Transcriptome analysis revealed misregulated gene expression in blastoderms of interspecific chicken and Japanese quail F1 hybrids.

Hybrid incompatibility, such as sterility and inviability, prevents gene flow between closely-related populations as a reproductive isolation barrier. F1 hybrids between chickens and Japanese quail (hereafter, referred to as quail), exhibit a high frequency of developmental arrest at the preprimitive streak stage. To investigate the molecular basis of the developmental arrest at the preprimitive streak stage in chicken-quail F1 hybrid embryos, we investigated chromosomal abnormalities in the hybrid embryos using molecular cytogenetic analysis. In addition, we quantified gene expression in parental species and chicken- and quail-derived allele-specific expression in the hybrids at the early blastoderm and preprimitive streak stages by mRNA sequencing. Subsequently, we compared the directions of change in gene expression, including upregulation, downregulation, or no change, from the early blastoderm stage to the preprimitive streak stage between parental species and their hybrids. Chromosome analysis revealed that the cells of the hybrid embryos contained a fifty-fifty mixture of parental chromosomes, and numerical chromosomal abnormalities were hardly observed in the hybrid cells. Gene expression analysis revealed that a part of the genes that were upregulated from the early blastoderm stage to the preprimitive streak stage in both parental species exhibited no upregulation of both chicken- and quail-derived alleles in the hybrids. GO term enrichment analysis revealed that these misregulated genes are involved in various biological processes, including ribosome-mediated protein synthesis and cell proliferation. Furthermore, the misregulated genes included genes involved in early embryonic development, such as primitive streak formation and gastrulation. These results suggest that numerical chromosomal abnormalities due to a segregation failure does not cause the lethality of chicken-quail hybrid embryos, and that the downregulated expression of the genes that are involved in various biological processes, including translation and primitive streak formation, mainly causes the developmental arrest at the preprimitive streak stage in the hybrids.

Introduction

Speciation, the process by which populations evolve into distinct species, is often associated with hybrid incompatibility, such as reduced fertility and viability of hybrid progeny [1-5]. Hybrid incompatibility may facilitate speciation by preventing gene flow between sympatric populations and also reinforce prezygotic reproductive isolation between populations through the promotion of the divergence of mating behavior or gametic interactions [6-8]. Hybrid incompatibility genes are defined as those that measurably decrease the fitness in F1, F2, or BC1 generations of interspecific hybrids [9]. According to the Dobzhansky and Muller (DM) model [10-12], genetic diversification occurs at multiple loci in populations originating from the same ancestral population, and incompatible allelic interactions in hybrids cause hybrid abnormalities as by-products of the evolution: alleles that effectively function in pure-species genetic backgrounds may cause adverse effects in the genetic background of interspecific hybrids. Hybrid incompatibility genes have been identified in many interspecific or intersubspecific hybrids in a wide range of taxa, including Saccharomyces, Arabidopsis, Oryza, Drosophila, Xiphophorus, and Mus [9,13,14]. However, the molecular basis of hybrid incompatibility remains poorly understood.

Although chicken (Gallus gallus domesticus) and Japanese quail (Coturnix japonica) belong to different genera that diverged approximately 35 million years ago (MYA) [15], interspecific hybrids can be generated by artificial insemination (AI) of chicken semen into the quail oviduct [16,17]. However, most hybrids die before hatching, and only a few male hybrids, which account for approximately 4% of the fertilized eggs can hatch [18], which is consistent with Haldane’s rule, “When in the F1 offspring of two different animal races one sex is absent, rare, or sterile, that sex is the heterozygous (heterogametic) sex” [19-24]. Our previous observation of chicken–quail hybrid embryos revealed that hybrid lethality occurs at various stages of embryonic development, including blastoderm, somite, and postsomite stages [18]. Most hybrid embryos (approximately 75% fertilized eggs) displayed developmental arrest during extraembryonic membrane formation and blood island formation stages (0–2 days of incubation). Furthermore, a substantial fraction of the hybrid embryos incubated for 8.5–36 h died at the Eyal-Giladi and Kochav XI–XIV stages, which are known as the preprimitive streak stage [25]: hybrid embryos with abnormal morphology accounted for 48.1% at 21–36 h after starting incubation, and 46.2% of the abnormal embryos were arrested at the XI–XIV-like stages.

The primitive streak is an organizing center of gastrulation in amniotes [26]. During the preprimitive and subsequent primitive streak stages, cell migration, proliferation, and differentiation occur, resulting in the formation of the second body axis and three germ layers [27-29]. The abnormal morphologies of the stage XI–XIV-like blastoderms may be due to aberrant migration, proliferation, and/or differentiation of epiblast and/or hypoblast cells during the preprimitive and subsequent primitive streak stages. Early embryonic lethality has also been observed in other avian interspecific hybrids such as hybrids between chicken and ring-necked pheasant and between chicken and turkey [30-32]. Therefore, developmental arrest at the early embryonic stages may be a common feature of interspecific hybrids of Phasianidae. In mammals, males are the heterogametic sex; by contrast, the heterogametic sex is females in birds [23,33]. In addition, genomic imprinting has not been found in birds, unlike in mammals [34-36]. Thus, bird hybrids would provide new insight into the molecular basis of hybrid incompatibility.

Numerical chromosome abnormalities due to a failure of segregation of chromosomes is associated with embryonic lethality in some interspecific hybrids of fish, frogs, and other organisms [37-39]. Uniparental chromosome elimination has been demonstrated in hybrid cells of various organisms, including plants [40-42], insects [43,44], and mammals [45-49]. Several types of molecular processes, including transcriptional regulation, post-transcriptional regulation, and protein-protein interactions, may cause hybrid incompatibility phenotypes [50]. Inappropriate transcriptional regulation (overexpression and/or underexpression) is associated with lethality, abnormal growth, and sterility in hybrids of Mus, Phodopus, and Xiphophorus [51-54].

In the present study, to enhance our understanding of the cause of lethality at the preprimitive streak stage of chicken–quail hybrid embryos, we initially performed chromosome analysis of the hybrid embryos at the early blastoderm stage (stage X) and in 3-day and 7-day-old hybrid embryos. Subsequently, to investigate the molecular basis of the hybrid inviability, we performed gene expression analyses of the embryos at the stage X and preprimitive streak stages (stage XIII/XIV) for parental species and their F1 hybrids. We generated expression profiles of genes at the two stages, determined the directions of expression changes (upregulation, downregulation, and no change) from the stage X to the stage XIII/XIV, and then compared the expression profiles between the hybrids and parental species.

Materials & methods

General

No statistical methods were used to predetermine sample size, and the experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.

Ethics statement

Animal care and all experimental procedures were approved by the Animal Experiment Committee, Graduate School of Bioagricultural Sciences, Nagoya University (approval number: 2018031334). Experiments were conducted in line with the Regulations on Animal Experiments at Nagoya University.

