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. 2025 Feb 20;104(4):104932. doi: 10.1016/j.psj.2025.104932

A comprehensive transcriptional profiling of developing gonads reveals the role of TGFβ signaling in female gonadal asymmetry in chickens

Hong Jo Lee a,b, Jeong Hoon Han a, Brittany Chapman a, Kyung Min Jung c, Isabella Rudd a, Jae Yong Han c, Tae Hyun Kim a,⁎
PMCID: PMC11910708  PMID: 40014972

Abstract

Asymmetrical gonadal development is an intriguing phenomenon observed in the majority of female birds. In chickens, the left gonad of female embryos develops into a functional ovary, while the right gonad undergoes degeneration during embryogenesis. This sexually dimorphic trait is primarily induced by the spatial differential expression of the paired like homeodomain 2 (PITX2) gene. However, a comprehensive understanding of the transcriptional profile of the developing gonads during asymmetric development is still lacking. To elucidate the molecular mechanism of asymmetric gonadal development in chickens, we compared the transcriptomes between left and right gonads of female chickens using bulk- and single cell-RNA sequencing (scRNA-seq) approaches. Our bulk RNA-seq analysis of the female chicken gonads at E5 (HH26), E6.5 (HH30), E8 (HH34), and E9.5 (HH36) revealed significant differential gene expression between the left and right female chicken gonads, particularly in signaling pathways, cell cycle, and metabolic processes. Moreover, scRNA-seq analysis revealed that coelomic epithelial, interstitial, and pre-granulosa cells of the left gonads at E5 show a highly proliferative status compared to the right gonad, contributing to the asymmetric gonadal cell proliferation, which may be regulated by the transforming growth factor beta (TGFβ) signaling pathway. Our findings demonstrate that dynamic cell-type-specific transcriptional profiles during embryogenesis play a vital role in the asymmetric gonadal development of female chickens.

Keywords: Asymmetry, Chicken, Embryonic gonad, Transcriptome analysis, Single-cell RNA sequencing

Introduction

Most avian species exhibit an intriguing phenomenon of asymmetrical gonadal development. In male birds, both the left and right gonads undergo proliferation and differentiation into functional testes. However, in female birds, only the left gonad and Mullerian duct undergo proliferation and differentiate into functional reproductive organs, i.e., ovary and oviduct, respectively, while the right gonad undergoes degeneration during embryogenesis. This sexually dimorphic trait is notably observed in chicken gonads starting at embryonic day 6.5 (E6.5, corresponding to Hamburger & Hamilton stage (HH) 30) (Hamburger and Hamilton, 1951). The left gonad of female chickens progressively increases in length and thickness as embryogenesis proceeds, and histological analysis has revealed the development of a cortical region of stratified epithelial-like cell structure that exclusively forms in the left gonad. In contrast, the right gonad regresses from the developmental stage lacking cortical formation and eventually undergoes complete degeneration at a later stage of gonadogenesis (Carlon and Stahl, 1985).

The morphological differentiation of bird gonads is dictated by sex chromosomes (ZZ for males and ZW for females). Previous studies aimed at identifying testis-determining factors demonstrated that the suppression of the doublesex and mab-3 related transcription factor 1 (DMRT1) gene, situated on the chicken Z chromosome, through RNA interference (RNAi) during embryogenesis results in the thickening of the left gonads and subsequent degeneration of the right gonads in male chickens. (Smith et al., 2009). The testis feminization associated with cortex development in the left gonads was further demonstrated in knock-out studies using clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) technology, indicating that genetic sex is a critical factor for left and right gonad asymmetry development, though it was an incomplete gonad feminization in terms of the transcriptome profile (Lee et al., 2021; Ioannidis et al., 2021). Estrogen has been shown to be a critical factor in the formation of the ovarian cortex in chickens, with alterations in estrogen levels leading to changes in both the morphology of the gonads and the differentiation patterns of germ cells in both sexes (Guioli et al., 2020). Recent research has suggested that estrogen also induces the expression of TGFB Induced Factor Homeobox 1 (TGIF1), which is necessary for proper ovarian development in chickens (Estermann et al., 2021).

Molecular analysis of the asymmetric gonadal development in female chickens has revealed that the morphological changes observed in embryonic gonads, especially in chickens, are caused by the differential spatial expression of PITX2 between left and right female gonads, which affects the size and shape of the gonads. Ectopic expression of PITX2 has been shown to modify the mitotic orientation of cells parallel to the cortex plane, resulting in cortex thickening by activating cyclin D1 (Rodriguez-Leon et al., 2008). Asymmetric expression of PITX2 in the left gonads of female chickens leads to downregulation of retinaldehyde dehydrogenase 2 (RALDH2), an enzyme responsible for synthesizing retinoic acid (RA). Furthermore, the activation of the adrenal binding protein 4/steroidogenic factor 1 (Ad4BP/SF-1) and estrogen receptor α (ERα) cascade in the left gonads promotes the expression of cyclin D1, which is crucial for cell proliferation (Ishimaru et al., 2008). Further exploration of the differential gene expression in chicken female gonads has shown that several genes involved in the bone morphogenetic protein (BMP) subfamily, Z-linked a disintegrin and metalloproteinase with thrombospondin motifs like 1 (ADAMTSL1), Gremlin 1 (GREM1), follicle stimulating hormone receptor (FSHR), cytochrome P450 family 19 subfamily A member 1 (CYP19A1), LIM homeobox protein 9 (LHX9), and various apoptotic genes exhibit asymmetric expression profiles, suggesting their possible involvement in the asymmetric development of female chicken gonads (Carre et al., 2011; Li et al., 2022; Shaikat et al., 2018).

