Conservation, Development, and Function of a Cement Gland-Like Structure in the ﬁsh Astyanax Mexicanus

Conservation, development, and function of a cement gland-like structure in the ﬁsh Astyanax mexicanus

Karen Pottin, Carole Hyacinthe, and Sylvie Rétaux1

NeD UPR2197, Centre National de la Recherche Scientiﬁque (CNRS), Institut A. Fessard, 91198 Gif/Yvette, France

Edited by Sean B. Carroll, University of Wisconsin, Madison, WI, and approved August 17, 2010 (received for review April 19, 2010) The larvae of the fish Astyanax mexicanus transiently develop to be carried out to support homology between the frog CG and a flat and adhesive structure on the top of their heads that we these organs, which appear widely diversified in number, size, have called “the casquette” (cas, meaning “hat”). We hypothesized shape, structure, and location on larval bodies. that the cas may be a teleostean homolog of the well-studied Astyanax mexicanus, the Mexican tetra, belongs to the order of Xenopus cement gland, despite their different positions and struc- characiforms. Astyanax has become increasingly popular for evo- tures. Here we show that the cas has an ectodermal origin, se- lutionary developmental studies because the species includes Bmp4 cretes mucus, expresses bone morphogenic protein 4 ( ) and many populations of blind and depigmented cavefish (CF), allow- Pitx1/2 pituitary homeobox 1/2 ( ), is innervated by the trigeminal ing the study of mechanisms of morphological and behavioral ganglion and serotonergic raphe neurons, and has a role in the evolution in adaptive context (18, 19). Here, we present evi- control and the development of the larval swimming behavior. fi Astyanax These developmental, connectivity, and behavioral functional data dence that both CF and surface sh (SF) larvae possess support a level of deep homology between the frog cement gland an adhesive organ, and we gather developmental, connectivity, and the Astyanax cas and suggest that attachment organs can and behavioral functional data to compare that organ with the develop in varied positions on the head ectoderm by recruitment frog CG, despite their major differences in morphology and po- of a Bmp4-dependent developmental module. We also show that sition. We also show that the attachment organ of the cichlid the attachment organs of the cichlid Tilapia mariae larvae display Tilapia mariae larvae shares some of these features. some of these features. We discuss the possibility that these highly diversified attachment glands may be ancestral to chordates and Results have been lost repetitively in many vertebrate classes. A Sticky, Mucus-Secreting, and Transient Organ on the Head of Astyanax Larvae. In the course of our studies on Astyanax,we bone morphogenic protein 4 | pituitary homeobox 1/2 | trigeminal | fish observed a peculiar structure located on top of the dorsal brain, behavior | homology overlying the midbrain–hindbrain boundary (Fig. 1 A–C, live larvae), which is not seen in other model fishes such as zebra- any vertebrate aquatic larvae possess an adhesive organ fish or medaka. We have called that structure the “casquette” Mthat secretes sticky mucus. The most popular among these (cas, meaning “hat”). The cas is very sticky and retains any par- organs is the amphibian cement gland (CG), which has been ticle in the swimming medium (Fig. 1B). It is a transient structure, studied widely from functional and developmental points of view, visible as a thickening of the epidermis as early as 20 h post- mainly in Xenopus (1, 2). The mature gland is composed of elon- fertilization (hpf) and well seen from 24 hpf (hatching time for gated secretory cells with basal cells underlying them (3). This Astyanax)to∼50 hpf. The cas is composed of elongated and organ allows the newly hatched larvae to hang motionless from parallel large cells containing a basally located nucleus and nu- the water surface or from plant leaves before they can swim or merous granules (Fig. 1C). These elongated cells are supported feed. Because it is innervated by the trigeminal nerve, the CG by