Figure 2. An A. willeyana GABARAPL2 ortholog is expressed exclusively in spherulite-forming cells. (A) A lateral view of a mature A. willeyana illustrating the calcified stalk (arrow) upon which the living part of the animal grows. (B) A magnified view of the boxed region in (A) highlighting the individual spherulites that comprise the calcified skeleton. (C) A series of SEMs illustrating the growth and change in morphology of spherulites (reproduced from).⁷ (D) A representative “apical” view of a decalcified and sagittally sectioned A. willeyana individual following WMISH against Awi-GABARAPL2. (E) Magnification of the boxed region in (D) to illustrate the distribution and density of Awi-GABARAPL2-positive cells. (F) A representative lateral view of the sectioned face of a decalcified and sagittally sectioned A. willeyana individual following WMISH against Awi-GABARAPL2. Awi-GABARAPL2-positive cells are clearly most abundant in the distal-most spherulite-forming region of the sponge. (G) Magnification of the boxed region in (F) reveals an absence of these transcripts from developing larvae (arrows). The blue dot present on the left-most larvae in this panel is overlying the larva. (H) Paraffin sections of WMISH material show the location of Awi-GABARAPL2-positive cells in more detail. These cells are located just below the distal-most layer of ectosomal cells (indicated by an arrow). (I) A higher magnification of this sectioned material reveals the distinctive morphology of spherulite-forming cells, and the exclusive signal of Awi-GABARAPL2 in these cells. (J) A magnified view of several Awi-GABARAPL2-positive cells. The texture of the IOM (cf. M) that remains following decalcification is visible within these cells. (K) Cells with the same morphology as those expressing Awi-GABARAPL2 also express Spherulin, a gene that was horizontally transferred from a bacterium into the genome of A. willeyana and is most likely now involved in biomineralization in A. willeyana.⁶ The texture of the IOM that remains following decalcification is also visible within these cells (cf. M). (L) Cells with the same morphology as those expressing Awi-GABARAPL2 also express Astrosclerin, an α-CA homolog that is involved in biomineralization in A. willeyana.⁷ The texture of the IOM that remains following decalcification is clearly visible within these cells (arrow, cf. Fig. 4F). (M) A representative section of A. willeyana material that was subjected to WMISH using a sense GABARAPL2 control probe. This section was counterstained with Nuclear Fast Red.