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We examined hypothalamic activation in chicken embryos from embryonic day (ED) 12\u201317 following experimental stimulation consisting of a localized foot injection of paraformaldehyde (PFA) together with the associated handling procedures required for its administration. Hypothalamic activation was quantified as the proportion of c-Fos-positive cells within the paraventricular nucleus (PVN). Generalized linear modelling revealed significant effects of both embryonic age and stimulation condition on c-Fos expression, with no evidence of an Age \u00d7 Stimulation interaction. Across development, stimulated embryos exhibited higher proportions of c-Fos-positive cells than age-matched controls. Independent of stimulation, the only significant difference between adjacent developmental stages occurred between ED14 and ED15, suggesting a candidate transition in hypothalamic maturation. Exploratory analysis indicated that these findings are consistent with an early (ED12\u2013ED14) and a late (ED15\u2013ED17) developmental phase and suggest that ED14\u2013ED15 may represent a candidate phase boundary in hypothalamic maturation.<\/jats:p>","DOI":"10.1038\/s41598-026-65626-x","type":"journal-article","created":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T07:12:26Z","timestamp":1788592346000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Hypothalamic c-Fos expression changes during late chicken embryonic development and increases following experimental stimulation"],"prefix":"10.1038","volume":"16","author":[{"given":"Leonhard","family":"Kowalczyk","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tobias","family":"Kowalczyk","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yile","family":"Huang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maik","family":"Hintze","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Longfei","family":"Cheng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bernd\u00a0","family":"Hilgers","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stefanie","family":"Kuerten","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qin","family":"Pu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Inga","family":"Tiemann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruijin","family":"Huang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,9,5]]},"reference":[{"key":"65626_CR1","doi-asserted-by":"publisher","first-page":"327","DOI":"10.1007\/s11065-010-9148-4","volume":"20","author":"J Stiles","year":"2010","unstructured":"Stiles, J. & Jernigan, T. 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