1. Developmental Biology
  2. Evolutionary Biology
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Bichir external gills arise via heterochronic shift that accelerates hyoid arch development

  1. Jan Stundl
  2. Anna Pospisilova
  3. David Jandzik
  4. Peter Fabian
  5. Barbora Dobiasova
  6. Martin Minarik
  7. Brian D Metscher
  8. Vladimir Soukup  Is a corresponding author
  9. Robert Cerny  Is a corresponding author
  1. Charles University in Prague, Czech Republic
  2. National Museum, Czech Republic
  3. Comenius University in Bratislava, Slovakia
  4. University of Vienna, Austria
Research Article
Cite this article as: eLife 2019;8:e43531 doi: 10.7554/eLife.43531
5 figures and 1 additional file


External gills of the Senegal bichir derive from the accelerated epidermal outgrowth of the hyoid segmental origin.

(A) Budgett’s illustration (Kerr, 1907) of a 3 cm long bichir larva with prominent external gills (exg). (B) Lateral view of an early pharyngula stage, SEM image showing external gills and cement glands (asterisk) as the first forming cranial structures. (C) SEM image of an early neurula stage with emerging bulge within the hyoid domain (hd). (D) Hoxa2 expression in the neural tube at the level of the presumptive hyoid arch. (E, G) SEM images of a tailbud embryo with external gills anlage. (F, H) Hoxa2 expression pattern in a tailbud stage, with highlighted position of external gills. (I–L) SEM images showing developmental morphogenesis of external gills. (C–F, I–K) Dorsal view. (G–H, L) Lateral view. e, eye primordium; ot, otic vesicle; r3, rhombomere 3; r5, rhombomere 5.

Accelerated formation and heterochronic development of the hyoid neural crest cells supply mesenchyme for the bichir external gills.

(A, D, G) Sox9 expression pattern in NC cells, from neurulation until early tailbud stages, dorsal views. Notice that the population of hyoid NC cells (marks as H) forms very early, and it later represents the most prominent cranial NC stream. (B–C, E–F) Sox9 expression pattern in the mandibular and the hyoid domain, respectively, transversal sections. White arrowheads mark the ventral position of the NC cells. Dotted lines represent boundaries of neural- (red) and non-neural (yellow) ectoderm. DAPI (blue) shows cell nuclei. (H) Pseudocolored SEM image, lateral view on an embryo with the partially removed surface ectoderm (blue). NC cells are green, notice the amount of hyoid NC cells. Mesodermal mesenchyme is reddish, endodermal pouches are yellow, and the neural tube is violet. (I–K) Sox9 antibody visualizes individual neural crest cells. Lateral views, with small insets representing dorsal views. Black arrowheads in I show the advanced position of the hyoid NC cells. (L–O) Hyoid NC cell fate mapping (DiI red). Superimposed fluorescent and dark-field images at successive stages of development. (L) Lateral view, stage 25 embryo showing the hyoid NC stream. Small inset (dorsal view) represents an embryo at stage 20 immediately after the focal DiI injection into the rhombomere 4 (r4). (M–N) DiI signal at developing external gills, dorsal views. (O) Transversal section through the external gill (exg) at the level indicated in O. White arrow shows DiI signal in the primary branch of the external gill. Asterisk, cement gland; e, eye primordium; H, hyoid NC stream; Ma, mandibular NC stream; np, neural plate; ot, otic vesicle; r3, rhombomere 3; r4, rhombomere 4.

The premature differentiation of the external gill-associated cranial muscle complex in the Senegal bichir larva.

(A–C) Dorsal view on bichir embryos, developing skeletal muscles are revealed by 12/101 antibody (red). The red signal in A (st. 27) refers to the post-otic somites. The first cranial muscle is associated with the external gills (B, stage 29). (C) Superimposed fluorescent and SEM image showing the context of the external gill muscles. (D) Transversal section through the external gills at the level indicated in B. DAPI (blue) stains cell nuclei. (E, F) Stage 30 bichir embryo, lateral view with (E) cranial nerves fibres labeled with anti-acetylated tubulin, and with (F) cranial muscles stained with 12/101 antibody (red). (G) Stage 33 bichir embryo, lateral view, with developed cranial muscles stained with 12/101 antibody (red). Asterisk, cement gland; am, adductor mandibulae; ah/ao, complex of adductor hyomandibulae and adductor operculi; b, brain; ba, branchial arches; bm, branchiomandibularis; cd,constrictor dorsalis; cement gland; e, eye primordium; lb/db, complex of levator branchiarum and depressor branchiarum; hh, hyohyoideus; ih, interhyoideus; im, intermandibularis; ot, otic vesicle; pf, pectoral fin; y, yolk; V., nervus trigeminus; VII., nervus facialis.

