TY - JOUR
T1 - Identification of lineage-specific zygotic transcripts in early Caenorhabditis elegans embryos
AU - Robertson, Scott M.
AU - Shetty, Premnath
AU - Lin, Rueyling
N1 - Funding Information:
The authors would like to thank Susan Mango for the plasmid pAP10, Morris Maduro and Joel Rothman for providing med-1, end-1, and end-3 cDNAs, Geraldine Seydoux for pes-10 cDNA, Keith Blackwell for skn-1 cDNA, Stuart Kim and the Stanford Microarray Facility for the microarray hybridizations, Norman Iscove for discussion of experimental details, Jim Waddle for the custom program os4D, and Manisha Patel for technical assistance. We are also grateful to the Japanese knockout consortium National BioResource Project ( http://www.nbrp.jp/index.jsp ), especially Shohei Mitani, which provided the tbx-11(tm278) mutant allele for analyses. All other strains used in this study were provided by the C. elegans Genome Consortium. This work is in part supported by grants from March of Dimes Birth Defect Foundation (1-FY01-265), American Heart Association (0455081Y) and NIH (HD37933) to R.L.
PY - 2004/12/15
Y1 - 2004/12/15
N2 - During Caenorhabditis elegans embryogenesis, a maternally supplied transcription factor, SKN-1, is required for the specification of the mesendodermal precursor, EMS, in the 4-cell stage embryo. When EMS divides, it gives rise to a mesoderm-restricted precursor, MS, and an endoderm-restricted precursor, E. To systematically identify genes that function as key regulators of MS and/or E-derived tissues, we identified, by microarray analyses, genes that are newly transcribed within a short developmental window (approximately 30 min) encompassing the generation and fate specification of the MS and E blastomeres. By comparing total cDNAs generated from individual, carefully staged embryos, we identified 275 genes up-regulated in 12-cell embryos compared to 4-cell embryos. Fifty of these 275 genes are down-regulated in 12-cell skn-1 mutant embryos and are designated skn-1-dependent zygotic (sdz) genes. The spatial and temporal expression patterns in C. elegans embryos of 10 randomly selected sdz genes were analyzed by a nuclear GFP reporter driven by the endogenous 5′ regulatory sequence of each gene. GFP expression, although absent at the 4-cell stage, was detected at the 12- to 16-cell stage for all 10 genes and was restricted to EMS-derived lineages for 7 of the 10. Among the seven lineage-specific genes, three genes are expressed equally in both MS and E lineages, two are expressed exclusively or predominantly in the MS lineage, and two are expressed exclusively in the E lineage. Depletion of skn-1 by RNAi abolishes the expression of all seven reporter transgenes in vivo, confirming that these genes are indeed skn-1 dependent. These results demonstrate the successful combination of single-staged embryo cDNAs, genetic mutants, and whole transcriptome microarray analysis to identify stage- and lineage-specific transcripts in early C. elegans embryos.
AB - During Caenorhabditis elegans embryogenesis, a maternally supplied transcription factor, SKN-1, is required for the specification of the mesendodermal precursor, EMS, in the 4-cell stage embryo. When EMS divides, it gives rise to a mesoderm-restricted precursor, MS, and an endoderm-restricted precursor, E. To systematically identify genes that function as key regulators of MS and/or E-derived tissues, we identified, by microarray analyses, genes that are newly transcribed within a short developmental window (approximately 30 min) encompassing the generation and fate specification of the MS and E blastomeres. By comparing total cDNAs generated from individual, carefully staged embryos, we identified 275 genes up-regulated in 12-cell embryos compared to 4-cell embryos. Fifty of these 275 genes are down-regulated in 12-cell skn-1 mutant embryos and are designated skn-1-dependent zygotic (sdz) genes. The spatial and temporal expression patterns in C. elegans embryos of 10 randomly selected sdz genes were analyzed by a nuclear GFP reporter driven by the endogenous 5′ regulatory sequence of each gene. GFP expression, although absent at the 4-cell stage, was detected at the 12- to 16-cell stage for all 10 genes and was restricted to EMS-derived lineages for 7 of the 10. Among the seven lineage-specific genes, three genes are expressed equally in both MS and E lineages, two are expressed exclusively or predominantly in the MS lineage, and two are expressed exclusively in the E lineage. Depletion of skn-1 by RNAi abolishes the expression of all seven reporter transgenes in vivo, confirming that these genes are indeed skn-1 dependent. These results demonstrate the successful combination of single-staged embryo cDNAs, genetic mutants, and whole transcriptome microarray analysis to identify stage- and lineage-specific transcripts in early C. elegans embryos.
KW - C. elegans
KW - Embryos
KW - Lineage specific
KW - Microarray
KW - Zygotic
KW - skn-1
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U2 - 10.1016/j.ydbio.2004.09.015
DO - 10.1016/j.ydbio.2004.09.015
M3 - Article
C2 - 15581881
AN - SCOPUS:9944221653
SN - 0012-1606
VL - 276
SP - 493
EP - 507
JO - Developmental Biology
JF - Developmental Biology
IS - 2
ER -