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PubMed2002

Stromal cells from murine embryonic aorta-gonad-mesonephros region, liver and gut mesentery expand human umbilical cord blood-derived CAFC(week6) in extended long-term cultures.

The first definitive long-term repopulating hematopoietic stem cells (HSCs) emerge from and undergo rapid expansion in the embryonic aorta-gonad-mesonephros (AGM) region. To investigate the presumptive unique characteristics of the embryonic hematopoietic microenvironment and its surrounding tissues, we have generated stromal clones from subdissected day 10 and day 11 AGMs, embryonic livers (ELs) and gut mesentery. We here examine the ability of 19 of these clones to sustain extended long-term cultures (LTCs) of human CD34(+) umbilical cord blood (UCB) cells in vitro. The presence of in vitro repopulating cells was assessed by sustained production of progenitor cells (extended LTC-CFC) and cobblestone area-forming cells (CAFC). The embryonic stromal clones differed greatly in their support for human HSCs. Out of eight clones tested in the absence of exogenous cytokines, only one (EL-derived) clone was able to provide maintenance of HSCs. Addition of either Tpo or Flt3-L + Tpo improved the long-term support of about 50% of the tested clones. Cultures on four out of 19 clones, ie the EL-derived clone mentioned, two urogenital-ridge (UG)-derived clones and one gastrointestinal (GI)-derived clone, allowed a continuous expansion of primitive CAFC and CFU-GM with over several hundred-fold more CAFC(week6) produced in the 12th week of culture. This expansion was considerably higher than that found with the FBMD-1 cell line, which is appreciated by many investigators for its support of human HSCs, under comparable conditions. This stromal cell panel derived from the embryonic regions may be a powerful tool in dissecting the factors mediating stromal support for maintenance and expansion of HSCs.

Animals↗
PubMed2002

Histochemical demonstration of the heterogeneity of the epithelium of proximal tubules in the chick mesonephros.

Proximal tubules (PT) in 7-10-day old chick mesonephros were examined histochemically to evaluate their structural and functional properties related to the absorption capacity of the epithelium and its possible alterations leading to cystically dilated tubules (CDT). Alkaline phosphatase activity at the apical cell membrane was demonstrated in varying intensities in large PT. A similar heterogeneity was also detected in the expression of proteoglycans (Lewis(x) antigen) localized in the apical part of the epithelial cell membrane but not in the basolateral membrane parts (beta-catenin, Na(+),K(+)-ATPase). In analogy, the ability to accumulate trypan blue was found in the same cell population. We hypothesize that epithelial cells in proximal tubules of nephrons with a defective apical cell membrane cause reduced fluid absorption and subsequent overfilling and dilation of the tubules.

Alkaline Phosphatase↗
PubMed2004

Spred-2 suppresses aorta-gonad-mesonephros hematopoiesis by inhibiting MAP kinase activation.

In midgestation mouse embryos, the aorta-gonad-mesonephros (AGM) region generates hematopoietic stem cells and definitive hematopoiesis is regulated by cell-cell interaction and signaling molecules. We showed that a Ras/mitogen-activated protein (MAP) kinase signaling-specific inhibitor and a dominant negative mutant Ras blocked the production of CD45+ hematopoietic cells in embryonic day 11.5 AGM culture, indicating an essential role for the MAP kinase pathway in AGM hematopoiesis. Overexpression of the Ras/MAP kinase pathway regulator, Spred-2, in the AGM culture significantly reduced the number of CD45+ cells. In contrast, production of CD45+ cells from the AGM region of Spred-2-null mice was up-regulated as compared with wild-type littermates. Furthermore, Spred-2-deficient mice exhibited elevated hematopoietic colony formation from vascular endothelial-cadherin+ cells. These data indicate that Spred-2 functions as a negative regulator of AGM hematopoiesis by inhibiting hematopoietic cytokine signaling.

