Chicken embryos share mammalian patterns of apoptosis in the posterior placodal area

Abstract

In the posterior placodal area (PPA) of C57BL/6N mice and primate-related Tupaia belangeri (Scandentia), apoptosis helps to establish morphologically separated otic and epibranchial placodes. Here, we demonstrate that basically identical patterns of apoptosis pass rostrocaudally through the Pax2⁺ PPA of chicken embryos. Interplacodal apoptosis eliminates unneeded cells either between the otic anlage and the epibranchial placodes 1, 2 and/or 3, respectively (type A), or between neighbouring epibranchial placodes (type B). These observations support the idea that in chicken embryos, as in mammals, interplacodal apoptosis serves to remove vestigial lateral line placodes (Washausen & Knabe, 2018, Biol Open 7, bio031815). A special case represents the recently discovered Pax2^- /Sox2⁺ paratympanic organ (PTO) placode that has been postulated to be molecularly distinct from and developmentally independent of the ventrally adjacent first epibranchial (or 'geniculate') placode (O'Neill et al. 2012, Nat Commun 3, 1041). We show that Sox2⁺ (PTO placodal) cells seem to segregate from the Pax2⁺ geniculate placode, and that absence of Pax2 in the mature PTO placode is due to secondary loss. We further report that, between Hamburger-Hamilton (HH) stages HH14 and HH26, apoptosis in the combined anlage of the first epibranchial and PTO placodes is almost exclusively found within and/or immediately adjacent to the dorsally located PTO placode. Hence, apoptosis appears to support decision-making processes among precursor cells of the early developing PTO placode and, later, regression of the epibranchial placodes 2 and 3.

Keywords: apoptosis; chicken embryos; epibranchial placodes; paratympanic organ (PTO) placode; posterior placodal area.

Figure 1

Pax2 protein expression and apoptosis in the posterior placodal area (ppa) of chicken embryos, Hamburger–Hamilton (HH) stages HH8 to HH10. (A‐C) Schematic reconstructions [top views, not drawn to scale; orientation mark indicating medial (m), lateral (l), rostral (r) and caudal (c) positions; paired maps, lettered left and mirrored right body sides] demonstrate strong or weak Pax2 immunopositivity (magenta, dark or light shades, respectively), different grades of thickened surface ectoderm (specified below) and apoptotic cells (red with black margin). Neuroepithelium (grey), cranialmost somites (yellow) and caudal parts of the Pax2⁺ isthmic organizer (io) were also included. Sectioning planes indicate the positions of anti‐Pax2 (A’, B’, C’) or anti‐cleaved caspase‐3 (C’’) stained sections (A’: mirror‐imaged left body side). (A, A’) In HH8 chicken embryos, Pax2⁺ placodal precursor cells are scattered within or adjacent to the thickened ppa, which mostly consists of at least one row of columnar epithelium (circumscribed by thin black lines). (B‐C’’) From HH9 to HH10, Pax2 expression intensifies within the thickened ppa (thin black lines), including the otic placode (ot), which consists of at least three rows of columnar epithelium (circumscribed by thick black lines in B, C; thick dashed line in C’). From HH8 to HH10, apoptosis is virtually absent from the developing ppa (A, B, C, C’’). nf, neural fold; np, neural plate; nt, neural tube; s1/s2, first/second somite. Scale bar: 20 μm.

Figure 2

Apoptosis in the posterior placodal area of chicken embryos as revealed by anti‐cleaved caspase‐3 immunohistochemistry. (A) Hamburger–Hamilton (HH) stage HH11 shows apoptosis at the ventral margin of the otic placode (ot), enlarged in the inset (arrowheads). (B) HH17 demonstrates apoptosis (arrow) at the detachment site of the otic vesicle (ov), interplacodal apoptosis type A (black arrowheads) between the developing otic vesicle and the combined anlage of the first epibranchial and paratympanic organ (PTO) placodes (e1/pt), and apoptosis (white arrowheads) in dorsal parts of this anlage. Asterisk, geniculate ganglion; hb, hindbrain; p1, pharyngeal pouch 1. Scale bars: 20 μm.

