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. 2013 Dec 12;10(3):408–415. doi: 10.4161/auto.27319

graphic file with name auto-10-408-g2.jpg

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.