Rescue of the embryonic lethal hematopoietic defect reveals a critical role for GATA-2 in urogenital development

Abstract

Mutations resulting in embryonic or early postnatal lethality could mask the activities of any gene in unrelated and temporally distinct developmental pathways. Targeted inactivation of the transcription factor GATA-2 gene leads to mid-gestational death as a consequence of hematopoietic failure. We show here that a 250 kbp GATA-2 yeast artificial chromosome (YAC) is expressed strongly in both the primitive and definitive hematopoietic compartments, while two smaller YACs are not. This largest YAC also rescues hematopoiesis in vitro and in vivo, thereby localizing the hematopoietic regulatory cis element(s) to between 100 and 150 kbp 5Ј to the GATA-2 structural gene. Introducing the YAC transgene into the GATA-2 -/-genetic background allows the embryos to complete gestation; however, newborn rescued pups quickly succumb to lethal hydroureternephrosis, and display a complex array of genitourinary abnormalities. These findings reveal that GATA-2 plays equally vital roles in urogenital and hematopoietic development.

Defining the Functional Boundaries of the Gata2 Locus by Rescue with a Linked Bacterial Artificial Chromosome Transgene

“…However, there are at least an equal number of examples of tissues in which this transcription factor is robustly expressed but also for which its in vivo requirement has not yet been completely elucidated. Because it is well established that Gata2 null mutants expire from hematopoietic failure (2) and that Gata2 YAC-rescued null mutants, after surmounting the lethal hematopoietic block, expire from urogenital patterning failures (6), it seemed reasonable to ask: if both of these embryonic deficiencies were complemented by a transgene bearing activities that would correct both deficiencies, would the animals survive? The alternative, which might have been expected in this case, is that the animals would probably not survive to reproductive age, but expire of a second perinatal lethal urogenital failure (due to the anticipated absence from the transgene of any urogenital epithelia-complementing activity), or even from a deficiency in some new tissue that was regulated by an enhancer located outside the boundaries circumscribed by the linked BAC.…”

“…We showed several years ago that Gata2 null mutant embryonic lethality could be rescued by complementation with a 247-kbp transgenic yeast artificial chromosome (YAC) 3 bearing sequences from Ϫ174 to ϩ73 kbp (revised endpoints relative to the Gata2 translational initiation site); the YAC contains all of the regulatory information required to rescue Gata2 function in primitive and definitive hematopoiesis (6). We subsequently found that Gata2 hematopoietic regulatory activity is conferred by at least two distinct cooperative cis elements, one lying about 170 kbp 5Ј to the gene and another lying somewhere between 40 and 100 kbp 5Ј.…”

“…We subsequently found that Gata2 hematopoietic regulatory activity is conferred by at least two distinct cooperative cis elements, one lying about 170 kbp 5Ј to the gene and another lying somewhere between 40 and 100 kbp 5Ј. 4 However, compound mutant mice bearing this YAC, but lacking endogenous Gata2, expired perinatally of kidney failure and uretero-vesicular obstruction, because the YAC-rescued Gata2 null mutant mice failed to develop a patent connection between the ureters and the bladder, causing the mice to develop cystic, non-functional kidneys and megaureters (6). Because the rescuing YAC was not expressed in the urogenital system, while endogenous GATA-2 is prominently expressed there, we speculated that the development of hydroureters and cystic kidneys was due to the absence of tissue-specific enhancers that were responsible for directing proper Gata2-mediated patterning of the developing urogenital system in the YAC transgene.…”

“…Although YACs have been generated that contain genomic inserts of the size required, numerous technical problems with YACs have also come to light, including chimerism (6,14) and innate replicative instability (15). BACs, while surmounting many of the problems encountered with YACs, posed a different problem, because the average genomic DNA insert size in BACs is only ϳ200 kbp (16), which is insufficient in this case to span the interval between the most distant Gata2 hematopoietic (Ϫ170 kbp) and urogenital (ϩ113 kbp) enhancers.…”

Expression of GATA-2 in Human Pituitary Adenomas

“…And the interaction between GATA-2 and Pit-1 can lead to genespecific action and differentiation of TSH-secreting adenomas. Thus, GATA-2, which earlier in development is required for the expansion of hematopoietic precursors (33) and the development of the placental trophoblast (34) and urogenital system (35), also plays a critical role in pituitary cell type determination (15).…”