattern’’ in the retina andtectum of anamniotes is also characterized by continuousgrowth through life and continuous adjustment of theprojection maps by shifting of the afferent terminals(Easter and Stuermer, 1984; Raymond, 1986). Mechani-cally, such an adjustment would be extremely difficult toconceive in the case of a ‘‘tangential morphogenetic pat-tern.’’ It therefore seems that 1) continuous growth ofcortical structures over life, 2) adjustment of projectionmaps, 3) ‘‘radial morphogenetic pattern,’’ and 4) absence offoliation in the implicated structures are events thatsomehow are evolutionary linked in anamniotes. Con-versely, 1) folding, 2) ‘‘tangential morphogenetic pattern,’’and 3) growth restricted to a given developmental periodcould also somehow be linked in the cortices of birds andmammals.

It could be objected that the differences discussed aboveare not characteristic of animal groups (anamniotes versusamniotes), but of the part of the CNS the cortices arederived from (midbrain for the OT versus forebrain for thecerebral cortex or hindbrain for the cerebellum). Thisseems quite unlikely, however, since the structure homolo-gous to the OT in mammals (the superior colliculus) doesnot exhibit a radial morphogenetic pattern, but rather atangential one, with both inside-out (in the deep region)and outside-in (in the superficial region) sequences (Alt-man and Bayer, 1981). The optic tectum of birds also growsaccording to a tangential pattern, albeit a complex one,with many co-existing neurogenetic gradients (La Vail andCowan, 1971a,b). In both cases (rat and chicken), thedevelopment of the tectum is limited to a well-defined timewindow in embryonic life, and no growth takes place inpost-embryonic or adult life.

Conversely, not all the cortical structures in teleostsgrow radially. A case in point is the cerebellum, theontogenesis of which has been described in several species,such as the trout (Pouwels, 1978a; 1978b) and the gymno-tiform Apteronotus (Zupanc et al., 1996). In these species


the cerebellum develops according to a pattern that ismore or less tangential: although it does not seem to be aswell defined as it is in birds and mammals, it is definitelynot radial, in the sense that we use here for the OT growth.It can therefore be concluded that the radial morphoge-netic pattern is a characteristic of the medaka (and,presumably, of most teleosts) OT; other cortical structuresin fishes grow differently, and the tectum of the amniotesalso follows a different (tangential) morphogenetic pat-tern.

The very peculiar geometry of growth in the medakatectum, where proliferation is restricted to a marginalzone, makes it quite easy to recognize, with in toto ISHtechniques, if a gene is expressed in the proliferativeneuroepithelium, or in zones where cells are differentiat-ing (Fig. 5C). The medaka tectum therefore appears anexcellent system to study the genetic regulation of cellproliferation in a cortical structure.

ACKNOWLEDGMENTS

We thank Dr. S.S. Easter for his helpful comments andscientific advice and Dr. S. Brown for careful reading of themanuscript. We are also grateful to Dr. A. Shima for thekind gift of medaka strains, to Mrs. P. Lafaux and M.

Vandeputte for skillful maintenance of the fish facility, andto Mr. F. Fort for excellent photographic assistance.

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Fig. 11. The building of different cortical (layered) structures.A: Schematic drawing of the building of the medaka optic tectum.B: Schematic drawing of the development of the mammalian cerebralcortex. In the medaka (and also in the trout and goldfish; see text),proliferation occurs in a marginal zone (mpz), and the tectum is builtby addition of columns of cells spanning the whole thickness of thestructure. Thus, cells in a tectal radial row all have the samebirthdate. Radial migration is proposed to occur from the deep (pgz) tothe external (cz and sz) layers, perhaps following radial glia (rg). Thisis a ‘‘radial morphogenetic pattern.’’ In mammals, layers are built by aproliferative neuroepithelium located underneath the developing struc-

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Morphogenesis of the optic tectum in the medaka (Oryzias latipes): A morphological and molecular study, with special emphasis on cell proliferation