Although there have been numerous investigations on the morphological and molecular profiles of gonadal asymmetric development in chickens, a direct comparison between left and right gonads using high-throughput sequencing analyses has yet to be fully conducted. This lack of comparison has limited the understanding of the molecular mechanisms underlying asymmetric gonad development in female chickens. In light of this, we conducted a bulk RNA sequencing (RNA-seq) study to explore the transcriptome profiles during chicken gonadal development, with a focus on the asymmetric development of the left and right gonads at various embryonic stages (E5, HH26; E6.5, HH30; E8, HH34; and E9.5, HH36). Additionally, we conducted scRNA-seq analysis on the left and right gonads at the early embryonic stage of E5 to investigate the molecular mechanisms underlying asymmetric gonadal development at the single-cell level, as well as to explore cell heterogeneity.

Materials and methods

Animal usage ethics statement

In this study, we utilized White Leghorn chicken embryos collected before embryonic day 16 (E16). According to the guidelines set forth by the Institutional Animal Care and Use Committee (IACUC), research involving avian embryos before E16 is not subject to formal IACUC approval. Therefore, this study did not require IACUC oversight. All procedures involving the handling of chicken embryos were conducted in accordance with ethical standards to minimize any potential harm. We ensured that the embryos were treated humanely and with care throughout the course of the study.

Chicken sexing and gonad sample preparation

Female chickens were determined by Z and W chromosome-specific PCR using blood extracted from HH stage 14-17 embryos (Itoh et al., 2001; Ogawa et al., 1997). For PCR analysis, 50 µl of PBS containing 1 µl of blood extracted from the embryonic blood vessel was used. This sample was then boiled at 95 °C for 10 minutes and used as the PCR template. The PCR reaction mixture consisted of 2 µl of the boiled blood/PBS mixture and 1 µl of 10 pM primers targeting chicken Z and W chromosomes in a final volume of 20 µl. PCR cycling conditions included an initial denaturation step at 95 °C for 3 min, followed by 30 cycles of denaturation at 95 °C for 30 sec, annealing at 60 °C for 30 seconds, and extension at 72 °C for 30 sec. A final extension step was performed at 72 °C for 5 minutes. The amplified PCR products were subsequently separated by electrophoresis on a 1.0 % agarose gel. Then, the left and right gonads were separately collected from the abdomens of more than 5 female chicken embryos at Hamburger and Hamilton (HH) stages 26, 30, 34, and 36 (Hamburger and Hamilton, 1951). The primers used in the sexing PCR were listed in Supplementary Table S1.

RNA extraction and quantitative RT-PCR

Total RNAs from each gonad were extracted using the Direct-Zol RNA Mini or Microprep kit (ZymoResearch, Irvine, CA, USA) and reverse-transcribed using the High-Capacity cDNA Reverse Transcription (RT) Kit (Thermo Fisher–Invitrogen). For qPCR, the PowerUp SYBR Green Master Mix and protocol (Thermo Fisher–Invitrogen) were used. Primers were designed using Primer3 algorithm and validated by measuring Ct values and analyzing melting curves. For each reaction, 2 µl of cDNA and 1 µl of 10 pM forward and reverse primers were used for a 20 µl qPCR reaction. Cycling conditions were as follows: 50 °C for minutes, 95 °C for 2 min, 40 cycles of 95 °C for 15 seconds, and 60 °C for 1 min, followed by a melting cycle. Each gene expression level was normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression level using the ΔΔCt method (Livak and Schmittgen, 2001). All qPCR was performed using at least 3 biological replicates, and a significant difference between the left and right gonads was evaluated by the t-test. The primers used in the qPCR were listed in Supplementary Table S1.