Figure 4 with 2 supplements
Considerable expansion of the hyoid pharyngeal endoderm contributes to the development of external gills in the Senegal bichir.

(A, F) SEM images, dorsal view of bichir embryos with developing external gills (exg), showing the level of virtual sections in B and G. Notice the correspondence of the hyoid pharyngeal endoderm (B, G) and the external gills (A, F). (B–D, G–H) 3D models of pharyngeal endoderm (yellow) from dorsal (C, H), and lateral (D, I) view, respectively. (E, J) Transversal sections show prominent lateral expansion of hyoid pharyngeal endoderm (white arrow). (K–M) Transversal sections show wild-type expression of Fgf8, Pea3, and Dusp6 (black arrow) in the external gills primordium. (N) Immunostaining of anti-activated MAP kinase antibody on transversal section of the external gills primordium. (O) Scheme summarizing Fgf8, Dusp6, and Pea3 (K–M) expression patterns in the external gills formation at stage 26. Violet indicates Fgf8 expression; blue marks Dusp6 expression in the endoderm and adjacent mesenchyme of the external gills; yellow depicts expression of Pea3 in the mesechyme of the external gills. (P–U) Inhibition of pouch-like endodermal outpocketings (P, R, T–U), dorsal view. (P–Q) Control larvae treated with DMSO develop normal pouch-like endodermal outgrowths (white arrow). (R) Larvae exposed to SU5402 from stage 20 till stage 26. (S) Transversal section shows loss of external gill anlagen. (T–U) SU5402 treated larvae fixed at stage 26 and probed for Pea3 (T) and Dusp6 (U). Nuclei are stained with DAPI (blue), basal laminae with anti-fibronectin (red). White arrowheads mark spiraculum (hyomandibular cleft) and black arrowhead marks hyo-branchial pouch. Asterisk, cement gland; b, brain; green, otic vesicle; e, eye primordium; nt, notochord; ot, otic vesicle; ph, pharynx.

Figure 4—figure supplement 1
Fgf8 expression during the course of bichir hyoid arch and external gill development.

(A–J) Fgf8 expression patterns at the indicated stages from dorsal (A–E), and lateral (F–J) views, respectively. (K–O) Sections at the level of the external gills (exg). White arrowheads mark spiraculum (hyomandibular cleft) and black arrowheads mark hyo-branchial pouch. White arrow indicates presence of Fgf8 transcripts in the hyoid endodermal outpocketings. Asterisk, cement gland; b, brain; nt, notochord; ph, pharynx.

Figure 4—figure supplement 2
Expression patterns of bichir Fgf8 and transcriptional readouts of Fgf signaling, Dusp6 and Pea3.

(A, D, G, J) Fgf8 expression patterns at the indicated stages from dorsal (A, G), and lateral (D, J) views, respectively. (B, E, H, K) Dusp6 expression patterns at the indicated stages from dorsal (A, G), and lateral (D, J) views, respectively. (C, F, I, L) Pea3 expression patterns at the indicated stages from dorsal (A, G), and lateral (D, J) views, respectively. Asterisk, cement gland; exg, external gills.

Bichir embryos diverge from the common anteroposterior differentiation scheme by accelerated development of the entire hyoid segment.

(A, B) A cartoon of cranial neural crest migration (green), the first mesoderm (red), and pharyngeal pouches (yellow) in a typical vertebrate (A) and a bichir (B). Top are left lateral views, below are left horizontal sections. (A) In vertebrates, the sequential anteroposterior formation of cranial segments is well conserved, including pharyngeal pouches and cranial neural crest streams. (B) In bichirs, the entire hyoid segment is accelerated with earlier formation of the endodermal, mesodermal, and neural crest tissues, what constitutes a developmental basis for the appearance their external gills. Surface ectoderm in horizontal sections is shown in blue and primitive gut in ochre; B, branchial NC stream; H, hyoid NC stream; Ma, mandibular NC stream; pp I.-pp VI., pharyngeal pouches.


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All data generated and analysed during this study are included in the manuscript and providing files. All sources are cited in the Methods chapter.

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