Differences in Gene Expression between Wild Type and Hoxa1 Knockout Embryonic Stem Cells after Retinoic Acid Treatment or Leukemia Inhibitory Factor (LIF) Removal

Eduardo Martinez-Ceballos,Pierre Chambon,Lorraine J Gudas

doi:10.1074/jbc.m414397200

Abstract

Homeobox (Hox) genes encode a family of transcription factors that regulate embryonic patterning and organogenesis. In embryos, alterations of the normal pattern of Hox gene expression result in homeotic transformations and malformations. Disruption of the Hoxa1 gene, the most 3' member of the Hoxa cluster and a retinoic acid (RA) direct target gene, results in abnormal ossification of the skull, hindbrain, and inner ear deficiencies, and neonatal death. We have generated Hoxa1(-/-) embryonic stem (ES) cells (named Hoxa1-15) from Hoxa1(-/-) mutant blastocysts to study the Hoxa1 signaling pathway. We have characterized in detail these Hoxa1(-/-) ES cells by performing microarray analyses, and by this technique we have identified a number of putative Hoxa-1 target genes, including genes involved in bone development (e.g. Col1a1, Postn/Osf2, and the bone sialoprotein gene or BSP), genes that are expressed in the developing brain (e.g. Nnat, Wnt3a, BDNF, RhoB, and Gbx2), and genes involved in various cellular processes (e.g. M-RAS, Sox17, Cdkn2b, LamA1, Col4a1, Foxa2, Foxq1, Klf5, and Igf2). Cell proliferation assays and Northern blot analyses of a number of ES cell markers (e.g. Rex1, Oct3/4, Fgf4, and Bmp4) suggest that the Hoxa1 protein plays a role in the inhibition of cell proliferation by RA in ES cells. Additionally, Hoxa1(-/-) ES cells express high levels of various endodermal markers, including Gata4 and Dab2, and express much less Fgf5 after leukemia inhibitory factor (LIF) withdrawal. Finally, we propose a model in which the Hoxa1 protein mediates repression of endodermal differentiation while promoting expression of ectodermal and mesodermal characteristics.

Highlights

Homeobox (Hox) genes encode a family of transcription factors that regulate embryonic patterning and organogenesis
We have characterized in detail these Hoxa1؊/؊ embryonic stem (ES) cells by performing microarray analyses, and by this technique we have identified a number of putative Hoxa-1 target genes, including genes involved in bone development (e.g. Col1a1, Postn/Osf2, and the bone sialoprotein gene or Bone sialoprotein gene (BSP)), genes that are expressed in the developing brain (e.g. Nnat, Wingless-related MMTV integration site 3A (Wnt3a), Brain-derived neurotrophic factor (BDNF), RhoB, and Gastrulation brain homeobox 2 (Gbx2)), and genes involved in various cellular processes (e.g. M-RAS, SRY-box containing gene 17 (Sox17), Cyclin-dependent kinase inhibitor 2B (Cdkn2b), LamA1, Col4a1, Foxa2, Foxq1, Kruppel-like factor 5 (Klf5), and Igf2)
Low levels of Hoxa1 expression in Hoxa1ϩ/Ϫ Cells Are Sufficient for a Normal Activation of the Hoxa1 Pathway— the levels of Hoxa1 mRNA expression after retinoic acid (RA) treatment in Hoxa1 heterozygous (ϩ/Ϫ) ES cells were between 3- and 4-fold lower than in Hoxa1-1 (Wt) ES cells under the same conditions, in general, these two cell lines displayed similar gene expression profiles as demonstrated by Northern blot assays

Summary

Introduction

Homeobox (Hox) genes encode a family of transcription factors that regulate embryonic patterning and organogenesis. The ectopic expression of Hoxa in transgenic mice leads to the ectopic activation of Hoxb in r2, produces anterior abnormalities, including the reorganization of the developing hindbrain, and results in embryonic death [27] These observations highlight the importance of the Hoxa gene and suggest that the regulatory effects of retinoids on cell growth and on embryonic patterning may be chain; LIF, leukemia inhibitory factor; LIFR, leukemia inhibitory factor receptor; Nnat, neuronatin; Postn/Osf periostin/osteoblast-specific transcription factor 2; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; Wnt3a, wingless-related MMTV integration site 3A; Wt, wild type; RT, reverse transcriptase; BDNF, brain-derived neurotrophic factor. In this research we have generated embryonic stem (ES) cells from wild-type, Hoxa ϩ/Ϫ, and Hoxa1Ϫ/Ϫ blastocyststage mouse embryos to use as a model system in which to characterize in detail the putative genes involved in the Hoxa signaling pathway and to identify Hoxa target genes by microarray analyses

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Results

Conclusion