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First published online 26 January 2005
doi: 10.1242/dev.01648

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Thyroid hormone T3 acting through the thyroid hormone receptor is necessary for implementation of erythropoiesis in the neonatal spleen environment in the mouse

¹ Laboratory of Molecular Cell Biology, Ecole Normale Supérieure de Lyon, UMR CNRS 5161, INRA 1237, IFR128 Biosciences Lyon-Gerland, 46 Allée d'Italie, 69364 Lyon Cedex 07, France
² Department of Gynecologic Endocrinology and Reproductive Medicine, Medical University of Vienna, Vienna, Austria
³ Research Institute of Molecular Pathology (IMP), Dr Bohr Gasse 7, 1030 Vienna, Austria
⁴ Université Claude Bernard Lyon I, 43 bd du 11 Novembre 1918, 69622 Villeurbanne cedex, France

View larger version (29K): [in a new window]   Fig. 1. Spleen cellularity and composition of spleens in control and mutant mice. (A) Spleens of control, Pax8-/- and Pax8-/- TH-treated mice. (B,C) Spleen cellularity in controls and Pax8-/- mice, treated or not with TH, and in controls, TR0/0 and TRß-/- mice. Nucleated splenocytes from 15-day-old mice were counted and cell numbers plotted as a cellularity index expressed as 105 cells/g of body weight. [*, P<0.005 compared with control; **, P<0.005 compared with Pax8-/-, n=20 for control, n=20 for Pax8-/-, n=4 for Pax8-/- after 3 and 24 hours, n=10 for Pax8-/- after 48 hours, n=13 for TR0/0 and n=5 for TR-/-]. (D) Analysis of spleen populations in wild-type, Pax8-/- ß and TH-treated Pax8-/- mice. Splenocytes were analyzed by flow cytometry using anti-B220 and anti-TER119 antibodies to identify cells belonging to the B and erythrocytic compartments, respectively. Numbers in the FACS profiles indicate percentages of the respective populations. (E) Total number of TER119- and B220-positive spleen subpopulations in wild-type, Pax8-/- and TH-treated Pax8-/- mice, plotted as a cellularity index (**, P<0.002; *, P=0.05 compared with control; n=7 for control, n=9 for Pax8-/- and n=5 for Pax8-/- + TH). (F) Comparison of numbers of TER119-positive spleen subpopulations between 15-day-old and 21-day-old wild-type and TR0/0 mice (*, P=0.05 compared with control; n=3).

View larger version (64K): [in a new window]   Fig. 2. Analysis of erythrocytic cell development in the spleen of TR0/0 and Pax8-/- mice. (A,B) Distributions of erythrocytic populations in TR0/0 (A) and Pax8-/- (B) mice. For each type of mouse, data from mutant, mutant treated with TH and littermate controls are presented. Freshly dissociated mouse spleen cells obtained from 15-day-old mice were counted and stained with anti-CD71 and anti-TER119 antibodies to distinguish the different developmental stages of erythroblast maturation (stages I to IV, as indicated) (Socolovsky et al., 2001). Percentages of each subset are indicated in the gates. Dead cells and enucleated red blood cells (with low forward scatter) were excluded from FACS analysis. (C) Benzidine-MGG-stained cytospin preparations of freshly isolated spleen cells from wild-type, Pax8-/- and TH-treated Pax8-/- mice, as indicated. The red arrowheads point to representative late basophilic erythroblasts. (D) Graphical representation of the distribution of the respective erythrocytic populations relative to the BFU-E population. For each tested mouse, the frequency of each cell population was divided by that of the BFU-Es. Numbers of animals analyzed were 5 for the control and Pax8-/- and 4 for the Pax8-/- +TH. (*, P=0.003). (E,F) Total numbers of nucleated cells TER119+ KI67+ and TER119+ AnnexinV+, respectively, plotted as a cellularity index in control, Pax8-/- and TH-treated Pax8-/- mice. Spleen cells from 15-day-old mice were counted and analyzed by flow cytometry using anti-TER119 together with either anti-KI67 (E) or anti-Annexin V (F) antibody (* P<0.01; n=3 for each genotype and treatment).