Animals

Commercial quail were purchased from a local hatchery (Cyubu Kagaku Shizai, Nagoya, Japan), and fertilized chicken eggs of the Ehime-jidori (Japanese native chicken breed) [55] and the BL-E line (long-term closed colony of Brown Leghorn breed) [56] were supplied by the National BioResource Project Chicken/Quail, Nagoya University, Japan. For chromosome analysis, we used embryos of commercial quail and Ehime-jidori chickens and their F1 hybrid embryos that were obtained by AI of semen from male Ehime-jidori chickens into female quail. Embryos of commercial quail, BL-E chickens and their F1 hybrid embryos at stages X and XIII/XIV were used for gene expression analyses. The two analyses were conducted at different times using different chicken lines because of the availability of adult chickens of these lines when the analyses were carried out. Chickens and quail were maintained with free access to water and a commercially available diet. The photoperiod was 14:10 h L:D, and room temperature was maintained at approximately 25°C. After all experiments, adult chickens and quail were decapacitated after isoflurane anesthesia.

Artificial insemination (AI)

AI was performed twice a week. Chicken semen was collected just before AI from 5–10 adult males of each strain. After addition of gentamicin into pooled semen (final concentration of 10 μg/ml), 50–100 μl semen was injected into vaginas of quail using a syringe. To avoid the excretion of the injected semen from vaginas by oviposition, AI was performed during the last 1–2 h of a light period, when oviposition on that day was completed in most female quail.

Egg preservation and incubation

Laid eggs were stored at 12°C until use. Eggs were used for incubation within 14 days of storage. They were incubated at 37.6°C and 70% relative humidity, with rocking at an angle of 90° at 30-min intervals.

mRNA sequencing

To extract total RNAs from blastoderms at the stage X, blastoderms were collected from the eggs that were laid on each day, which were preserved at 12°C immediately after being laid. To extract total RNAs of blastoderms at the stage XIII/XIV, we began the incubation of the eggs within 3 d after they were collected and preserved at 12°C, and blastoderms were collected after 7.5–10.0 h of incubation. We classified the developmental stages of hybrid blastoderms with abnormal morphology using the following criterion: hybrid blastoderms at stages similar to stages XIII–XIV of chickens, at which the hypoblast is formed, were considered stage XIII–XIV-like blastoderms.

After removal of egg yolk from blastoderms in phosphate buffered saline (PBS), the tissues were minced in a 5–10 μl of PBS by pipetting. One μl of each cell suspension was lysed in a 50 μl buffer containing 10 mM Tris-HCl pH 7.5, 5 mM EDTA, 0.5% Tween-20, and 50 μg/ml Proteinase K, and incubated at 50°C for 15 min and then at 95°C for 5 min. After centrifugation of the lysates at 12,000 rpm for 5 min, supernatants were used for molecular sexing, which was performed by PCR analysis of sequence length polymorphism of the intron of CHD1 as described elsewhere [57]. Sequences of primers used for PCR were as follows: 2550F (5′-GTTACTGATTCGTCTACGAGA-3′) and 2718R (5′-ATTGAAATGATCCAGTGCTTG-3′). PCR products were visualized by 2% agarose gel electrophoresis. The 600-bp PCR fragment derived from the Z chromosome was detected in both sexes, whereas an additional W chromosomal 450-bp PCR fragment was amplified only in females.

The remaining cell suspensions were lysed in TORIZOL reagent (Life Technologies, Carlsbad, CA, USA) immediately after tissue sampling. The solutions including blastodermal tissues were transferred into QIAshredder Mini Spin Columns (Qiagen, Hilden, Germany) and spun down. The flow-through samples were stored at -80°C until use. The frozen samples were thawed on ice, and total RNAs were purified according to the manufacturer’s instructions. The aqueous phases were transferred into Buffer RLT of RNeasy Plus Micro Kit (Qiagen) and then total RNAs were purified. RNA quality was assessed using Bioanalyzer Pico Chips (Agilent Technologies, Santa Clara, USA). RNAs whose RNA Integrity Numbers were over 7.5 were used for mRNA sequencing.

We converted oligo(dT)-selected RNA into cDNA libraries for mRNA sequencing using the SureSelect Strand Specific RNA Library preparation kit (Agilent Technologies) according to the manufacturer’s instructions. The libraries were sequenced on an Illumina® HiSeq 2500 platform using paired-end sequencing (100 bp). A total of 174 GB was obtained from 48 libraries (average of 3.6 GB per sample). We trimmed the adapter sequences from the reads using Trimmomatic v0.33 [58], and then mapped the reads to the reference genome (Accession codes: GCF_000002315.5 for chickens and GCF_001577835.1 for quail) using HISAT2 v2.1.0 [59]. Multi-mapped reads and reads with >2 mismatches were filtered out using SAMtools v1.9 [60], and orphan reads were eliminated using a custom Perl script. Read counts per gene were calculated by HTSeq v0.11.2 [61] using concordantly aligned read pairs. For the analysis of allelic expression in the hybrids, reads that were mapped to both reference genomes of parental species were removed before the calculation of read counts per gene using Bash scripts. Before detecting differentially expressed genes between stage X and stage XIV embryos, we excluded the genes whose counts fell below the threshold (1) in any sample in the dataset. Afterward, we used the Wald test for significance testing using DESeq2 v1.18.1 [62]. Fold change (FC) of gene expression was calculated by comparing gene expression between stage X and stage XIV embryos using DESeq2 v1.18.1. P values were adjusted using the Benjamini–Hochberg method. Genes with adjusted P value (false discovery rate, FDR) less than 0.05 and FC more than 2 were considered upregulated and those with FDR less than 0.05 and FC less than 0.5 were considered downregulated. Genes with FDR more than or equals to 0.05 or FC more than or equals to 0.5 and less than or equals to 2 were considered unaltered.

GO term enrichment analyses

Gene Ontology (GO) term enrichment analyses were performed using the overrepresentation test (Released 20200728) of the PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification System [63]. The Gallus gallus database was used as the reference (GO Ontology database doi: 10.5281/zenodo.3954044). P values of Fisher’s exact test were adjusted using the Benjamini–Hochberg method. GO terms were considered significant if they had an FDR less than 0.05. We referred to the AmiGO 2 database (v2.5.13) for the relationship of GO terms [64,65].

Cell culture and chromosome analysis

We prepared chromosomes from stage X blastoderms according to methods described previously [66]. Briefly, blastoderms were incubated in Hank’s solution at 39°C for 1 h, incubated in hypotonic solution (0.9% sodium citrate) at room temperature (RT) for 30 min, and then fixed in 3:1 methanol: acetic acid fixative at RT for 30 min. After the removal of the fixative, tissues were suspended in 50% acetic acid at RT for 5–10 min. After pipetting gently, 5–10 μl of cell suspension was spread on glass slides on a hot plate at 50°C. The preparations were stained using 4% Giemsa solution for 10 min.