Bulk RNA sequencing and data analysis

Three replicates were analyzed, but each sample was pooled from at least 5 embryos (RIN 9.80 ± 0.29, minimum of 9.0 max 10.0). Strand-specific cDNA libraries were generated using NEBNext Ultra II Directional RNA Library Prep Kit (NEB) after mRNA purification using polyA selection method. A minimum of 20 million paired-end reads were generated for each sample on Illumina NovaSeq6000. Raw reads were quality trimmed using TrimGalore (0.6.7) and the trimmed reads were aligned against GRCg6a NCBI Gallus gallus Annotation Release 104 using STAR (2.7.10a). Raw read counts were derived using HTSeq-count (2.0.0) and used for differential gene expression analysis. The differentially expressed genes (DEGs) obtained from DESeq2 (1.36.0) and edgeR (3.38.4) R packages were combined, and the low read genes that fragments per kilobase per million reads (FPKM) value less than 1 were excluded from the list. We set the false discovery rate (FDR) to 5 % for comparing left vs. right and 1 % for comparisons between time points. To determine the functional implications of the differentially expressed genes, we performed functional annotations using DAVID. Heat maps and k-means clustering analysis diagrams were generated using custom R and Python scripts. The gap statistics and sum of squared error methods were applied to determine the optimal number of clusters (Tibshirani et al., 2001).

Single-cell RNA sequencing and data analysis

The left and right E5 (HH26) embryonic gonads were separately collected from ten females and were dissociated in 1 ml trypsin EDTA (Thermo Fisher–Invitrogen) for 10 min. Trypsin was inactivated by addition of 100ul fetal bovine serum followed by washing with PBS. Minimum of 85 % cell viability was achieved by eliminating dead cells using the Dead Cell Removal Kit and MiniMACS separator (Miltenyl Biotec). The scRNA-seq libraries were generated using the 10 × Chromium Next GEM Single Cell 3′ v3.1 protocol (10 × Genomics) and prepared using the Chromium controller. Illumina NextSeq High Output 150 cycle sequencing was performed at the Penn State Genomics Core Facility, which generated 159,687,745 reads in the left gonad and 130,348,214 reads in the right gonad.

Sequencing data was aligned against GRCg6a NCBI Gallus gallus Annotation Release 104 using Cell Ranger (6.1.1) and the expression matrices were generated using the “count” command and used for subsequent analysis using the Seurat V4 R package. Following quality filtering, which involved selecting cells with a minimum of 500 and a maximum of 4,000 unique genes and less than 10,000 detected molecules, as well as removing doublets, a total of 20,913 and 19,410 single cell transcriptomes from the left and right gonads, respectively, were analyzed. We integrated left and right gonad dataset using the IntegrateData function after anchor identification for subsequent analyses. After standard workflow for data reduction and clustering, data was visualized using Uniform Manifold Approximation and Projection (UMAP) dimensions. FindNeighbors and FindClusters commands are used to define clusters which resulted in 18 clusters. To determine the differential expression of these markers, we employed the Wilcox test for each cluster. Furthermore, the FindAllMarkers function was utilized to obtain a list of cluster marker genes, as well as differentially expressed genes (DEGs) between the left and right gonads within each cluster. To assign specific cell types to each cluster, we used canonical gene markers that are known to be specific to each cell type. For visualization of gene expression, FeaturePlot, DoHeatMap, and VlnPlot were utilized. To identify transcription factors among DEGs, we used the Animal transcription factor database (AnimalTFDB 3.0) (Hu et al., 2019). Functional enrichment analysis was conducted using DAVID, while the gene set enrichment analysis (GSEA) was performed using the clusterProfiler R package (4.4.4).

Whole-mount in situ hybridization

Hybridization probes for chicken LTBP1, TGFB2, and PITX2 were made from total RNA extracted from embryonic gonads, which was then reverse-transcribed using the SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). Next, cDNA was amplified using validated LTBP1-, TGFB2- or PITX2-specific primers (Antin et al., 2014; Yu et al., 2001). The PCR product was cloned into the pGEM-T Easy Vector System (Promega, Madison, WI, USA). After sequence verification by Sanger single-molecule sequencing, recombinant plasmids containing the target genes were amplified using T7- and SP6-specific primers to prepare the template for labeling the hybridization probes. Digoxigenin (DIG)-labeled sense and antisense LTBP1, TGFB2, and PITX2 hybridization probes were transcribed in vitro using a DIG RNA Labeling Kit (Roche Diagnostics, Indianapolis, IN, USA). Whole-mount in situ hybridization of female chicken embryonic gonads at E5 was performed using a standard protocol with an anti-DIG alkaline phosphatase-conjugated antibody and visualization by a 5-Bromo-4-chloro-39-indolyl phosphate p-toluidine salt (BCIP) and nitro blue tetrazolium chloride (NBT) colorimetric reaction. Then, the in situ–hybridized samples were paraffin-embedded, sectioned (thickness, 8-10 µm), and evaluated under a microscope (Nikon, Tokyo, Japan). The primers used in the whole-mount in situ hybridization were listed in Supplementary Table S1.

