Early patterning in a chondrichthyan model, the small spotted dogfish: towards the gnathostome ancestral state

Abstract

In the past few years, the small spotted dogfish has become the primary model for analyses of early development in chondrichthyans. Its phylogenetic position makes it an ideal outgroup to reconstruct the ancestral gnathostome state by comparisons with established vertebrate model organisms. It is also a suitable model to address the molecular bases of lineage-specific diversifications such as the rise of extraembryonic tissues, as it is endowed with a distinct extraembryonic yolk sac and yolk duct ensuring exchanges between the embryo and a large undivided vitelline mass. Experimental or functional approaches such as cell marking or in ovo pharmacological treatments are emerging in this species, but recent analyses of early development in this species have primarily concentrated on molecular descriptions. These data show the dogfish embryo exhibits early polarities reflecting the dorso-ventral axis of amphibians and teleosts at early blastula stages and an atypical anamniote molecular pattern during gastrulation, independently of the presence of extraembryonic tissues. They also highlight unexpected relationships with amniotes, with a strikingly similar Nodal-dependent regional pattern in the extraembryonic endoderm. In this species, extraembryonic cell fates seem to be determined by differential cell behaviors, which lead to cell allocation in extraembryonic and embryonic tissues, rather than by cell regional identity. We suggest that this may exemplify an early evolutionary step in the rise of extraembryonic tissues, possibly related to quantitative differences in the signaling activities, which shape the early embryo. These results highlight the conservation across gnathostomes of a highly constrained core genetic program controlling early patterning. This conservation may be obscured in some lineages by taxa-specific diversifications such as specializations of extraembryonic nutritive tissues.

Fig. 1

General characteristics of the early dogfish embryo. (A) Telolecithal dogfish egg. The blastoderm lies on top of the undivided yolk mass. (B–E) Dorsal view of early embryos. The organizer side of the blastoderm (posterior relative to the forming embryo axis) is to the bottom. (B) Late blastula embryo (stage 10). The most obvious characteristic of this stage is the appearance of a posterior thickening. (C) Early gastrula embryo (stage 11). This stage is marked by the beginning of mesendoderm involution at the posterior margin. (D) Axis formation (stage 12). The anterior neural plate becomes visible, as well as a characteristic posterior indentation corresponding to the organizer and referred to as notochordal triangle or node. Mesendoderm internalization takes place at the level of the posterior arms lying on each side of this structure. (E) Axis elongation and head enlargement formation (stage 14). (F) Cellular organization of a stage 11 embryo at a median sagittal section plane. Posterior is to the right. Histological sections provide indications of cell ingressions from the upper layer and of cells fusing with the yolk. Direct cell labeling highlights anterior displacements of cells detaching from the leading edge of the involuting layer and migrating as single cells. Colored arrows in (B–F) show prevailing cell movements at different levels of the embryo: dotted blue arrows: individual cell movements in the lower layer, taking place prior and anterior to the involution movement; red curved arrows: involution of the upper layer; orange arrows: epiboly; green arrows: posterior convergence. A red point indicates the organizer (notochordal triangle) in (D) and (E).

Fig. 2

Regionalization of the site of mesoderm internalization: comparison between the dogfish (A), xenopus/zebrafish (B), turtle (C) and chick (D). The sites of axial, paraxial and lateral mesoderm internalization are shown in red, orange and yellow, respectively. In the turtle, mesoderm internalization only takes place at early gastrulation stages at the level of the blastoporal plate (dotted line).

Fig. 3

Dynamic of gene expression at the posterior margin of stage 10–13 dogfish embryos. Organizer side (posterior referring to the orientation of the axis when it appears) is to the right. Lim1 (light blue), Hex (dark blue) and Chordin (red) chronological order of expression is translated into an ordered spatial organization of their territories along the antero-posterior axis at stages 11–12. When the morphogenesis of the foregut diverticulum takes place, Hex territory is found ventrally at this level.

Fig. 4

Similar specification events in the mouseVE between mouse VE and dogfish endoderm layer. Column 1, a dorsal projection of the mouse VE. Column 2, schematized sagittal section of the mouse conceptus from 4.5 to 5.5 dpc, with posterior to the right and distal to the bottom. Column 3, a schematized sagittal section of a dogfish embryo, with posterior to the right. Column 4, dorsal view with posterior to the bottom. In the mouse (columns 1–2), specification of Lim1 first (light blue) in the embryonic VE and then Hex (dark blue) in the distal VE successively takes place under the control of Nodal signaling (after Mesnard et al. 2006). In the dogfish (columns 3–4), Lim1 and Hex expression are expressed at the margin starting from late blastula stages, in the same chronological order and with the same nested spatial organization. These expressions are also dependent on Nodal/activin signaling.

Fig. 5

Expression territories of Brachyury, Otx2, LeftyA, LeftyB and Bmp4 in the dogfish embryo from blastula to early gastrula stages. (A) Early to mid-gastrula; (B) stages 10+ to 11; (C) stage 12. Brachyury expression (purple) is in the upper layer at stages 10+ to 11, at the margin and internalizing mesendoderm at stage 12. Otx2 expression (blue) is in the upper layer at all stages shown. LeftyB (orange) expression is restricted to the posterior margin and adjacent lower layer at blastula stages. LeftyA (red) and LeftyB expressions are located at the lateral and posterior margin, excluding the midline (upper layer) at stages 11 and 12. Bmp4 (green) expression is restricted to the upper layer at blastula stages and at stages 10+ to 11, and becomes expressed in an additional territory at the lateral and anterior margins at stage 12. A faint signal is also observed at this stage in the presumptive surface ectoderm, whether embryonic or extraembryonic.

Fig. 6

Relationships between the early polarities observed in the zebrafish (A), xenopus (B), mouse (C) and dogfish (D). Molecular characterizations highlight an early organizer–ab-organizer polarity in the dogfish, unambiguously related to the dorso-ventral polarity of teleosts and amphibians. Comparisons of the dynamics of expression in early endodermal tissues between the mouse and the dogfish suggest that, in the former, this polarity is translated into a radialized, proximal to distal organization.