Figure 1.
Malawi Cichlids Exhibit Toothed Oral and Pharyngeal Jaws
(A) Schematic drawing (lateral view) of the generalized cichlid cranial skeleton, showing the relative location of the oral jaws (purple) and the pharyngeal jaws of PA7 (blue).
(B) Dorsal view of an alizarin red skeletal preparation of the lower pharyngeal and oral elements of a juvenile D. compressiceps (DC) showing the series of branchial (pharyngeal) arches 1–7 and ceratobrachial elements CB1–5; the white asterisk indicates the toothed pharyngeal jaw. Scale bar represents 500 μm.
(C and D) Lateral views with expression of dlx2 labeling neural crest-derived cells in the pharyngeal arches of M. zebra [MZ] (C) and D. compressiceps [DC] (D). Both cichlids are 4 dpf and to the same scale; scale bar in (D) represents 200 μm. dlx2 expression is observed throughout the arches from the mandibular arch, PA1 (black arrowheads), throughout the pharynx to the most posterior arch, PA7 (white arrowheads). dlx2 expression is also present in neural crest-derived mesenchymal cells that populate the arches (white arrows).
(E and F) Sagittal sections of MZ (5 dpf) showing expression of bmp4 in multiple regions of the developing head and pharynx. bmp4 is expressed throughout the arches in neural crest-derived arch mesenchyme for each pharyngeal arch (PA1–7), including both the mesenchyme and epithelial components of the developing teeth (black arrowheads). (E) The medial sagittal section and (F) lateral sagittal section show the gill-bearing arches (PA3–6). Both (E and F) are to the same scale; scale bar in (E) represents 200 μm.
Figure 2.
Oral and Pharyngeal Tooth Number Is Correlated in Malawi Cichlids
(A–I) show three species of Malawi cichlid: D. compressiceps (DC) (A–C), M. zebra (MZ) (D–F), and L. fuelleborni (LF) (G–I). Dorsal views of (B, E, and H) show adult lower oral jaws, cleared and alizarin-stained bone/dentine preparation, and (C, F, and I) show adult lower (ceratobrachial [CB]5/PA7) pharyngeal jaws with the soft tissue removed. DC, MZ, and LF represent a range in oral and pharyngeal tooth number (Table S1): there are fewest teeth in DC, more teeth in MZ, many teeth in LF.
(J) Across Malawi cichlids, a positive correlation is observed between the number of teeth on the oral and pharyngeal jaws (r = 0.53 without P. nigra and r = 0.66 including P. nigra; p < 0.00001), see (A–I). Data points labeled DC, MZ, and LF refer to the three species in (A–I).
(K) Phylogenetically independent contrasts for tooth number across the Malawi cichlid flock without P. nigra (r = 0.39; p < 0.019; see Materials and Methods).
Scale bars in (B) and (H) represent 500 μm. (E) and (H) are to the same scale. (C, F, and I) show the pharyngeal jaws to the same scale.
Figure 3.
Multiple Hox Genes Are Expressed during Pharyngeal Jaw and Tooth Development in Malawi Cichlids
(A–C) hoxA2b (A2b) expression in M. zebra (MZ).
(D–F) hoxA5a (A5a) expression in C. conophorus (CC).
(G–I) hoxB5b (B5b) expression in MZ.
(J–L) hoxB6b (B6b) expression in MZ.
(M–O) hoxD4a (D4a) expression in CC.
(A, D, G, J, and M) show all whole-mount lateral views. (B, C, E, F, H, I, K, L, N, and O) show all coronal sections of the pharyngeal jaw (PA7/CB5).
(A) hoxA2b is expressed in the hindbrain (black arrow) and the pharyngeal arch mesenchyme (white arrowhead).
(B and C) show an oblique coronal section: (B) hoxA2b expression surrounds more mature upper pharyngeal dentition away from cells directly associated with the teeth (black arrow). Tooth germs at an earlier stage of development show the expression of hoxA2b in closer proximity within the mesenchymal cells enveloping the tooth germs (black arrowheads) and in (C) demarcated by red dashed circles.
(D) hoxA5a is expressed in the hindbrain (black arrow) and in the posterior pharyngeal mesenchyme (white arrowhead).
(E and F) show a coronal section: (E) hoxA5a expression underlying the teeth (black arrowheads) in the pharyngeal jaw (and in [F]). hoxA5a expression is present around the forming cartilages of the fifth ceratobranchial (CB5), (white arrow) and the upper fourth epi/pharyngobranchial.