Results

Transcriptomic profiles of left and right gonads during embryogenesis

Asymmetric gonadal development is evident in chicken embryos from embryonic day 6.5 (HH30) onwards (Hamburger and Hamilton, 1951). To investigate the underlying transcriptional dynamics, we performed bulk RNA-seq analyses before and after HH30. We collected and analyzed the left and right gonads at different developmental stages (E5, E6.5, E8, and E9.5), when the gonads were morphologically undifferentiated or differentiated (Fig. 1A). Then, we performed principal component analysis (PCA), which revealed that the transcriptome profiles between the left and right female gonads were not clearly separated at earlier stages (E5 and E6.5), but were separated at later stages (E8 and E9.5), indicating transcriptional differentiation initiates between E5.5 to E6.5 (Fig. 1B). Although the transcriptome profiles were similar at earlier stages, we found differentially expressed genes (DEGs) between left and right gonads at all time points. Specifically, at E5, we identified 274 up-regulated or 374 down-regulated genes in the right female gonads compared to the left gonads (FDR<5 %), and at E6.5, we identified 197 up-regulated and 472 down-regulated genes in the right gonads. The number of DEGs between left and right gonads dramatically increased at E8 and E9.5 (1,243 up-regulated and 1,274 down-regulated genes at E8, and 1,285 up-regulated and 1,139 down-regulated genes at E9.5), indicating an increasing transcriptional dissimilarity between left and right gonads in female chickens as embryogenesis progressed (Fig. 1C). The list of DEGs is in Supplementary Table S2.

Fig. 1.

Fig 1

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Transcriptomic analysis of female chicken gonads at HH26, HH30, HH34 and HH36. (A) Representative images of female chicken embryonic gonads. (B) Principal component analysis (PCA) of each cDNA library. Replicates from each experimental group are separated by developmental stage and side. (C) Volcano plot of transcriptomic differences between the left and right female gonads in each developmental stage; colored dots correspond to significant DEGs [False discovery rate (FDR) < 5 %]. (D) Top 5 gene ontology (GO) and KEGG pathway analyses of DEGs between left and right gonads in each developmental stage using DAVID. The number of genes enriched in each term is in parentheses. (E) Heatmaps showing the expression fold change of DEGs across timepoints in the TGF-beta signaling pathway. (F) Heatmaps showing the expression fold change of DEGs across timepoints in the cell cycle pathway. (G) Heatmaps showing the expression fold change of DEGs across timepoints in germ cell and stem cell markers.

To gain more insights into the molecular mechanisms underlying the differential development of the left and right female chicken gonads, we performed functional annotation of the differentially expressed genes (DEGs) at each time point using DAVID. Specifically, we used the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and gene ontology (GO) terms to functionally annotate the DEGs and identify their potential roles in various biological processes and pathways. At E5, the GO and KEGG analyses showed an average enrichment of more than two-fold in several biological processes, including “Positive regulation of MAPK cascade”, “piRNA metabolic process”, “Immune response”, “Neural crest cell migration”, and “Anterior/posterior pattern specification”. The significantly enriched KEGG pathways at this stage were “Neuroactive ligand-receptor interaction”, “Adrenergic signaling in cardiomyocytes”, “Cell adhesion molecules”, “Transforming growth factor β (TGFβ) signaling pathway”, and “ECM-receptor interaction”. At E6.5, the most enriched GO biological process term was “Axon guidance”, while the significantly enriched KEGG pathways were “Cytokine-cytokine receptor interaction”, “Neuroactive ligand-receptor interaction”, and “Jak-STAT signaling pathway”. At E8, the most enriched GO:BP terms were “Blood coagulation” and “Epithelial cell differentiation”, and the significantly enriched KEGG pathways were “Cell cycle” and ”Metabolism pathways”. At E9.5, the most enriched GO:BP term was “Metabolic process”, and the significantly enriched KEGG pathways were “Metabolic pathways”, “Cell cycle”, and “Biosynthesis of antibiotics”. Fig. 1D summarizes the GO and KEGG analyses for all time points, highlighting the significant pathways and processes associated with the DEGs between the left and right gonads.

To delve deeper into the molecular mechanisms underlying the asymmetric development of the left and right female chicken gonads, we specifically investigated the gene expression profiles of the TGFβ signaling pathway, which was identified as a significantly enriched KEGG pathway at E5. Consistent with previous studies, PITX2, a critical factor for asymmetric gonadal development, showed lower expression in the right gonads at all four time points. Moreover, several other TGFβ signaling pathway-related genes, including latent transforming growth factor beta binding protein 1 (LTBP1), transforming growth factor beta 2 (TGFB2), anti-Mullerian hormone (AMH), and bone morphogenetic protein 7 (BMP7), also exhibited lower expression in the right gonads compared to the left gonads, while suppressor of mothers against decapentaplegic (SMAD) family member 6 (SMAD6), SMAD family member 9 (SMAD9), bone morphogenetic protein receptor type 2 (BMPR2), transforming growth factor beta receptor 2 (TGFBR2), and bone morphogenetic protein 6 (BMP6) showed higher expression levels in the right gonads (Fig. 1E). Furthermore, we observed that most cell cycle-related genes were downregulated, and their expression was lower at E8 and E9.5 than at E5 and E6 in the right gonads, indicating a lower proliferation rate of the cells located in the right gonads as embryogenesis progressed (Fig. 1F). Additionally, we found that the expression of germ cell and stem cell markers, including deleted in azoospermia like (DAZL), DEAD-box helicase 4 (DDX4), piwi like RNA-mediated gene silencing 1 (PIWIL1), and C-X-C motif chemokine ligand 12 (CXCL12), was significantly lower in the right gonads at morphologically undifferentiated stages (Fig. 1G).

