The expression of Delta ligands in the sponge Amphimedon queenslandica suggests an ancient role for Notch signaling in metazoan development

Abstract

Background: Intercellular signaling via the Notch pathway regulates cell fate, patterning, differentiation and proliferation, and is essential for the proper development of bilaterians and cnidarians. To investigate the origins of the Notch pathway, we are studying its deployment in a representative of an early branching lineage, the poriferan Amphimedon queenslandica. The A. queenslandica genome encodes a single Notch receptor and five membrane-bound Delta ligands, as well as orthologs of many genes that enact and regulate canonical Notch signaling events in other animals.

Methods: In the present report we analyze the structure of the five A. queenslandica Deltas using bioinformatic methods, and characterize their developmental expression via whole mount in situ hybridization and histological staining.

Results: Sequence analysis of the A. queenslandica Delta ligands highlights the conservation of their extracellular domains. This contrasts with the divergence of their intracellular regions, each of which is predicted to bear a unique repertoire of protein interaction motifs. In keeping with this diversity, these ligands are expressed differentially and dynamically throughout A. queenslandica embryogenesis, both in cell type specific patterns and broader regional domains. Notably, this expression coincides with the development of the photosensitive larval pigment ring, the non-ciliated cuboidal cells located at the anterior pole of the larva, and the intraepithelial flask cells and globular cells that are presumed to have sensory and/or secretory roles.

Conclusions: Based on the dynamic and complex patterns of expression of these Delta ligands and the Notch receptor, we propose that the Notch signaling pathway is involved in regulating the development of diverse cell types in A. queenslandica. From these observations we infer that Notch signaling is a conserved feature of metazoan development, ancestrally contributing to cell determination, patterning and differentiation processes.

Figure 1

Molecular characteristics of Amphimedon queenslandica Deltas. (A) Alignment of the extracellular regions of A. queenslandica and Drosophila Deltas. All sequences possess a signal peptide and transmembrane (TM) domain, a Delta/Serrate/Lag (DSL) domain, epidermal growth factor (EGF) repeats (except AmqD4) and the conserved pattern of cysteine residues characteristic of the MNLL region. A series of basic residues (V) lies downstream of the TM domain, possibly representing a nuclear localization sequence. The region between EGF2 and the TM domain is omitted. Dashes indicate gaps, residues are shaded according to the level of conservation at each position: 100%, black; 80%, dark grey; 60%, light grey. (B) The diversification of the A. queenslandica Delta ligands is reflected in the number and organization of their extracellular EGF domains, and the distribution and identity of predicted interaction sites in their intracellular tails. EGF repeat identities and bilaterian consensus pattern follow [18]. Potential sites of protein binding, cleavage, phosphorylation and glycosyl modification based on [19]. PTB, phosphotyrosine-binding domain; SH2, Src homology 2; SH3, Src homology 3; TRAF, tumor necrosis factor receptor-associated factor.

Figure 2

AmqDelta1 developmental expression. (A) Expression of AmqDelta1 is first detected in cells in the outer layer in early cloud stage embryos (A’). (B) Expression remains in scattered cells (B’) in the outer layer, more densely occurring at the boundary between the two layers. (C) Late spot stage embryos express AmqDelta1 in the outer layer, as well as in cells under the center of the forming pigment ring (white arrow) (C’) and at the anterior pole (arrowhead) (C”). (D) Ring stage embryos display expression in the forming middle layer (white arrowhead), as well as the anterior pole (inset). (E) In late ring embryos, expression is detected in the subepithelial (middle) layer (white arrowhead), and in the globular cells (double arrowhead) that are migrating from the subepithelial layer to the outer margin (E’). (F) Expression in larvae persists in the globular cells (double arrowhead), now located around the outer margin (F’,F”), and within the pigment ring (white arrow). All panels display cleared, whole mount embryos, except (F”) in which the embryo was sectioned after staining. (C”), anterior view; (C’), posterior view; all remaining panels are lateral views with posterior to the top.

Figure 3

AmqDelta2 developmental expression. (A) Brown stage, isolated cells express AmqDelta2 in no discernable pattern. (B) At late cloud stage, expression is under the forming pigment spot (arrowhead) at the posterior pole. (C) Spot stage embryos show strong expression of AmqDelta2 under the spot (arrowhead) (C’), as well as some expression at the boundary between the middle and outer layers. (D) Ring stage embryos no longer display an expression domain under the posterior pole, expression is now localized to the forming subepithelial layer (white arrowhead) and to a small population of cells at the anterior margin of the embryo (arrow) (D). (E) In late ring stages, staining persists in the anterior margin (arrow) (E’) and subepithelial layer (white arrowhead) (E”) and is apparent in the inner cell mass. (F) Little expression is detected in the larva, except for some cells in the subepithelial layer (white arrowhead) (F’). All panels display cleared whole mount embryos, lateral views with posterior to the top.

