The skate spiracular organ develops from a unique neurogenic placode that is distinct from lateral line placodes

Abstract

The spiracular organ is an epithelial pouch or tube lined with mechanosensory hair cells that is found embedded in the wall of the spiracle in many non-teleost jawed fishes. It is innervated via a branch of the anterior lateral line nerve and usually considered a specialised lateral line organ, despite its presumed function as a proprioceptor for jaw movement. It is homologous to the paratympanic organ: a hair cell-lined epithelial pouch embedded in the wall of the middle ear of birds, alligators and Sphenodon. A previous study showed that the chicken paratympanic organ and its afferent neurons originate from a molecularly distinct placode immediately dorsal to the geniculate placode. Here, fate mapping in a cartilaginous fish (little skate, Leucoraja erinacea) shows that the spiracular organ derives from a previously unrecognised neurogenic placode immediately dorsal to the geniculate placode that is spatially and molecularly distinct from lateral line placodes. Retrograde labelling of the spiracular organ identified afferent neurons located within the geniculate ganglion, as reported previously for paratympanic organ afferents. These findings support the independence of this unique jawed-vertebrate mechanosensory organ from the lateral line system.

Keywords: Eya4; Sox2; Sox3; Chondrichthyan; Hair cells; Paratympanic organ.

Fig. 1.

The spiracular organ of cartilaginous fishes. MicroCT reconstruction of the head of a pre-hatchling embryonic shark (Scyliorhinus canicula). (A) The spiracle (sp) sits behind the eye; it is derived from the first embryonic pharyngeal cleft. (B-C′) The spiracular organ (SpO, magenta) is an epithelial diverticulum embedded in connective tissue between the hyomandibula (hm, blue) and the braincase (bc, lilac). The jaws, consisting of Meckel's cartilage (Mc) and the palatoquadrate (pq), are highlighted in green. Scale bar: 1 cm.

Fig. 2.

The spiracular organ and nearby cranial sensory ganglia in late-stage skate embryos. (A) Masson's trichrome-stained horizontal (frontal) section through the head of a S32 skate embryo at the level of the spiracle, showing the position of the SpO relative to the spiracle (sp), the geniculate ganglion (gen) and the vestibuloacoustic ganglion (va). The geniculate and vestibuloacoustic ganglia form composite ganglia, respectively, with the anteroventral (av) and anterodorsal (ad) lateral line ganglia (LLg). (B) Haematoxylin and Eosin-stained section of the S32 skate SpO. (C,D) At S32, immunofluorescence shows that (C) the SpO expresses the hair cell marker parvalbumin and the supporting cell marker Sox2 in a mutually exclusive pattern, and (D) the sensory epithelium of the SpO is innervated by nerve fibres expressing the neurofilament-associated antigen (3A10). (E-H) In situ hybridisation chain reaction (HCR) or in situ hybridisation (ISH) on sections shows that the SpO expresses the transcription factor genes (E) Atoh1, (F) Six1, (G) Eya4 and (H) Pou4f1. (I,I′) ISH on adjacent horizontal (frontal) paraffin sections at S32 shows (I) Phox2b expression in the geniculate ganglion and (I′) Eya4 expression in the anteroventral LLg. Eya4-positive cells are also scattered amongst the Phox2b-positive neurons in the geniculate ganglion. (J,J′) HCR shows Eya4 expression in the anteroventral LLg and in scattered cells within the geniculate ganglion (white arrows in J′). (K) Box and whisker plot (boxes show upper and lower quartiles; whiskers show upper and lower extremes) showing that Eya4-positive anteroventral LLg neurons (Eya4+ AV) are the same size (cell-body area) as Eya4-positive cell bodies within the geniculate ganglion (Eya4+ GEN), but both Eya4-positive cell types are significantly larger than Eya4-negative cell bodies in the geniculate ganglion (Eya4− GEN). Eya4-positive and Eya4-negative cell-body areas were measured using FIJI on sections stained post-HCR with Masson's trichrome (see L,L′). For each cell type, ten cell bodies were measured in each of three individuals, except for one individual that had only seven Eya4-positive geniculate cells. A Kruskal–Wallis H test indicated a significant difference between the different groups: χ²(2)=59.88, P<0.001, with a mean rank score of 63 for Eya4+ AV (n=30), 54.56 for Eya4+ GEN (n=27) and 15.5 for Eya4− GEN (n=30). A post-hoc Dunn's test using a Bonferroni corrected alpha of 0.017 indicated that the mean ranks of the following pairs are significantly different: Eya4+ AV versus Eya4− GEN and Eya4− GEN versus Eya4+ GEN. (L,L′) The same sections as in J,J′ stained post-HCR with Masson's trichrome. Black arrows in L′ indicate the same Eya4-positive geniculate cells indicated by white arrows in J′. (M-N′) Frontal sections through the geniculate/anteroventral lateral line ganglionic complex from two different S32 embryos after retrograde labelling of the SpO with CM-DiI. In both cases, multiple CM-DiI-positive cell bodies are present in the geniculate ganglion. ad, anterodorsal lateral line ganglion; av, anteroventral lateral line ganglion, gen, geniculate ganglion; hm, hyomandibula; sp, spiracle; SpO, spiracular organ; va, vestibuloacoustic ganglion. Scale bars: 50 µm in A; 25 µm in B-H; 25 µm in I-J′,L,L′; 25 µm in M-N′.

