Insulin receptor localization in the embryonic avian hypothalamus

Abstract

The hypothalamus is a brain region critical for the homeostatic regulation of appetite and energy expenditure. Hypothalamic neuronal activity that is altered during development can produce permanent physiological changes later in life. For example, circulating hormones such as insulin have been shown to influence hypothalamic neuronal projections, leading to altered metabolism in adult rodents. While insulin signaling in the post-hatch chicken has been shown to mirror that of mammals, the developmental role of insulin in the avian embryonic hypothalamus remains largely unexplored. Here we present the earliest known evidence for insulin receptor (InsR) expression in embryonic avian hypothalamic nuclei governing energy homeostasis. RT-PCR analysis reveals InsR mRNA in E8, E10, and E12 neurons while western blot data demonstrate protein expression in E12 avian whole brain and hypothalamic lysates. Immunohistochemical analysis of avian hypothalamic brain slices demonstrates a shift in InsR localization from paraventricular expression in E8 to a more defined concentration of InsR in developmental regions resembling the ventromedial hypothalamus (VMH) and arcuate nucleus (ARC) in E12 time points. In addition, InsR expression appears in a heterogeneous pattern, suggesting receptor localization to subpopulations of avian hypothalamic neurons as early as E8. With increasing evidence suggesting energy homeostasis pathways may be altered via the gestational environment, it is important to understand how insulin signaling may affect embryogenesis. Our research provides evidence for the earliest known embryonic expression of InsR protein in the avian hypothalamus and may suggest a developmental role for insulin signaling in the early patterning of metabolic pathways in the central nervous system.

Keywords: Avian embryo; Energy homeostasis; Hypothalamic development; Insulin receptor; Metabolism.

Fig. 1.. Insulin receptor expression in the embryonic avian brain.

A) Immunofluorescence staining of InsR protein in dissociated neuronal cultures (7 DIV) derived from embryonic day 12 (E12) avian whole brain tissue. Scale bar = 100μm; n=5. B) High power magnification of InsR labeled neuron from the dotted region indicated in A. Scale bar = 50μm. C) Western blot analysis of InsR protein detected in E12 whole brain tissue. M = marker; n = 4. D) RT-PCR analysis of InsR expressed at E8, E10, and E12 in the intact avian hypothalamus. A no reverse transcriptase (-RT) condition was performed in parallel as a negative control for genomic DNA contamination. M = marker; n = 3. E) RT-PCR analysis of InsR expressed in dissociated E12 avian hypothalamic cultures. M = marker; n = 3. F) Western blot analysis of InsR protein detected in E12 hypothalamic lysates. M = marker; n = 3.

Fig. 2.. Changes in insulin receptor localization during hypothalamic development.

Representative low power composite (5 × 5 array) fluorescence micrographs of E8 (top) and E12 (bottom) coronal hypothalamic sections labeled with DAPI nuclear staining (A, D) and anti-InsR antibody (B, E) and subsequently merged (C, F). Dotted lines highlight increased InsR intensity in areas that resemble the ventromedial hypothalamus (VMH) and arcuate nucleus (ARC) of E12 as compared to adjacent tissue. 3v = third ventricle; scale bar = 200μm; n = 6 (E8); n = 8 (E12).

Fig. 3.. Heterogeneous insulin receptor expression in the E8 hypothalamus.

Representative high power fluorescence micrograph of an E8 coronal hypothalamic section depicting the developing paraventricular (PVN) region labeled with A) DAPI nuclear staining and B) anti-InsR antibody and subsequently merged (C). Scale bar = 20μm; n = 6.

Fig. 4.. Insulin receptor expression in the E12 ventromedial nucleus of the hypothalamus.

Fluorescence micrographs of E12 coronal hypothalamic sections labeled with DAPI nuclear staining (A, D) and anti-InsR antibody (B, E) and subsequently merged (C, F). Dotted line depicts a clear border between InsR staining in the developing ventromedial hypothalamus (VMH) compared to adjacent tissue. 3v = third ventricle; top scale bar = 100μm; bottom scale bar = 20 μm; n = 8.