Figure 4

AmqDelta3 developmental expression. (A) Expression commences during late cleavage in cells scattered throughout the embryo (A’). (B) As pigment cells begin to migrate to the posterior pole, expression of AmqDelta3 is in cells found at the boundary between the two forming cell layers, including a group of cells located towards the anterior of the embryo (arrowhead). (C) Similar to the cloud stage, expression in spot embryos is in cells located around the boundary between cell layers, with an anterior condensation of expression (arrowhead) ((C’) note: this image is from an early spot stage embryo, not the embryo pictured in (C)). (D) At the early ring stage, expression has coalesced in the forming middle layer. (E) Expression in the late ring stage is localized to the subepithelial layer, as well as a small number of cells located within the outer layer at the anterior (arrow) and posterior (white arrow) poles (E’,E”). (F) Expression in the larval stage remains in the subepithelial layer. All panels display cleared whole mount embryos. (C’), anterior view; all remaining panels are lateral views with posterior to the top.

Figure 5

AmqDelta4 developmental expression. (A) At cloud stage, AmqDelta4 expression is in cells located near the boundary between the inner and outer layers of the embryo, as well as in a patch of cells that lie beneath the anterior pole (arrowhead) (A’). (B) At spot stage, expression persists in cells at the boundary between the inner and outer layers, and beneath the anterior pole (arrowhead), and a new expression domain arises around the outer edge of the pigment spot (arrow) (B’). (C) Ring stage expression is strong under the forming ring (arrow) (C’) and in the middle layer (white arrowhead) (C”). (D) Late ring embryos show no expression in the subepithelial layer; expression under the ring remains strong (arrow) (D’), and expression arises in cells located at the outer margin of the embryo, more densely occurring in the anterior third (double arrowhead) (D”,D”’). (E) Larval expression is predominantly under the pigment ring (arrow) (E”). All panels display cleared whole mount embryos. (A’), anterior view; (B’,C’,D’), posterior views; all remaining panels are lateral views with posterior to the top.

Figure 6

AmqDelta5 developmental expression. Across all developmental stages, AmqDelta5 is expressed in a small number of spatially isolated cells throughout the embryo. These are scattered through all layers in earlier stages (A-C), becoming more localized to the developing middle and inner layers in later stages (D-E). Fewer cells are apparent in the ring and larval stages (E,F). All panels display cleared whole mount embryos, lateral views with posterior to the top.

Figure 7

AmqNotch developmental expression. (A) In late cleavage stages, expression is detected in scattered cells (A’,A”). (B) Cloud stage, expression is diffuse throughout the embryo, with denser staining at the boundary between the inner and outer layers. (C) Expression remains diffuse throughout the spot stage embryo, although is somewhat concentrated in two areas (circled); a domain of higher expression is noted around the pigment spot (arrow). (D) Expression persists into the late spot stage, a domain of higher expression remains localized to cells surrounding the pigment spot (arrow) (D’). (E) In the ring stage, expression of AmqNotch has coalesced to the forming middle layer as well as remaining associated with the pigment ring (arrow) (E’); expression also is detected in the inner cell mass. (F) Expression in late ring embryos is within the subepithelial layer and outer layer. (G) AmqNotch is expressed throughout the larva. (Note: expression around the outer margin of the ring embryo (E) is in cells that make up the non-embryonic follicle layer.) All panels display cleared whole mount embryos, lateral views with posterior to the top.

Figure 8

Cytological features of Amphimedon queenslandica development. All panels show sectioned embryos stained with hematoxylin and eosin. (A-A”) Posterior ciliated cells. Cloud, no cell population uniquely lies in the future position of the posterior ciliated cells, dotted box (A). Spot, cells lateral to the pigment spot are morphologically distinct, box (A’). Late ring, these cells have apparently ingressed, and appear to be clustered, box (A”). (B-B”) Anterior pole. Cloud, distinct cells with small inclusions are present at the anterior pole, dashed line (B). Spot, the anterior-most cells are condensing at the pole, dashed line (B’). Ring, the anterior pole contains a group of non-ciliated cuboidal cells with apical nuclei, dashed line (B”). (C-C”) Cell layers. Cloud, the inner layer contains large granular macromeres, the outer layer contains micromeres and large globular macromeres (C). Ring, the outer layer contains epithelial cells, interspersed with flask cells, the inner layer is comprised of large macromeres among other cell types (C’). The larva has three cells layers; an outer epithelial layer interspersed with globular cells and flask cells, a subepithelial layer composed mostly of large macromeres, and the inner cell mass (C”). (D) Subposterior region. Inner layer macromeres are more densely packed directly under the forming pigment spot. Dashed line, approximates the area of higher cell density. (E,E’) Intraepithelial cells. Ring, the epithelial layer contains flask cells, dotted lines (E). Larva, both flask cells and globular cells (dashed lines) are present (E’). P, pigment cells; IL, inner layer; ICM, inner cell mass; OL, outer layer; EL, epithelial layer; SEL, subepithelial layer.

Figure 9

Summary of the expression of AmqDelta genes during Amphimedon queenslandica development. Schematic representation of A. queenslandica development highlighting the dynamic expression of AmqDeltas across multiple cell layers and cell types. Cell types are identified where possible; note that while each gene is represented with a unique territory of expression, coexpression of ligands in the same cell type is likely: for example, AmqDelta1 and AmqDelta4 in the anterior pole cells, spot stage.