Chromosomes from metaphase nuclei were also prepared from cultured fibroblast cells derived from 3-day-old female embryos of chickens and quail, and 3-day and 7-day-old male and female hybrid embryos. The embryonic fibroblast cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen-GIBCO, Carlsbad, CA, USA) supplemented with 15% fetal bovine serum (Invitrogen-GIBCO), 100 μg/ml kanamycin, and 1% Gibco® Antibiotic–Antimycotic (PSA) (Invitrogen-GIBCO) at 39°C under 5% CO2. Replication-banded chromosome slides were prepared for in situ hybridization as described previously [67]. The fibroblast cell cultures were treated with 5-bromodeoxyuridine (BrdU) (25 μg/ml) (Sigma-Aldrich, St Louis, MO, USA) at the late replication stage for 4.5 h including 0.5-h colcemid treatment. After staining the slides with Hoechst 33258 (3 μg/ml) for 5 min, they were heated at 65°C for 3 min and exposed to UV light at 65°C for 6.5 min. The slides were stored at -80°C until use.

Fluorescence in situ hybridization

Fluorescence in situ hybridization of centromeric DNA repeats and chromosome painting with DNA probes of chicken chromosomes 1–8 and Z [68] were conducted as described previously [69]. The DNA probes were labeled with biotin-16-dUTP (Roche Diagnostics, Basel, Switzerland) by nick translation and hybridized to metaphase spreads at 37°C for 4 days. After washing, the slides were incubated with FITC-avidin (Roche Diagnostics). For dual-color FISH, the biotin- and DIG-labeled probes were reacted with FITC-avidin and anti-DIG antibody (Roche Diagnostics), respectively [70].

Imaging

We used a cooled charge-coupled device (CCD) camera (Leica DFC360 FX, Leica Microsystems, Wetzlar, Germany) mounted on a Leica DMRA microscope for FISH and chromosome painting, and analyzed the data using the 550CW-QFISH program (Leica Microsystems Imaging Solutions Ltd., Cambridge, UK).

Statistical analysis

R v.3.4.3 (R Core Team) and MS Excel (Microsoft Corp., Redmond, WA, USA) were used for statistical analyses. In addition, we used the Tukey-Kramer test for pairwise comparisons of total number of chromosomes and for the comparison of the number of microchromosomes between parental species and chicken–quail hybrids. A P-value less than 0.05 indicated statistical significance. We also calculated Pearson’s correlation coefficient for correlational analyses of changes in gene expression.

Results

Chromosome analysis of embryos of chicken, quails, and their F1 hybrids

The number of chromosomes in both quail and chicken is 78 (2 n  =  78) [71]. We prepared chromosomes from stage X blastoderms and 3-day and 7-day-old embryos of chicken, quails, and their F1 hybrids (Fig 1A) for molecular cytogenetic analyses of hybrid nuclei. Macrochromosomes consisted of nine pairs of homologous chromosomes including ZZ or ZW sex chromosomes, some of which exhibited slight differences in size between chicken and quail chromosomes (Fig 1B). Chromosome painting with chicken macrochromosome-specific (chromosomes 1 to 8) and Z chromosome DNA probes [68], and hybridization with a chicken W-specific DNA repeat [72] confirmed that hybrid nuclei consisted of nine pairs of macrochromosomes and one pair of sex chromosomes (Fig 1C). One of each macrochromosome pair exhibited a larger size and stronger hybridization signal than the other, which suggested that the larger-sized chromosome in each pair originated from chicken. All the observed nuclei (96–158 nuclei for one DNA probe) showed two painted signals for each chromosome-specific DNA probe or one hybridization signal for W-specific DNA repeat (S1 Table).

Fig 1

Chromosome analysis of chicken–quail F1 hybrids.

A. Giemsa-stained metaphase spread of a blastodermal cell of the chicken-quail F1 hybrid, consisting of large-sized macrochromosomes and small-sized microchromosomes. B. Hoechst-stained chromosomes of cultured fibroblast cells from embryos of the chicken, chicken–quail hybrid, and quail, which show eight pairs of macrochromosomes and the Z and W sex chromosomes. The sizes of each pair of chromosomes differ between the chicken and quail chromosomes in the hybrid. C. Chromosome painting with macrochromosome-specific DNA probes and hybridization with the W-specific DNA repeat in fibroblast cells of the hybrids. Larger macrochromosomes with stronger hybridization signals are considered to be derived from chicken (GGA, Gallus gallus), and the others from quail (CJA, Coturnix japonica). Scale bars, 10 μm.

Subsequently, we attempted to discriminate the parental origins of microchromosomes on metaphase spreads of the hybrid nuclei. Centromeric DNA repeats that are predominantly localized to microchromosomes have been isolated from chicken (GGA-TaqI-8) and quail (CJA-BglII-M9) in previous studies [73,74]. GGA-TaqI-8 and CJA-BglII-M9 were hybridized into most of the microchromosomes derived from chicken and quail, respectively (Fig 2A and 2B), although GGA-TaqI-8 was also hybridized into two pairs of macrochromosomes (Fig 2A). We observed intense hybridization signals of GGA-TaqI-8 on chicken microchromosomes (Fig 2A) and very weak cross-hybridization signals of this probe on a part of quail microchromosomes (Fig 2B). CJA-BglII-M9 exhibited non-species-specific hybridization; the repeat was hybridized into quail microchromosomes (Fig 2B) and cross-hybridized into a part of chicken microchromosomes and a few macrochromosomes (Fig 2A). Therefore, CJA-BglII-M9 detected quail microchromosomes and chicken microchromosomes simultaneously (Fig 2C). We considered microchromosomes that were hybridized with GGA-TaqI-8 or with both of the two repeats as chicken-derived chromosomes, and those exhibiting hybridization signals of CJA-BglII-M9, with no or weak cross-hybridization signals of GGA-TaqI-8, as quail-derived chromosomes.

Fig 2

Chromosomal localization of chicken and quail centromeric repetitive sequences in chickens, quail, and their hybrids.

Fluorescence-labelled DNA probes of chicken and quail centromeric repeats (GGA-TaqI-8 and CJA-BglII-M9, respectively) were hybridized into chromosome spreads of chickens (A), quail (B), and their hybrids (C). A. GGA-TaqI-8 was localized to almost all microchromosomes (arrows in the left panel indicate representatives) and two pairs of macrochromosomes (arrowheads in the left panel). CJA-BglII-M9 was cross-hybridized into a part of microchromosomes and a few macrochromosomes (arrows and arrowheads, respectively, in the middle panel). B. GGA-TaqI-8 was cross-hybridized into quail chromosomes, which was observed as weak hybridization signals (arrows in the left panels). CJA-BglII-M9 was localized to almost all microchromosomes (arrows and arrowheads in the middle panel). C. Chromosomes that were hybridized only with GGA-TaqI-8 (arrows) and those that were hybridized with both GGA-TaqI-8 and CJA-BglII-M9 (arrowheads) were observed in the hybrid. These were considered as chicken-derived chromosomes. In addition, chromosomes that exhibited hybridization signals of CJA-BglII-M9, with no or weak hybridization signals of GGA-TaqI-8, were observed (asterisks). These were considered as quail-derived chromosomes. Scale bars, 10 μm.