Animals↗
PubMed1993

Ontogeny of the epidermal growth factor receptor during development of the fetal bovine mesonephros and associated organs of the urogenital tract.

A primary cell culture system was developed to study the epidermal growth factor (EGF) receptor in the fetal bovine mesonephros and its urogenital derivatives. Radioreceptor assays demonstrated EGF binding as early as Day 37 in mesonephric cells and in cells derived from the fetal reproductive ducts, gonads, and metanephros--all mesonephric derivatives. Immunocytochemical studies revealed that EGF receptors were localized in the ductal and tubular epithelium of these urogenital organs. EGF induced DNA synthesis and tyrosine phosphorylation in the bovine mesonephric cells, suggesting that EGF receptors detected in these cells were functional. In addition, transforming growth factor alpha, the putative fetal ligand for the EGF receptor, was found to specifically compete for EGF binding to the receptor, as well as to induce DNA synthesis in a manner similar to that of EGF. Estrogen did not regulate EGF receptors or specifically bind to bovine mesonephric cells. The development of estrogen receptors in the bovine species occurs markedly later than that of the EGF receptor, in contrast to the mouse, where both receptors are observed at very early stages of reproductive tract development.

Animals↗
PubMedDate not supplied

The embryonic aorta-gonad-mesonephros region as a generator of haematopoietic stem cells.

During mammalian embryonic development the definitive haematopoietic stem cells (HSCs) may arise either in the extra-embryonic mesoderm or in the aorta-gonad-mesonephros (AGM) region that forms in close proximity to the assembling urogenital system, generating the gonad, cortex of the adrenal gland and metanephros. Researchers have been attempting for a long time to define the region of importance for generating the definitive HSCs that colonize the fetal liver and bone marrow, the two major sites where haematopoiesis takes place in the adult. The fetal liver might gain HSCs from both of the primary haematopoietic sources, but the extra-embryonic HSCs seem not to be able to colonize adult bone marrow directly. It is known that the microenvironment around the HSCs is important for directing cell fates, but we do not yet have much idea about the cell-cell interactions, tissue interactions and molecules that regulate cell behaviour in the AGM. We will here discuss the contribution of the AGM to definitive haematopoiesis in mammals and review some of the cell-cell interactions and associated signalling systems involved in the development of AGM stem cells.

Animals↗
PubMed2001

Role of the microenvironment of the embryonic aorta-gonad-mesonephros region in hematopoiesis.

Although various cytokines, growth factors, and chemokines are known to regulate hematopoiesis, expansion of hematopoietic stem cells (HSCs) in vitro with the use of such agents has proved problematic. Stromal cells are major components of the microenvironment that surrounds hematopoietic cells and are thought to play an important role in hematopoiesis in vivo. Co-culture of HSCs with stromal cells promotes hematopoiesis and self-renewal of HSCs. Definitive hematopoietic cells first appear during mammalian embryonic development in the aorta-gonad-mesonephros (AGM) region, and it is therefore thought that the microenvironment of this region plays an important role in HSC ontogeny. We have adopted two approaches to studying the contribution of the AGM microenvironment to hematopoiesis. In the first approach, we have developed an in vitro culture system for mouse AGM explants. Hematopoiesis is enhanced in such cultures by the presence of the combination of stem cell factor (SCF), basic fibroblast growth factor, leukemia inhibitory factor, and oncostatin M (SFLO culture). However, transplantation assays revealed that HSCs capable of long-term reconstitution of the hematopoietic compartment of irradiated mice (LTR-HSCs) do not expand in AGM-SFLO cultures; rather, these cultures appear to provide a favorable microenvironment for hematogenic angioblasts that are precursors of both endothelial and hematopoietic cells. In our second approach, we have established various stromal cell lines from the mouse AGM region. The AGM-S3 cell line supports human and mouse primitive hematopoietic cells as well as mouse LTR-HSCs. Maintenance of LTR-HSCs is mediated by a mechanism other than SCF signaling through its receptor (c-Kit). These two in vitro approaches should prove useful for further elucidation of the mechanisms that underlie hematopoiesis and HSC self-renewal.