(G) hoxB5b is expressed in the hindbrain (black arrow) and the most posterior pharyngeal arch mesenchyme (white arrowhead).
(H and I) show a coronal section: (H) hoxB5b marks the dental mesenchyme surrounding each tooth germ (black arrowheads) in the pharyngeal jaw (CB5); higher magnification is shown in (I). NC, notochord.
(J) hoxB6b is present in the developing hindbrain (black arrow) and the posterior pharyngeal mesenchyme (white arrowhead).
(K and L) show a coronal section: (K) hoxB6b expression surrounds each tooth in the dental mesenchymal cells (black arrowheads); stronger expression is observed at the base of each tooth unit (black arrow), possibly related to the attachment between the mineralized tooth and the underlying cartilage of CB5. Expression surrounds the pharyngeal cartilages (white arrow); a higher magnification is shown in (L). pc, pharyngeal cavity.
(M) hoxD4a is expressed in the hindbrain (black arrow) and in the posterior pharyngeal mesenchyme (white arrowhead).
(N and O) show a coronal section: (N) hoxD4a is strongly expressed in the dental mesenchyme surrounding each tooth germ of the pharyngeal jaw. Each individual tooth is demarcated by the mesenchymal expression (black arrowheads). hoxD4a is also strongly up-regulated in the mesenchyme directly enveloping the cartilages of CB5 (white arrow). Expression of hoxD4a is identical between the upper (N) and lower (O) pharyngeal dentition.
(B, E, H, K, N, and O) are all to the same scale: scale bars represent 100 μm. Scale bar in (C) represents 20 μm. Scale bars in (F, I, and L) represent 50 μm. Embryos shown are 6–7 dpf.
Figure 4.
A Dental Gene Network Exhibits Conserved Expression in Oral and Pharyngeal Teeth
(A, C, E, G, I, K, M, and O) show gene expression in the oral dentition, all dorsal views of the lower jaw. (D, F, H, J, N, and P) show dorsal views of the developing lower pharyngeal dentition; (B and L) are coronal sections through the pharyngeal teeth. M. zebra (MZ) (A, B, and I–N) and L. fuelleborni (LF) (C–H, O, and P) are represented here at the three to four oral tooth stage (∼6–7 dpf); the pharyngeal dentition develops ahead of the oral teeth, and so at this time point there are approximately seven pharyngeal teeth per pharyngeal quadrant.
(A and B) show pax9 expression in MZ. (A) In the lower oral jaw, pax9 marks mesenchymal cells surrounding teeth (black arrowheads). (B) shows a coronal section showing upper pharyngeal teeth with pax9 lateral to (black arrows) but not associated with teeth (red dashed circles). Scale bar in (B) represents 20 μm. EB4, epibranchial 4; pc, pharyngeal cavity.
(C and D) show barx1 expression in LF. (C) In the lower oral jaw (outlined in blue dashes), barx1 is expressed in a band of mesenchymal cells (black arrow) lingual to the tooth sites (red dashed regions), and also in lateral cell clusters (black arrowhead) away from tooth sites. (D) barx1 is localized to mesenchymal cells underlying tooth sites in developing lower pharyngeal jaw (CB5, black arrowhead). CB5, ceratobranchial 5.
(E and F) show runx2 expression in LF. (E) runx2 labels both mesenchymal cells of the oral mesiodistal field for tooth competence and mesenchymal cells that surround the epithelial tooth germs (black arrowhead). (F) Equivalent expression is observed in the developing pharyngeal jaw (tooth germs, black arrowheads) and in tooth-competent regions of mesenchyme (black arrow).
(G and H) show shh expression in LF. (G) shh is expressed in the epithelial cells of the developing oral dentition (black arrowheads; differences in expression from spots to open circles show variation in the stage of tooth development). shh also labels the epithelial odontogenic band for posterior tooth rows (black arrow). (H) shh is up-regulated in the pharyngeal dentition (black arrowheads) as in oral teeth.
(I and J) show pitx2 expression in MZ. (I) pitx2 marks the dental-competent oral epithelium around the tooth sites and is up-regulated in tooth germs, from the thickened epithelium to the maturing tooth germ (black arrowheads). (J) Similarly, pitx2 labels both dental competent epithelia and epithelial tooth germs (black arrowheads) of PA7/CB5.