We counted the number of chromosomes on metaphase spreads of the 0-h blastoderms and 3- and 7-day-old embryos. The total number of chromosomes in the hybrid cells was mostly 78, which was nearly equal to that in parental species (Fig 3A and 3B, S1 Data, Tukey-Kramer test, P > 0.05). We counted chicken- and quail-derived microchromosomes on metaphase spreads of the hybrids using two repeated sequences. The numbers of chicken- and quail-derived microchromosomes with positive signals in the hybrids were 25 and 23 on average, respectively, which were nearly equal to half the number of microchromosomes that could be detected with the chicken- and quail-specific centromeric repeats (Fig 3C and 3D, S1 Data, Tukey-Kramer test, P > 0.05). The total number of microchromosomes was 58 (29 pairs) in both parental species; therefore, our method using centromeric DNA repeats could not fully discriminate the parental origins of microchromosomes. However, the results collectively suggest that the hybrid nuclei consist of a fifty-fifty mixture of chicken and quail chromosomes, and that numerical abnormality, such as chromosome loss and/or duplication, hardly occurred in the hybrids.

Fig 3

Number of chromosomes in parental species and their F1 hybrids.

A. Total number of chromosomes in blastodermal cells of quail and the hybrids. Three males and three females were used for each group. The number of chromosomes was not different among individuals (Tukey-Kramer test, P > 0.05). NS, not significant. B. Total number of chromosomes in fibroblast cells from 3- or 7-day-old male and female hybrid embryos and those from 3-day-old female embryos of parental species. The number of chromosomes was not different among them (Tukey-Kramer test, P > 0.05). C. The number of chromosomes hybridized with GGA-TaqI-8 in 3-day and 7-day-old male and female hybrid embryos. In 3-day female chickens, a half of the total number of microchromosomes per nucleus, which were detected with GGA-TaqI-8, is shown. The number of chicken-derived microchromosomes in the hybrids did not deviate from half the number of GGA-TaqI-8-positive microchromosomes in chicken cells (Tukey-Kramer test, P > 0.05). D. The number of chromosomes hybridized with CJA-BglIII-M9 in 3-day and 7-day-old male and female hybrid embryos. In 3-day female quail, a half of the total number of microchromosomes per nucleus, which were detected with CJA-BglIII-M9, is shown. The number of quail-derived microchromosomes in hybrid cells did not deviate from half the number of CJA-BglII-M9-positive microchromosomes in quail cells (Tukey-Kramer test, P > 0.05).

Expression changes in chicken and quail genes in the hybrids and the parental species

To study the molecular basis of developmental arrest in chicken–quail hybrid embryos at the preprimitive streak stage (Fig 4A and 4B), we performed whole-transcriptome analyses of the embryos of parental species and their F1 hybrids at stages X and XIII/XIV by mRNA sequencing (Fig 4C, S1 and S2 Figs). Average numbers of mapped reads in the stage X embryos were 31.1 million in chickens, 26.5 million in quails, and 8.9 million for chicken-derived alleles and 11.1 million for quail-derived alleles in the hybrids, and 17.7 million in chickens, 18.3 million in quail, and 7.5 million for chicken-derived alleles and 8.9 million for quail-derived alleles in the stage XIII/XIV embryos. We used species-specific reads to avoid apparent increases in gene expression owing to redundant mapping of the reads that were mapped to both reference genomes. Fig 4D shows mapping rates of sequence reads in a male hybrid at the XIII/XIV-like stage. The number of species-specific reads was the maximum when two mismatches were allowed in read mapping. Therefore, we estimated allelic expression of genes using species-specific reads obtained under such mapping conditions.

Fig 4

Experimental scheme of gene expression analysis.

A. Quail, chicken, and hybrid embryos at stages X and XIII/XIV. Stage XIII embryos of parental species showed hypoblast cells in the middle region (asterisks). Hybrid embryos showed extensively proliferated hypoblast cells (arrows). B. Numbers and sexes of embryo samples used for mRNA sequencing. C. Schematic diagrams of mRNA sequencing and read mapping. Sequence reads from the hybrids were mapped to reference genomes of parental species. The reads that were mapped to both reference genomes were removed before counting the number of mapped reads for each gene. D. Percentage of reads that were mapped to chicken reference genome (chicken-specific), quail reference genome (quail-specific), or both genomic sequences (common). Number of mismatches indicate the maximum number of allowed mismatches per read.

Relative gene expression levels cannot be compared directly between chicken and quail because the efficiency of read mapping is considered to vary between two species owing to differences in the reference genome sequences. Therefore, we investigated changes in gene expression from the stage X to the stage XIII/XIV for chicken (G) and quail (Q) genes in each parental species and chicken-derived alleles (HG) and quail-derived alleles (HQ) in the hybrids, and then compared changes in expression between G and Q, between G and HG, and between Q and HQ (S2 Data). The correlation coefficients of the expression change were much higher in Q vs. HQ (0.521) and G vs. HG (0.537) than in G vs. Q (0.107) in males (Fig 5A–5C). Similar results were also obtained from transcriptome analysis of female embryos (S3 Fig). We then determined the directions of change in expression (upregulated, downregulated, or unaltered expression) for each gene by differential gene expression analysis between the stage X and XIII/XIV male blastoderms (Fig 5D). We revealed that 8,376 (72.4%) out of a total of 11,575 genes exhibited similar changes in expression between quail (Q) and chickens (G) (Fig 5E). We also showed that 9,253 (79.9%) and 8,572 (74.1%) genes exhibited similar changes in expression between quail (Q) and hybrids (quail-derived alleles, HQ) and between chickens (G) and hybrids (chicken-derived alleles, HG), respectively, in males (Fig 5F and 5G). The numbers were higher than that between parental species. Similar results were also obtained in females (S3 Fig). The results suggest that expression profiles of chicken and quail alleles in the hybrids retain considerable patterns of gene expression from the parental species.

Fig 5

Gene expression changes from the stage X to the stage XIII/XIV in male embryos.

A–C. Comparison of gene expression changes [log2(fold change)] between quail (‘Q’) and chickens (‘G’) (A), between quail (‘Q’) and quail-derived alleles in the hybrids (‘HQ’) (B), and between chickens (“G”) and chicken-derived alleles in the hybrids (‘HG’) (C). Pearson’s correlation efficient (r) is indicated above the graphs. D. Pattern classification of gene expression changes from the stage X to the stage XIII/XIV. E–G. Comparison of the direction of gene expression changes between quail and chickens (E), between quail and quail-derived alleles in the hybrids (F), and between chickens and chicken-derived alleles in the hybrids (G). The number in each rectangle indicates the percentage of genes. Percentages of genes that exhibited the same directions of expression changes are indicated in bold-lined rectangles. Numbers and percentages of genes exhibiting the same or different directions of expression change are shown in the tables. Color scale at the far right shows the percentage of genes.

