ZIP8 zinc transporter: indispensable role for both multiple-organ organogenesis and hematopoiesis in utero

Abstract

Previously this laboratory characterized Slc39a8-encoded ZIP8 as a Zn(2+)/(HCO(3)(-))(2) symporter; yet, the overall physiological importance of ZIP8 at the whole-organism level remains unclear. Herein we describe the phenotype of the hypomorphic Slc39a8(neo/neo) mouse which has retained the neomycin-resistance gene in intron 3, hence causing significantly decreased ZIP8 mRNA and protein levels in embryo, fetus, placenta, yolk sac, and several tissues of neonates. The Slc39a8(neo) allele is associated with diminished zinc and iron uptake in mouse fetal fibroblast and liver-derived cultures; consequently, Slc39a8(neo/neo) newborns exhibit diminished zinc and iron levels in several tissues. Slc39a8(neo/neo) homozygotes from gestational day(GD)-11.5 onward are pale, growth-stunted, and die between GD18.5 and 48 h postnatally. Defects include: severely hypoplastic spleen; hypoplasia of liver, kidney, lung, and lower limbs. Histologically, Slc39a8(neo/neo) neonates show decreased numbers of hematopoietic islands in yolk sac and liver. Low hemoglobin, hematocrit, red cell count, serum iron, and total iron-binding capacity confirmed severe anemia. Flow cytometry of fetal liver cells revealed the erythroid series strikingly affected in the hypomorph. Zinc-dependent 5-aminolevulinic acid dehydratase, required for heme synthesis, was not different between Slc39a8(+/+) and Slc39a8(neo/neo) offspring. To demonstrate further that the mouse phenotype is due to ZIP8 deficiency, we bred Slc39a8(+/neo) with BAC-transgenic BTZIP8-3 line (carrying three extra copies of the Slc39a8 allele); this cross generated viable Slc39a8(neo/neo)_BTZIP8-3(+/+) pups showing none of the above-mentioned congenital defects-proving Slc39a8(neo/neo) causes the described phenotype. Our study demonstrates that ZIP8-mediated zinc transport plays an unappreciated critical role during in utero and neonatal growth, organ morphogenesis, and hematopoiesis.

Figure 1. Characterization of Slc39a8(neo/neo) phenotype.

(A) Body weight of embryos and fetuses of the three genotypes–from GD13.5 through GD18.5. N = 15–20 per genotype. * P<0.001, compared with values in Slc39a8(+/+) and Slc39a8(+/neo). In this and subsequent figures, bars and brackets denote means ± S.E.M. Brackets denoting S.E.M. are present in Fig. 1A but are mostly covered up by the size of the symbols. (B) Photographs of (genotyped) Slc39a8(+/+) and Slc39a8(neo/neo) pups at GD11.5 (upper left panel), GD13.5 (upper right), GD16.5 (middle both left & right), and at PND1 (bottom panel). The GD16.5 fetuses (middle left) represent a single litter of 13 pups; the two asterisks in middle left panel denote Slc39a8(neo/neo) homozygotes; the other 11 were Slc39a8(+/+) wild-type and Slc39a8(+/neo) heterozygotes.

Figure 2. Relative ZIP8 mRNA levels in Slc39a8(

+ / + ) , Slc39a8( + /neo) and Slc39a8(neo/neo) genotypes. (A) At three gestational ages, mRNA levels were determined in whole embryo/fetus, yolk sac, and placenta. (B) The mRNA was examined in PND1 liver, lung, kidney (Kidn), cerebrum (Cereb), cerebellum (Cblm), thymus (Thym), heart, and carcass (Carc). (“Carcass” is what remains after removal of liver, lungs, kidneys, heart, head and GI system.) Samples between five and 12 individual mice were examined. Mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed as the normalization control. The Slc39a8(+/+) wild-type ZIP8 mRNA/GAPDH mRNA ratio (left-most in each panel) and mRNA expression levels were expressed as linear fold-changes–relative to the normalized wild-type control. To lessen clutter in this figure or figure legend, all statistics are detailed in Supplementary Data S1 online.

