A. Appearance of mask mice at 4 weeks of age. B. Anemia and iron deficiency in mask homozygotes 6-8 weeks of age. Hemoglobin (Hb), mean red blood cell volume (MCV) and plasma iron levels in mask homozygote and control mice. N=4 for each column. Error bars represent 1 standard deviation. C. and D. Retention of radioactive iron administered intragastrically. In C, mice were maintained on a normal diet prior to instillation of iron. In D, mice were iron depleted for one month prior to instillation of iron. E. After measuring retention of ⁵⁹Fe in iron-depleted mice, hepcidin mRNA levels were measured in the liver of the same five animals, normalized to mRNA levels of ribosomal protein S18. Color coding preserved for individual mice. Re-growth of hair in the mouse shown in panel A, one week (F) and two weeks (G) after initiation of a high-iron diet.

Four founders (designated 49-52 OQ) were produced by microinjecting a BAC clone bearing the WT Tmprss6 sequence into single-cell embryos homozygous for the mask mutation. Hemoglobin and serum iron levels in each of the founders, in blood sampled at 4 weeks of age. B6 controls, age-matched C57BL/6J mice, N=4. Non-transgenic controls, Tmprss6^msk/msk littermates that lacked the transgene, N=4. Error bars indicate 1 SEM.

A. Schematic illustration of the transcripts resulting from the mutated Tmprss6^mask allele. The mutation site, at the acceptor splice junction upstream from exon 15, is indicated by black arrowhead; a premature stop codon is predicted as indicated by the asterisk. B. Illustration of the coding change that results from the mutation (SMART diagram).

HepG2 cells were transfected with a full-length Hamp promoter reporter construct in all experiments shown in A-D, and with a normalizing renilla luciferase construct. Tmprss6, full length construct encoding the native protein. Tmprss6^msk, the truncated mutant sequence expressed by mask mice. Tmprss6^S762A, protease-dead Tmprss6 mutant. delN, GFP cytoplasmic domain, TMPRSS6 transmembrane and ectodomain. delC, GFP ectodomain and TMPRSS6 transmembrane and cytoplasmic domains. A. The effect of Tmprss6 and Tmprss6^msk on Hamp promoter activity in cells cotransfected with a hemojuvelin-encoding vector or stimulated with BMP2, BMP4, BMP9, IL-6, or IL-1α. B. Comparison of the Hamp promoter-suppressing effects of Tmprss6, Tmprss6^msk, and Tmprss6^S762A in cells subjected to the stimuli indicated. C. Localization of delN and delC constructs in HepG2 cells by confocal fluorescence microscopy. D. Comparison of the Hamp promoter-suppressing effects of Tmprss6, Tmprss6^msk, delN and delC constructs in cells subjected to the stimuli indicated. E. Response of Hamp promoter deletion constructs to induction by IL-6 (length indicated with respect to the cap site), and the relative suppression of responses by co-transfection with Tmprss6 or Tmprss6^msk constructs. In all experiments (A-E), duplicate transfections were performed. Incubations with cytokines were performed for 16 hours before luminescence was read to estimate hepcidin promoter activity. Error bars indicate 1 SEM.