Animals↗
PubMed2003

Regulation of hematopoietic development in the aorta-gonad-mesonephros region mediated by Lnk adaptor protein.

Development of hematopoietic cells in the aorta-gonad-mesonephros (AGM) region in the midgestation mouse embryo involves a multistep process, sequentially changing from endothelial cell-like cells, including hemangioblasts, into hematopoietic stem cells, progenitors, and/or lineage-committed cells. An adaptor molecule, Lnk, is known to negatively control the production of pro- and pre-B cells and hematopoietic progenitor cells in adult bone marrow. Here we show a role of Lnk in hematopoietic development in the AGM region. Lnk was predominantly expressed in the endothelial cells lining the dorsal aorta at embryonic day 11.5 (E11.5). Overexpression of Lnk in the primary culture of the AGM region at E11.5 suppressed the emergence of CD45+ hematopoietic cells. Point mutation in the SH2 domain of Lnk, which abolishes the binding capability of Lnk to c-Kit upon stimulation with stem cell factor (SCF), led to loss of Lnk-dependent inhibition of hematopoietic cell development in AGM cultures, suggesting Lnk-mediated inhibition of the SCF/c-Kit signaling pathway. In cultured AGM cells from Lnk homozygous mutant mouse embryos, the number of emerged CD45+ cells was 2.5-fold larger than that from heterozygous littermates. Furthermore, aorta cells of E11.5 Lnk homozygous mutant mice also showed enhanced hematopoietic colony-forming activity. Thus, Lnk is a negative regulator of hematopoiesis in the AGM region.

Adaptor Proteins, Signal Transducing↗
PubMed1986

Scanning electron microscopical observations on the differentiating mesonephros of the chick embryo.

Chick embryos were staged according to the method of Hamburger and Hamilton [1951] and fixed. Cross sections through the cephalic fourth of the mesonephric ridges were examined by scanning electron microscopy. The steps in glomerular differentiation could be observed with ease. The first foot processes to appear in podocytes arose directly from the basal surface of the cell body. In a second step, lateral branches appeared and gave off secondary or even tertiary branches that interdigitated with those from neighbouring podocytes, following a pattern that was very similar to the one previously described by other authors in metanephric nephrons. Endothelial pores appeared in the glomerular capillaries at very early stages of the glomerular differentiation. The differentiation of the epithelium of proximal tubules was characterized by the growth of apical microvilli and of finger-like evaginations from the lateral membranes. At stages 20 and 21, the most differentiated glomeruli had only basal foot processes; only after stage 25 did the first generation nephrons reach full maturity. Because during this period the mesonephros is known to produce urine, our results indicate that nephrons start to function before they have completed their differentiation.

Animals↗
PubMed2002

Identification of 2 novel genes developmentally regulated in the mouse aorta-gonad-mesonephros region.

The first adult-repopulating hematopoietic stem cells (HSCs) emerge in the mouse aorta-gonad-mesonephros (AGM) region at embryonic day 10.5 prior to their appearance in the yolk sac and fetal liver. Although several genes are implicated in the regulation of HSCs, there are gaps in our understanding of the processes taking place in the AGM at the time of HSC emergence. To identify genes involved in AGM HSC emergence, we performed differential display reverse transcriptase-polymerase chain reaction (DD RT-PCR). Differentially expressed genes included beta-catenin and homologs of human TM9SF2 and TAB2. We characterized the expression pattern of Wnt/beta-catenin signaling, mTM9SF2, and mTAB2 in the embryo and adult. Interestingly, the expression of mouse TAB2 (mTAB2) in the E11 dorsal aorta endothelium suggests a role for mTAB2 in HSC emergence and/or regulation. The identification of differentially expressed genes in the AGM region should yield further insights into the development of this tissue and into the emergence and regulation of HSCs.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Generation of definitive hematopoietic stem cells from murine early yolk sac and paraaortic splanchnopleures by aorta-gonad-mesonephros region-derived stromal cells.