Identification of candidate genes responsible for developmental arrest

We postulated that genes whose expression is upregulated from the stage X to the stage XIII/XIV in both parental species play essential roles in the developmental process of embryos; the downregulated or unaltered expression (hereafter, referred to as misregulated expression) of such genes could cause the developmental arrest in the hybrid embryos. Subsequently, we searched for genes exhibiting misregulated expression in the hybrids (referred to as pattern D in Fig 6A). We found that 285 genes were upregulated from the stage X to the stage XIII/XIV in male and/or female embryos of parental species (S3 Data). Out of these 285 genes, 60 exhibited misregulated expression in males and/or females (S2 Table; Fig 6A, S4 Fig). Only four genes, encoding BMP binding endothelial regulator (BMPER), gap junction protein alpha 1 (GJA1, also known as Connexin43), ribosomal protein SA (RPSA), and Wnt family member 5B (WNT5B), exhibited pattern D of expression in both sexes.

Fig 6

GO term enrichment analysis of genes whose expression was misregulated in the male and/or female hybrids.

A. Number of genes whose expression was upregulated from the stage X to the stage XIII/XIV in male and/or female embryos of parental species. In pattern D, 60 genes showed no upregulation of their chicken- and quail-derived alleles in male and/or female hybrids. B. Overrepresented GO-BP terms and the number of genes that are associated with these terms. Eleven GO-BP terms that involve numerous ribosomal protein genes are indicated in gray. C. GO-BP terms related to primitive streak formation and the number of genes associated with these terms.

GO term enrichment analysis using PANTHER [63] revealed that 23 GO terms of biological process (hereafter, referred to as GO-BP terms) were overrepresented (FDR < 0.05) in the 285 genes whose expression was upregulated in both parental species (S3 Table). We show GO-BP terms that are related to primitive streak formation and chromosome segregation in S4 Table. Although none of these GO-BP terms were significantly overrepresented, the 285 genes included genes that are associated with primitive streak formation (S5 Table). GO term enrichment analysis of the 60 misregulated genes showed 14 overrepresented GO-BP terms (FDR < 0.05); 11 of these GO-BP terms, including peptide biosynthetic process, translation, amide biosynthetic process, nitrogen compound biosynthetic process, and ribosome biogenesis, involved numerous ribosomal protein genes (indicated in gray in Fig 6B and S6 Table). The other overrepresented GO-BP terms included regulation of sprouting angiogenesis and cell population proliferation. No GO-BP terms related to the formation of primitive streak (S4 Table) were not nominated in this enrichment analysis; however, several of the 60 misregulated genes were associated with primitive streak formation-related GO-BP terms, including gastrulation and anterior/posterior axis specification (Fig 6C; S5 Table). One of the 60 genes, HORMAD2, was associated with chromosome segregation-related GO-BP terms (S6 Table); however, HORMAD2 has been known to play a role in meiosis, not mitosis [75]. Thus, the finding is consistent with the finding from chromosome analyses in the present study, in that chromosome segregation may not be affected hybrid embryos.

We then examined the expression of nine genes that are widely used as molecular markers of embryonic polarity before, during, and after primitive streak formation (Fig 7A) [27,76-85]. Most of the genes displayed similar changes in expression between parental species and their F1 hybrid males (Fig 7B) and females (S5 Fig). Only WNT5B exhibited the pattern D of gene expression in both sexes (Fig 7B, S5 Fig).

Fig 7

Expression changes of primitive streak formation-related genes.

Genes that function before (WNT8A and PITX2), during (NODAL, CHRD, and WNT5A/5B), and after (TBXT, GSC, and CNOT1) the formation of the primitive streak (A) and changes of their expression from the stage X to the stage XIII/XIV in male embryos (B). Four-digit numbers shown on the right side of the column indicate the direction of expression changes in quail, chickens, and their hybrids (quail- and chicken-derived alleles), respectively. Only WNT5B displays the pattern D of gene expression. Color scale on the far right shows the degree of gene expression change.

Discussion

Elucidation of the molecular basis of hybrid incompatibility in birds may enhance our understanding of their speciation process. In the present study, we investigated the cause of lethality of chicken–quail F1 hybrid embryos at the preprimitive streak stage by focusing on chromosome segregation and gene expression. This is, to the best of our knowledge, the first study investigating the cause of lethality of chicken-quail F1 hybrids by molecular cytogenetic analysis and mRNA sequencing. We have demonstrated that nuclei of the chicken–quail hybrid embryos had a fifty-fifty mixture of parental chromosomes and that numerical abnormalities due to a failure in chromosomal segregation hardly occurred, which is consistent with the observations of previous cytological studies [86,87]. In the present study, we examined only 0-h-old blastoderms and cultured fibroblast cells from 3-day and 7-day-old embryos; therefore, it remains unclear whether chromosome abnormalities occur at different developmental stages and/or in different types of cells in the hybrids. However, the results of the present study suggest that numerical chromosomal abnormalities due to a segregation failure, which have been observed for interspecific hybrids of other organisms [37-39], were not a major cause of the lethality in the chicken–quail hybrid embryos.

Transcriptome analysis of stage X and stage XIII/IV embryos revealed that out of the genes whose expression was upregulated from the stage X to the stage XIII/XIV in the embryos of parental species, neither chicken- nor quail-derived alleles were upregulated for 60 genes in the hybrid males and/or females. Such misregulated genes are potentially responsible for developmental arrest at the preprimitive streak stage. GO term enrichment analysis of the 60 misregulated genes revealed that GO-BP terms, including peptide biosynthetic process, translation, cell population proliferation, and sprouting angiogenesis, were significantly overrepresented. These results suggest that biological processes, such as translation and expansion of cell population, could be affected considerably in the hybrids, with adverse effects on cell migration, proliferation, and/or differentiation in the embryos at the preprimitive streak stage, resulting in developmental arrest. Many ribosomal protein genes were misregulated in the hybrids, suggesting the presence of incompatibilities between chicken- and quail-derived genetic elements that regulate the expression of these ribosomal protein genes. It is unconceivable that the developmental arrest is caused by abnormal sprouting angiogenesis because blood vessels are not formed at the preprimitive streak stages [88].

Alternatively, the developmental arrest may result from the dysregulation of other biological processes. Out of the 60 genes, there were several genes that are associated with primitive streak formation-related GO-BP terms, such as gastrulation and anterior/posterior axis specification. For instance, CRB2 is essential for normal mesoderm formation and is involved in the ingression of epiblast cells during the epithelial-to-mesenchymal transition at gastrulation [89]. WNT3A may mediate the formation of paraxial mesoderm in the anterior primitive streak [90]. RPS6 haploinsufficiency induces embryonic death during gastrulation in mice [91]. Furthermore, out of the well-known primitive streak formation-related genes, WNT5B expression was misregulated in hybrids of both sexes. WNT5B is required for normal cell migration through primitive streak during gastrulation [79]. Expression of WNT8A and PITX2, which are involved in the initiation of primitive streak formation [27, 76], was not misregulated in the hybrids. Therefore, the misregulated expression of the genes involved in primitive streak formation and gastrulation could inhibit the formation, but not the initiation, of the primitive streak, which may block the normal progression of primitive streak formation, resulting in the developmental arrest at the preprimitive streak-like stage.

