Elsevier

Life Sciences

Volume 76, Issue 22, 15 April 2005, Pages 2559-2568
Life Sciences

Gene expression analysis of Gata3−/− mice by using cDNA microarray technology

https://doi.org/10.1016/j.lfs.2004.10.054Get rights and content

Abstract

Transcription factor Gata3 is implicated in the formation of autosomal dominant hypoparathyroidism, sensorineural deafness, and renal anomaly (HDR) syndrome. We pursued to identify the potential Gata3 target genes by profiling the gene expression pattern in E9.5 Gata3−/− mouse embryos. Altogether four independent microarray hybridizations were carried out on NIA Mouse15K cDNA arrays. We discovered two hundred and sixty one genes that are downregulated in Gata3 mutant embryos at E9.5 (with a minimal 2.0-fold change). The majority of the differentially expressed genes belong to two functional groups — genes involved in transcription regulation and cellular signaling.

One of the genes discovered to be downregulated in Gata3 mutant embryos was tumor suppressor gene Disabled 2. The validity of this finding was checked by using the whole mount in situ hybridization technology. This study revealed that the sites, where Dab2 is downregulated in the mutant embryos partly overlap with the Gata3 expression domains, including the mid-embryo region, branchial arches and facio-acoustic (VII–VIII) neural crest complex. This is the first time when tumor supressor gene Dab2 is shown to be implicated in the defective phenotype of Gata3 mutant mice.

Introduction

Gata3 belongs to a family of transcription factors that bind with high affinity to the consensus sequence (A/T)GATA(A/G) and share a steroid hormone receptor super family C4 zinc-finger DNA binding motif (Ko and Engel, 1993). In vertebrates the Gata factor family is composed of six members: Gata1 - Gata6 (Lakshmanan et al., 1999). Gata factors have been shown to play critical roles in development, including in cell-fate specification, regulation of differentiation, and control of cell proliferation and movement (Patient and McGhee, 2002). Based on their expression patterns, the Gata proteins have been divided into two subfamilies, Gata-1, -2, and -3 and Gata-4, -5, and -6. Gata-1, -2, and -3 genes are prominently expressed in haematopoietic stem cells where they regulate differentiation-specific gene expression in T-lymphocytes, erythroid cells, and megakaryocytes (Ko and Engel, 1993). Gata-4, -5, and -6 genes are expressed in various mesoderm- and endoderm-derived tissues such as heart, liver, lung, gonad, and gut where they play critical roles in regulating tissue-specific gene expression (Molkentin, 2000).

In addition to the haematopoietic system, Gata3 is expressed in a great variety of tissues in spatio-temporally controlled manner (Lakshmanan et al., 1999). Its expression in mouse is first detected in the ectoplacental cone, branchial arches, and cloaca around E8.5 (George et al., 1994). Subsequently, Gata3 expression can be observed in the central nervous system (CNS - midbrain, hindbrain, and spinal cord), in the otic vesicle, the developing eye, the heart, the mesonephric or Wolffian duct, and in ganglia of the peripheral nervous system (PNS), including trigeminal ganglia and ganglia of the facio-acoustic complex (George et al., 1994, Lakshmanan et al., 1999).

Gene ablation studies have revealed that Gata3 null-mutation is embryonic lethal around E11.0 (Pandolfi et al., 1995). Although the direct cause of lethality has remained elusive, it has been suggested that defects in noradrenaline signaling and heart formation have an important role to play (Lim et al., 2000). However, given the pleiotropic expression pattern of transcription factor Gata3, the mutant phenotype has multiple developmental and physiological defects throughout the whole embryo (Pandolfi et al., 1995). By the time of termination mutants display massive internal bleeding, malformations along the spinal cord, defective microvasculature, and marked growth retardation (Pandolfi et al., 1995).

We have also observed that Gata3 mutant embryos have several developmental defects in their caudal region (area behind the first limb buds), apart from those described by Pandolfi et al. (1995). These include the Wolffian duct elongation and path finding defects, deformities in the ducts' epithelial positioning and aberrations in its surrounding microvasculature (Kärner et al., unpublished). This leads us to hypothesize that the lethal phenotype of Gata3 mutant embryos might have more widespread defects at the molecular level than previously thought and that they have simply gone unnoticed by using the conventional molecular biology methods. Therefore, in this study we assessed the impact of Gata3 deficiency on the mouse E9.5 embryo caudal region transcriptome by comparing the respective regions from wild-type and Gata3 mutant embryos. cDNA microarray technology was chosen as the most appropriate method to address this complex problem. Microarray hybridization data were analyzed for genes with significant differences in expression pattern between mutant and wild type embryos. It appeared that two hundred and sixty one genes were downregulated in the Gata3 mutant embryos at E9.5 (with a minimal 2.0-fold change). The majority of the differentially expressed genes belong to two functional groups — genes involved in transcription regulation and cellular signaling.

Whole mount in situ hybridization was used as an independent method to validate the microarray-based observations. One gene, Disabled 2 (Dab2) was chosen to screen E9.5 wild-type and Gata3 mutant mouse embryos for its expression pattern.

Dab2 belongs to a family of adapter proteins that are involved in cellular signaling, oncogenesis, and development (Morris et al., 2002a, Morris et al., 2002b, Mishra et al., 2002, Hocevar et al., 2003). It has been shown to function as a tumor suppressor gene, and to be implicated in the formation of many mammary, ovarian, and prostate carcinomas (Mok et al., 1998, Schwahn and Medina, 1998, Tseng et al., 1998, Fazili et al., 1999, He et al., 2001).

Consistent with our array results is the observation that Dab2 is significantly downregulated in the caudal region of mutant embryos. This is the first time when the tumor supressor gene Disabled 2 is shown to be implicated in the defective phenotype of Gata3 mutant mice.

Section snippets

Embryo manipulation and tissue dissection

We used genetically homogeneous, inbred strains of wild-type (C57Bl/6) and Gata3/-NLS (C57Bl/6) mouse embryos. Embryos were staged by counting the number of somites (Kaufman, 1992). For experimental purposes only embryos at embryonic age E9.5 (21–25 pairs of somites) were used.

Material was collected either as whole embryos (for in situ hybridization reactions) or, alternatively, only the posterior part of the embryos was preserved for studies (cDNA array hybridization reactions). Dissected

Experimental outline

To identify genes that were altered in Gata3 mutant strain, total RNA was first isolated from the posterior portion of wild-type (C57Bl/6) and Gata3-NLS transgenic (C57Bl/6) E9.5 embryos. The extracted material was pooled and labeled either with the Cy5 dye (wild-type RNA) or Cy3 dye (knock-out RNA). Differently labeled cDNA from independently pooled samples was cohybridized on four arrays.

Determining differentially expressed genes

Hybridization data from four arrays were used to identify genes that are differentially expressed between

Discussion

Mouse embryos mutant to transcription factor Gata3 display severe developmental defects at the midgestational stage, which leads to embryonic death around E10.5. This study focused on the molecular nature of the caudalmost development of E9.5 embryos, the region where Gata3 is expressed in the Wolffian ducts and cloaca (George et al., 1994, Lakshmanan et al., 1999). Apart from the tissues that express Gata3, structures not complementary to Gata3 expression are also affected in the mutants.

Acknowledgement

This study was supported by a research grant from Estonian Science Foundation ESF 4894.

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