a thin monolayer of small cells with horizontally oriented nu- mediates a stopping response when the larva encounters a surface clei that we interpret as basal supporting cells (Figs. 1H and 2 J or is safely attached (4, 5). It thus is a transient organ that fulfils and K). The cas undergoes massive and specific cell death (Fig. both an attachment and a mechano-sensory function. The Xenopus CG derives from the outer ectoderm at the 1D) but no proliferation (Fig. 1E) and disappears completely anteroventral side of the head and is visible by pigmentation by after 3 d of development. The high granule content and adhesive neurula stage (6). By that time, the CG starts to express proteins nature of the cas suggested that it may secrete substances such as such as anterior gradient (xAgr) that are used as CG markers and mucus. Indeed, it is stained selectively and strongly by the pe- are important for the gland’s secreting function (7, 8). The posi- riod acid-Schiff (PAS) histological method, which labels mucus- tioning and inducing of the CG relies on the expression of or- secreting cells (Fig. 1 F–H). The intensity of PAS staining of the thodenticle homeobox 2 (Otx2) and pituitary homeobox (Pitx) cas increases as development proceeds (Fig. 1 F–H), and the la- genes (2, 8–12), whereas adequate levels of bone morphogenic beling is concentrated at the apical/external surface of the elon- protein 4 (Bmp4) signals are permissive for its development (13). gated cas cells (Fig. 1 G and H). There are no differences between The study of the amphibian CG has been confined mainly to CF and SF larvae in these events, so the data presented are taken the Xenopus laevis species, although a recently published, com- collectively from both forms, except as otherwise indicated. plete survey of the existence and morphology of the CG in 20 anuran species discusses the CG with regard to the species’ hab- itat, behavior, and mode of development (14). The balancers of Author contributions: K.P. and S.R. designed research; K.P., C.H., and S.R. performed re- urodele amphibians have been suggested to be related evolu- search; K.P., C.H., and S.R. analyzed data; and S.R. wrote the paper. tionarily to the anuran CG, and some support for this idea comes The authors declare no conflict of interest. from the description of trigeminal innervation and Otx5 expres- This article is a PNAS Direct Submission. sion in the balancers (5, 15, 16). Among other chordates, a similar Data deposition: The sequences reported in this paper have been deposited in the Gen- transient adhesive organ has been described in ascidians (17) and Bank database (accession nos. HQ225730 to HQ225734). in some fishes belonging to the basal actinopterygians and to the 1To whom correspondence should be addressed. E-mail: [email protected]. cichlids (Discussion). However, relevant comparative morpho- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. logical, molecular, developmental, and functional studies have yet 1073/pnas.1005035107/-/DCSupplemental.

17256–17261 | PNAS | October 5, 2010 | vol. 107 | no. 40 www.pnas.org/cgi/doi/10.1073/pnas.1005035107 Downloaded by guest on October 2, 2021 Fig. 2. The cas expresses high levels of Bmp4 and Pitx genes. In all panels, a thick black arrow indicates the cas. (A–G) Bmp4 expression (blue) in toto (A, B, D, and E) and on sections (F and G). C′ Inset shows a high magnification of a section double-labeled for Bmp4 (black) and Hnk1 (green). (A and B) Lateral views. (D and E) Dorsal views. (H–K) Pitx1/2 expression in toto (H, dorsal; I, lateral) and in sections (J and K). In H the dashed line delineates the cas contours. In I the asterisks indicate the expressing lamina below the posterior cas. In J and K (high power), DAPI-stained nuclei are blue. White arrows point to the nuclei of secretory cells; white arrowheads point to nuclei of basal cells. Stages are indicated. Cp, choroid plexus; e, eye; ot, otic Fig. 1. The cas is a sticky, transient, and mucus-secreting structure. (A) vesicle; rl, rhombic lip; rp, roof plate; tb, tailbud; tc, tela choroida. General