In addition to WNT5B, BMPER, GJA1, and RPSA exhibited pattern D expression in both sexes. BMPER functions in gastrulation though inhibiting BMP signaling [92]. GJA1 mediates gap junctional communication for morphogenesis during gastrulation [93]. Therefore, the misregulated expression of BMPER and GJA1 may also cause the developmental arrest at the preprimitive streak-like stage through inhibiting gastrulation. RPSA is a component of the 40S subunit and also acts as a membrane receptor [94]. The protein is required for pre-rRNA processing and spleen formation in Xenopus [95]; however, its role in gastrulation remains unknown.

Male sterility in M. m. musculus × M. m. domesticus hybrids is one of the most intensively studied models of hybrid incompatibility [96,97]. The hybrid male sterility in Mus musculus subspecies arises from abnormal expression of X-linked genes in testes, which is caused by incompatibility between X chromosome-linked genes and autosomal genes [53,98-101]. Therefore, misregulated gene expression in chicken-quail F1 hybrids may also be caused by incompatible interaction between chicken- and quail-derived genes in hybrid embryos. Further investigation of the causal relationship of the 60 misregulated genes to developmental arrest in the hybrid embryos and the molecular mechanisms of misregulated expression of these genes may provide important information to understand the molecular basis of hybrid incompatibility in birds.

Conclusions

We hypothesized that chromosomal abnormality is associated with the developmental arrest of chicken–quail F1 hybrid embryos; however, it may be not the case considering the findings of the present study. We revealed misregulated expression of genes that are involved in various biological processes including translation, cell proliferation, and gastrulation as a potential cause of developmental arrest at the preprimitive streak-like stage in the hybrid embryos. Further functional analyses of the genes whose expression was misregulated in the hybrid blastoderms could facilitate the uncovering of the molecular basis of hybrid lethality.

Number of chromosomes in parental species and their hybrids.

(XLSX)

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Count data obtained from mRNA sequencing.

(TXT)

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List of 285 genes that were upregulated in parental species.

(XLSX)

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Stage X blastoderms of chicken, quail, and their hybrids used for mRNA sequencing.

Images of blastoderms are shown with their sample numbers.

(TIF)

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Stage XIII/XIV blastoderms of chicken, quail, and their hybrids used for mRNA sequencing.

Images of blastoderms are shown with their sample numbers.

(TIF)

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Gene expression changes from the stage X to the stage XIII/XIV in female embryos.

A–C. Comparison of gene expression changes [log2(fold change)] between quail (‘Q’) and chickens (‘G’) (A), between quail (‘Q’) and quail-derived alleles in the hybrids (‘HQ’) (B), and between chickens (“G”) and chicken-derived alleles in the hybrids (‘HG’) (C). Pearson’s correlation efficient (r) is indicated above the graphs. D–F. Comparison of the direction of gene expression changes between quail and chickens (D), between quail and quail-derived alleles in the hybrids (E), and between chickens and chicken-derived alleles in the hybrids (F). The number in each rectangle indicates the percentage of genes. Percentages of genes that exhibited the same direction of expression changes are indicated in bold-lined rectangles. Numbers and percentages of genes exhibiting the same or different directions of expression change are shown in the tables. Color scale at the far right shows the percentage of genes.

(TIF)

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Patterns of expression changes of 60 genes whose expression was misregulated in the male and/or female hybrids.

A, B. Patterns of expression changes of the 60 genes in male (A) and female (B) embryos.

(TIF)

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Patterns of expression changes of primitive streak formation-related genes in female blastoderms.

(TIF)

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(PDF)

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Summary of patterns of gene expression changes.

(PDF)

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Overrepresented GO-BP terms in 285 genes that were upregulated in male and/or female embryos of parental species.

(PDF)

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GO-BP terms including those related to primitive streak formation and chromosome segregation.

(PDF)

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Result of GO term enrichment analysis of 285 genes that were upregulated in male and/or female embryos of parental species (primitive streak formation- and chromosome segregation-related GO-BP terms).

(PDF)

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Summary of GO term enrichment analysis of 60 genes showing pattern D.

(PDF)

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29 May 2020

PONE-D-20-09946

Transcriptome analysis revealed misregulated gene expression in blastoderms of interspecific chicken and Japanese quail F1 hybrids

PLOS ONE

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Reviewer #1: Comments

In this manuscript, Ishishita et al describes that the numerical chromosomal abnormalities due to segregation failure does not cause the lethality of chicken–quail hybrid embryos, and that the developmental arrest at the preprimitive streak stage in the hybrids is mainly caused by the downregulated expression of the genes involved in various biological processes. The later part is the important part of this manuscript, however it raises several questions in its current form. Please see below for my specific comments on this manuscript.

Methods section:

In methods section, the chicken management, semen collection, and AI is not clear. Line 125 indicates that the chicken eggs and semen were supplied by the National BioResource Project…Line 131 indicates that the chickens were maintained (locally?). Line 135 indicates that the semen was collected just before AI…. If the chicken egg and semen were supplied, no need of chicken maintenance, as the paper just deals with chicken embryos. In this case, authors need to write semen preservation. If the semen was collected by authors, statement in line 125 should be excluded.

Lines 127-131. While both chromosome and gene expression analyses included in a single paper, it is not clear why Ehime-jidori chickens used for chromosome analysis only, and BL-E chickens used for gene expression analysis only.

Please expand the molecular sexing procedure. When and how the sexing of early embryos (stage X – XIII/XIV) detected?

Results section:

In figure 1, please use different arrows to indicate CJA-Bgl II-M9 signal in 1E, and quail-derived chromosomes in 1F.

In figure 4D, what does mean FC 1/2?

Lines 345-353. Please indicate clearly that you are describing male embryos here. I understood only after reading the corresponding figure legend (Fig. 4). The female counterpart (shown in S1 figure only) is not described and cited in this paragraph.

Lines 349-352. “The numbers of genes whose directions…” Please simply this sentence for clear understanding, and write the number of genes.

Line 371-376. “genes whose expression is upregulated …. referred to as pattern D)”. It is little difficult to connect this message, Fig. 5A, and S2 Fig. in the current form. I suggest to split the S2 Fig. with grouping of those 28 male genes, 28 female genes and 4 common genes fall on pattern D in Fig.5A.

Lines 370-409. This should be the major vital portion of the manuscript as the authors primarily claim in the title. However, it does not strengthen the manuscript, and raises many questions.

1. While avian/chicken specific GO terms are available in many databases including AmiGO, why the authors used Homo sapiens database as the reference? (stated in the methods section, lines 187-190).

2. What is the functional classification of genes listed in Fig. 5B, and how they differ from Fig.5D? does the other genes have no functional classification?

