Kit regulatory elements required for expression in developing hematopoietic and germ cell lineages

Abstract

IntroductionStem cells are traditionally considered to be either multipotent (eg, embryonic stem [ES] cells) or restricted in their differentiation potential (tissue stem cells). Reports on "transdifferentiation" and the discovery of multipotent adult progenitor cells in bone marrow, brain, and muscle have recently challenged this view. [1][2][3][4][5] One central issue underlying the debate on developmental options of stem cells concerns the molecular mechanisms responsible for establishing and maintaining their transcriptional programs and decisions to differentiate. So far, few of the key regulatory genes active in stem cells and/or multipotent progenitors have been studied at the level of transcriptional regulation. [6][7][8][9][10][11][12][13][14][15][16][17] Identification and comparison of several such genes will provide important insights into the transcriptional programs of stem and multipotent progenitor cells.The Kit (White spotting locus) gene, encoding the transmembrane receptor of the cytokine stem cell factor/Kit ligand (KL), is an important regulator of proliferation/survival and/or migration of several stem cell types such as the primordial germ cells (PGCs), [18][19][20][21][22] the multipotent hematopoietic stem cells (HSCs), 20,23 the neural crest, and the intestinal Cajal cells. 20 Null mutations in the Kit or the KL (Steel) gene result in severe hematopoietic and germ cell defects and in utero or perinatal death, whereas mutations that diminish Kit tyrosine kinase activity or KL production affect mainly hematopoiesis and the development of germ cells, melanocytes, and the intestinal Cajal cells. 20 During mouse developmentKit is expressed at a low level in pluripotent inner cell mass cells (and in epiblast-derived ES cells in culture) 24,25 and, at relatively higher levels, in PGCs, early hematopoietic progenitors, and other cells. 25 In adults, Kit is expressed in a variety of cell types including HSCs, immature hematopoietic progenitors and mast cells, oocytes, a subpopulation of male germ cells, and melanocytes. [18][19][20][21][22][23]25 In the mouse embryo, hematopoietic progenitors are detected both extraembryonically in the yolk sac, after embryonic day 7 (E7), and intraembryonically, first in the para-aortic splanchnopleura and aorta-gonad-mesonephros (AGM) regions, then in fetal liver and vitelline vessels, [26][27][28] and finally in bone marrow. The earlier hematopoietic cells (primitive hematopoietic cells) are morphologically and biologically distinct from the later cells (definitive hematopoietic cells). Definitive hematopoiesis is seeded by HSCs, which arise intraembryonically in the AGM region and in the vitelline vessels around E11. [28][29][30] Immature precursors to definitive HSCs, however, have also been detected both intraembryonically 31,32 and in the yolk sac 32 at earlier stages of development. Murine PGCs first become visible around E7 in the extraembryonic mesoderm, then migrate through the allantois (E8) to the hindgut, from where they move to reach the gonadal ...

Essential Role of Sox2 for the Establishment and Maintenance of the Germ Cell Line

“…We have identified a region within the first Kit intron, which is specifically bound by Sox2 in PGCs. Notably, the first Kit intron was previously shown to be essential for Kit expression in these cells as well as in hematopoietic precursors [22,47]. Since Kit signaling is essential for survival and proliferation of germ cells [25,27,48], we can hypothesize that one of the mechanisms by which Sox2 ablation affects PGCs is through downregulation of Kit transcription.…”

“…Homozygous YFPRosa26 loxP/loxP and LacZ-Rosa26 loxP/loxP mice were kindly provided by GianGiacomo Consalez (University of San Raffaele, Milan, Italy). Kit Egfp mice have been previously described [22]. For staging embryos, 0.5 dpc corresponded to the day of vaginal plug.…”

“…In Kit Egfp transgenic animals, expression of the reporter EGFP is driven by Kit regulatory regions of the promoter and the first intron (corresponding to the genomic region depicted in the scheme of Fig. 6D), which have been previously shown to be specifically expressed in forming PGCs in the ExM [22]. As shown in Figure 7, in control embryos at 7.5 dpc, PGCs can be easily identified by the expression of the EGFP transgene driven by Kit transcriptional regulatory sequences, whereas, in Sox2 conditionally deleted mutant embryos we found a dramatic reduction or even complete absence of labeled cells.…”

ATRA and KL promote differentiation toward the meiotic program of male germ cells.

“…Spermatogonia were obtained as we previously reported 21 by differential enzymatic digestion of testes from 4 to 7 days post natum (dpn) CD1 albino mice. To precisely define the temporal appearance of Kit expressing spermatogonia, a transgenic line (p18) expressing EGFP under the control of the c-Kit promoter and expanded on a CD1 background, 25,26 was used. Isolation of Kit positive spermatogonia was performed by using magnetic-activated cell sorting (MACS) with CD117 conjugated microbeads (Miltenyi Biotec, Germany).…”

A Distant Upstream Locus Control Region Is Critical for Expression of the Kit Receptor Gene in Mast Cells

“…DNase I-hypersensitive sites were also observed in the vicinity of the Kit transcription start site in the chromatin of Kit-expressing hematopoietic, melanocytic, and embryonic stem cells. In addition, a cluster of four hypersensitive sites has been detected in the middle of intron 1 in hematopoietic cells (12). In order to investigate the roles of the distant upstream sequences in Kit receptor expression in various cell systems, we have made transgenic mice carrying bacterial artificial chromosome (BAC) reporter constructs containing 200 kb upstream and 60-kb Kit coding sequences.…”