Dynamic temporal gene expression profiling

In order to gain insight into the temporal gene expression patterns during gonad development, we compared the differential gene expression profiles at different time points within the same tissue (FDR <1 %). Specifically, we found that 1,252 (E5 vs E6.5), 3,807 (E5 vs E8) and 3,931 (E5 vs E9.5) genes showed significant differential expression in the left gonad, while 1,697 (E5 vs E6.5), 4,659 (E5 vs E8) and 4,445 (E5 vs E9.5) genes showed significant differential expression in the right gonad (Fig. 2A). Of these genes, 914 were shared between the left and right gonads, while 338 and 783 were specific to the left and right gonads, respectively, among the 1,252 and 1,697 differentially expressed genes at E5 vs E6.5. At E5 vs E8, there were 2,813 shared DEGs and 994 and 1,845 left or right gonad specific DEGs, while at E5 vs E9.5, there were 2,796 shared DEGs and 1,135 and 1,649 left or right gonad-specific DEGs (Fig. 2B). Subsequently, k-means clustering was performed among the DEGs, which identified a total of eight clusters based on their expression patterns. Among these, clusters 1, 4, 5, and 7 showed higher expression at the early stages of embryogenesis (E5 and E6.5). In particular, these clusters contained genes involved in various signaling pathways, including the MAPK signaling pathway, insulin signaling pathway, and TGFβ signaling pathway, indicating their important roles in the early stage of gonad asymmetric development. Meanwhile, clusters 2, 3, 4, and 8 included many genes involved in diverse metabolic pathways as well as cell adhesion, suggesting dramatic metabolic and cellular structural changes at the later stages of development (Fig. 2C). It is worth noting that cell cycle-related genes were found to belong to different clusters in the left and right gonads, indicating differential growth rates between the two sides. This is a crucial factor in ovary morphogenesis, specifically cortex formation (Fig. 2D). The list of DEGs can be found in Supplementary Table S3.

Fig. 2.

Fig 2

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Temporal gene expression dynamics profiling in gonad asymmetry. (A) The number of DEGs (FDR < 1 %) is shown for left and right gonads during development. Each horizontal comparison indicates the number of DEGs at each time point compared to embryonic day 5. (B) Venn diagrams show the DEGs between left and right gonads during development at each time point. (C) The k-means clustering of total differentially expressed genes is based on the expression dynamics and expression cluster shift of genes between left and right gonads. Respective gene expression is shown in gray, and the representative expression pattern is highlighted in different colors. A chord diagram indicates the number of DEGs that shifted clusters from left to right. (D) DEGs in the cell cycle pathway and their expression clusters between left and right gonads are shown on the left panel. The colors of the clusters correspond to the colors used in (C). A heatmap of gene expression for the pathway is on the right panel.

Identification of gonadal cell types in embryonic gonads at the early stage of gonadogenesis by single-cell RNA sequencing (scRNA-Seq)

To understand the transcriptional mechanisms underlying the onset of left-right gonadal asymmetry, which becomes apparent at HH30, we performed single-cell RNA sequencing (scRNA-seq) on undifferentiated embryonic gonads (E5) using the 10x Genomics Chromium platform (as shown in Fig. 3A). We filtered a total of 40,323 cells from both left and right gonads (20,913 and 19,410 cells, respectively), which were then clustered based on gene expression using the uniform manifold approximation projection (UMAP) algorithm. This allowed us to identify 19 distinct clusters (c0-c18) that covered all cells from both gonads (Fig. 3B and 3C). Cluster-specific gene expression patterns were also assessed, and a heatmap showed differentially expressed genes (DEGs) for each cluster (Fig. 3D). Based on the expression of marker genes, we were able to annotate 11 different cell types from the left and right gonads of female chicken embryos at E5. Clusters 0 (c0) and 3 (c3) were found to correspond to coelomic epithelial cells that expressed epithelial somatic markers, LHX9. Clusters 4 (c4), 5 (c5), and 6 (c6) were annotated as pre-granulosa based on odd-skipped related transcription factor 1 (OSR1) expression, which is a marker for avian pre-granulosa cells. Cluster 17 (c17) was identified as theca cells by thecal-related steroidogenic genes cytochrome P450 family 11 subfamily A member 1 (CYP11A1) expression. Clusters 1 (c1), 2 (c2), 7 (c7), 8 (c8), and 9 (c9) were annotated as interstitial cell populations enriched in the expression of extracellular matrix-related gene decorin (DCN). Cluster 16 (c16) was identified as germ cells showing DAZL expression, which is a germ cell marker. Furthermore, we detected glial and neuronal populations (cluster 12, transgelin 3 (TAGLN3)), erythrocytes (cluster 10, hemoglobin subunit alpha 1 (HBA1)), macrophages (cluster 18, colony stimulating factor 1 receptor (CSF1R)), kidney (cluster 14, myo-inositol oxygenase (MIOX)), glomeruli (cluster 11, NPHS2 podocin (NPHS2)), and endothelial cells (cluster 13, cadherin 5 (CDH5)) based on each cell marker expression. However, we assumed contamination from neighboring tissues due to the difficulty in dissecting embryonic gonads at E5 (Fig. 3E).

