Figure 1.
Hierarchical Clustering of the biological samples.
Hierarchical clustering was performed using complete linkage algorithm applied on the similarity matrix based on the Pearson correlation coefficient. LO = left ovary; RO = right ovary; LT = left testis; RT = right testis.
Figure 2.
Hierarchical clustering of 110 gene expression patterns in right and left male and female chicken gonads.
Each row represents a gene, and each column represents a sample. The samples at the top of the heatmap are set out according to sex (male and female), according to side (left or right) and according to development stage. Each cell in the matrix corresponds to an expression level, with blue for underexpression, yellow for overexpression, black for gene expression close to the median (see color scale) and grey for missing values. Genes are grouped according to their expression pattern across all samples using hierarchical clustering based on the Pearson correlation coefficient as a similarity matrix between genes, and complete linkage algorithm. Particular clusters are quoted on the right side of the picture. Differential analysis was performed with the Limma R package. Genes are considered differentially expressed if the adjusted p values by the Benjamini and Hochberg's method are below a threshold fixed at 0.05 for the contrast tested: A) Analysis of all left female samples vs all left male samples. B) Analysis of early left female samples vs early left male samples. C) Analysis of late left female samples vs late left male samples. D) Analysis of early left female samples vs late left female samples. E) Analysis of early left male samples vs late left male samples. F) Analysis of left female samples vs right female samples. G) Analysis of left vs right early female samples. H) Analysis of left vs right later female samples. I) Analysis of left male samples vs right male samples. J) Analysis of left vs right early testis samples. K) Analysis of left vs right later testis samples. Red asterisks indicate overexpression in female gonads, blue asterisks indicate overexpression in male samples, brown asterisks indicate overexpression in late group of samples, orange asterisks indicate overexpression in early group of samples, dark green asterisks indicate overexpression in left gonads and light green asterisks indicate overexpression in right group of samples.
Figure 3.
Expression profiles of some representative genes from the four main clusters (C1 to C4) identified during male and female sex differentiation.
Cluster C1.2: INHA (inhibin alpha) and CLDN11 (claudin 11 (oligodendrocyte transmembrane protein)); cluster C1.3: ADAMTS12 (ADAM metallopeptidase with thrombospondin type 1 motif, 12) and LOC427192 (similar to hypothetical protein MGC42105); cluster C2: BMP2 (bone morphogenetic protein 2) and AR (Androgen receptor); cluster C3: BMP3 (bone morphogenetic protein 3) and HSD17B4 (Hydroxysteroid (17-beta) dehydrogenase 4); Cluster C4: PAX2 (paired box 2) and GREM1 (gremlin 1). For each histogram, female samples are represented by black squares and black lines and male samples by empty squares and dotted line. Results are represented with an arbitrary scale as the ratio between the expression of the specific gene and the mean expression of EEF1A1and RPL15. Each square represents the mean of two different measurements from different biological samples for the same male or female population and the same sampling date.
Figure 4.
Expression profiles of some representative genes presenting asymmetric expression between left and right gonads.
FST (Follistatin); GNRH1 (gonadotropin-releasing hormone 1 (luteinizing-releasing hormone); GREM1 (Gremlin 1, cysteine knot superfamily, homolog (Xenopus laevis)); IGF1 (insulin-like growth factor 1 (somatomedin C)); BMP3 (bone morphogenetic protein 3); PAX5 (paired box 5); GJB1 (gap junction protein, beta 1, 32 kDa). Male and female expression levels are represented by empty circles and black squares, respectively. Expression levels in the right and left gonads are represented by a dotted line and black line, respectively. Each dot represents the means of two different measurements corresponding to two different biological samples for the same male or female population or the same sampling date.
Figure 5.
Expression of CLDN11, ADAMTS12 and BMP4 in left male and female embryonic gonads analyzed by whole mount in situ hybridization.
Gonads with undelaying mesonephros and 50 µM gelatin sections of the same gonads are presented for each probe. g (gonad), m (medulla), c (cortex). Day of incubation is indicated in the figure for each analysis. Scale Bars = 50 µM.