Figure 3

Apoptosis in the posterior placodal area of chicken embryos, Hamburger–Hamilton (HH) stages HH11 to HH17. Schematic reconstructions [lateral views, not drawn to scale; orientation mark indicating dorsal (d), ventral (v), rostral (r) and caudal (c) positions; paired maps, lettered left and mirrored right body sides] demonstrate high‐grade thickenings (grey) revealing the otic placode (ot), the combined anlage of the first epibranchial and paratympanic organ (PTO) placodes (e1/pt), and the epibranchial placodes 2 and 3 (e2, e3). Patterns of apoptosis (red), pharyngeal membranes/pouches (dotted black lines), borders of the branchial arches (thin black lines in B‐F) and cranialmost somites (yellow) are also shown. (A‐D) Between HH11 and HH14, apoptosis in the thickened posterior placodal area (at least one row of columnar epithelium, circumscribed by a thin black line in (A) is centred on the ventral margin of the invaginating otic placode (circumscribed by thick black line). Thereafter, apoptosis shifts to the detachment site (‘otic pore’, again circumscribed by a thick black line) of the developing otic vesicle (thin dashed black line in E, F). Interplacodal apoptosis type A between the otic anlage and the combined anlage of the first epibranchial and PTO placodes, and/or between the otic anlage and the epibranchial placodes 2 and 3 starts around HH14 (D) and peaks around HH17/HH18 (F). Apoptosis in dorsal parts of the combined anlage of the first epibranchial and PTO placodes and/or in dorsal parts of the epibranchial placode 2 arises between HH15 (E) and HH17 (F). b2, branchial arch 2.

Figure 4

Apoptosis in the posterior placodal area of chicken embryos, Hamburger–Hamilton (HH) stages HH18 to HH23. Schematic reconstructions [lateral views, not drawn to scale; orientation mark indicating dorsal (d), ventral (v), rostral (r) and caudal (c) positions; paired maps, lettered left and mirrored right body sides] demonstrate high‐grade thickenings (grey) revealing the combined anlage of the first epibranchial (e1) and paratympanic organ (PTO) placodes (pt), and the epibranchial placodes 2 and 3 (e2, e3 or e3₁ and e3₂, respectively). Patterns of apoptosis (red), immunopositivity for Sox2 in the PTO placode (green, with dark or light shades indicating strong or weak immunopositivity, respectively), pharyngeal membranes/pouches (dotted black lines) and borders of the branchial arches (thin black lines) are additionally shown. Interplacodal apoptosis type A between the position of the internalized otic vesicle (thin dashed black lines in A‐D, also see solid otic stalk: thick dashed black lines in A, B) and the combined anlage of the first epibranchial and PTO placodes, and/or between the otic anlage and other epibranchial placodes, peaks around HH17/HH18 (A) and regresses from HH19 onwards (B–D). Interplacodal apoptosis type B between neighbouring epibranchial placodes starts around HH21 (C) and peaks on HH23 between the epibranchial placodes 2 and 3₁ (D). (A–C) Between HH18 and HH21, apoptosis is present within and/or adjacent to the developing (Sox2⁺) PTO placode and/or in dorsal parts of the epibranchial placode 2. (D) From HH23 onwards, apoptosis slightly decreases within the PTO placode, and centres on dorsal and/or ventral parts of the epibranchial placodes 2, 3₁ and 3₂, the latter two demonstrating a pit‐like invagination (circumscribed by thick black lines). b1/b2, branchial arches 1 and 2 that, from HH23 onwards, overgrow e1 and e2, respectively (D).

Figure 5

Apoptosis contributes to the development of the paratympanic organ (PTO) placode, which appears to segregate from the Pax2⁺ chicken first epibranchial placode. (A, B) Schematic reconstructions, lateral views, not drawn to scale; orientation mark indicating dorsal (d), ventral (v), rostral (r) and caudal (c) positions; paired maps, left and mirrored right body sides. Strong or weak Pax2 (magenta) and Sox2 (green) immunopositivity is indicated by dark or light shades, respectively. (C‐F) Double immunofluorescence stainings for Pax2 (magenta) and Sox2 (green), each row showing an overview (merged channels) and three images of the magnified placode (merged and individual channels) with relative z‐positions indicated. (A, B) Apoptosis (red) is centred on the PTO placode (pt), but is almost absent from the first epibranchial placode (e1). (A, C‐F) During Hamburger–Hamilton (HH) stages HH19 and HH21, both the dorsally located Sox2⁺ PTO placode and the ventrally located Sox2⁻ epibranchial placode 1 originate from a combined Pax2⁺ anlage. Later (HH23, HH25), downregulation of Pax2 in the PTO placode helps to distinguish between the Pax2⁻/Sox2⁺ PTO placode and the Pax2⁺/Sox2⁻ epibranchial placode 1 (B). Arrows, migrating neuroblasts; dotted black lines (A, B) or p1 (C–F), pharyngeal membrane/pouch 1; gg, geniculate ganglion; ov, otic vesicle. Scale bars in (C–F): 50 μm (overviews) or 20 μm (details).