view and (B) close-up of the cas (black arrow in A) of a live CF embryo at 24 hpf. The asterisk indicates a particle adhering to the cas. (C) terior aspect (Fig. 2 D and E). Observation of transverse sections Nomarski image of a live CF at 36 hpf. White arrows show basally located nuclei of elongated cells; arrowheads point to basal cells. Both types of cells shows Bmp4 expression in both the elongated and the basal cas and their nuclei are delineated by dotted lines. Note the high density of cells, as well as in the forming and underlying choroid plexuses granules. (D) LysoTracker live fluorescent imaging of an SF at 40 hpf. Cell (neurohemal organs that secrete the cerebrospinal fluid) of the death is seen in the cas (white arrows). (E) Section from a 36-hpf SF stained brain ventricle (Fig. 2 F and G). for proliferating cell nuclear antigen (PCNA, brown). (F–H) Sections of CF cas We also tested whether the cas expresses Pitx genes, consid- after PAS staining (pink) at indicated stages. (G) High magnification image. ered as CG specifiers in Xenopus (1). We cloned Pitx1- and Pitx2- The asterisk indicates the PAS-negative adjacent skin. (H) DAPI nuclear type sequences, both of which showed complex, placodal-related fi staining is blue. In all gures, anterior is to the left, and dorsal is up. Cp, expression patterns at neurula stages. Slightly later, when the cas choroid plexus; e, eye; fb, forebrain; hb, hindbrain; mb, midbrain; mhb, midhindbrain boundary; ot, otic vesicle; pi, pineal gland. is well developed, Pitx1 and Pitx2 are heavily transcribed in the cas, exclusively in its posterior half, in a lamina underlying the large cas cells (Fig. 2 H and I). Examination of sections coun- The Casquette Expresses High Levels of Bmp4 and Pitx, but Not terstained with DAPI demonstrates that Pix1 and Pitx2 expres- Anterior Gradient 2 or Muscle Segment Homeobox (Msx) Genes. sion is restricted to the small basal cas cells underlying the large The characteristics described above suggested that the Astyanax secreting cells (Fig. 2 J and K). cas may be an equivalent to the Xenopus CG, although the two Agr2 (also known as “XCG”) is a specific and early CG marker structures differ critically in the position where they develop. in Xenopus (8). Astyanax Agr2 was transcribed exclusively at the Moreover, the cas, being an ectodermal thickening closely asso- rostral tip of the embryonic head, just overlying the yolk, in the ciated with the neural tube, also may show placodal-like prop- position of the hatching gland, but never in the cas itself (Fig. erties. Thus, we sought to obtain genetic developmental data to S2). Msx homeodomain-containing genes are downstream effec- test our hypothesis. We analyzed expression of Bmp4, anterior tors of Bmp signaling in several organs, including the choroid gradient 2 (Agr2), Msx1/2, and Pitx1/2 transcripts, because they plexuses and roof plate, where Bmp4 also is expressed in Asty- are recognized CG and/or placodal-related markers in Xenopus anax. We thus asked whether Msx factors are part of the cas gene and other teleost species, respectively (cloning and phylogeny are regulatory network. Neither MsxE/Msx1 nor MsxD/Msx2 is ex- shown in Fig. S1). pressed in the cas, although both genes show strong expression in Bmp4 is one of the earliest molecules expressed in the Xen- the roof plate and choroid plexuses as well as in the tela choroida opus CG primordium, at the anterior tip of the animal axis at the covering the hindbrain (Fig. S3). neurula stage (13). In equivalent-stage Astyanax embryos, Bmp4 transcripts are found in a very different position, because they The Casquette Is Innervated by Fibers Originating from the Trigeminal localize to the ectoderm overlying the anterior neural tube ap- Ganglion. In Xenopus, the CG is innervated by the trigeminal nerve, proximately at the midbrain level, i.e., at the presumptive cas a connection that allows the tadpole to stop swimming when it level (Fig. 2 A and B) (16 hpf corresponds to 15 or 16 somites at encounters an obstacle. Observation of live Astyanax larva under 23 °C). Despite their peculiar aspect and localization, Bmp4- Normarski optics identified the large trigeminal ganglion neu- expressing cells are not migrating neural crest cells, because they rons and detected thick fibers running dorsally toward the basal do not express the crest marker Hnk1 (Fig. 2 C and Inset C′). surface of the cas (Fig. 3A). To confirm this observation, we Later on, the Bmp4 pattern becomes complex (note expression stained neurons and axonal projections of the developing ganglia in the roof plate in Fig. 2D), but one prominent site of expression with an mAb recognizing neurofilaments, mAb 3A10 (20). Neu- EVOLUTION always is the developing and mature cas, particularly in its pos- rons of the trigeminal ganglion were stained by 3A10 and dis-

Pottin et al. PNAS | October 5, 2010 | vol. 107 | no. 40 | 17257 Downloaded by guest on October 2, 2021 Fig. 3. The cas is innervated by the trigeminal ganglion. In A, C, and E,the cas contours are indicated by a dashed line. (A) Nomarski photograph of a live CF larva at 36 hpf. A trigeminal neuron (tg) sends a thick fiber (arrows) toward cas cells,which are recognizable by their size and granule content. (B–F) Neurofilament staining (brown) in toto (B, C, and E)orofsections(D and D’). In D and D’, arrows point to fibers entering the lateral part of the cas. In E,cas cells show degenerating aspect and background coloration. Trigeminal fibers have a collapsing punctuate aspect (arrowheads), are thin, and seem to re- ′ tract from the cas. (F) Immunostaining against serotonin (brown) on a dis- Fig. 4. Cas ablation affects larval swimming behavior. (A ) Completeness of ′ ′′ – sected brain of a CFat 36 hpf. Arrowheads follow the 5-HT fasciculated fibers, cas removal on an SF embryo at 24 hpf (A , control; A , poked control). (B E) and asterisks indicate bouton-like terminals underlying the cas. Tracking of swimming behavior of SF (B)orCF(E). Experimental conditions and time of recordings are indicated. C, D, and F show quantification of swimming activity in SF or CF in controls (cont, black bars), poked controls played typical fibers of nerve V running anteriorly around the eye (pok, gray bars) or after cas ablation (abl, white bars). Asterisks indicate toward the face and jaws and also a number of “nonconventional” statistical significance after Student’s t test (*P < 0.05; **P < 0.005; ***P < projections traveling dorsally toward the cas (Fig. 3B). These fibers 0.001), and error bars indicate SEM. entered the lateral aspect of the cas, exhibiting axonal branching and swellings and encircling the base of the cas cells (Fig. 3 C, D, “ ” “ and D′). Cas innervation by the trigeminal ganglion was most de- tomical relationship between the cas system and the 5-HT system” in Astyanax. Surprisingly, we found a conspicuous innervation veloped around 36 hpf and regressed in the following hours. At 48 of the cas by 5-HT fibers originating in the hindbrain raphe nucleus hpf, when the cas itself is degenerating and reduced in size, tri- (Fig. 3F). These fasciculated fibers establish a pathway that runs geminal fibers showed a retracting and collapsing aspect (Fig. 3E), dorsally toward the cas and is highly unusual for raphe 5-HT pro- suggesting a parallel transient nature for both the target and the jections. These data show that the cas, albeit transient, is strongly fibers that innervate it. interconnected to the nervous system and may play more complex The Casquette Regulates Astyanax Larval Swimming Behavior and roles in the control of larval behavior than expected. Receives Serotonergic Innervation. Because the cas is innervated, Adhesive Gland of the Cichlid T. Mariae: Similarities to and we next sought to challenge its mechanosensory function. To this Astyanax fi Differences from the Casquette. We searched the litera- end, we ablated the cas at 24 hpf, when the structure is suf - ture for descriptions of attachment organs in other fish species. ciently developed to ensure a complete removal and before tri- Among teleosts, several species of cichlids have long been reported geminal