There is controversy as to whether murine definitive hematopoiesis originates from yolk sac (YS) or the intraembryonic region. This study reports the generation of definitive hematopoietic stem cells (HSCs) from both early YS and intraembryonic paraaortic splanchnopleures (P-Sp) on AGM-S3 stromal cells derived from the aorta-gonad-mesonephros (AGM) region at 10.5 days post coitum (dpc). YS and P-Sp cells at 8.5 dpc generated no definitive hematopoiesis-derived colony-forming cells in cocultures with AGM-S3 cells, but spleen colony-forming cells and HSCs capable of reconstituting definitive hematopoiesis in adult mice simultaneously appeared on day 4 of coculture. Precursors for definitive HSCs were present in YS and P-Sp at 8.0 dpc, a time when YS and embryo were not connected by blood vessels. It is proposed that precursors with the potential to generate definitive HSCs appear independently in YS and intraembryonic P-Sp and that the P-Sp or AGM region affords the microenvironment that facilitates generation of definitive hematopoiesis from precursors.

Animals↗
PubMed2002

Stromal cell lines from mouse aorta-gonads-mesonephros subregions are potent supporters of hematopoietic stem cell activity.

The aorta-gonads-mesonephros (AGM) region autonomously generates the first adult repopulating hematopoietic stem cells (HSCs) in the mouse embryo. HSC activity is initially localized to the dorsal aorta and mesenchyme (AM) and vitelline and umbilical arteries. Thereafter, HSC activity is found in the urogenital ridges (UGs), yolk sac, and liver. As increasing numbers of HSCs are generated, it is thought that these sites provide supportive microenvironments in which HSCs are harbored until the bone marrow microenvironment is established. However, little is known about the supportive cells within these midgestational sites, and particularly which microenvironment is most supportive for HSC growth and maintenance. Thus, to better understand the cells and molecules involved in hematopoietic support in the midgestation embryo, more than 100 stromal cell lines and clones were established from these sites. Numerous stromal clones were found to maintain hematopoietic progenitors and HSCs to a similar degree as, or better than, previously described murine stromal lines. Both the AM and UG subregions of the AGM produced many supportive clones, with the most highly HSC-supportive clone being derived from the UGs. Interestingly, the liver at this stage yielded only few supportive stromal clones. These results strongly suggest that during midgestation, not only the AM but also the UG subregion provides a potent microenvironment for growth and maintenance of the first HSCs.

Animals↗
PubMed2002

Cultivation of aorta-gonad-mesonephros-derived hematopoietic stem cells in the fetal liver microenvironment amplifies long-term repopulating activity and enhances engraftment to the bone marrow.

During mammalian development, definitive hematopoietic stem cells (HSCs) arise in the aorta-gonad-mesonephros (AGM) region and colonize the fetal liver (FL) before hematopoiesis occurs in the bone marrow. The FL is a unique hematopoietic organ where both HSCs and mature blood cells are actively generated along with functional maturation of hepatic cells as a metabolic organ. To characterize HSCs and FL microenvironments during development, this study establishes a coculture system composed of AGM-originated HSCs and FL nonhematopoietic cells. The results demonstrate that FL cells support significant expansion of lineage-committed hematopoietic cells as well as immature progenitors. More important, long-term repopulating activity was amplified from AGM-originated HSCs in this coculture system. Engraftment of HSCs to the bone marrow was strongly enhanced by coculture. In addition, AGM HSCs produced significantly more hematopoietic cells than E14.5 and E18.5 FL HSCs in vitro. These results suggest that the FL microenvironment not only stimulates expansion of the hematopoietic system, but also possibly modifies the characteristics of AGM HSCs. Thus, this coculture system recapitulates the developmental process of HSCs and the FL microenvironment and provides a novel means to study the development of hematopoiesis.