(K and L) show bmp2 expression in MZ. (K) bmp2 is localized to the epithelial tooth thickenings (black arrowheads) and the competent epithelia along the mesiodistal axis of the oral jaw. bmp2 becomes coexpressed to both the developing epithelial (teleost enameloid cell cluster [TEC], black arrow in [L]) components and mesenchymal papilla of the tooth germs [49] and in epithelial cells in a band lingual to the first teeth (black arrow in [K]) for new tooth rows. (L) shows a coronal section of lower pharyngeal teeth; bmp2 labels both the epithelial cells of the TEC (black arrow) and mesenchymal cells of the dental papilla (black arrowhead) of the same tooth (red dashed circle). In epithelial thickenings, bmp2 is present in the thickened epithelial cells and is also expressed in the condensing cells of the underlying mesenchyme (black arrowheads). The black dashed line indicates the pharyngeal cavity. Scale bar in (L) represents 50 μm.
(M and N) show bmp4 expression in MZ. (M) In the oral jaw (OJ) and (N) the pharyngeal jaw (PJ), bmp4 is expressed in dental epithelial cells of tooth germs (OJ, black arrowheads; PJ, white arrowheads); later, bmp4 is coexpressed in the mesenchymal cells of the dental papilla. Along the mesiodistal axis of the oral jaw, bmp4 labels the mesenchymal field of dental competence, and for new tooth rows lingually (black arrow). In addition, bmp4 is expressed throughout the pharyngeal arch mesenchyme (see also Figure 1E and 1F).
(O and P) show dlx2 expression in LF. dlx2 labels the first tooth (in the series) for each tooth row in both the oral jaw (PA1, black arrowhead in [O]) and the pharyngeal jaw (PA7, black arrowheads in [P]). dlx2 is also present in the mesenchymal cells along the mesiodistal axis of the oral jaw and pharyngeal jaw (white arrows). (P) The black arrowheads show new row initiation. R1, row 1; R2, row 2.
(A, C, E, G, I, K, M, and O) are all to the same scale. Scale bar in (E) represents 100 μm. (D, F, H, J, N, and P) are all to the same scale. Scale bar in (J) represents 100 μm.
For details of expression in thin section for most of these markers, see [49].
Figure 5.
Ectodysplasin Pathway Genes, eda and edar, Are Expressed in Skeletal Structures throughout the Oropharynx
(A, B, and C) show edar expression in the lower pharyngeal elements of L. fuelleborni (LF) (6 dpf); dorsal views. (A) shows edar expression in the entire lower pharyngeal series. edar is localized to the epithelium of the tooth germs (black arrow) of the oral jaws (B) (arrowheads), in the surrounding epithelium along the mesiodistal axis in (B) (arrow), and in the pharyngeal endoderm including the hyoid arch (asterisk in [A]). edar is expressed in gill raker bud epithelium (similar to tooth germs) on pharyngeal arches PA3–6 and in a band of gill raker initiation along the mesiodistal axes of each gill-bearing arch (white arrows in [A] and in [C]). edar also labels in the epithelial cells of the pharyngeal tooth germs on PA7 (white arrowheads in [A] and in [C]).
(D, E, and F) show eda in the lower pharyngeal elements of M. zebra (MZ) (7dpf); dorsal views. (D) shows eda expression in the lower pharyngeal series of MZ. (D) is composed of two images of the same specimen; the boxed area of the oral jaw (separate image) is shown in (E). eda expression marks the mesenchymal cells surrounding and separating the tooth germs of the oral jaw (black arrow in [D]; open circles surrounded by eda expression represent the epithelial tooth germs; also see [49]), through to the equivalent cells surrounding the tooth germs of the pharyngeal jaw, PA7 (white arrowheads in [D] and in [F]). Between these separated dental sites, eda also labels the medial pharyngeal mesenchyme from the hyoid (PA2), including the second arch extension that will form the opercular flap (black arrowhead), through the series to the most posterior arch (PA7). eda is also expressed in relation to the initiating gill rakers lining each of the gill-bearing arches, PA3–6 (white arrows). Breaking up the bands of expression are eda-negative sites of gill raker bud initiation (red dashed circles) that express edar in (C).
Scale bar in (B) represents 100 μm; (B, E, C, and F) are all to the same scale. Scale bars in (A and D) both represent 100 μm.
CB5, ceratobranchial 5.
Figure 6.
Teleost Pharyngeal Arches Exhibit Teeth on Old and New Jaws
(A) Schematic diagram of the lower pharyngeal arches in an embryonic (∼7 dpf) cichlid fish (dorsal view).
(B) Cartoon of the cichlid head showing pharyngeal arches (representing a general embryonic cichlid ∼7 dpf).
