Transcriptional Profiling of Zebra Finch Hypothalamus After Chronic Heat Call Exposure

Late-stage zebra finch embryos were exposed to chronic playback of parental heat calls or control sounds to test how acoustic cues of extreme heat reshape hypothalamic gene expression. RNA-seq of medial hypothalamic punches revealed modest changes in neuroendocrine genes but strong downregulation of muscle and cytoskeletal programs, with evidence of altered isoform usage and enrichment in neurovascular cell populations. These findings indicate that parental heat calls drive a non-neuroendocrine transcriptional program underlying early-life developmental plasticity and acoustic-mediated adaptation to environmental extremes in birds. Overall design: Embryonic zebra finch (Taeniopygia guttata) eggs from breeding pairs were incubated at 37.5°C and, on embryonic day 10 (E10) in the morning, were randomly allocated to either heat-call playback (whine + heat calls; n=10) or control-call playback (whine + tet calls; n=9) using a within-clutch design that balanced laying order. Playbacks were delivered via speakers attached to incubators from E10-E13 for 9 h/day over 4 days, with eggs and playbacks swapped daily between incubators to minimize incubator-specific effects. On the night of E13, all embryos were transferred to a new incubator without playback so both groups experienced identical conditions immediately prior to sampling and the experimenter could be blind to treatment. On E14 (10:00-12:00), embryos were removed from the egg and decapitated, and one 1 mm medial hypothalamic punch was collected from 100 um coronal cryosections at the level where the third ventricle first became visible; punches were frozen on dry ice and stored at -80°C. RNA was sequenced on an Illumina NextSeq 500 (75 bp, paired-end) following adapter/quality trimming (Trimmomatic v0.39), and expression was analyzed to test effects of playback condition (and sex, where applicable), with downstream co-expression network, pathway enrichment, and cell-type deconvolution analyses to link transcriptional changes to hypothalamic cell populations.