innervation is established (Fig. 4A). Cas ablation did not to possess such specializations (23, 24). We obtained eggs and affect survival in laboratory conditions (n = 75 ablated embryos, hatched larvae after natural breeding of T. mariae specimens from survival rate 92% 7 d after operation), and the cas did not re- our laboratory hall aquarium. Just after hatching, these larvae have generate following ablation. The larval swimming behavior then six large glands on their heads: one pair on top of the forebrain and was analyzed 4, 24, and 48 h postablation (i.e., in larvae 28, 48, two pairs on top of the midbrain (Fig. 5A). The number, the lo- and 72 hpf, respectively). We compared the time spent swimming cation, and the conical shape of these glands (Fig. 5B) are different and the distance swum during 10-min periods in cas-ablated, cas- from the Astyanax cas, but they show identical sticky properties poked (sham ablation; SI Materials and Methods), and control (asterisk in Fig. 5A) and also are transient. Tilapia larvae use them ′ ′′ larvae (Fig. 4 A and Insets A and A ). The results were quan- to form rosettes (Fig. 5C) and to stay grouped under parental care, titatively different for SF and CF and therefore are presented in the vicinity of the nest built by the parents, for 2 or 3 d after separately. Tracking patterns show that the basal swimming ac- hatching (details are given in the legend of Fig. S4). We next tivity of control CF is about 10 times higher than that of SF performed neurofilament staining on Tilapia larvae. The three (compare Fig. 4 B vs. E and C vs. F; see scales). In both pop- pairs of organs were innervated by a complex pattern of trigeminal ulations, however, the effect of cas ablation was the same: a sig- branches (Fig. 5D). In particular, each of the two pairs of adhesive nificant increase in swimming activity 4 h and 24 h after ablation glands located above the midbrain received one major, thick in SF and 24 h after ablation in CF (Fig. 4 C, D, and F). Larvae branch together with thinner fibers, which formed bouton-like whose cas was poked but not ablated behaved like controls, swellings at the base of the glands (Fig. 5E). This pattern was demonstrating that the operation had no nonspecific effect on symmetric, because the left ganglion was connected to the left the larval behavior. These results are consistent with an inhib- gland, and vice-versa, and we did not observe fibers crossing the itory role of the cas on larval swimming activity, at the stages when midline. Thus, innervation by the trigeminal ganglion seems to be the adhesive organ is well developed and functional. At a later adefining feature of CG-like organs. stage (48 h after operation, when the cas normally is regressed), cas ablation no longer has any effect, and the swimming activity of Discussion all of the larvae tends to increase. We present morphological, developmental, hodological, and In zebrafish, the serotonergic (5-HT) neurotransmitter sys- functional behavioral evidence supporting the homology of the tem regulates the establishment of locomotor behavior (21, 22). As Astyanax casquette and the Tilapia adhesive glands with the a first insight into this issue, we explored the possibility of an ana- Xenopus cement gland. This molecular and functional analysis

17258 | www.pnas.org/cgi/doi/10.1073/pnas.1005035107 Pottin et al. Downloaded by guest on October 2, 2021 Fig. 5. Morphology, function, and trigeminal innervation of Tilapia mariae adhesive glands. (A) General view of the head and (B) close-up of the conical- shaped glands (three pairs, indicated by arrowheads) of live Tilapia 4–5 dpf. The asterisk indicates a particle sticking to the gland. (C) A rosette formed by nine larvae. (D and E) Neuroﬁlament staining. In D, arrowheads point to the right pair of glands over the midbrain; arrows point to the thick branches of nerve V (trigeminal) innervating each gland. In E, arrows point to large, bouton-like structures at the base of the glands. E, eye; fb, forebrain; hb, hindbrain; mb, midbrain; ot, otic vesicle; tg, trigeminal ganglion.

