Figure 6.

Adoptively transferred hemocytes from Percoll gradient layer 1 enter the neurogenic lineage

Hemolymph from donor crayfish containing EdU-labeled hemocytes was separated into two layers with Percoll gradients. Layer 1 cells (hyaline and semigranular hemocytes) were injected into recipient crayfish. Brains and associated niches are shown with EdU labeling that indicates the progression of donor hemocytes through the neurogenic lineage. Scale bars: 10 μm.

(A) EdU-labeled cells are observed in the streams (arrows) and proliferation zones (arrowhead) of Cluster 10 at 7 days after transfer of donor hemocytes from layer 1. A, Hoechst 33,342 (cyan); Ai, EdU cell labeling (red); Aii, anti-GS (blue). The GS antibody labeled all cells in the niche, streams and proliferation zones in P. clarkii. EdU-labeled cells are also observed in the vicinity that are not in the streams or cell cluster. The merged image (Aiii) illustrates the positions of these cells, as well as the location of the enlarged image shown in Aiv-Avi (dotted line box). These highly magnified images show the labeled cells (arrows) and their placement in the streams. The ‘holes’ in GS labeling (Av) are the positions of the EdU labeled cells (Aiv), shown merged in Avi.

(B and C) Three weeks after transfer of layer 1 cells from donor crayfish, single cells (B-Bii) and pairs of cells (C-Cii) are observed in Cluster 10. This pair of cells contains no SIFamide labeling (blue, Ci, Cii).

(D) Six weeks after transfer of layer 1 cells from donor crayfish, EdU-labeled cells are frequently found in clusters (Di), and cells in these clusters contain immunoreactivity for the neurotransmitter SIFamide (Dii, Diii). (E) In these higher magnification images taken six weeks after layer 1 transfer to a recipient crayfish, EdU-labeled cells are observed with anti-SIFamide labeling in the cytoplasm (arrows; Eii shows the merged image illustrating this feature).