Fig. 3.

Fig 3

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Gonadal cell subpopulations determined by scRNA-seq analysis of female chicken gonads at HH26. (A) A graphical representation of the methodology utilized to produce single-cell RNA-seq of embryonic day 5 female chicken gonads. (B,C) A combined total of 40,323 cells (20,913 from the left and 19,410 from the right) were analyzed utilizing Aggregated UMAP (Uniform Manifold Approximation and Projection) clustering, which recognized 18 unique clusters. (D) Heatmap exhibiting marker genes across 18 clusters of E5 female gonadal cells. (E) Characterization of cell types based on canonical marker genes.

Differential gene expression of each cell type in the left and right gonads at a single-cell level

Based on scRNA-seq data, we conducted a comparative analysis of differentially expressed genes (DEGs) between the left and right gonads of 11 annotated cell types to explore the transcriptome profile inducing asymmetric gonadal development in female chickens. Coelomic epithelial, interstitial, and pre-granulosa cells exhibited the most distinct differential transcriptome profiles, with 1,426 (1,208 down and 218 up, FDR < 0.5 %), 1,239 (1,128 down and 111 up, FDR < 0.05 %), and 880 (848 down and 32 up, FDR < 5 %) genes differentially expressed in the left and right gonads, respectively (Fig. 4A and 4B, Supplementary Table S4). The DEGs were then analyzed using DAVID to identify KEGG pathways, and we found that these three cell types shared several pathways, such as “Ribosome”, “Oxidative phosphorylation”, “Spliceosome”, “Proteasome”, “Cardiac muscle contraction”, “Nucleocytoplasmic transport”, and “Cell cycle”, indicating their similarity in terms of cell transcriptional profile. However, each cell type also exhibited cell-specific enriched pathways, such as “Oocyte meiosis” for coelomic epithelial cells, “Focal adhesion” for interstitial cells, and “Carbon metabolism” for pre-granulosa cells (Fig. 4C). Further analysis of individual genes revealed that polypyrimidine tract binding protein 3 (PTBP3) and musculin (MSC), involved in RNA processing, and cyclin-dependent kinase 1 (CDK1) and ring-box 1 (RBX1), cell cycle-related genes, were significantly upregulated in the left gonadal tissues, including coelomic epithelial, interstitial, pre-granulosa, theca, and germ cells (Fig. 4D and 4E). However, genes related to cell junction, gap junction protein alpha 1 (GJA1) and cadherin 11 (CDH11), and cell self-renewal and apoptosis, mitogen-activated protein kinase kinase kinase 1 (MAP3K1) and tissue factor pathway inhibitor 2 (TFPI2), showed significant differential expression patterns between the left and right gonads depending on cell types, indicating differential cell status at this early stage of gonadal development (Fig. 4F and 4G).

Fig. 4.

Fig 4

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Differential Gene Expression (DEG) in Cell Types Between Left and Right Female Chicken Gonads. (A) Analysis of DEG (FDR < 0.05 % for Coelomic epithelial & Interstitial, and < 5 % for Pre-granulosa) between left and right gonads in three major cell types. (B) Volcano plot showing the transcriptomic differences between the left and right female gonads in each cell type, with colored dots indicating significant DEGs. (C) KEGG pathway analysis of DEGs in each cell type. (D-G) Violin plots depicting the expression of selected genes in different cell types between left (blue) and right (purple) gonads.

TGFβ signaling at the early stage of gonadal development

To further investigate the molecular cascades underlying asymmetric gonadal development in female chicken embryos, we analyzed transcription factors (TFs) that showed differential gene expression between the left and right gonads. We identified 102 TFs with significant differential expression patterns in coelomic epithelial, interstitial, and pre-granulosa cells of the left and right gonads. Further analysis using DAVID revealed that five TFs (inhibitor of DNA binding 1 (ID1), ID2, ID4, PITX2, and SMAD family member 5 (SMAD5)) were involved in the TGFβ signaling pathway and exhibited significant differential expression between the left and right gonads (Fig. 5A). To better understand the role of the TGFβ signaling pathway in asymmetric gonadal development, we performed gene set enrichment analysis (GSEA) on genes belonging to this pathway. The results indicated that the genes in this pathway were significantly enriched in the left gonads compared to the right gonads (Fig. 5B). In particular, the ligands of the TGFβ signaling pathway (LTBP1 and TGFB2) and one of the downstream genes (PITX2) showed significant differential gene expression at both the whole gonadal level (Fig. 5C) and single-cell level (Fig. 5D) during early gonadal development. Moreover, these genes displayed asymmetric spatial expression between the left and right gonads, further supporting their putative roles in asymmetric gonadal development in female chickens (Fig. 5E).

