Lateral line placodes of aquatic vertebrates are evolutionarily conserved in mammals

Abstract

Placodes are focal thickenings of the surface ectoderm which, together with neural crest, generate the peripheral nervous system of the vertebrate head. Here we examine how, in embryonic mice, apoptosis contributes to the remodelling of the primordial posterior placodal area (PPA) into physically separated otic and epibranchial placodes. Using pharmacological inhibition of apoptosis-associated caspases, we find evidence that apoptosis eliminates hitherto undiscovered rudiments of the lateral line sensory system which, in fish and aquatic amphibia, serves to detect movements, pressure changes or electric fields in the surrounding water. Our results refute the evolutionary theory, valid for more than a century that the whole lateral line was completely lost in amniotes. Instead, those parts of the PPA which, under experimental conditions, escape apoptosis have retained the developmental potential to produce lateral line placodes and the primordia of neuromasts that represent the major functional units of the mechanosensory lateral line system.

Keywords: Apoptosis; Lateral line; Mouse embryos; Neuromasts; Posterior placodal area; Vestigial placodes.

Fig. 1.

Pharmacological inhibition of apoptosis in the posterior placodal area (PPA) of embryonic mice. (A) Summary scheme of in utero-developed control embryos including ectoderm (light grey), otic vesicle with detachment site (dark grey), epibranchial placodes (orange), and apoptosis (purple) demonstrates the peak of PPA apoptosis (compiled from Washausen and Knabe, 2013, ; n=44 body sides). (B) Levels of apoptosis in the PPA (black contour in the schematized embryo; section interval evaluated=10 µm) of in utero-developed embryos (n=20 body sides) or specimens developed for 24 h in whole embryo culture (wec). Embryos were cultured either in the presence of only the solvent DMSO (control; n=20), or in the presence of 10–100 µM of the pan-caspase inhibitor Q-VD-OPh (n=8 for 10, 20, or 50 µM, respectively; n=20 for 100 µM), or in the presence of 200 µM of the more narrow spectrum caspase inhibitor Z-VAD-fmk (n=12). It turned out that Q-VD-OPh treatment reduces PPA apoptosis in a dose-dependent manner. Furthermore, inhibition with 50 µM Q-VD-OPh or 200 µM Z-VAD-fmk is significantly less efficient compared with treatments using 100 µM Q-VD-OPh. Significant differences were measured with unpaired Mann–Whitney test (*P<0.001). Box plots indicate medians (centre lines), 25th and 75th percentiles (box limits), lower and upper extremes (whiskers), data points evaluated separately for each body side (purple dots), and outliers (open circles). (C) Micrographs (standardized sectioning plane) taken from anti-cleaved caspase-3 (Casp3) stained serial sections of mouse embryos treated with 10, 20, 50 or 100 µM Q-VD-OPh or 200 µM Z-VAD-fmk. It turned out that the more effective reduction of PPA apoptosis (arrowheads) is, the better rudiments of the lateral line placodes are preserved. Scale bars: 50 µm (overviews) and 10 µm (magnified insets). E, embryonic day; e1, e2, e3, epibranchial placodes 1, 2, 3, respectively; ot, otic anlage; ov, optic vesicle; p1, p2, pharyngeal pouch 1, 2, respectively.

Fig. 2.

Pharmacological inhibition of apoptosis generates lateral line placodes in embryonic mice. (A) Micrographs of representative serial sections used to compile schematic reconstructions of Q-VD-OPh-treated mouse embryos. Exemplarily, topography and dimensions of a middle lateral line placode (m) are shown (embryo #125; relative z-positions indicated). (B,C) Reconstructions of mouse (B) and Xenopus laevis (C) embryos show ectoderm (light grey), otic vesicle with detachment site (dark grey), epibranchial placodes (orange), apoptosis (purple), and lateral line placodes (blue). (B) Inhibition of apoptosis for 24 h in whole embryo culture (wec) generates lateral line placodes (embryo #030, mirror-imaged right side). (C) Lateral line placodes in a Xenopus laevis embryo, stage 27 (adapted from Schlosser and Northcutt, 2000). (D) Frequency of anterodorsal (ad), middle (m), and posterior (p) lateral line placodes either found unilaterally or bilaterally in the posterior placodal area of mouse embryos exposed to Q-VD-OPh for 18, 24, or 36 h. Scale bar: 100 µm. ad, av, m, p, anterodorsal, anteroventral, middle, and posterior lateral line placode, respectively; asterisk and triangle, pharyngeal pouches 1 and 2, respectively; e1, e2, e3, e3₁, e3₂, epibranchial placodes 1, 2, 3, 3₁, 3₂, respectively; ot, otic anlage; ov, optic vesicle; Q-VD-OPh, pan-caspase inhibitor; v, migratory primordia of ventral trunk lines.

Fig. 3.

