RAG-1 and Ly6D independently reflect progression in the B lymphoid lineage.

Common lymphoid progenitors (CLPs) are thought to represent major intermediates in the transition of hematopoietic stem cells (HSCs) to B lineage lymphocytes. However, it has been obvious for some time that CLPs are heterogeneous, and there has been controversy concerning their differentiation potential. We have now resolved four Flt3(+) CLP subsets that are relatively homogenous and capable of forming B cells. Differentiation potential and gene expression patterns suggest Flt3(+) CLPs lacking both Ly6D and RAG-1 are the least differentiated. In addition to B cells, they generate natural killer (NK) and dendritic cells (DCs). At the other extreme is a subset of the recently described Flt3(+) Ly6D(+) CLPs that have a history of RAG-1 expression and are B lineage restricted. These relatively abundant and potent CLPs were depleted within 48 hours of acute in vivo estrogen elevation, suggesting they descend from hormone regulated progenitors. This contrasts with the hormone insensitivity of other CLP subsets that include NK lineage progenitors. This progenitor heterogeneity and differentiation complexity may add flexibility in response to environmental changes. Expression of RAG-1 and display of Ly6D are both milestone events, but they are neither synchronized nor dependent on each other.

Hoxa9 and Flt3 signaling synergistically regulate an early checkpoint in lymphopoiesis.

Hoxa9 and Flt3 signaling are individually important for the generation of lymphoid lineage precursors from multipotent hematopoietic progenitors (MPP) in bone marrow. Mice deficient for Hoxa9, Flt3, or Flt3 ligand (FL) have reduced numbers of lymphoid-primed multipotential progenitors (LMPP), common lymphoid progenitors (CLP), and B/T cell precursors. Hoxa9 regulates lymphoid development, in part, through transcriptional regulation of Flt3. However, it was unclear whether Hoxa9 has functions in lymphopoiesis independent of, or alternatively, synergistically with Flt3 signaling. In this study, we show that Hoxa9(-/-)Flt3l(-/-) mice have more severe deficiencies in all B lineage cells, CLP, LMPP, and total Flt3(+) MPP in bone marrow than the single knockouts. Although LMPP and Flt3(+) CLP contain precursors for NK and dendritic cell lineage cells, no deficiencies in these lineages beyond that in Flt3l(-/-) mice was found. Thymocyte cellularity was significantly reduced in the compound knockout, although peripheral T cell numbers mirrored Flt3l(-/-) mice. Analysis of the hematopoietic progenitor compartment revealed elevated numbers of CD150(+hi)CD34(-)CD41(+) myeloid-biased stem cells in Hoxa9(-/-)Flt3l(-/-) mice. In contrast, CD150(-) MPP enriched for lymphoid potential were synergistically reduced, suggesting Hoxa9 and Flt3 signaling function coordinately to regulate lymphopoiesis at a very early stage. Real-time PCR analysis of CD150(-)Flt3(+) cells from wild-type control, Hoxa9(-/-), and Flt3l(-/-) single knockouts revealed decreased lymphoid transcripts, corroborating the importance of these regulators in lymphoid development. Taken together, these studies reveal a very early checkpoint in lymphopoiesis dependent on the combinatorial activities of Hoxa9 function and Flt3 signaling.

Magnetic resonance imaging of contrast-enhanced polyelectrolyte complexes.

Polyelectrolyte complexes (PECs) assembled from oppositely charged polymers were endowed with gadolinium (Gd) and evaluated to determine cytotoxicity and magnetic resonance image (MRI) contrast enhancement in vivo. Chitosan grafted with Gd diethylenetriaminepentaacetic acid (DTPA) was electrostatically complexed with dextran sulfate, producing particles of about 300 nm possessing a negative surface charge. Alternatively, Gd was ionically trapped within PECs by mixing PECs (with or without Gd-DTPA graft) with gadolinium chloride (GdCl(3)). Combinations of these two approaches for including Gd resulted in three particle types: Gd-loaded PECs, Gd-DTPA-conjugated PECs, and PECs containing both ionically trapped Gd and Gd-DTPA grafts. Polyelectrolytes, Gd-DTPA, and PECs were all found to have relatively low cytotoxicity (IC(50) > 1 mg/mL) in human umbilical cord vascular endothelial cells. In vivo, MRI revealed that the contrast-enhanced PECs were found to accumulate rapidly in the rat kidney. Some accumulation was also noted in the rat liver; however, negligible enhancement occurred in other tissues. Contrast enhancement was especially intense in or near the renal pelvis. MRI detection of PECs provides a potential approach to rapidly evaluate parameters such as the biodistribution and pharmacokinetics of these established drug and gene delivery vehicles.

Expression and regulation of SLC39A family zinc transporters in the developing mouse intestine.

