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Use of RDA analysis of knockout mice to identify myeloid genes regulated in vivo by PU.1 and C/EBPalpha.

ABSTRACT: PU.1 and C/EBPalpha are transcription factors essential for normal myeloid development. Loss-of-function mutation of PU.1 leads to an absolute block in monocyte/macrophage development and abnormal granulocytic development while that of C/EBPalpha causes a selective block in neutrophilic differentiation. In order to understand these phenotypes, we studied the role of PU.1 and C/EBPalpha in the regulation of myeloid target genes in vivo . Northern blot analysis revealed that mRNAs encoding receptors for M-CSF, G-CSF and GM-CSF, were expressed at low levels in PU.1(-/-) fetal liver compared with wild type. To identify additional myeloid genes regulated by PU.1 and C/EBPalpha, we performed representational difference analysis (RDA), a PCR-based subtractive hybridization using fetal livers from wild type and PU.1 or C/EBPalpha knockout mice. By introducing a new modification of RDA, that of tissue-specific gene suppression, we could selectively identify a set of differentially expressed genes specific to myeloid cells. Differentially expressed genes included both primary and secondary granule protein genes. In addition, eight novel genes were identified that were upregulated in expression during myeloid differentiation. These methods provide a general strategy for elucidating the genes affected in murine knockout models.

SUBMITTER: Iwama A

PROVIDER: S-EPMC147647 | biostudies-other | 1998 Jun

REPOSITORIES: biostudies-other

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Use of RDA analysis of knockout mice to identify myeloid genes regulated in vivo by PU.1 and C/EBPalpha.

Iwama A A Zhang P P Darlington G J GJ McKercher S R SR Maki R R Tenen D G DG

Nucleic acids research 19980601 12

PU.1 and C/EBPalpha are transcription factors essential for normal myeloid development. Loss-of-function mutation of PU.1 leads to an absolute block in monocyte/macrophage development and abnormal granulocytic development while that of C/EBPalpha causes a selective block in neutrophilic differentiation. In order to understand these phenotypes, we studied the role of PU.1 and C/EBPalpha in the regulation of myeloid target genes in vivo . Northern blot analysis revealed that mRNAs encoding recepto ...[more]

PMID: 9611252

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Project description:BackgroundIn humans, cleft palate (CP) accounts for one of the largest number of birth defects with a complex genetic and environmental etiology. TGF?3 has been established as an important regulator of palatal fusion in mice and it has been shown that TGF?3-null mice exhibit CP without any other major deformities. However, the genes that regulate cellular decisions and molecular mechanisms maintained by the TGF?3 pathway throughout palatogenesis are predominantly unexplored. Our objective in this study was to analyze global transcriptome changes within the palate during different gestational ages within TGF?3 knockout mice to identify TGF?3-associated genes previously unknown to be associated with the development of cleft palate. We used deep sequencing technology, RNA-Seq, to analyze the transcriptome of TGF?3 knockout mice at crucial stages of palatogenesis, including palatal growth (E14.5), adhesion (E15.5), and fusion (E16.5).ResultsThe overall transcriptome analysis of TGF?3 wildtype mice (C57BL/6) reveals that almost 6000 genes were upregulated during the transition from E14.5 to E15.5 and more than 2000 were downregulated from E15.5 to E16.5. Using bioinformatics tools and databases, we identified the most comprehensive list of CP genes (n = 322) in which mutations cause CP either in humans or mice, and analyzed their expression patterns. The expression motifs of CP genes between TGF?3+/- and TGF?3-/- were not significantly different from each other, and the expression of the majority of CP genes remained unchanged from E14.5 to E16.5. Using these patterns, we identified 8 unique genes within TGF?3-/- mice (Chrng, Foxc2, H19, Kcnj13, Lhx8, Meox2, Shh, and Six3), which may function as the primary contributors to the development of cleft palate in TGF?3-/- mice. When the significantly altered CP genes were overlaid with TGF? signaling, all of these genes followed the Smad-dependent pathway.ConclusionsOur study represents the first analysis of the palatal transcriptome of the mouse, as well as TGF?3 knockout mice, using deep sequencing methods. In this study, we characterized the critical regulation of palatal transcripts that may play key regulatory roles through crucial stages of palatal development. We identified potential causative CP genes in a TGF?3 knockout model, which may lead to a better understanding of the genetic mechanisms of palatogenesis and provide novel potential targets for gene therapy approaches to treat cleft palate.

Dataset Information

Use of RDA analysis of knockout mice to identify myeloid genes regulated in vivo by PU.1 and C/EBPalpha.

Publications

Use of RDA analysis of knockout mice to identify myeloid genes regulated in vivo by PU.1 and C/EBPalpha.

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