Figure 1. Strategy for identifying genes required for neoblast-dependent formation of the anterior pole.

(A) in situ hybridizations to detect expression of notum after amputation in untreated animals or animals exposed to gamma irradiation (6000 Rads) 48 hours prior to surgery. Late (72 hours) expression of notum at the anterior pole is irradiation sensitive (arrow), but not early (18 hours) expression of notum in disperse cells near the wound site. Cartoon shows surgery (red lines) and enlarged region (green box). Bars, 300 microns. (B) Cartoon showing strategy to identify genes expressed differentially within neoblasts in head and tail regeneration. Animals were amputated transversely at either planes (i) or (ii), and X1 neoblasts purified by FACS from macerated tissue fragments from regions near injury sites engaged in either head or tail regeneration in a time series. Expression profiling was performed to identify genes whose expression significantly changed at 1, 2, or 3 days after amputation as compared to neoblasts isolated from regionally matched non-regenerating tissue. (C) Heat map showing only genes upregulated as detected by microarray (yellow, log₂ fold-change (FC), chosen with false-discovery rate <10%) in head and/or tail regeneration. (D) qPCR experiment showing expression of 21/24 candidate genes upregulated in neoblasts due to regeneration whose expression behavior was confirmed upregulated. (C–D) Heat maps show microarray and qPCR data for genes ordered using hierarchical clustering with both microarray and qPCR expression values as inputs and broadly categorize into those upregulated primarily in anterior or posterior regeneration, or both (yellow, log₂ fold-change versus 0 hour timepoint for each anterior or posterior series). We further analyzed a subset of genes that reproducibly increased in expression specific to anterior regeneration.