Lateral line placodes: morphological comparison between Q-VD-OPh-treated mice and axolotl embryos. (A–C,E) Micrographs taken from serially sectioned mouse embryos. (A) Cubic to columnar, single-row to pseudostratified epithelium of a middle lateral line placode. (B) Six1⁺ middle lateral line placode with neuromast primordium. (C) Neuromast primordium of a posterior lateral line placode with kinocilium (arrow). (E) Neuromast primordium of a middle lateral line placode revealing an apical cavity (arrowhead) that is covered by a flat superficial ectodermal cell. (D,F) Comparison images taken from axolotl embryos (Ambystoma mexicanum). (D) Primary neuromast with kinocilium (arrow). Reproduced from Schlosser, 2002b © 2002, with permission from Elsevier. (F) Neuromast primordium with apical cavity (ac; arrowhead) and flat covering cell. Reproduced with permission from Northcutt et al., 1994 © 1994 Wiley-Liss, Inc. All images were adjusted for brightness (including slight gamma changes), colour balance, and sharpness. Scale bars, 5 µm. h, H, hair cell; Haem, haematoxylin staining (Mayer); m, mantle cell; Q-VD-OPh, pan-caspase inhibitor; s, support cell; wec, whole embryo culture; MS, mantle type of supporting cell; S, supporting cell.

Fig. 4.

Lateral line placodes of Q-VD-OPh-treated mice reveal the specific molecular properties of posterior placodes. (A–C,G) Micrographs taken from serially sectioned mouse embryos, with their positions being shown in the preceding reconstructions that demonstrate ectoderm (light grey), otic pit (A,C) or otic vesicle (B) with detachment site (dark grey), epibranchial placodes (orange), lateral line placodes (blue), plane of sectioning (black line in reconstructions). (A) Pax8 immunopositivity is present in epibranchial placode 1, in the prospective anterodorsal lateral line placode as well as in remaining parts of the thickened PPA. (B) Close apposition of Pax8⁺ epibranchial placode 2 and Pax8⁻ posterior lateral line placode. (C) Anterodorsal lateral line and otic placodes spring from a common Sox10⁺ domain. (D) Neuromast primordium of an anterodorsal lateral line placode with Sox10⁺ mantle (m) and support cells (s). (E,F) During the peak period of PPA apoptosis, in utero-developed control embryos [embryonic day 9.5 (E9.5)] demonstrate disorganized Sox10⁺ (E) or predominantly Sox10⁻ ectodermal cells (F, but see arrow) as well as high numbers of apoptotic cells (arrowheads) in the positions of vestigial lateral line placodes, here shown for an anterodorsal placode. (G) Scattered Tbx3⁺ cells in a middle lateral line placode (boxed area enlarged in H). (I) Neuromast primordium of an anterodorsal lateral line placode with Tbx3⁺ mantle (m) and support cells (s). Images A–C are stitched from two micrographs using Corel Photo-Paint. All micrographs were adjusted for brightness (including slight gamma changes), colour balance, and sharpness. Scale bars: 20 µm in A–C,G, 5 µm in D–F,H and I. ad, m, p, anterodorsal, middle, and posterior lateral line placode, respectively; e1, e2, e3, epibranchial placodes 1, 2, 3, respectively; gg, geniculate ganglion; ot, otic anlage; ov, optic vesicle; pg, petrosal ganglion; PPA, posterior placodal area; Q-VD-OPh, pan-caspase inhibitor; wec, whole embryo culture.

Fig. 5.

Neurogenesis in the lateral line placodes of Q-VD-OPh-treated mice. (A,C,F,G) Reconstructions of serially sectioned mouse embryos (section interval evaluated=10 µm) show ectoderm (light grey), otic vesicle with detachment site (dark grey), epibranchial placodes (orange), lateral line placodes (blue), immunopositive cells (brown dots), developing cranial nerves (partly numbered brown contours in G). (B,D,E) Micrographs taken from serially sectioned mouse embryos. (A) Sox2⁺ cells are present in epibranchial placodes and, to a much lesser extent, in lateral line placodes (embryo #021). (B) Sox2⁺ mantle (m) and support (s) cells in a neuromast primordium of a middle lateral line placode. Note the apical cavity (arrowhead). (C) Ngn1⁺ cells are present in epibranchial placodes and, to a much lesser extent, in lateral line placodes (embryo #039, mirror-imaged right side). (D) Ngn1⁺ neuroblasts in an anterodorsal lateral line placode. (E,F) Ngn2⁺ neuroblasts are present in epibranchial placodes, but absent from lateral line placodes (embryo #001). (G) Tubb3⁺ neurons constitute the anlagen of the cranial nerves 7 and 8 (VII/VIII), 9 (IX), 10 (X), 11 (XI) and 12 (XII). Tubb3⁺ candidates for vestigial lateralis ganglia (asterisks) reside in close proximity to the middle lateral line placode (embryo #014). All micrographs were adjusted for brightness (including slight gamma changes), colour balance, and sharpness. Scale bars: 5 µm. ad, m, p, anterodorsal, middle, and posterior lateral line placode, respectively; e1, e2, e3, epibranchial placodes 1, 2, 3, respectively; ot, otic anlage; ov, optic vesicle; Q-VD-OPh, pan-caspase inhibitor; wec, whole embryo culture.