3. Except a very few term, most of the terms shown in Fig. 5B-D are not relevant to embryonic development at the analysis period, stage X – stage XIII/XIV. Authors need to screen terms closely related to stage X – XIII/XIV development. You may also include terms related to shortly before and after this time point. In addition, you can include the terms related to chromosomal properties to support the first section of this manuscript.

4. I suggest the authors to search and include the signaling pathways of those 60 misregulated genes and include as Fig. 5E. It will be interesting if you restrict the signaling pathways related to early embryonic development.

5. Then, Fig 5E can be moved to Fig 6. This information is also inadequate. Please extend this figure by showing a table containing critical/exact role of these markers during embryonic polarity before and during gastrulation.

Discussion section:

This section should be modified according to the revision of results section (lines 370-409, and Fig 5) after considering my review comments, primarily focusing on GO and signaling terms affected in stage X – XIII/XIV hybrid embryos.

Reviewer #2: The manuscript is about identifying the mis-regulated gene expression in blastoderms of interspecific chicken-Japanese quail F1 hybrids. Using transcriptome analysis, they have found that numerical abnormalities due to a segregation failure does not lead to the lethality of chicken–quail hybrid embryos, and that the developmental arrest at the pre-primitive streak stage in the hybrids is mainly caused by the downregulated expression of the genes involved in various biological processes such as translation and gastrulation.

The manuscript lacks the idiographic information in several parts and needs extensive restructuring.

Below are my specific comments mainly on the arrangement of materials and methods.

1. The manuscript is studying chicken (Male) and Japanese quail (Female) F1 hybrids. As well known, the fertilization and lethal rate of hybrids from the different combination is discrepancy. Have you looked at the gene expression of chicken (Female) and Japanese quail (Male) hybrids? If so, how about the result? If not, why?

2. Figures included in this manuscript are too blurry to look at the details. Please reupload figures with high resolution, especially Figure 1 and Figure 5.

3.The Figure legends in this manuscript should be corrected.

4. The statistics analysis needs to be corrected. Why were two different statistic methods used in the same analysis? For example, in Figure 2: the Tukey-Kramer test was used in panel A and B, while the Dunnet’s test was used in panel C and D. Besides, please explain why not use the Pearson’s test but Spearman’s rank in correlation coefficient analysis in Figure 4.

5. In Figure 2A, how do you explain the outliers in the Hybird group in panel A? In addition, the “A” is smaller than other alphabets, please correct the format of figure.

6. Discussion needs to be restructured explaining more about the impact of current findings in order to make the paper compelling for readers.

**********

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Reviewer #1: Yes: Deivendran Rengaraj

Reviewer #2: No

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Submitted filename: Comments.docx

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13 Sep 2020

Response to Reviewer #1

We wish to express our appreciation to the Reviewer for his or her insightful comments, which have helped us significantly improve the paper.

Methods section:

In methods section, the chicken management, semen collection, and AI is not clear. Line 125 indicates that the chicken eggs and semen were supplied by the National BioResource Project…Line 131 indicates that the chickens were maintained (locally?). Line 135 indicates that the semen was collected just before AI…. If the chicken egg and semen were supplied, no need of chicken maintenance, as the paper just deals with chicken embryos. In this case, authors need to write semen preservation. If the semen was collected by authors, statement in line 125 should be excluded.

Response: Line 117–118. We have removed this part because we used semen for artificial insemination immediately after collection of semen from the strain Ehime-jidori. We have also corrected Acknowledgement as follows. “Chicken semen and fertilized eggs were provided by the Avian Bioscience Research Center,” has been changed to “Chicken fertilized eggs were provided by the Avian Bioscience Research Center,” (Line 525¬–526).

Lines 128-131. While both chromosome and gene expression analyses included in a single paper, it is not clear why Ehime-jidori chickens used for chromosome analysis only, and BL-E chickens used for gene expression analysis only.

Response: Line 123–125. The two analyses were conducted at different times using different lines because of the availability of adult chickens when the experiments were carried out.

Please expand the molecular sexing procedure. When and how the sexing of early embryos (stage X ? XIII/XIV) detected?

Response: Line 148–158. Embryonic tissues, which were collected at stage X to XIII/XIV, were minced, and a part of each tissue was used for molecular sexing. The remaining cell suspensions were used for RNA extraction. The description for the molecular sexing method has been included in the item of mRNA sequencing in the revised manuscript.

Results section:

In figure 1, please use different arrows to indicate CJA-Bgl II-M9 signal in 1E, and quail-derived chromosomes in 1F.

Response: Line 258–267. We have corrected Fig 1D–F and the corresponding figure caption for readability. Microchromosomes that were hybridized only with CJA-BglII-M9 were considered quail-derived chromosomes and are indicated by asterisks in Fig 1F.

In figure 4D, what does mean FC 1/2?

Response: Line 182–188. Fold change (FC) of gene expression of stage XIV embryos was calculated by comparing with that of stage X embryos. When the expression level of stage XIV embryos is less than half that of stage X embryos, it has been described as FC is lower than 1/2 (FC < 1/2). We have added description regarding this issue in Methods. “1/2” has been change to “0.5”.

Lines 345-353. Please indicate clearly that you are describing male embryos here. I understood only after reading the corresponding figure legend (Fig. 4). The female counterpart (shown in S1 figure only) is not described and cited in this paragraph.

Response: Line 367–369. We have added the underlined phrase and sentence in this part as follows:

The correlation coefficients of the expression change were much higher in G vs. HG (0.502) and Q vs. HQ (0.558) than in G vs. Q (0.124) in males (Fig 4A–C). Similar results were also obtained from transcriptome analysis of female embryos (S1 Fig).

Lines 349-352. “The numbers of genes whose directions…” Please simply this sentence for clear understanding, and write the number of genes.

Response: Line 3723–377. We have corrected this part as follows:

We revealed that 8,376 (72.4%) out of a total of 11,575 genes exhibited similar changes in expression between quail (Q) and chickens (G) (Fig 4E). We also showed that 9,253 (79.9%) and 8,572 (74.1%) genes exhibited similar changes in expression between quail (Q) and hybrids (quail-derived alleles, HQ) and between chickens (G) and hybrids (chicken-derived alleles, HG), respectively, in males (Fig 4F and G). The numbers were higher than that between parental species. Similar results were also obtained in females (S1 Fig).

Line 371-376. “genes whose expression is upregulated …. referred to as pattern D)”. It is little difficult to connect this message, Fig. 5A, and S2 Fig. in the current form. I suggest to split the S2 Fig. with grouping of those 28 male genes, 28 female genes and 4 common genes fall on pattern D in Fig.5A.

Response: As suggested by the reviewer, we have split the data in S2 Fig and classified 28 male genes, 28 female genes and 4 common genes into pattern D.

Lines 370-409. This should be the major vital portion of the manuscript as the authors primarily claim in the title. However, it does not strengthen the manuscript, and raises many questions.