of adhesive organs in teleosts allows us to discuss the ancestral dium, a change in expression pattern that may be imposed by nature of the CG in chordates. Bmp4 (27). It will be informative to analyze further the expression patterns of Otx family members in the head ectoderm of Morphological Diversity of Adhesive Glands Across Species. The Astyanax (one CG on top of the head) and in Tilapia (six CGs significantly different appearance of the cas/CG in the two fish distributed on the head) to reinforce with evolutionary devel- we have studied illustrates the great diversification of adhesive opmental arguments the hypothesis that Otx2 induces CG for- organ(s) in the chordate species that possess one (references are mation in a Bmp4-positive competent ectodermal domain. In given in the legend of Fig. S4). In these species, the form of the this respect, an additional argument worth considering is that in glands can vary from a conical crater (Tilapia) to a fold or groove, ascidians (nonvertebrate chordates; Fig. S4) the papilla, which a horseshoe, or a flat shape (Astyanax). They can be paired (Ti- derives from the anterior ectoderm and is used to attach to lapia) or unpaired (Astyanax). Their number can range from one the substrate, expresses both Bmp4 and Otx (Aniseed database, to eight. Their morphogenesis often includes invagination of http://139.124.8.91/). the primordium (most frogs, or Tilapia shown here) but some- In many organs, Msx transcription factors act downstream times does not (X. laevis, or Astyanax shown here). Their po- of Bmp4 signaling (e.g., in the development of teeth, epidermis, sition is not a defining feature, because it can vary from ventral, to facial mesenchyme, limbs, or neural tube dorsal midline) (28– the mouth in X. laevis [but not in all anuran species; e.g., the frog 31). In particular, in mammals, Msx1 colocalizes with Bmp4 in Hyla minuta has two CG on each side of the mouth (14)], to the choroid plexuses that develop adjacent to the roof plate of a more posterior dorsal or lateral position on the head. Hence the the neural tube. Choroid plexus development is almost unknown names these structures were given in the various species in which in fish, but a pioneer study using GFP enhancer-trap zebrafish they were described vary: “CG,”“adhesive organ,”“sticky gland,” lines (32) allowed us to identify unambiguously the Bmp4- and “attachment gland,”“sucker,”“sucking disk,” and so forth. In Msx1/2-expressing structure below the cas as the Astyanax cho- Astyanax we named the structure “casquette” (“hat”) because it roid plexuses. This Bmp–Msx relationship therefore is conserved is unusual when compared with other model fish (zebrafish or in the fish roof plate and choroid plexuses but not in the cas medaka), is located on the top of the head, and is flat. itself. We cannot exclude the possibility that another Msx member [there are five Msx genes in zebrafish (33)] is expressed in the cas. Versatile Developmental Module for CG Formation. The cellular Indeed, the involvement of a Bmp4/Msx2 pathway in the positive structure of the Astyanax cas (two layers, one of large, elongated control of cell death during mammalian embryogenesis has been cells and one of small, supporting cells), its ectodermal origin, and reported repeatedly (30, 34, 35) and would be particularly likely in its mucus-secreting and adhesive properties are features favor- the context of the cas, which is a very transient and rapidly apo- ing its homology with the well-studied Xenopus CG. However, ptotic structure, as we have shown. common genetic-specification mechanisms during embryonic Agr2 is a thioredoxin-like domain containing disulfide isom- development are considered more definitive arguments support- erase involved in the processing of secreted molecules, including ing homologies. Therefore we cloned and analyzed in Astyanax mucin (36). As such it is an excellent marker of the CG in Xen- the expression of several factors that are hallmarks of CG de- opus. In zebrafish, which lacks a CG, Agr2 is expressed first in velopment in Xenopus. ectodermal derivatives such as the otic vesicle, the hatching gland, Bmp4 is considered a permissive factor for Xenopus CG de- skin mucus cells, and olfactory epithelium and then in the endo- velopment. Its expression defines a competent region where Otx2 dermal gut (37). We found in Astyanax Agr2 is expressed in the is able to induce CG formation (13). Our findings that Astyanax hatching gland, as in zebrafish, but not in the cas, as in Xenopus. Bmp4 is expressed at neurula stage at a very unusual position There are three Pitx genes and several splice-variant forms that corresponds precisely to the location of the future cas and for Pitx2, which are differentially involved in “anterior extreme” that Bmp4 expression persists at high levels in the mature gland (i.e., pituitary, stomodeal, and CG) development in Xenopus (1). cannot be a mere coincidence. These data are consistent with the Only two, Pitx1 and Pitx2c, are expressed in the CG, and their idea that Bmp4 is permissive