The zinc-binding protein chordc1 undergoes complex diurnal changes in mRNA expression during mouse brain development

Abstract

Diurnal changes in Chordc1 mRNA were recently described in mouse hypothalamus. This report shows that Chordc1 mRNA changes rhythmically throughout the entire adult brain with highest expression levels occurring around the dark-light transition. The rhythmic cycling pattern of Chordc1 was retained under various light-dark schedules and analysis of adult whole brain revealed diurnal patterns that were different than young animals (postnatal day (P) 6). Analysis of adult hippocampus, prefrontal cortex and cerebellum confirmed these observations and a comparison between adult and P6 animals using in situ hybridization indicated that Chordc1 underwent coordinated but altered diurnal changes in mRNA abundance during development. Further, a developmental profile of Chordc1 expression beginning at embryonic day 17 revealed a regional distribution of Chordc1 consistent with its adult pattern. These results suggest that Chordc1 mRNA is under complex and widespread transcriptional regulation during development and implicate Chordc1 in circadian and/or homeostatic mechanisms in mammalian brain.

Fig. 1

Diurnal expression of Chordc1 mRNA in adult mouse brain during two photic-entrainment schedules. (A) Densitometric analysis of Chordc1 mRNA at four timepoints in a 12:12 light-dark (LD) schedule (blue diamonds and blue solid lines; ZT0, ZT6, ZT12, ZT18) and four timepoints in a 14:10 LD schedule (red circles and red dashed lines; ZT2.5, ZT8.5, ZT14.5, ZT20.5) using in situ hybridization. Each value represents the average ± S.E.M. N = 2 per timepoint. Data from each region on 12:12 LD schedule were subjected to one-way ANOVA and a statistically significant diurnal variation was observed (Hippocampus P < 0.001; Prefrontal Cortex P < 0.001; Cerebellum P < 0.001). *** P < 0.001 versus ZT12 (post hoc Bonferroni). Data from each region on 14:10 LD schedule were also subjected to one-way ANOVA and a statistically significant diurnal variation was observed (Hippocampus P < 0.05; Prefrontal Cortex P < 0.05; Cerebellum P < 0.05). * P < 0.05 versus ZT8.5 (post hoc Bonferroni). (B) Representative sagittal sections from adult mouse brain hybridized to Chordc1 cRNA probe and analyzed for densitometry as described in A and Methods. N = 2 per timepoint in each 12:12 and 14:10 LD schedule

Fig. 2

Diurnal rhythm of Chordc1 mRNA is differentially regulated in P6 compared to adult mouse whole brain. (A) Chordc1 mRNA levels from adult mouse whole brain expressed relative to the average value of all samples normalized to glyceraldehyde-3-phoshate dehydrogenase (Gapdh) from four timepoints on 14:10 LD schedule (ZT2.5, ZT8.5, ZT14.5, ZT20.5). N = 2 per timepoint. Data were subjected to one-way ANOVA and a statistically significant diurnal effect was revealed (P = 0.002). *P < 0.05, ***P = 0.001 (post hoc Bonferroni). The inset panel illustrates representative diurnal Northern blots of Chordc1 and Gapdh mRNA from adult mouse whole brain. Chordc1, 2200 bp; Gapdh, 1240 bp. (B) Chordc1 mRNA levels from P6 mouse whole brain expressed relative to the average value of all samples normalized to Gapdh from four timepoints on 14:10 LD schedule (ZT2.5, ZT8.5, ZT14.5, ZT20.5). N = 3 per ZT2.5 and ZT14.5 timepoints, N = 2 per ZT8.5 and ZT20.5 timepoints. Data were subjected to one-way ANOVA and a statistically significant diurnal effect was not observed (P = 0.114). t-test of highest and lowest expression points (ZT14.5 and ZT8.5, respectively) approached significance (P = 0.065). The inset panel illustrates representative diurnal Northern blots of Chordc1 and Gapdh mRNA from P6 mouse whole brain. Chordc1, 2200 bp; Gapdh, 1240 bp