1. While avian/chicken specific GO terms are available in many databases including AmiGO, why the authors used Homo sapiens database as the reference?

Response: Line 189–195. Fig 6B, C and S3, 5, and 6 Tables.

We used the Gene Ontology (GO) Annotation database for human for the reason that the GO database is more substantial in human compared with that of the chicken. We have carried out GO term enrichment analysis again using the chicken database according to the reviewer’s comment. We have used PANTHER classification system, but not DAVID in this analysis because PANTHER is used in AmiGO. The result of the overrepresentation test for GO terms of biological process is shown in Fig 6B, C and S3, 5, and 6 Tables.

2. What is the functional classification of genes listed in Fig. 5B, and how they differ from Fig.5D? does the other genes have no functional classification?

Response: We have deleted the gene list shown in Fig. 5B of the original manuscript because the functional classification analysis is specific to DAVID.

3. Except a very few term, most of the terms shown in Fig. 5B-D are not relevant to embryonic development at the analysis period, stage X ? stage XIII/XIV. Authors need to screen terms closely related to stage X ? XIII/XIV development. You may also include terms related to shortly before and after this time point. In addition, you can include the terms related to chromosomal properties to support the first section of this manuscript.

Response: Line 400–403, 417–435. Fig 6C and S3–6 Tables.

No primitive streak formation-related GO terms came up by the overrepresentation test in the present study. However, as suggested by the reviewer, we picked up GO biological process terms related to primitive streak formation and chromosome segregation and show them and relevant genes in Fig. 6C and S4–6 Tables. Overrepresented GO terms of molecular function categories (shown in Fig. 5D of the original manuscript) have been removed because the result is redundant with that of the overrepresentation test regarding GO terms of biological process categories. We also performed GO analysis of 285 genes that were upregulated in male and/or female embryos of parental species and show the result in S3 Table.

Response: We could not find the terms related to shortly before and after this time point.

Response: Line 425¬–426. We have added the following sentence.

Consistent with the results of chromosome analysis in the present study, none of the 60 genes were associated with mitotic chromosome segregation-related GO-BP categories.

4. I suggest the authors to search and include the signaling pathways of those 60 misregulated genes and include as Fig. 5E. It will be interesting if you restrict the signaling pathways related to early embryonic development.

Response: We have conducted the overrepresentation test regarding signaling pathways. We noticed that there were no categories associated with early embryonic development, such as mesoderm formation and gastrulation, in the reference database.

5. Then, Fig 5E can be moved to Fig 6. This information is also inadequate. Please extend this figure by showing a table containing critical/exact role of these markers during embryonic polarity before and during gastrulation.

Response: Line 436–440. This data has been shown in Fig 6 following the reviewer’s suggestion. We have picked up nine marker genes that are well-known for being functional before, during and after the formation of primitive streak. We have added a table describing functions of these markers in Fig 6. Furthermore, we made discussion about this issue in line 486-493.

Discussion section:

This section should be modified according to the revision of results section (lines 370-409, and Fig 5) after considering my review comments, primarily focusing on GO and signaling terms affected in stage X ? XIII/XIV hybrid embryos.

Response: We have revised the Discussion section. First, we have added brief description for the aim and significance of this study as the first paragraph (line 451–456). According to the reviewer’s suggestion, we have discussed biological processes that may affect the development of hybrid embryos, focusing on overrepresented GO terms of biological process categories (line 466–478) and GO terms of biological process categories that are related to primitive streak formation (line 479–485).

We wish to thank the Reviewer again for his or her valuable comments.

Response to Reviewer #2

We wish to express our appreciation to the Reviewer for his or her insightful comments, which have helped us significantly improve the paper.

1. The manuscript is studying chicken (Male) and Japanese quail (Female) F1 hybrids. As well known, the fertilization and lethal rate of hybrids from the different combination is discrepancy. Have you looked at the gene expression of chicken (Female) and Japanese quail (Male) hybrids? If so, how about the result? If not, why?

Response: Because of a low amount of semen that can be obtained from male quail, generation of F1 hybrids between chicken females and quail males by artificial insemination is difficult. To our knowledge, no one has succeeded.

2. Figures included in this manuscript are too blurry to look at the details. Please reupload figures with high resolution, especially Figure 1 and Figure 5.

Response: As suggested, we have reuploaded high-resolution figures. In addition, Fig 1 has been divided into two Figures (Fig 1 and Fig 2 in the revised manuscript)

3.The Figure legends in this manuscript should be corrected.

Response: We have corrected the figure legends.

4. The statistics analysis needs to be corrected. Why were two different statistic methods used in the same analysis? For example, in Figure 2: the Tukey-Kramer test was used in panel A and B, while the Dunnet’s test was used in panel C and D. Besides, please explain why not use the Pearson’s test but Spearman’s rank in correlation coefficient analysis in Figure 4.

Response: Line 230–232, line 304–311, line 319–327, and line 367¬–368. As the reviewer pointed out, Tukey-Kramer test was conducted for both panel A and B, and no significant difference was detected. We have also conducted Pearson’s tests and Fig 5 and S3 Fig shows the results of Pearson’s tests.

5. In Figure 2A, how do you explain the outliers in the Hybird group in panel A? In addition, the “A” is smaller than other alphabets, please correct the format of figure.

Response: The outliers may be caused by the artifact when chromosome spreads were prepared, which cause the loss of chromosomes. We have corrected the size of the letter “A” in Fig 3 (Fig 2 in the original manuscript).

6. Discussion needs to be restructured explaining more about the impact of current findings in order to make the paper compelling for readers.

Response: We have revised the Discussion section as follows.

We have added brief description for the aim and significance of this study and as the first paragraph (line 451–456). Subsequent to the discussion about chromosome analysis (line 456–465), we have discussed about biological processes that may affect the development of hybrid embryos, based on overrepresented GO biological process categories (line 466–479), expression of genes associated with GO biological process categories that are related to primitive streak formation (line 479–485), and expression of primitive streak formation-related genes (line 486-493). We have made discussion about four genes that are misregulated in hybrids of both sexes (494–501). Then, we have made the discussion regarding the cause of misregulatd gene expression and future perspective for elucidating the molecular basis of embryonic lethality in chicken-quail hybrids (502–511).

We wish to thank the Reviewer again for his or her valuable comments.

Submitted filename: Response_letter_sishishita_200913.docx

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22 Sep 2020

Transcriptome analysis revealed misregulated gene expression in blastoderms of interspecific chicken and Japanese quail F1 hybrids

PONE-D-20-09946R1

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Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: No

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: Although the author's answer to the first question is vague, it does not affect the quality of the whole manuscript . In addition, I focused on the quality of the Figures in the manuscript, which were too blurry. Hope the author can provide clear original figures.

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Reviewer #1: Yes: Deivendran Rengaraj

Reviewer #2: No

1 Oct 2020

PONE-D-20-09946R1

Transcriptome analysis revealed misregulated gene expression in blastoderms of interspecific chicken and Japanese quail F1 hybrids

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