for CG formation but conflict with misexpression induces an ectopic CG (38). Moreover the ex- the proposal that the CG forms “at a conserved anterior posi- pression of different members also differs between germ layers tion, where embryonic ectoderm and endoderm touch” (25). In (inner or outer ectoderm layer or endoderm). Pitx1/2 expression fact, the position of the CG is not conserved even among dif- in the basal supporting cells of the posterior half of the cas is an ferent frog species (14). The CG happens to correspond to the additional element that supports a common genetic specification “anterior extreme” in Xenopus (1, 2), but its position probably is for the cas and the Xenopus CG. not a defining feature—although the anterior position is indeed We also show that CG innervation by trigeminal fibers is a conserved feature for the stomodeum (26). Of note, in Eleu- a characteristic Astyanax and Tilapia share with Xenopus. Given therodactylus coqui, a direct-developing frog which lacks a CG, the changing position and number of the CGs in these species, EVOLUTION Otx2 expression does not expand anteriorly to the CG primor- a neurodevelopmental correlate of this finding is that axonal

Pottin et al. PNAS | October 5, 2010 | vol. 107 | no. 40 | 17259 Downloaded by guest on October 2, 2021 guidance cues probably are provided by the target, which is the one species of mormyre, Hyperopisus bebe, possesses six glands on CG itself. In Xenopus, CG ablation results in failure of trigeminal its larval head (40). Attachment glands have been reported re- axons to stop and arborize (20). This attraction is driven by peatedly in the cichlids: various Tilapia species (T. tholloni, BDNF, which is expressed in the CG and provides an attractive T. mariae, T. nilotica, T. macrocephala) (24), the angelfish Pter- signal to trigeminal growth cones (39). Such a system, in which ophylum scalare (23), Etroplus maculatus (45), and Cichlasoma the guidance is provided by the target, appears very flexible and dimerus (46) have three pairs of glands. Among cichlids, most but would allow trigeminal fibers to reach their targets anywhere on not all mouth-brooding species (which originate from substrate- the head of the larva. brooding species) have lost their cement glands: Tropheus moorii lacks a CG (47), but Geophagus jurupari still has one (48). Function of the CG and Larval Behavior. The proposed general In Astyanax, we have found a single, median CG-like organ in function of the CG in aquatic larvae is their protection and both SF and CF larvae, indicating that, unlike eyes and pigmen- safety. This idea comes from observations of animals in the wild. tation, this gland has not been lost after 1 million years of CF Using their sticky glands, larvae can stay attached safely to leaves evolution. It is hard to imagine, however, that its function could be or to the water surface (frogs) or can stay grouped together exactly the same in the two populations. Extrapolating from what under parental care (ref. 24 presents data for Tilapia). In this way we know about their respective behaviors, we imagine that the SF larvae hide from predators and save energy before they can feed. CG is used to attach to a safe place hidden from predators, whereas However, the CG has properties beyond simple adhesive prop- the CF CG is used to hang from the water surface to escape from erties. Here we show that neural connection also is a feature the adults, which are exclusive bottom-feeders. Interestingly, the lit- Astyanax cas, and Tilapia CG share with their Xenopus homolog, erature carries one example of another characiform, Sarcodaces because they all receive fibers from the trigeminal ganglion. odoe, with a single median gland on the head (40). Electrophysiological recordings in Xenopus have shown that a It seems unlikely that a CG would have arisen independently stopping response is elicited upon excitation of pressure-sensitive in at least 11 distantly related groups of chordates (Fig. S4), sug- trigeminal receptors that innervate the CG. These receptors gesting a scenario in which the CG is an ancestral chordate char- release glutamate to stimulate brainstem GABAergic reticulo- acter and has been lost in many groups. It is impossible, however, spinal neurons, which in turn inhibit spinal neurons to turn off with present methods, to rule out the possibility of parallel evo- the central pattern generator for swimming (4, 5). Here, we took lution. We show here that the actinopterygian CG shares with the advantage of the fact that the Astyanax cas is single, accessible, CG of amphibians and urochordates not only adhesive properties and relatively easy to remove without affecting embryo survival but also a genetic specification program during development, to- to challenge directly its function on the regulation of larval be- gether with neural connection and function. These shared char- havior. Our results indicate a powerful inhibitory function of acteristics constitute strong support for homology (and therefore the cas on swimming behavior, because