Fig. 5.

Fig 5

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Analysis of transcription factors and transforming growth factor (TGF)-beta (TGFβ) signaling pathway in left and right gonads at E5. (A) Enrichment analysis of differentially expressed transcription factors in three major cell types (coelomic epithelial, interstitial, and pre-granulosa cells) in left and right gonads. (B) Gene set enrichment analysis (GSEA) of the TGFβ signaling pathway using differentially expressed genes in coelomic epithelial cells. (C) Quantitative real-time PCR (qRT-PCR) analysis of relative gene expression levels of paired like homeodomain 2 (PITX2), latent transforming growth factor beta binding protein 1 (LTBP1), and transforming growth factor beta 2 (TGFB2) between left and right gonads. (D) Uniform Manifold Approximation and Projection (UMAP) visualization of the differential expression of the three genes at the individual cell level. (E) In situ hybridization analysis of the three genes in female embryonic gonads at embryonic day 5.

Discussion

The primary objective of this study is to investigate the transcriptome profiles of female chicken embryos displaying asymmetric gonadal development, which is a characteristic feature in most avian species. Through the analysis of RNA-seq data, we identified significant differential gene expression between the left and right gonads of female chickens, accompanied by dynamic changes in transcriptional profiles. Asymmetric gonadal development led to an increase in the number of genes showing significant differential expression between the left and right gonads. Our findings also suggest that various signaling pathways, including the TGFβ, Jak-STAT, and Toll-like receptor pathways, exhibit differential expression during the early stages of embryogenesis (E5 and E6.5), indicating their potential involvement in asymmetric gonadal development in female chickens.

In particular, we observed significant upregulation of PITX2, a member of the TGFβ signaling pathway, in the left gonads, which has been established as a crucial determinant of asymmetric gonadal development (Guioli and Lovell-Badge, 2007; Ishimaru et al., 2008; Rodriguez-Leon et al., 2008). The results support the notion that the identified signaling pathways play significant roles in the process of asymmetric gonadal development in female chickens. Indeed, Pitx2 gene is involved in diverse left-right signaling including asymmetrical cardiac morphogenesis and atrial fibrillation (Franco et al., 2014) and genetic mutations of Pitx2 in mice causes developmental defects in pituitary gland, lung, tooth, eyes, heart, abdominal viscera, and limbs (Gage et al., 1999; Kitamura et al., 1999; Lin et al., 1999; Lu et al., 1999), suggesting its conserved role in organogenesis during early development. In the later stages of embryogenesis (E8 and E9.5), the RNA-seq data indicated significant differential gene expression of cell cycle-related or metabolic pathway-related genes, suggesting differences in cell status between the left and right gonads. Specifically, the majority of cell cycle-related genes were found to be downregulated in the right gonads compared to the left gonads, indicating a lower level of cell proliferation rather than cell apoptosis. This may be influenced by the differential expression of pathway-related genes, as a previous study found that the PITX2-cyclin D cascade regulates mitotic spindle orientation of the gonad cortex, inducing cell-type-specific proliferation (Rodriguez-Leon et al., 2008).

The analysis of temporal gene expression profiling demonstrated that there were dynamic changes in gene expression during embryogenesis. There was an increased number of differentially expressed genes (DEGs) in both the left and right gonads, with some DEGs specific to each gonad. This suggests that there is a differential cell status between the left and right gonads. Although we did not observe a major cluster shift of expression dynamics of DEGs among the 8 clusters, the temporal gene expression profiling analysis showed that cell cycle-related genes were differently categorized in the left and right gonads. Specifically, cell cycle-related genes in the left gonads belonged to clusters 2, 3, and 7, which showed higher expression levels at the later stage of embryogenesis. Additionally, we observed a significant downregulation of germ cell markers in the right gonads compared to the left gonads, consistent with a previous study (Intarapat and Stern, 2013). This lower expression of germ cell markers suggests that there are significantly fewer germ cells in the left gonads in chickens (Nakamura et al., 2007).

To investigate the molecular profiles of individual cells in chicken female gonads, scRNA-seq was conducted during the early stage of embryogenesis (E5). The scRNA-seq results revealed that both left and right gonads were composed of 18 clusters, categorized into 11 cell types based on marker expression, with varying cell numbers in each cluster (Estermann et al., 2020). Three cell types, including coelomic epithelial, interstitial, and pre-granulosa cell populations, displayed distinct gene expression differences between the left and right gonads. However, relatively differentiated cell types, such as theca cells and germ cells, did not exhibit significant differences in transcription profiles between the left and right gonads at the early stage of embryogenesis (E5) since the gonads were not yet fully differentiated at this stage, consistent with previous studies (Guioli et al., 2014). These results suggest that the development and differentiation of gonadal cells occur asynchronously during embryogenesis.