View larger version (28K): [in a new window]   Fig. 3. Bone marrow erythropoiesis in Pax8-/- and TR0/0 mutants. (A) Bone marrow cellularity in Pax8-/-, TH-treated Pax8-/- mice, TR0/0 mice and respective controls. Bone marrow nucleated cells obtained from 15-day-old mice were counted and cell numbers plotted as a cellularity index (n=3 for each genotype). (B) Analysis of resident BM populations in Pax8-/- mice (n=3 except for Pax8-/-, where n=2). (C) Distribution of the various erythrocytic cell subpopulations in the bone marrow of Pax8-/- and TR0/0 mice. Due to the different genetic backgrounds, each mutant was compared with its appropriate control littermate in the same column. Bone marrow cells obtained from 15-day-old mice were analyzed by FACS using the anti-CD71 and anti-TER1119 antibodies to distinguish the different developmental stages of erythroblast maturation (stages I to IV). Percentages of each subset are indicated in the gates. Dead cells and enucleated red blood cells (with low forward scatter) were excluded from FACS analysis. One representative example, out of four, is shown here.

View larger version (20K): [in a new window]   Fig. 4. Establishment of primary cultures of spleen erythroblasts in proliferation conditions. (A) Freshly dissociated mouse splenocytes obtained from 10-day-old wild-type mice were stained with anti-CD71 and anti-TER119 antibodies to estimate the amount of TER119+/CD71+ erythroblasts before plating in serum-free medium under conditions that favor proliferation of erythroblasts. (B) Cumulative cell number of erythroblasts determined daily by Trypan Blue exclusion. (C) MGG and neutral benzidine staining of cytospin preparations obtained from proliferating erythroblasts analyzed on day 7.

View larger version (49K): [in a new window]   Fig. 5. Effect of T3 on the differentiation of spleen and bone marrow erythroblast primary cultures. After 7 days in culture, in proliferation conditions, erythroblasts derived from either spleen or bone marrow were switched to medium that favors their differentiation in the presence or absence of 10-7 mol T3. (A,C) Growth curves of respectively spleen- and bone-marrow-derived erythroblasts. Cell numbers during differentiation were determined at the indicated timepoint by Trypan Blue exclusion. (B) Spleen culture and (D) bone marrow culture show the morphology of differentiating cells after staining with MGG and neutral benzidine together with the corresponding cumulative numbers of hemoglobin-positive (red bars) and hemoglobin-negative (white bars) cells in the presence or absence of T3 (*P<0.05; **P<0.01; n=3 for each condition). The numerous small brownish bodies in B at 42 hours in the presence of T3 represent orthochromatophilic erythroblasts with a thin rim of cytoplasm and a highly condensed nucleus and expelled nuclei. These orthochromatophilic erythroblasts were scored as hemoglobin-positive cells. (E,F) Cumulative numbers of differentiating cells and hemoglobin-positive and hemoglobin-negative cells in cultures of TR0/0 spleen (E) and bone marrow (F) cultures. (No statistical differences were observed in hemoglobin-positive cell numbers between -T3 and +T3 at the respective times; n=3 for each condition).

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View larger version (79K): [in a new window]   Fig. 6. Erythropoietic reconstitution in lethally irradiated mice. Donor spleen cells from 10-day-old wild-type or TR0/0 mice were injected into irradiated adult mice. Eleven days after the grafting the adult recipient mice (wild type or TR0/0) reconstituted with wild-type or TR0/0 cells, as indicated, were sacrificed and the hematopoietic reconstitution in the spleen was analyzed by examination of spleen paraffin sections after hematoxilin and eosin coloration to show the size of the hematopoietic colonies (colonies are delineated by white dotted lines in A-C), and by examination of cytospin cells after neutral benzidine and MGG staining. Arrows in D and F point to hemoglobin-positive erythroblasts.