Homotypic signalling regulates Gata1 activity in the erythroblastic island

Laura Gutirrez 0 1 Fokke Lindeboom 0 1 An Langeveld 0 1 Frank Grosveld 0 1 Sjaak Philipsen ) 0 1 David Whyatt 0 1 0 Erasmus MC, Department of Cell Biology , PO Box 1738, 3000 DR Rotterdam , The Netherlands 1 Homotypic signalling in the erythroid system - Gata1 is a transcription factor essential for erythropoiesis. Erythroid cells lacking Gata1 undergo apoptosis, while overexpression of Gata1 results in a block in erythroid differentiation. However, erythroid cells overexpressing Gata1 differentiate normally in vivo when in the presence of wild-type cells. We have proposed a model, whereby a signal generated by wild-type cells (red cell differentiation signal; REDS) overcomes the intrinsic defect in Gata1overexpressing erythroid cells. The simplest interpretation of this model is that wild-type erythroid cells generate REDS. To substantiate this notion, we have exploited a Erythropoiesis in mammals goes through two distinct stages, a primitive and a definitive stage. In the mouse, primitive erythropoiesis begins in the yolk sac at around 7 days post coitus (dpc) and produces nucleated erythrocytes. Shortly after 10 dpc, erythropoiesis switches from the primitive to the definitive stage, and at around 12 dpc enucleated erythrocytes begin to replace nucleated erythrocytes in the circulation (Rifkind et al., 1969; Russell, 1979; Wong et al., 1985). The primary site of definitive erythropoiesis is the foetal liver, followed by the spleen and bone marrow later in development (Medvinsky and Dzierzak, 1998; Moore and Metcalf, 1970). Definitive erythropoiesis takes place in erythroblastic islands, which consist of a central macrophage surrounded by erythroid precursors located further towards the periphery of the island in progressive stages of differentiation (Bessis et al., 1983). Gata1 belongs to the GATA family of zinc-finger transcription factors (Evans and Felsenfeld, 1989; Patient and McGhee, 2002; Tsai et al., 1989; Yamamoto et al., 1990). It is mainly expressed in haematopoietic cells (erythroid cells, megakaryocytes, eosinophils and mast cells) (Hannon et al., 1991; Martin and Orkin, 1990; Patient and McGhee, 2002; Romeo et al., 1990; Weiss and Orkin, 1995a) but also in Sertoli cells of the testis (Ito et al., 1993; Yomogida et al., 1994). Gata1 recognises a consensus binding motif that is present in the regulatory elements of all erythroid-specific genes examined, including the Gata1 gene itself (Ohneda and Yamamoto, 2002). Correct regulation of Gata1 levels appears crucial for normal primitive and definitive erythropoiesis. Erythroid cells null for Gata1 undergo apoptosis at the relatively immature proerythroblast stage (Pevny et al., 1995; Pevny et al., 1991; Weiss et al., 1994; Weiss and Orkin, 1995b) and Gata1 tissue specific Cre/loxP system and the process of Xinactivation to generate mice that overexpress Gata1 in half the erythroid cells and are Gata1 null in the other half. The results show that the cells supplying REDS are erythroid cells. This study demonstrates the importance of intercellular signalling in regulating Gata1 activity and that this homotypic signalling between erythroid cells is crucial to normal differentiation. knockout mice die of anaemia at around 11.5 dpc (Fujiwara et al., 1996). The Gata1 gene is X-linked (Zon et al., 1990) and, owing to X-inactivation, female mice heterozygous for a functional Gata1 gene have two populations of erythroid cells with respect to Gata1 expression, one that is wild type and one that is Gata1 null. These mice are transiently anaemic during gestation, but recover during the neonatal period, probably owing to the in vivo selection of progenitors able to express Gata1. Mutations resulting in reduced levels of Gata1 also inhibit erythroid differentiation (McDevitt et al., 1997; Takahashi et al., 1997). Interestingly, overexpression of Gata1 in erythroid cells inhibits erythroid differentiation both in vitro and in vivo (Whyatt et al., 2000; Whyatt et al., 1997). In order to study overexpression in vivo, mice were generated that express Gata1 from an X-linked transgene under the control of the erythroidspecific b -globin gene promoter and locus control region. Transgenic males display pancellular Gata1 overexpression in the erythroid lineage and die of anaemia at around 13.5 dpc, because of a block in definitive erythroid differentiation (Whyatt et al., 2000). Furthermore, Gata1-overexpressing erythroid colonies grown from single precursors (colony forming units-erythroid, CFU-Es) fail to differentiate normally in vitro. By contrast, heterocellular overexpression of Gata1, as occurs in chimeric mice or in the heterozygous transgenic females because of X-inactivation, results in live transgenic mice that are phenotypically normal. Remarkably, all erythroid cells, both wild type and overexpressing Gata1, contribute normally to the differentiated erythrocyte pool in these animals. This shows that the defect generated by overexpression of Gata1 is ce (...truncated)