Figure 6

Apoptosis in the posterior placodal area of chicken embryos, Hamburger–Hamilton (HH) stage HH26. Schematic reconstructions [lateral views, not drawn to scale; orientation mark indicating dorsal (d), ventral (v), rostral (r) and caudal (c) positions; paired map, lettered left and mirrored right body sides] demonstrate the high‐grade thickened (grey) combined anlage of the first epibranchial (e1) and paratympanic organ (PTO) placodes (pt), the epibranchial placode 2 (e2), the invaginated epibranchial placodes 3₁ and 3₂ (e3₁, e3₂, circumscribed by a thick black line), and interplacodal remnants of the posterior placodal area. Patterns of apoptosis (red), pharyngeal membranes/pouches (dotted black lines), and borders of the branchial arches (thin black lines) are also shown. Interplacodal apoptosis type B peaks between the epibranchial placodes 2 and 3₁ as well as between the epibranchial placodes 3₁ and 3₂. Large‐scale apoptosis is also present in the regressing epibranchial placode 2 and in the invaginated epibranchial placodes 3₁ and 3₂. Residual apoptosis is present within and/or adjacent to the Sox2⁺ (green, with dark or light shades indicating strong or weak immunopositivity, respectively) PTO placode. Thin dashed black lines, otic vesicle; b1/b2, branchial arches 1 and 2 that overgrow e1 and e2, respectively.

Figure 7

Apoptosis in the posterior placodal area of chicken embryos as revealed by anti‐cleaved caspase‐3 immunohistochemistry. Hamburger–Hamilton (HH) stage HH23, relative z‐positions indicated. Overviews (A, C, E) with boxed areas enlarged in (B, D, F) demonstrate intraplacodal apoptosis (white arrowheads) in the epibranchial placode 2 (e2: A‐D) and interplacodal apoptosis type B (black arrowheads) between the epibranchial placodes 2 and 3₁ (A‐F). Branchial arch 2 (b2) has overgrown epibranchial placode 2, and pharyngeal pouch 3 (p3) opens to the amniotic cavity (asterisk in E). Arrow (in A), migrating neuroblasts; b3, b4, branchial arches 3 and 4, respectively; da, dorsal aorta; pg, petrosal ganglion; ph, pharynx; III, IV, branchial arteries 3 and 4, respectively. Scale bars: 100 μm (overviews) or 20 μm (details).

Figure 8

Apoptosis in the posterior placodal area of chicken embryos as revealed by anti‐cleaved caspase‐3 immunohistochemistry. Hamburger–Hamilton (HH) stage HH23, relative z‐positions indicated (continued from Fig. 7). Overviews (A, C) with boxed areas enlarged in (B, D) demonstrate interplacodal apoptosis type B (black arrowheads) between the epibranchial placode 2 and the deeply invaginated epibranchial placode 3₁ (e3₁). Arrow (in C), migrating neuroblasts; b4, branchial arch 4; cv, cardinal vein; da, dorsal aorta; ng, nodose ganglion; ph, pharynx; p4, pharyngeal pouch 4; va, ventral aorta; white arrowheads, apoptosis in epibranchial placode 3₁; IV, branchial artery 4. Scale bars: 100 μm (overviews) or 20 μm (details).

Figure 9

Apoptosis in the posterior placodal area of chicken embryos as revealed by anti‐cleaved caspase‐3 immunohistochemistry. Hamburger–Hamilton (HH) stage HH26, relative z‐positions indicated. Sections demonstrate interplacodal apoptosis type B (black arrowheads) between the invaginated epibranchial placodes 3₁ (e3₁) and 3₂ (e3₂, A–C) as well as intraplacodal apoptosis (white arrowheads) in these two placodes (A, B, D). Arrows (in D), migrating neuroblasts; b4, b6, branchial arches 4 and 6, respectively; ng, nodose ganglion; p3, p4, pharyngeal pouches 3 and 4, respectively; IV, branchial artery 4. Scale bar: 20 μm.