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.