Figure 3

Thin sections of the developmental program of Malawi cichlid dentitions. (A)-(C) Metriaclima zebra (MZ) pitx2 expression within the oral and dental epithelium. (A), (B) 3–4-teeth stage, 7 dpf; (C) first-tooth stage. (A) Sagittal section (dentary), expression in the dental epithelium (tooth number 1 in the series, inner dental epithelium (IDE) and outer dental epithelium (ODE); black arrowhead). Continued dental competence for the next row (odontogenic band (OB) 2) is shown by expression in the lingual extent of the oral epithelium (arrow, OB2). (B) Sagittal section (upper jaw); pitx2 is expressed in the IDE and ODE (arrowhead, tooth number 1 in the series). Expression within the basal epithelial cells denoting the competent oral epithelium for future tooth initiation (OB2; arrow). (C) Coronal section (dentary) initial thickened epithelia of the first-tooth germs (1, thickened dental epithelium; black arrowheads) labelled by pitx2. The competent field (OB1) continues laterally from which new adjacent tooth germs will develop (arrow). (D)-(F) MZ pax9 in the dental/oral mesenchyme underlying the thickened epithelium (D) (thickening stage of the first tooth) and surrounding the developing dental units (E), (F) (3–4-teeth stage, 7 dpf). (D) pax9 (arrowhead) in mesenchyme (asterisk, dental epithelium, first tooth). Continued lateral expression in mesenchyme underlies the odontogenic band of the first row (OB1, arrow). (E), (F) Oblique coronal sections (dentary) of MZ. (E) pax9 expression is observed within the dental mesenchyme (arrowhead) of tooth 3 in the series (medial, on each half of the dentary) and surrounding the developing epithelial tooth germ (arrow) within the mesenchyme and in (F); for orientation, the thickened epithelium is forming down into the page; epithelium (E), mesenchyme (M, arrowheads), tooth 1 (arrow, 1, developing down into the page). (G)-(I) LF shh expression in the dental epithelium (3–4-teeth stage, 6 dpf). (G) Sagittal section (dentary), expression in the IDE of the first tooth in the series (1, black arrowhead). Black arrow, competent OB already marking the presumptive second row (OB2). (H) Epithelial thickening stage of tooth 3 in the series (3), sagittal section shh in the thickened dental epithelium (black arrowhead). The red dashed line demarcates the epithelium joining the second OB (OB2, black arrow) with the tooth germ. (I) Coronal section (dentary) showing the first three tooth germs in the series (1–3 in order of development) shh is restricted to the IDE of tooth 1, the epithelial germ in tooth 2 and the thickened epithelium in tooth 3. Directly above the tooth germs (in this plane of section) is the second OB for the initiation of subsequent teeth. (J)-(L) MZ bmp2 in both the dental epithelium and dental papilla (mesenchyme) (3–4-teeth stage, 7 dpf). (J) Oblique coronal section (dentary) expression within thickened epithelium for teeth 2 and 3 in the series (epithelium; arrowhead). More mature developing tooth (number 1, red dashed line) showing dental papillary expression (mesenchyme). The region between the 3 tooth units of varying stages, the ZOI, contains no bmp2 expression (arrow). Dental epithelium of tooth 3 initiating epithelial thickening stage, (arrowhead with asterisk, 3) well spaced from the neighbouring germ. (K) Sagittal section (dentary) the maturing (cytodifferentiation) first tooth (1) showing bmp2 in the dental papilla (mesenchyme; arrowhead) and simultaneous expression in IDE cells at the tip of the tooth (arrow), equivalent to the primary enamel knot in mammals. (L) Coronal section (dentary); order of initiation of the first three teeth (1–3) showing the variety of bmp2 expression. First tooth (1) shows dental papillary expression, second and third tooth to initiate (2, 3) show epithelial expression, more so in (2) as developing tooth germ is present. All images are to the same scale; scale bar in (A) is 20 μm. Sections cut to a thickness of 25 μm. E, epithelium; M, mesenchyme; Mc, Meckel's cartilage; pmc, premaxillary cartilage.