Fig. 3.

A putative spiracular organ placode in the skate, immediately dorsal to the geniculate placode. Given the homology of the amniote PTO and the SpO, the putative SpO placode in skate is expected to lie immediately dorsal to the geniculate (first epibranchial) placode, above the first pharyngeal (spiracular) cleft. (A-D) Whole-mount in situ hybridisation at S24 for Pax2 (A) and Sox3 (B) identifies the epibranchial placodes dorsocaudal to each pharyngeal cleft. Ectoderm immediately dorsal to the geniculate placode expresses Eya4 (C) and is distinct from Sox2-positive elongating lateral line primordia (D). Insets show higher-power views of the first pharyngeal (spiracular) cleft region. (E,E′) In situ hybridisation on adjacent transverse paraffin sections at S24 reveals a domain of Eya4-positive placodal ectoderm (red asterisk) and subjacent mesenchyme, presumably neuroblasts, immediately dorsal to the geniculate placode, here identified as a placodal source of Phox2b-positive neuroblasts in the ventral region of the adjacent ganglion. (F,F′) This domain of placodal ectoderm (red asterisk) is also a source of Tbx3-positive neuroblasts, which emigrate immediately dorsal to Phox2b-positive geniculate placode-derived neuroblasts. (G-K) Selected transverse histological sections at S24 in a caudal-to-rostral sequence, including the putative SpO placode (red asterisk and red dotted line in all sections). Successively, the sections show: (G) the geniculate placode (g) with emigrating neuroblasts; (H,I) the putative SpO placode with emigrating neuroblasts lying immediately dorsal to the geniculate placode; (J) a break in the placodal ectoderm; (K) a dorsal neurogenic placode with emigrating neuroblasts, expected to be the neurogenic pole of the anteroventral lateral line placode (av). All these regions of neurogenic placodal ectoderm contribute neuroblasts to the same ganglion. The complete series of histological sections is provided as Movie 1. (L) Schematic illustrating the spatial organisation, relative locations and gene expression of the geniculate, SpO and anteroventral lateral line placodes, and their neuroblasts, at S24. Red asterisk indicates the SpO placode. av, anteroventral lateral line placode; e, eye; g, geniculate placode; ot, otic vesicle; sp, spiracle. Scale bars: 500 µm in A-D; 25 µm in E-K.

Fig. 4.

Embryonic origin of the spiracular organ of the skate. (A-D′) Labelling of the geniculate placode with CM-DiI at S24 (A,B) does not label the spiracular organ at S32 (C,C′), but abundant CM-DiI-positive neurons are seen in the geniculate ganglion (D,D′). (E-H′) Labelling of placodal ectoderm dorsal to the geniculate placode (i.e., the putative SpO placode) with CM-DiI at S24 (E,F) results in abundant CM-DiI-positive cells within the spiracular organ (G,G′), as well as CM-DiI-positive neurons within the geniculate and anteroventral lateral line ganglia (H,H′). (I) Schematic summary of the location of and gene expression in the lateral line, SpO and epibranchial placodes in skate (left) and the PTO and epibranchial placodes in chicken (right). (J) Phylogenetic distribution of sensory hair cell-containing sense organs in vertebrates points to an origin of the spiracular organ along the jawed vertebrate stem and its independence from the lateral line system. ad, anterodorsal lateral line placode; av, anteroventral lateral line placode or ganglion; e, eye; g, geniculate placode; gen, geniculate ganglion; m, middle lateral line placode; n, nodose placode; n_1-3, nodose placodes; o, otic lateral line placode; ot, otic vesicle; p, posterior lateral line placode; pt, petrosal placode; st, supratemporal lateral line placode. Scale bars: 500 µm in A,E; 25 µm in B-D′,F-H′.