Animals↗
PubMed2005

Genetic and functional characterization of isolated stromal cell lines from the aorta-gonado-mesonephros region.

The hematopoietic system interacts with a supportive stromal environment allowing maintenance and differentiation of hematopoietic stem cells (HSCs). The aorta-gonado-mesonephros (AGM) region serves as a unique embryonic microenvironment, generating the first adult repopulating HSCs in the mouse embryo. To eludicate factors involved in hematopoietic support and induction of hematopoietic differentiation, we isolated more than 100 stromal cell clones derived from the AGM region of embryonic day (E) 10.5 mouse embryos for functional and genetic analysis. Selected isolated AGM stromal cell lines are highly efficient in supporting maintenance and expansion of mouse and human hematopoietic stem and progenitor cells. In addition, we can demonstrate for the first time that AGM stromal cell lines are also potent inducers of hematopoietic differentiation of murine embryonic stem cells. Stromal gene array analysis has identified genes that could play a role in hematopoietic support.

Animals↗
PubMed2006

Down-regulation of endogenous Wt1 expression by Sry transgene in the murine embryonic mesonephros-derived M15 cell line.

Wt1 is one of numerous candidate genes comprising the hypothetical chain of gene expression essential for male sex differentiation of the bipotential indifferent gonads during embryogenesis. However, the evidence in the literature is ambivalent regarding the position of Wt1 relative to Sry in this scheme; Wt1 might act either upstream or downstream of Sry. In the present study, the effects of Sry expression upon Wt1 were investigated using M15 cells (XX karyotype), which are derived from murine embryonic mesonephros and express endogenous Wt1. In 3 stably-transformed Sry-expressing M15 cell lines, we showed that the expression levels of the mRNAs coding for all 4 isoforms of the WT1 proteins were down-regulated. Similarly, Wnt 4 expression was down-regulated in these cell lines. Silencing of Sry in the transformed cell lines using ribozymes or short hairpin RNAs (shRNAs) resulted in elevated levels of Wt1 and Wnt4 expression. These results strongly indicate that Wt1 might be under the control of Sry during gonadal differentiation in the mouse. In electrophoretic mobility shift assays (EMSA), we demonstrated that the 3.7 kb 5'-upstream DNA stretch of Wt1 containing potential Sry binding sites was capable of forming molecular complexes with nuclear protein(s) from Sry expressing cells but not with those from control non-Sry expressing cells. In summary, our present results support the notion that Wt1 is located downstream of Sry and down-regulated by the sex determining gene. Although the precise biological meaning of the present findings have yet to be clarified, it is possible that Wt1 plays a dual role during gonadal differentiation, i. e., turning on Sry expression on one hand, and being down-regulated by its product, Sry, on the other, possibly forming a type of negative feed-back mechanism. Further work is needed to substantiate this view.

Animals↗
PubMed1990

Pronephros and mesonephros--Cohnheim revisited.

Erroneous quotations in the literature and Cohnheim's statement, in his Lectures on General Pathology, that the Wolffian body or mesonephros is the first anlage of the urogenital system prompted this description of the growth of our knowledge of the early development of the kidney. Some of the pertinent older literature is reviewed, and the concept of the holonephros, as opposed to the trinephric view of kidney development, is discussed. Emphasis is placed on the decreasing functional significance of the pronephros with evolutionary development, to the extent that the role of the pronephros in the human is questioned. Cohnheim's seemingly erroneous reference to the development of the kidney is shown to be merely a reflection of the views current at his time.

History, 19th Century↗
PubMed2001

Immature multipotent hemopoietic progenitors lacking long-term bone marrow-reconstituting activity in the aorta-gonad-mesonephros region of murine day 10 fetuses.

