Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Oct 31;21(21):8170.
doi: 10.3390/ijms21218170.

Whole-Transcriptome Sequencing-Based Analysis of DAZL and Its Interacting Genes during Germ Cells Specification and Zygotic Genome Activation in Chickens

Deivendran Rengaraj  1 , Sohyoung Won  2 , Jong Won Han  1 , DongAhn Yoo  2 , Heebal Kim  1   2   3 , Jae Yong Han  1
Affiliations

Whole-Transcriptome Sequencing-Based Analysis of DAZL and Its Interacting Genes during Germ Cells Specification and Zygotic Genome Activation in Chickens

Deivendran Rengaraj et al. Int J Mol Sci. .

Abstract

The deleted in azoospermia like (DAZL) is required for germ cells development and maintenance. In chickens, the mRNA and protein of DAZL, a representative maternally inherited germ plasm factor, are detected in the germ plasm of oocyte, zygote, and all stages of the intrauterine embryos. However, it is still insufficient to explain the origin and specification process of chicken germ cells, because the stage at which the zygotic transcription of DAZL occurs and the stage at which the maternal DAZL RNA/protein clears have not yet been fully identified. Moreover, a comprehensive understanding of the expression of DAZL interacting genes during the germ cells specification and development and zygotic genome activation (ZGA) is lacking in chickens. In this study, we identified a set of DAZL interacting genes in chickens using in silico prediction method. Then, we analyzed the whole-transcriptome sequencing (WTS)-based expression of DAZL and its interacting genes in the chicken oocyte, zygote, and Eyal-Giladi and Kochav (EGK) stage embryos (EGK.I to EGK.X). In the results, DAZL transcripts are increased in the zygote (onset of transcription), maintained the increased level until EGK.VI, and decreased from EGK.VIII (possible clearance of maternal RNAs). Among the DAZL interacting genes, most of them are increased either at 1st ZGA or 2nd ZGA, indicating their involvement in germ cells specification and development.

Keywords: DAZL; DAZL interacting genes; germ cells development; intrauterine embryos; zygotic genome activation.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The gene ontology enrichment of deleted in azoospermia like (DAZL) interacting genes. DAZL and a set of DAZL interacting genes predicted in chickens using the search tool for the retrieval of interacting genes/proteins (STRING) database and motifs analysis are subjected to the AmiGO 2 gene ontology database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Genes in the germ plasm component and germ cells development categories are identified using the AmiGO database. Genes in all other categories (processes related to zygotic genome activation (ZGA)/maternal-to-zygotic transition (MZT)) are identified using the KEGG database.
Figure 2
Figure 2
Interaction network and intrauterine embryonic expression of DAZL interacting genes identified in the germ plasm category. The confidence-based direct interaction of DAZL with genes identified in the germ plasm category is prepared using the STRING database (A). The expression patterns of DAZL interacting genes (germ plasm category) in the chicken oocyte, zygote, and Eyal-Giladi and Kochav (EGK) stage intrauterine embryos (EGK.I to EGK.X) are examined using the whole-transcriptome sequencing (WTS) data. log2 trimmed mean of M-values (TMM)-normalization is used to better visualize the gene expression through heatmap (B) and line graph (C).
Figure 3
Figure 3
Interaction network and intrauterine embryonic expression of DAZL interacting genes identified in the germ cells development category. The confidence-based direct interaction of DAZL with genes identified in the germ cells development category is prepared using the STRING database (A). The expression patterns of DAZL interacting genes (germ cells development category) in the chicken oocyte, zygote, and EGK stage intrauterine embryos (EGK.I to EGK.X) are examined using the WTS data. log2 TMM-normalization is used to better visualize the gene expression through heatmap (B) and line graph (C).
Figure 4
Figure 4
Interaction network and intrauterine embryonic expression of DAZL interacting genes identified in the transcription factors category. The confidence-based direct interaction of DAZL with genes identified in the transcription factors category is prepared using the STRING database (A). The expression patterns of DAZL interacting genes (transcription factors category) in the chicken oocyte, zygote, and EGK stage intrauterine embryos (EGK.I to EGK.X) are examined using the WTS data. log2 TMM-normalization is used to better visualize the gene expression through heatmap (B) and line graph (C).
Figure 5
Figure 5
Differentially expressed DAZL interacting genes between consecutive stages of intrauterine embryonic development in chicken. The differentially expressed genes between stages are detected using a negative binomial-based GLM. In total, six statistical tests are performed: oocyte versus zygote, zygote versus EGK.I, EGK.I versus EGK.III, EGK.III versus EGK.VI, EGK.VI versus EGK.VIII, and EGK.VIII versus EGK.X. FDR-adjusted p < 0.05 is considered for significant genes. logFC > 1 is considered for upregulated genes (green arrow), logFC < −1 is considered for downregulated genes (red arrow), and genes fall between these logFC conditions is considered as unchanged (yellow arrow). The logFCs of genes listed at the top and bottom in a box are mentioned.
Figure 6
Figure 6
Exon–intron specific RT-qPCR validation of DAZL interacting genes during the intrauterine embryonic development in chicken. Total RNAs from the oocyte, zygote, and EGK.I to EGK.X stage embryos are reverse transcribed using the random hexamer. No reverse transcriptase (no-RT) negative control of EGK.X embryos are also prepared to monitor the genomic DNA contamination. Then, the cDNAs are amplified using the gene-specific forward and reverse primers. The forward primer (F) on a suitable exon region and the reverse primer (R) on the following intron region is designed with the GenBank sequences of each gene. qPCR is performed in triplicate with three independent samples. The relative quantification of the gene expression is normalized with the chicken GAPDH and a reference sample (oocyte) and analyzed by the 2−ΔΔCt method. Significant differences between the oocyte and other test samples are determined using the Student’s t-test. Statistical significance is ranked as * p < 0.05, ** p < 0.01, or *** p < 0.001. ns—not significant.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Mitalipov S., Wolf D. Totipotency, pluripotency and nuclear reprogramming. Adv. Biochem. Eng. Biotechnol. 2009;114:185–199. doi: 10.1007/10_2008_45. - DOI - PMC - PubMed
    1. Jamieson-Lucy A., Mullins M.C. Current Topics in Developmental Biology. Volume 135. Elsevier; Amsterdam, The Netherlands: 2019. The vertebrate Balbiani body, germ plasm, and oocyte polarity; pp. 1–34. - DOI - PubMed
    1. Tang W.W., Kobayashi T., Irie N., Dietmann S., Surani M.A. Specification and epigenetic programming of the human germ line. Nat. Rev. Genet. 2016;17:585–600. doi: 10.1038/nrg.2016.88. - DOI - PubMed
    1. Hird S.N., Paulsen J.E., Strome S. Segregation of germ granules in living Caenorhabditis elegans embryos: Cell-type-specific mechanisms for cytoplasmic localisation. Development. 1996;122:1303–1312. - PubMed
    1. Lehmann R. Germ-plasm formation and germ-cell determination in Drosophila. Curr. Opin. Genet. Dev. 1992;2:543–549. doi: 10.1016/S0959-437X(05)80169-8. - DOI - PubMed

Substances