(A and B) Oral jaw (PA1, lilac) houses a dentition (dark-blue circles) that develops in an ectoderm-influenced environment; dashed line in (A) represents the ectoderm/endoderm interface; a strict boundary may not exist due to cell mixing across this interface [36]. This dashed line also reflects the border between Hox gene–positive and Hox gene–negative oropharyngeal regions among vertebrates; we do not suggest a functional relationship between the two. The unique hyoid arch (PA2, green) is the only arch in the series that has no teeth (not a general rule for all fish) or gills/gill rakers; however, it does extend to cover all posterior arches as the opercular flap. PA3–6 (CB1–4, light blue) are gill-bearing arches (black triangles in [A]); these arches also house gill rakers (purple circles) that express a similar suite of genes during development in a similar temporospatial manner to the teeth on PA1 and PA7. Gill rakers will feature a secondary tooth/denticle set later in development. Oral and pharyngeal tooth development is generally governed by the same genetic regulators (see Figures 4 and 5); this corresponds to a positive correlation in the number of tooth units across the two disparate sites (tooth number regulation; see Figure 2). Pharyngeal tooth sites (PA7, blue) represent the first sites of tooth formation in vertebrates. The oral jaw and the teeth of PA1 develop devoid of hox gene expression (-ve), whereas the pharyngeal jaw and the teeth of PA7 (and all other arches in the series PA3–6) develop with hox genes strongly expressed in the pharyngeal mesenchyme around the forming pharyngeal jaw cartilages and teeth (hox positive; see Figure 3). pax9 is only expressed in relation to the dentition of the oral jaw, whereas barx1 is only expressed in relation to the dentition of the pharyngeal jaw (B). eda and edar are both expressed in a similar pattern throughout the entire pharyngeal arch series from the dentitions of the oral and pharyngeal jaw to the organization of the gill raker buds along the cartilage bars of PA3–6 (see Figure 5). Colors in (A) correspond to those in (B).
Figure 7.
The Coevolutionary History of Jaws and Dentitions
Panels 1 and 2 show a simplified evolutionary progression showing the hypothetical advent of the vertebrate ancient gene network (blue icon), core dental network (yellow icon), dentitions, and jaws.
Point A indicates the origin of the ancient dental gene network and pharyngeal teeth in extinct (†) jawless fish.
Point B indicates the origin of oral jaws in concert with the co-option of the core dental gene network allowing the development of teeth on the recently acquired oral jaws. This was accompanied by the loss of some genes occupying the ancient gene network, including the hox genes and barx1 (Barx1 was then regained in mammalian molar formation); Placoderms (the earliest jawed vertebrates) evolved an oral dentition independently in derived groups [74]; however, we suggest that the networks are common for teeth and were in place prior to the advent of oral jaws.
Point C: the branch leading to the tetrapods shows a reduction in the sites that are occupied by a dentition and thus the pharyngeal dentition (ancient dental network) is lost (X in panel 2C). In addition to this loss, the oral dentition is greatly modified in tetrapods. Some vertebrates have lost the entire oral dentition (birds, turtles, etc.), and some have extreme modifications of the oral dentition (e.g., mammals).
Point D: advanced groups of teleost fish, including the cichlids, have evolved a modified set of toothed pharyngeal jaws, further co-opting the ancient site of the first teeth and ancient dental gene network for involvement on a new functional jaw.
Panel 2: schematic representation of the evolution of teeth and jaws in vertebrates.
Panel 3: a schematic developing generalized tooth germ showing localization of the genes in the ancient vs. the core dental network (1, outer dental epithelium; 2, inner dental epithelium; 3, dental papillary mesenchyme; and 4, dental mesenchymal envelope). Color scheme is then represented on panel 1. Bracketed genes represent those with different cellular localization (mesenchyme or epithelium) in alternative vertebrates. Teleosts express fgf3 in the dental epithelium, whereas mammalian Fgf3 is mesenchymal; teleosts express eda in the mesenchyme, whereas mammalian Eda is epithelial.
Panel 4: schematic representation of a generalized vertebrate tooth germ showing the putative interactions between the dental epithelial (pink) and dental mesenchymal (light blue) genetic players of the core dental network; those genes in blue ovals represent elements of the ancient dental network (e.g., Hox and barx1); those in green (pax9) represent molecules of neither the core nor the ancient dental network, present during oral dentitions of the mouse and cichlids (Table 1). eda is in brackets due to the differential expression between fish (mesenchyme) and mammals (epithelium). Regionalized signaling in an enamel organ such as that originating from the enamel knot has been purposely omitted from this generalized diagram. See Table S2 for references documenting each interaction.
Table 1.
Identifying the Core versus Ancient Dental Network across Vertebrates