A tissue-specific knockout reveals that Gata1 is not essential for Sertoli cell function in the mouse

Abstract

The transcription factor Gata1 is essential for the development of erythroid cells. Consequently, Gata1 null mutants die in utero due to severe anaemia. Outside the haematopoietic system, Gata1 is only expressed in the Sertoli cells of the testis. To elucidate the function of Gata1 in the testis, we made a Sertoli cell-specific knockout of the Gata1 gene in the mouse. We deleted a normally functioning 'floxed' Gata1 gene in pre-Sertoli cells in vivo through the expression of Cre from a transgene driven by the Desert Hedgehog promoter. Surprisingly, Gata1 null testes developed to be morphologically normal, spermatogenesis was not obviously affected and expression levels of putative Gata1 target genes, and other Gata factors, were not altered. We conclude that expression of Gata1 in Sertoli cells is not essential for testis development or spermatogenesis in the mouse.

Figure 1

A conditional allele of the Gata1 gene. (A) The mouse Gata1 locus, with the testis-specific first exon (IT) ∼8 kb upstream of the erythroid-specific first exon (IE). The coding exons (II–VI) are common to both mRNAs. After homologous recombination with the targeting vector, the Gata1 locus has two loxP sites and the splice acceptor/GFP cassette inserted, and is referred to as the ‘floxed’ locus. After recombination of the floxed Gata1 locus, the coding exons are deleted and the GFP transcript should be expressed. (B) Southern blot of BamHI-digested DNA from ES cells harbouring the floxed Gata1 locus, before and after Cre transfection. Left panel: the blot was hybridised with a Gata1 cDNA probe. Because the coding sequences are removed upon recombination, no band is detected with the cDNA probe after recombination. Right panel: same blot, re-hybridised with the 5′ probe (A). The size of the 10 kb band is reduced to 3.4 kb after recombination.

Figure 2

Expression of Cre in Sertoli cells driven by Dhh genomic sequences. (A) Dhh-Cre construct used to make transgenic mice. Dhh exons are in green; Cre-encoding sequences in red. B = BamHI; N = NotI. (B) (a) LacZ-stained section of E18 Dhh-Cre/R26R transgenic testis. CE = coelomic epithelium; TC = testis cords. Arrows point to pre-Sertoli cells; arrowheads indicate endothelial cells. Original magnification 25×. (b) LacZ-stained whole mount of P15 Dhh-Cre/R26R transgenic testis. All testis cords are stained intensely blue. Original magnification 2.5×.

Figure 3

The Dhh-Cre transgene abolishes testis-specific Gata1 expression from the floxed Gata1 allele. Testes were isolated from P14 Gata1^floxed:y and Gata1^floxed:y::Dhh-Cre mice. RNA was extracted from total testis, and expression of Gata1 was assessed by RT–PCR analysis. The size of the expected product for Gata1 cDNA is 297 bp. RT–PCR analysis of Hprt expression was used as an internal control; expected product size of cDNA is 247 bp.