Several ZIP genes (SLC39A family of metal transporters) play roles in zinc homeostasis. Herein, the temporal and spatial patterns of expression of the mouse ZIP1, 3, 4, and 5 genes in the developing intestine and the effects of maternal dietary zinc deficiency on these patterns of expression were examined. ZIP1 and ZIP3 genes, conserved members of the ZIP subfamily II, were found to be coexpressed during development. Expression of these genes was detected on day 14 of gestation in smooth muscle and the pseudostratified endoderm. By 5 days post-partum, prominent expression became restricted to muscle and connective stroma. In contrast, expression of ZIP4 and ZIP5 genes, members of the ZIP subfamily called LIV-1, coincided with epithelial morphogenesis. ZIP5 expression was detected on d16 of gestation and localized to the basolateral membranes of the single-layered epithelium. ZIP4 expression was detected on d18 of gestation and localized to the apical membrane of villus epithelial cells. When dams were fed a zinc-deficient diet beginning at parturition, ZIP4 expression in the nursing neonate was greatly induced. In contrast, neonatal ZIP5 expression remained unchanged, but this protein was removed from the basolateral membrane of the enterocyte. These responses to dietary zinc deficiency mimic those found in the adult intestine. These studies reveal cell-type-specific expression of ZIP genes during development of the intestine, and suggest that the mouse intestine can elicit an adaptive response to dietary zinc availability at birth.

Mouse ZIP1 and ZIP3 genes together are essential for adaptation to dietary zinc deficiency during pregnancy.

Subfamily II of the solute-linked carrier 39A superfamily contains three well-conserved zinc transporters (ZIPs1, 2, 3) whose physiological functions are unknown. We generated mice homozygous for knockout alleles of ZIP1 and both ZIP1 and ZIP 3 (double-knockout). These mice were apparently normal when dietary zinc was replete, but when dietary zinc was limited during pregnancy embryos from ZIP1 or ZIP3 knockout mice were two to three times more likely to develop abnormally than those in wildtype mice, and 91% (71/78) of embryos developed abnormally in ZIP1, ZIP3 double-knockout mice. Analysis of the patterns of expression of these genes in mice revealed predominate expression in intestinal stromal cells, nephric-tubular epithelial cells, pancreatic ductal epithelial cells, and hepatocytes surrounding the central vein. This suggests that these zinc transporters function, at least in part, in the redistribution and/or retention of zinc rather than its acquisition from the diet. In conclusion, mutations in the ZIP1 and ZIP3 zinc transporter genes are silent when dietary intake of zinc is normal, but can dramatically compromise the success of pregnancy when dietary intake of zinc is limiting.

Generation and characterization of mice lacking the zinc uptake transporter ZIP3.

The mouse ZIP3 (SLC39A3) gene encodes an eight-transmembrane-domain protein that has been conserved in mammals and can function to transport zinc. To analyze the expression of ZIP3 in the early embryo and neonate and to determine its in vivo function, we generated ZIP3 null mice in which the ZIP3 open reading frame was replaced with that of the enhanced green fluorescent protein (EGFP) reporter. EGFP fluorescence revealed that ZIP3 was expressed in the inner cell mass of the blastocyst and later during embryonic development in many tissues. Elevated expression was apparent in the embryonic brain and neurotube and neonatal gonads. Homozygous knockout mice were viable and fertile and under normal growth conditions exhibited no obvious phenotypic abnormalities. Deletion of ZIP3 did not alter zinc homeostasis at the molecular level as assessed by essential metal levels and the expression of zinc-responsive genes. In knockout mice stressed with a zinc-deficient diet during pregnancy or at weaning, a subtle increase in the sensitivity to abnormal morphogenesis of the embryo and to depletion of thymic pre-T cells, respectively, was noted. These results suggest that this protein plays an ancillary role in zinc homeostasis in mice.

Chronic consumption of a moderately low protein diet does not alter hematopoietic processes in young adult mice.

The current studies examined whether hematopoiesis in the bone marrow and T-cell development in the thymus were attenuated in young adult A/J mice fed a moderately low protein diet (MPD, 50 g protein/kg) for 15 wk compared with mice fed a control protein diet (CPD, 180 g protein/kg). Flow cytometric analyses using antibodies against CD31 and Ly-6C as well as CD4 and CD8 were performed to identify stem, mixed progenitor, erythroid, lymphoid, granuloid and monocytic compartments in the bone marrow and four thymocyte subsets, respectively. Chronic restriction of young adult mice to MPD neither decreased the cellularity nor altered the distribution of subpopulations in either primary tissue. Subsequently, a new set of mice were provided with CPD and a low protein diet (LPD, 25 g protein/kg). After 5 wk, body and thymus weights in LPD group were reduced 26 and 30%, respectively, which was accompanied by a 505% increase in serum corticosterone. Surprisingly, LPD did not alter the number or distribution of cells in the bone marrow and the percentages of thymocyte subsets, supporting the findings from the MPD group. We conclude that chronic consumption of a marginal protein diet by young adult mice does not disrupt hematopoietic processes.