Notably, the scRNA-seq analysis revealed that the DEGs in each cell type had common KEGG pathways related to RNA or protein processing and cell cycle pathways, indicating that these cell types share a similar cell status, which is highly proliferating. Regarding gene function, RNA processing (MSC and PTBP3) and cell cycle (CDK1 and RBX1) related genes exhibited left gonad-enriched patterns, suggesting differential cell proliferation status that may result in asymmetric gonadal development in female chickens. Additionally, at the single-cell level, we observed differential gene expression of cell junction-related genes (GJA1 and CDH11) between the left and right gonads. This finding suggests that cell adhesion is critical for epithelial morphogenesis, and the asymmetry in cell adhesion and extracellular matrix components of female chicken gonads may contribute to the asymmetry between the left and right gonads (Guioli and Lovell-Badge, 2007; Guioli et al., 2014; Rodriguez-Leon et al., 2008). Hence, the scRNA-seq analysis revealed that the differential expression of cell junction-related genes in specific cell types may control the distinct cell adhesion properties in the left and right gonads, leading to asymmetrical development. Additionally, the scRNA-seq data identified cell type-specific expression patterns of genes related to self-renewal and apoptosis, such as MAP3K1 and TFPI2. MAPK pathways have been shown to play a crucial role in embryonic developmental processes, including cell growth and migration, with MAP3K1 being strongly associated with various cancers (Xue et al., 2018). On the other hand, our bulk RNA-seq analysis, together with previous research, supports the notion that the observed asymmetric cortical development is primarily due to increased proliferation in the left cortex, rather than increased apoptosis in the right cortex (Ishimaru et al., 2008). However, our scRNA-seq data showed that TFPI2, known as a tumor suppressor gene in several cancers by promoting apoptosis and inhibiting angiogenesis and invasion (George et al., 2007; Wang et al., 2018), was significantly upregulated in the coelomic epithelial cell populations, suggesting the coelomic epithelial cell-specific TFPI2 expression may induce apoptosis in the right gonads, causing asymmetric gonadal development of female chicken gonads.

To understand the molecular mechanisms that induce asymmetric gonadal development, we further investigated the gene expression of TFs with significantly differential expression in three cell types: coelomic epithelial, interstitial, and pre-granulosa cells. Using DAVID and GSEA analysis tools, we identified that the TGFβ signaling pathway was significantly enriched in the left gonads compared to the right gonads. Additionally, the differential spatial gene expression of the transcription factors of TGFβ signaling pathway supports its putative roles in asymmetric gonadal development. Indeed, the TGFβ signaling pathway is involved in many different developmental processes in vertebrates, including maintaining germ stem cells and patterning the eggshell (Monsivais et al., 2017), particularly, axis formation and patterning during early vertebrate embryonic development (Zinski et al., 2018). In addition, Nodal-Pitx2 cascade, which is belong to TGFβ signaling pathway, is indispensable for asymmetric tissue or organ morphogenesis in vertebrates and in some invertebrates (Jia and Meng, 2021), suggesting the role of TGFβ signaling pathway in asymmetric gonadal development in chickens. The investigation of the cell-type-specific knockdown or overexpression of the TGFβ signaling pathway, followed by the analysis of cell proliferation, could offer compelling evidence relating the roles of the TGFβ signaling pathway in the asymmetric gonadal development.

Conclusions

In this study, we comprehensively investigated the transcriptional profiles during gonadal development using RNA-seq and scRNA-seq. Our findings revealed a dynamic transcriptional profile of female chicken gonads, and a cell-type-specific transcriptional landscape at the early stage of gonadogenesis. We observed that the left gonads exhibited a highly proliferating cell status compared to the right gonads, and the TGFβ signaling pathway appeared to play a crucial role in the asymmetric gonadal development. To further enhance our understanding of the asymmetric gonadal development in female chickens, functional validation studies are warranted. Additionally, performing scRNA-seq analysis at different developmental time points, particularly earlier stage of gonadogenesis (before E5), and validating the spatial and temporal gene expression profiles of TGFβ signaling in wild bird species which have two bilaterally symmetrical ovaries (Rodler et al., 2015) could provide valuable insight into the temporal gene expression profiles and cell fate determination of both left and right gonads.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The research was supported in part by United States Department of Agriculture (USDA), National Institute of Food and Agriculture (NIFA) #2023-67015-39264, National Science Foundation (NSF/BIO) #1645331 and National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. RS-2024-00418297). We thank the Poultry Education and Research Center and their staff at Penn State University for preparing and providing fertilized eggs for this study.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.psj.2025.104932.

Appendix. Supplementary materials

mmc1.docx (15.6KB, docx)
mmc2.xlsx (502.1KB, xlsx)
mmc3.xlsx (2.4MB, xlsx)
mmc4.xlsx (24.3MB, xlsx)

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Supplementary Materials

mmc1.docx (15.6KB, docx)
mmc2.xlsx (502.1KB, xlsx)
mmc3.xlsx (2.4MB, xlsx)
mmc4.xlsx (24.3MB, xlsx)

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