Sexually dimorphic microRNA expression during chicken embryonic gonadal development

Abstract

MicroRNAs are a highly conserved class of small RNAs that function in a sequence-specific manner to posttranscriptionally regulate gene expression. Tissue-specific miRNA expression studies have discovered numerous functions for miRNAs in various aspects of embryogenesis, but a role for miRNAs in gonadal development and sex differentiation has not yet been reported. Using the chicken embryo as a model, microarrays were used to profile the expression of chicken miRNAs prior to, during, and after the time of gonadal sex differentiation (Embryonic Day 5.5 [E5.5], E6.5, and E9.5). Sexually dimorphic miRNAs were identified, and the expression patterns of several were subjected to further validation by in situ hybridization and Northern blot analysis. Expression of one chicken miRNA, MIR202*, was observed to be sexually dimorphic, with upregulation in the developing testis from the onset of sexual differentiation. Additional data from deep sequencing of male and female embryonic gonad RNA samples also indicated upregulation of MIR202* in male gonads. These findings provide the first evidence of sexually dimorphic miRNA expression during vertebrate gonadal sex differentiation and suggest that MIR202* may function in regulating testicular development.

FIG. 1.

The miRNAs expressed in embryonic chicken gonads during sexual differentiation. Heat map displays all 92 miRNAs detected with expression in chicken embryonic gonads from E5.5, E6.5, and E9.5. Expression is color coded to reflect average normalized signal intensity per miRNA, per sex and stage. Males are shown on the left and females on the right. Signal intensity values represented by heat map are given in Supplemental Table S1 under the averages columns.

FIG. 2.

Average number of miRNAs upregulated greater than 2-fold during gonadal differentiation. Averages are calculated from the number of miRNAs expressed 2-fold higher in either sex for at least three replicate gonad miRNA arrays per sex, per stage. Error bars represent SD. **P < 0.01 between grouped samples. Total number of miRNAs differentially upregulated in embryonic gonads was 50. Five of these miRNAs showed switching in upregulation between male and female at varying stages.

FIG. 3.

Analysis of MIR101, MIR449, and MIR193B expression by WISH. Whole-mount chicken UGSs from E6.5 and E9.5 embryos probed with LNA probes for MIR101, MIR193B, MIR499, MIR17-5P (positive control), and MIRscrambled (miR-Scram; negative control). Positive detection of miRNA expression is indicated by blue/purple staining. Images were taken approximately 4 h after addition of color solution. Bars = 500 μm.

FIG. 4.

Expression of MIR202 and MIR202* detected by WISH. Whole-mount and transverse-sectioned UGSs showing detection of MIR202 (A–L) and MIR202* (M–X). Gonads in MIR202 whole mount are outlined in gray dashed lines. In female E6.5 panels, white dashed line separates cortex (C) and medulla (M). White arrowheads indicate testis cords, and black arrowheads indicate interstitium. Bars = 500 μm (whole mount) and 100 μm (sections). Area represented in zoomed, high-power (original magnification ×400) images (I–L and U–X) of sectioned gonads is indicated by white/black dashed-line box in low-power/ unzoomed (E6.5, original magnification ×400; E9.5, original magnification ×200) images (E–H, Q–T).

FIG. 5.

Deep sequencing profile of MIR202* and MIR202 expression in chicken embryo gonads. Normalized read counts are shown for MIR202* (black bars) and MIR202 (gray bars) in male and female gonad RNA samples. Normalized read counts were calculated by dividing the number of raw read counts for exact miRBase sequences of MIR202* and MIR202 by the total number of tagged reads obtained, per sample. Ratio of miRNA:total tagged reads was then converted to whole numbers by multiplying by a constant value of 1 × 10⁶.

FIG. 6.

Northern blot detection of MIR202 and MIR202* expression. RNA samples purified for miRNA fractions from male and female chicken embryonic gonads (G) at E5.5, E6.5, and E9.5, and at E9.5 in embryonic kidney (K). a) Northern blot (1 μg RNA/lane) probed with LNA for MIR202* and MIR202, and chicken U6 snRNA probed as loading control. b) Normalized expression of MIR202* in male versus female gonad and kidney samples, as detected by Northern blot, showing male upregulation at E6.5 and E9.5. c) Normalized expression of MIR202 as detected by Northern blot. Normalized values are calculated as a ratio of signal for chicken U6 snRNA loading control per sample and multiplied by a constant of 100 to give whole numbers. nt, nucleotide.