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SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure 1.
Analysis of the frs anemic phenotype and the
expression of mfrn in zebrafish. a, Flow-cytometry showed deficiency of
mature erythrocytes (red, arrow) in frs mutants (right panel). Cells from the
kidney marrow were separated by forward scatter (FSC) and side scatter
(SSC), also showing a compensatory increase in hematopoietic progenitors
(purple, wt 6% vs. frs 40%) and a relative decrease in myeloid cells (green, wt
24% vs. frs 13%). b, The frs locus is transected by the deletion breakpoint in
the deficiency allele, sptb333. Genomic DNA’s from wt (lanes 1-4) and sptb333
(lanes 5-8) siblings were tested for the presence of mfrn exons 1 and 4,
showing the 3’-end of the mfrn gene is deleted.
c, Zebrafish mfrn RNA
expression pattern parallels the expression pattern of gata-1 in the lateral
plate mesoderm and ICM. Wild type zebrafish embryos from developmental
stages, 5-somites to 36-hours post fertilization (hpf), were subjected to in situ
hybridization with gata-1 and mfrn probes.
The expression of gata-1
precedes that of mfrn at the 5-somite stage; however, mfrn expression
persists after gata-1 mRNA is no longer detected at 36-hpf.
Supplementary Figure 2. Expression pattern of murine Mfrn in cultured
cells and zebrafish mfrn2 in developing embryos. a, Mouse Mfrn mRNA
is induced with erythroid maturation in cultured murine cells. Mouse Mfrn
mRNA expression was strongly up-regulated in differentiating mouse
erythroleukemia (MEL) cells (DS19 clone) induced with 5 mM hexamethylene
bis-acetic acid (HMBA), coincident with the induction of -globin mRNA and
the appearance of cells that stain with o-dianisidine, a marker of hemoglobin.
The expression of Mfrn2 mRNA did not vary with erythroid maturation in MEL
cells. Total RNA samples from MEL cells treated with HMBA for varying
lengths of time were isolated, transferred to nylon, and sequentially hybridized
with Mfrn, Mfrn2, -globin, and GAPDH probes. b, Mfrn mRNA expression
was strongly induced by GATA-1 in GATA1-null G1ER22 cells after exposure
to -estradiol. We used G1ER cells, which undergo terminal maturation upon
activation of an estradiol-inducible form of GATA-1, to study the regulation of
Mfrn by GATA-1. Mfrn was detected at 7 hours post -estradiol treatment and
reached maximal expression at 21 hours post treatment, mimicking the
expression kinetics of -globin22. This places Mfrn downstream of GATA-1 in
temporal epistasis during erythroid maturation, paralleling their relationship
during zebrafish development (Supplementary Fig. 1c). Ethidium bromide
staining for the 28S rRNA is shown as a control for RNA loading. c, Zebrafish
mfrn2 is ubiquitously expressed at low levels in the developing embryo. Wild
type embryo at 24 hpf was hybridized with mfrn2 anti-sense (AS) and control
sense-strand (S) probes.
Supplementary Figure 3. Biochemical characterization of mfrn-deficient
mouse hematopoietic cells and yeasts.
a, Immortalized hematopoietic
cells derived from Mfrn-/- mouse ES cells are deficient in Mfrn mRNA. Total
RNA from wild type (wt) and Mfrn-/- hematopoietic cells were subjected to RTPCR analysis with Mfrn and control GAPDH primers.
b, Biochemical
restoration of the Fe-S clusters in mrs3/4 mutant by zebrafish mfrn.
Mitochondrial aconitase activity was assayed from wild type (wt), mrs3/4
mutant, and mrs3/4 mutant transformed with zebrafish mfrn cDNA (mrs3/4
+ mfrn), which shows full biochemical correction of aconitase activity.
Supplementary Figure 4. Zebrafish mfrn corrects the mitochondrial iron
deficiency of yeast mrs3/4 mutant. a, Zebrafish mfrn fully restored the
activity of mitochondrial iron-dependent superoxide dismutase (Fe-SOD). FeSOD from E. coli was stably expressed and properly targeted to the
mitochondria in wild type (wt) and mutant mrs3/4 yeast strains. Cell lysates
from wt, mrs3/4 mutant, and mutant transfected with zebrafish mfrn were
isolated and separated on a non-denaturing polyacrylamide gel.
In situ
staining to detect Fe-SOD activity in the native polyacrylamide gel was
performed with a fluorogenic substrate. b, Immunoblot of the cell lysates was
probed with antisera against zebrafish mfrn. c, Immunoblot of the cell lysates
was probed with antisera against the endogenous yeast mitochondrial porin
and FLAG-tagged E. coli Fe-SOD.