Figure 3. Western immunoblot analysis of ZIP8 protein (50.082 kDa) in the same tissues described in Fig. 2

. (A) Whole embryo/fetus and yolk sac; (B) Four tissues at PND1. Antibody to β-actin protein (ACTB; 41.7 kDa) was used as the lane-loading control. To maximize contrast, exposure times ranged from 1 min to 6 h. (C) Densitometric semi-quantification of Western immunoblots: whole embryo/fetus, yolk sac and four tissues at PND1. The Slc39a8(+/+) wild-type ZIP8 protein/ACTB protein ratio (left-most in each panel) and protein expression levels were expressed as linear fold-changes–relative to the normalized wild-type control.

Figure 4. Relative ZIP8 mRNA levels in untreated Slc39a8(

+ / + ) , Slc39a8( + /neo) and Slc39a8(neo/neo) MFF and fetal liver cultures. As in Fig. 2, the Slc39a8(+/+) wild-type ZIP8 mRNA/GAPDH mRNA ratio (left-most in each panel) and mRNA expression levels were expressed as linear fold-changes–relative to the normalized wild-type control. The MFFs were prepared from GD14.5 fetuses whose livers had been removed. Livers were trypsinized and cultured until they approached ∼80% confluency (<2 weeks). *P<0.0001, compared with (+/+) or (+/neo) genotypes. These data represent duplicate results from three independent experiments.

Figure 5. Hematopoiesis parameters.

(A) Histology of GD16.5 liver from Slc39a8(+/+) vs Slc39a8(neo/neo) pups at GD16.5; toluidine blue O stain was used. Red arrows denote hematopoietic islands; blue arrows glycogen. Magnification = 100X. Bar = 100 microns. (B) Histology of GD13.5 yolk sac. Bar = 50 microns. (C) Histogram of red cell count (RBC; 10⁶ per µL), hemoglobin (g/dL), and hematocrit (percent) in PND1 mice. N = 8 for Slc39a8(+/+), N = 13 for (+/neo), and N = 7 for (neo/neo). Student’s t-test was used: * P = 0.016. ^† P = 0.008. At far right, volume density (V_d) of hematopoietic (Hem) islands/total cell number was measured in GD16.5 liver. N = 3 for Slc39a8(+/+), N = 7 for (+/neo), and N = 3 for (neo/neo). These V_d differences did not reach statistical significance (P<0.05) between Slc39a8(+/+) vs Slc39a8(neo/neo) pups.

Figure 6. Flow cytometry.

Expression of erythroid, myeloid and lymphoid markers in GD16.5 liver. These data represent typical findings from an individual animal of each genotype. (A) Classification and comparison of erythroid developmental stages using CD71 (transferrin receptor) and TER119 (erythroid series). Note Regions 2, 3, 4 & 5 are delineated. (B) Flow-cytometry analysis of the expression of CD3e (lymphoid T-cells) and CD11b (myeloid series).

Figure 7. Divalent cation uptake in MFF and fetal liver cells of the three genotypes in culture.

Uptake was linear for 20 min, at which time these values were recorded. These cell cultures were the same as those described in Fig. 4. At 8.0 µM zinc in MFFs, (+/+) vs (neo/neo) P = 0.01 and (+/+) vs (+/neo) P = 0.044. At 2.0 µM cadmium in MFFs, (+/+) vs (neo/neo) P = 0.002 and (+/+) vs (+/neo) P = 0.012. At 16.0 µM iron in MFFs, (+/+) vs (neo/neo) P = 0.003 and (+/+) vs (+/neo) P = 0.015. At 16.0 µM iron in fetal liver cells, (+/+) vs (neo/neo) P = 0.031.

Figure 8. Divalent cation content (determined by ICP-MS) in four tissues at PND1.

(A) zinc; (B) iron. Comparing two, we used Student’s t-test. For more than two, we used all-pairwise multiple-comparison procedures (Holm-Sidak method). To lessen clutter in the figure, all statistics are detailed in Supplementary Data S1 online.

Figure 9. Comparison of ALAD specific activity from PND1 liver among the three genotypes for the non-activated vs zinc-activated assays.

Values shown at top of each set of bars correspond to the Relative Index (ratio reflecting proportion in which the activity might increase upon zinc addition). Brackets denoting S.E.M. cannot be added because only two pools per genotype were assayed.