Fig. 3

Diurnal expression of Chordc1 mRNA in P6 mouse brain. (A) Densitometric analysis of Chordc1 mRNA at four timepoints in a 14:10 LD schedule (ZT2.5, ZT8.5, ZT14.5, ZT20.5) using in situ hybridization. Each value represents the average ± S.E.M. N = 3 per ZT2.5 and ZT14.5 timepoints, N = 2 per ZT8.5 and ZT20.5 timepoints. Data from each region on 14:10 LD schedule were also subjected to one-way ANOVA and a statistically significant diurnal variation was observed (Hippocampus P = 0.011; Prefrontal Cortex P < 0.01; Cerebellum P < 0.05). *P < 0.05 versus ZT8.5 (post hoc Bonferroni). (B) Representative sagittal sections from mouse brain hybridized to Chordc1 cRNA probe and analyzed for densitometry as described in A and Methods. N = 3 per ZT2.5 and ZT14.5 timepoints, N = 2 per ZT8.5 and ZT20.5 timepoints

Fig. 4

Distribution of Chordc1 mRNA in developing mouse brain In situ hybridization with ³⁵S-labeled antisense riboprobe was used to detect the distribution of Chordc1 mRNA in sagittal sections from (A) postnatal day (P) 1, (B) P5, (C) P14 and (D) adult mouse brain. Insets show control brain sections at each age hybridized to ³⁵S-labeled sense riboprobe. Images were color rendered to enable visualization of labeling (red, highest and blue, lowest levels). Bar in A–D = 1 mm. BS, brain stem; CB, cerebellum; CC, cerebral cortex; H, hippocampus; P, postnatal day

Fig. 5

Distribution of Chordc1 mRNA in developing cerebral cortex. (A) Dark field photomicrographs (top panel) of coronal sections from embryonic day 17 mouse brain depicting Chordc1 mRNA labeling primarily within the cortical plate (CP) with lighter labeling in the subventricular zone (SVZ) and intermediate zone (IZ). Bottom panel is a light field micrograph of the same region counterstained with cresyl violet. (B) Dark field micrograph (top) of postnatal day (P1) cortex revealing a disperse pattern of Chordc1 labeling throughout the cortical layers. Bottom panel is section counterstained with cresyl violet. (C) By P14, label to Chordc1 mRNA was present throughout the cortex in a pattern suggesting the presence of Chordc1 mRNA in individual cells. Top, dark field, bottom, counterstained light field micrograph. (D) In adult brain, a more punctuate distribution of Chordc1 was evident and heavy relative labeling was present in large foci suggestive of pyramidal cells in layer V. Top, dark field, bottom, counterstained light field micrograph. Bar in A, B = 100 μm; in C, D = 200 μm. E, embryonic; P, postnatal

Fig. 6

Chordc1 mRNA is expressed in both pre- and post-migratory cells in developing cerebellum. (A) In situ hybridization using Chordc1 riboprobe to postnatal day (P) 1 sagittal brain sections revealed robust labeling throughout cerebellum that did not distinguish layers in the cerebellar cortex (CC). DCN, deep cerebellar nuclei. Image is a dark field micrograph. (B) At P5, label corresponding to Chordc1 was present in the premigratory external granule cell layer (EGL) and the postmigratory Purkinji cell (PCL) and internal granule cell (IGL) layer. Note that in this dark field micrograph heavier Chordc1 labeling was present in PCL. (C) By P10, Chordc1 mRNA (dark sliver grains) was evident in the Purkinji (P) and granule cell layer (GL) but was absent from the cell-sparse molecular layer (ML). Section was counterstained with cresyl violet. Inset shows low magnification dark field micrograph with highest intensity of labeling in PCL. (D) In sagittal sections from P14 brain, silver grain accumulation could be identified over individual Purkinji cells (P). Chordc1 mRNA was also present in GL and in cells within the ML. Inset shows low magnification dark field micrograph. Bar in A = 200 μm; B = 250 μm; C = 30 μm; D = 25 μm

Fig. 7

Expression of Chordc1 mRNA in developing hippocampus. (A) A diffuse labeling pattern corresponding to Chordc1 mRNA was evident in dark field micrographs from P1 hippocampus suggestive of Chordc1 in hippocampal neurons (CA1, CA3) in the final stages of migration and maturation. Only faint labeling was present in dentate gyrus (DG). (B) Chordc1 mRNA labeling was more concentrated in pyramidal cell layers of hippocampus (CA1, CA3) and granule cells of dentate gyrus (DG) at P5 (dark field micrograph). (C) By P14, labeling corresponding to Chordc1 mRNA followed the major cell layers of hippocampus and by (D) adulthood, Chordc1 labeling followed a neuronal cell-like pattern with intense labeling in CA1–3 pyramidal cells of the hippocampus. Bar in A, B = 100 μm; C = 230 μm; D = 200 μm