Regionalization of the shark hindbrain: a survey of an ancestral organization

Abstract

Cartilaginous fishes (chondrichthyans) represent an ancient radiation of vertebrates currently considered the sister group of the group of gnathostomes with a bony skeleton that gave rise to land vertebrates. This out-group position makes chondrichthyans essential in assessing the ancestral organization of the brain of jawed vertebrates. To gain knowledge about hindbrain evolution we have studied its development in a shark, the lesser spotted dogfish Scyliorhinus canicula by analyzing the expression of some developmental genes and the origin and distribution of specific neuronal populations, which may help to identify hindbrain subdivisions and boundaries and the topology of specific cell groups. We have characterized three developmental periods that will serve as a framework to compare the development of different neuronal systems and may represent a suitable tool for comparing the absolute chronology of development among vertebrates. The expression patterns of Pax6, Wnt8, and HoxA2 genes in early embryos of S. canicula showed close correspondence to what has been described in other vertebrates and helped to identify the anterior rhombomeres. Also in these early embryos, the combination of Pax6 with protein markers of migrating neuroblasts (DCX) and early differentiating neurons (general: HuC/D; neuron type specific: GAD, the GABA synthesizing enzyme) revealed the organization of S. canicula hindbrain in both transverse segmental units corresponding to visible rhombomeres and longitudinal columns. Later in development, when the interrhombomeric boundaries fade away, accurate information about S. canicula hindbrain subdivisions was achieved by comparing the expression patterns of Pax6 and GAD, serotonin (serotoninergic neurons), tyrosine hydroxylase (catecholaminergic neurons), choline acetyltransferase (cholinergic neurons), and calretinin (a calcium-binding protein). The patterns observed revealed many topological correspondences with other vertebrates and led to reconsideration of the current view of the elasmobranch hindbrain segmentation as peculiar among vertebrates.

Keywords: HoxA2; Wnt8; calretinin; cartilaginous fishes; development; evolution; rhombomeres; shark embryo.

Figure 1

Rhombomeres are visible in early embryos as the interrhombomeric limits are evident. (A–H) Whole-mount in situ hybridization for ScHoxA2 (A–C), ScWnt8 (D–G), and ScPax6 (H) in the early developing embryo in lateral view at the indicated developmental stages. Asterisks in (A–D) label the rostralmost intrarhombomeric ventricular ridge at r1. (F,G) are panoramic and detail of a sagittal section of a S24 embryo whole-mount processed for in situ hybridization showing ScWnt8 expression in r4 (in blue) where DCX-immunoreactive fibers of the acoustic-facial nerve roots emerge. (I–K) Parasagittal sections immunolabeled for Pax6 (I), GAD (J), and DCX (K) showing ventricular ridges that coincide with interrhombomeric limits. At these boundaries low density of Pax6 cells (I), radially oriented GAD-ir (J) and DCX (K) cells are observed. Scale bars: 500 μm (A–E, H); 100 μm (F,G, I,J).

Figure 2

Schematic representation of the embryonic brain at stage 26 showing the distribution of GAD-ir (green dots), 5-HT-ir (dark blue dots), and TH-ir (red dots) cells and the relation to transverse segments. The dotted line indicates the alar–basal boundary. The vertical bars indicate the approximate levels of sections of Figures 5 and 6. For abbreviations, see list.

Figure 3

The distribution of Pax6, GAD, and DCX cells reveals the r0/r1 boundary. (A–C) Panoramic views of sagittal sections of embryos at stage 26 to show the distribution of Pax6 (A), GAD (B), and DCX (C) in the rostral rhombencephalon. Note the absence of Pax6 cells in r0 and the dorsoventral band of GAD and DCX cells caudally to r0 (white arrow in B,C). (D,E) Detail of this cell band to show colocalization of both substances in some cells (yellow in F). Asterisk in A indicates the rostralmost intrarhombomeric boundary at r1. For abbreviations, see list. Scale bars: 500 μm (A); 300 μm (B,C); 50 μm (D–F).

Figure 4

Sagittal sections of embryos at stage 26 to show the position of nerve roots in relation to rhombomeres (A–E) and the caudal limit of the rhombencephalon (F,G). (A,B) Panoramic view and detail to show the roots of several branchiomeric nerves and the distribution of GAD-ir cells at the r2–r3 interrhombomeric boundary (arrows). (C–E) DCX labeled processes at the roots of V (C,D), VII–VIII (C), and VI (E) nerves. (F,G) Panoramic view and detail showing the rostral extension of the spinal TH-ir CSF-contacting neurons. Arrows indicate the possible rhombencephalon–spinal cord boundary. For abbreviations, see list. Scale bars: 200 μm (A,C,D,F,G); 100 μm (B,E).

Figure 5

Sagittal (A–C) and transverse (D–I) sections of embryos at stage 25 to show the distribution of Pax6- (A–F) and Sonic hedgehog-immunoreactive cells (G–I). The levels of (D–I) are indicated in (A–C) and in Figure 2. Scale bars: 500 μm (A–C); 200 μm (D–I).

Figure 6

Sagittal (A–D) and transverse (E–H) sections of embryos at stage 26 showing the columnar distribution of GAD-ir populations. The levels of (E–I) are indicated in (A) and in Figure 2. Dorsolateral and ventrolateral columns are continuous along the rhombencephalon from rostral to caudal levels (A–H) but from r4 these columns appeared subdivided into dorsal and ventral subgroups (F–H). Scale bars: 500 μm (A); 100 μm (B–H).

Figure 7

Transverse sections from rostral (A,E,I) to caudal (D,H,L) rhombencephalic levels of embryos at stage 29 showing the comparative distribution of Pax6 (A–D) and GAD (E–H) cells. Double labeling (I–L) reveals a band of GAD-negative cells coinciding with the interface between intense and weak Pax6 cells that may corresponds to the alar–basal boundary (white arrows). (M–O) Detail of this boundary. Scale bar: 200 μm (A–L); 100 μm (M–O).

Figure 8

Schematic representation of the embryonic brain at stage 29 showing the distribution of CR- (bright blue dots), 5-HT- (dark blue dots), TH- (red dots), and ChAT-ir (yellow dots) immunoreactive cells and the relation to transverse segments. The dotted line indicates the alar–basal boundary. The vertical bars indicate the approximate levels of sections of Figure 9. For abbreviations, see list.

Figure 9

Sagittal (A–E) and transverse (F–J) sections of embryos at stage 29 showing the columnar distribution of CR-ir populations. The levels of (F–J) are indicated in (A) and in Figure 8. Note that CR-ir cells are more abundant in the basal than in the alar rhombencephalon and form midline (Ra) and lateral (Re) populations of the reticular formation. Scale bars: 500 μm (A); 200 μm (B–E); 100 μm (F–I).

Figure 10

Sagittal sections showing the distribution of ChAT-ir cell groups in the rhombencephalon at the stage 29. (A–D) Panoramic view (A) and details (B–D) of ChAT-ir cells of the oculomotor and trochlear nucleus (B), trigeminal motor nucleus (C), and magnocellular octaval nucleus (D). (E,F) Panoramic view and detail of ChAT-ir cells in the visceromotor column. Asterisks label the negative roots of the glossopharyngeal and vagal nerves. For abbreviations, see list. Scale bars: 200 μm.