Previous studies indicated that multipotent progenitors exist in early fetuses that do not contain long-term reconstituting (LTR) activity. However, it remained unclear whether these multipotent progenitors are committed to the hemopoietic lineage or are immature mesodermal cells or hemangioblasts. In this study, we have succeeded in enriching the multipotent progenitors that are capable of generating myeloid, T, and B cells in the LFA-1(-) subpopulation of TER-119(-)c-kit(+)CD45(+) cells from the aorta-gonad-mesonephros (AGM) region of day 10 fetuses. We found that these day 10 AGM LFA-1(-) cells do not show the LTR activity, whereas day 11 AGM LFA-1(-) cells do have such an activity. These results strongly suggest that multipotent progenitors lacking LTR activity emerge as CD45(+) hemopoietic progenitor cells in the AGM region on the 10th day of gestation, and such p-Multi mature into hemopoietic stem cells by acquiring LTR activity.

Animals↗
PubMed1999

In vitro hematopoietic and endothelial cell development from cells expressing TEK receptor in murine aorta-gonad-mesonephros region.

Recent studies have shown that long-term repopulating hematopoietic stem cells (HSCs) first appear in the aorta-gonad-mesonephros (AGM) region. Our immunohistochemistry study showed that TEK+ cells existed in the AGM region. Approximately 5% of AGM cells were TEK+, and most of these were CD34(+) and c-Kit+. We then established a coculture system of AGM cells using a stromal cell line, OP9, which is deficient in macrophage colony-stimulating factor (M-CSF). With this system, we showed that AGM cells at 10.5 days postcoitum (dpc) differentiated and proliferated into both hematopoietic and endothelial cells. Proliferating hematopoietic cells contained a significant number of colony-forming cells in culture (CFU-C) and in spleen (CFU-S). Among primary AGM cells at 10.5 dpc, sorted TEK+ AGM cells generated hematopoietic cells and platelet endothelial cell adhesion molecule (PECAM)-1(+) endothelial cells on the OP9 stromal layer, while TEK- cells did not. When a ligand for TEK, angiopoietin-1, was added to the single-cell culture of AGM, endothelial cell growth was detected in the wells where hematopoietic colonies grew. Although the incidence was still low (1/135), we showed that single TEK+ cells generated hematopoietic cells and endothelial cells simultaneously, using a single-cell deposition system. This in vitro coculture system shows that the TEK+ fraction of primary AGM cells is a candidate for hemangioblasts, which can differentiate into both hematopoietic cells and endothelial cells.

Animals↗
PubMed1999

The mouse GATA-2 gene is expressed in the para-aortic splanchnopleura and aorta-gonads and mesonephros region.

We previously reported that the mouse GATA-2 gene is regulated by two alternative promoters (Minegishi et al, J Biol Chem, 273:3625, 1998). Although the more proximal IG (general) promoter is active in almost all GATA-2-expressing cells, the distal IS (specific) promoter activity was selectively detected in hematopoietic tissues but not in other mesodermal tissues. We report here in vivo analysis of the GATA-2 locus and its regulatory characteristics in hematopoietic tissues of transgenic mice. Transgenes containing 6 or 7 kbp of sequence flanking the 5' end of the IS first exon direct expression of beta-galactosidase or green fluorescent protein (GFP) reporter genes specifically to the para-aortic splanchnopleura, aorta-gonads, and mesonephros (AGM) region, and in the neural tissues. In situ hybridization analysis showed that reporter gene expression specifically recapitulates the endogenous expression profile of GATA-2 in these tissues. The flk-1, CD34, c-kit, and CD45 antigens were identified in the GFP-positive cells from the AGM region and fetal liver, indicating that GATA-2 is expressed in immature hematopoietic cells. Deletion of 3.5 kbp from the 5' end of the 6.0 kbp IS promoter construct, including one of the DNase I hypersensitive sites, completely abolished hematopoietic expression. These experiments describe an early developmental GATA-2 hematopoietic enhancer located between 6.0 and 2.5 kbp 5' to the IS exon.

Animals↗