cas removal induces an ancestrality) among all these organs/glands. The various shapes ∼10-fold increase in swimming activity in both SF and CF 24 h and forms of the teleost CGs do not contradict their being ho- after ablation. Our behavioral analysis in fish therefore is con- mologous, because they contain the same type of secretory, mucus- sistent with the previous electrophysiological analysis in Xenopus producing, elongated cells and are all ectodermal derivatives. and shows that the functional connectivity of the CG is con- Their varying numbers and positions are more difficult to reconcile served in fish and amphibians, adding to the evidence supporting with true homology, even if they always emerge from the ante- homology between their organs. rodorsal part of head ectoderm and never in a ventral or post- We also were surprised to discover a 5-HT innervation of the erior position. This variation in number and position suggests cas. Such innervation has not been described in any other spe- an interesting example of deep homology (49) in which a Bmp4- cies, including Xenopus. Serotonin recently has been identified as dependent genetic network would be recruited to various parts of a key neurotransmitter that controls swimming behavior in the head ectoderm to build analogous structures. If Bmp4 really zebrafish (21, 22)—but zebrafish larvae do not possess a CG. Our is the permissive factor for CG formation, then the precise regu- finding therefore leads to many interesting questions about the lation of its expression in a particular area of the head non- conservation and the function of this CG/5-HT connectivity in neural ectoderm is the key to the morphogenesis of a CG in various species. a particular location. This supposition fits well with the cur- rent ideas that form evolves largely by altering the expression of Is the CG an Ancestral Larval Chordate Character? The avail- functionally conserved molecules and that the modularity in cis- able molecular data on anuran CG are derived exclusively from regulatory transcriptional control of such molecules with pleio- studies of X. laevis. However, most frogs possess a CG, even tropic and morphogen effects (such as Bmp or Pitx) is responsible though some species that are direct developers, hatch late, or for morphological variations in evolution (50, 51). A case study spend the posthatching period in foam nests do not (14), most recently has reported a Pitx enhancer that is repeatedly responsible probably because they have lost it. In urodeles (anuran sister for pelvic spine reduction in populations of freshwater stickle- group), there is no CG per se, but the balancers are de- backs (52, 53). In the case of the CG, subtle changes or loss in velopmentally related, if not homologous, to the CG (16). By a regulatory element(s) directing Bmp4 expression to the head contrast, there is no CG-like organ in amniotes. Among non- ectoderm at neurula stage would generate the diversity in CG tetrapod sarcopterygians, a larval CG has long been described position, size, and form across species. in some species of lungfish/dipnoi such as Lepidosiren paradoxa and Protopterus annectans (40, 41). Materials and Methods Among other chordates, the adhesive papilla of ascidians is Astyanax and Tilapia samples were obtained from our Fessard Institute fish an important developmental feature in many Ciona larvae (42, facility and processed using classical histological and immunological proce- 43). The papilla expresses Bmp4 and Otx, is neurally connected to dures. Molecular cloning, in situ hybridization, and phylogenetic recon- the sensory vesicle (17), and may be involved in triggering meta- structions were performed as previously described (54). Cas ablations were morphosis. Among basal actinopterygians, a CG was described performed on anesthetized embryos, and their swimming behavior was long ago in several species (sometimes called “ganoids”) (44). recorded individually during 10-min time periods. Detailed procedures are These species include sturgeons (e.g., Acipenser ruthenus), bichirs given in SI Materials and Methods. (e.g., Polypterus senegalus), and bowfins (e.g., Amia calva). Of ACKNOWLEDGMENTS. We thank D. Stock (University of Colorado, Boulder, note, sturgeons are the only species in which CG do not disappear, CO) for the Bmp4 probe, P. Herbomel for Nomarski imaging, Y. Elipot for because they transform into the adult barbels. Among teleosts, 5-HT staining, S. Père for taking care of our Astyanax colony, L. Legendre

17260 | www.pnas.org/cgi/doi/10.1073/pnas.1005035107 Pottin et al. Downloaded by guest on October 2, 2021 and M. Simion for their idea of decorating the laboratory hall with an la Recherche (ANR-Neuro [Midline]) and the Centre National de la Recherche aquarium hosting several species of cichlids, and F. Bourrat for critical read- Scientiﬁque (S.R). and a Ph.D. Fellowship from the French Ministry of Re- ing of the manuscript. This work was supported by the Agence Nationale de search and ARC (K.P.).

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