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Supplementary Information
The transcription factor Hmx1 and growth factor receptor
activities control sympathetic neurons diversification
Alessandro Furlan, Moritz Lübke, Igor Adameyko, Francois Lallemend and Patrik
Ernfors
Supplemental Figures
Supplementary Figure 1
Supplementary Figure 2
Supplementary Figure 3
Supplementary Figure 4
Supplementary Figure 5
Supplementary Figure 6
Supplementary Figure 7
Supplementary Figure 1. Temporal expression of TrkC in the sympathetic ganglion
during development
Double immunostaining for TRKC and ISL1 at E13.5 and E14.5 on SG sections from
wild-type embryos and triple immunostaining for TRKC, ISL1 and CFP (i.e. CFP in
RetCFP mice) or TRKA at E15.5 on SG sections from RetCFP/+;Hmx1LcZ/+ embryos. Note
TrkC expression in all ISL1+ SG neurons at E13.5 but not at E14.5. Scale bar
represents 50μm. At E15.5 TrkC expression is largely mutually exclusive with both
RET+ and TRKA+ neurons. Scale bar represents 10μm.
Supplementary Figure 2. Establishment of Hmx1LcZ mice
(A) Scheme showing the strategy to target the insertion of the LacZ reporter into the
murine Hmx1 gene by homologous recombination. Endogenous gene function is
abolished and the nuclear β-galactosidase protein is expressed in neurons expressing
the Hmx1 gene. A HUbCpro-neof-p(A) cassette was placed downstream LacZ. ES cells
containing this allele were purchased and used for germline transmission of the
allele. (B) Hmx1LcZ genotyping strategy. (C) Genotyping results from wild-type and
Hmx1LcZ/+ genomic tail DNA.
Supplementary Figure 3. Hmx1 expression is initiated in TRKC+ neurons
Triple immunostaining for TRKC, Hmx1-βgal and ISL1 at E13.5 and for TRKC, Hmx1βgal and TRKA at E14.5. Note βgal expression onset (arrowhead) in TRKC+ neurons at
E13.5. At E14.5 βgal+/TrkA+ neurons are TRKC- (arrowheads) whereas βgal+ only
neurons are TRKC+ (arrows). Scale bar in all images represents 10μm.
Supplementary Figure 4. E15.5 is a critical stage for the segregation of cholinergic
and noradrenergic sympathetic neurons
Triple immunostaining for CFP (i.e. CFP in RetCFP mice), VAChT and PRPH (A), and for
CFP, Hmx1-βgal and VMAT2 (B) on SG sections from E15.5 RETCFP/+;Hmx1LcZ/+
embryos. Note segregation of RET and PRPH in the cholinergic population (defined
by VAChT expression) and of HMX1 in the noradrenergic population (defined by
Vmat2 expression) at E15.5. Scale bar represents 50μm.
Supplementary Figure 5. Generation of Hmx1 conditional null mice
(A) Genomic organization of Hmx1. (B, C) Hmx1 targeting strategy. (B) Exon 2
(including 3’UTR) was flanked by LoxP sites. Positive selection cassette (NeoR) was
flanked by FRT sites and inserted into intron 1. Homologous recombinant clones (C)
were isolated using positive (G418 resistance) and negative (thymidine kinase - Tk)
selection. (D) Conditional KO allele after Flp-mediated removal of selection marker
by crossing to the FlpE mouse line. (E) KO allele after Cre-mediated recombination.
Deletion of Exon 2 results in loss of function of the Hmx1 gene by deleting the
homeobox domain and the 3’ untranslated region. (F) Hmx1fl/fl genotyping strategy.
(G) Genotyping results from Hmx1fl/fl, wild-type and Hmx1fl/+ genomic tail DNA.
(H) Expression of (mRNA) Hmx1 by in situ hybridization in ISL1+ neurons on SG
sections from E15.5 wild-type (upper panels) and Hmx1fl/fl;Wnt1-Cre (lower panels)
mice. Hmx1 mutant mice display complete loss of HMX1 in SG neurons. Scale bar
represents 50μm. (I) A significant increase in ISL1+ neurons was observed in
Hmx1fl/fl;Wnt1-Cre compared to WT SG at P0. n=3 animals/group, 14-44 ganglia were
analyzed per animal. Statistical analysis was performed using Student’s t-test. * p <
0.05. (J) Double immunostaining for ISL1 and NPY, ChAT, VAChT, DBH, VMAT2, TRKC
or CGRP on SG sections from WT and Hmx1fl/fl;Wnt1-Cre animals at P0. A dorsal root
ganglion section is on the right and serves as positive control for CGRP. Expression of
all markers is either not affected in the mutant SG or completely absent (i.e. CGRP)
in both WT and mutant SG. Scale bar represents 20μm. (K)
Double immunostaining for RET and Hmx1-βgal on SG section from control
(Hmx1LcZ/+;Wnt1-Cre) and mutant (Hmx1LcZ/fl;Wnt1-Cre) animals at P5. Scale bar
represents 20μm.
Supplementary Figure 6. Absence of RET signaling leads to precocious Hmx1βgal+/TRKA+ neurons and failure of ChAT expression already at E14.5
(A) Double immunostaining for Hmx1-βgal and TRKA on SG sections from control
(RetCFP/+;Hmx1LcZ/+) and RetCFP/CFP;Hmx1LcZ/+ embryos at E14.5. Note βgal (pointed
with arrowheads) expressed in TRKA+ neurons in the mutant. (B) Double
immunostaining for TH and ChAT on SG sections from control (RetCFP/+;Hmx1LcZ/+) and
RetCFP/CFP;Hmx1LcZ/+ embryos at E14.5. Note the marked reduction of ChAT+ neurons
in the Ret KO (lower panel, arrowhead) compared to control condition (upper panel).
Scale bar in all images represents 50μm.
Supplementary Figure 7. Innervation deficits of sympathetic target in the back skin
of P19 mice lacking HMX1
(A-D) The arrector pili muscle of Hmx1fl/fl;Wnt1-Cre mice is poorly innervated.
(A,B) Double immunostaining for TH and PGP9.5 on back skin sections from 19-daysold control Hmx1fl/fl (A) and Hmx1fl/fl;Wnt1-Cre (B) mice. Arrows indicate the arrector
pili muscle. Note TH+/PGP9.5+ fibers contacting the arrector pili muscle of every hair
follicle of control but not HMX1-deficient mice. An increase of cutaneous PGP9.5+
fibers was also observed in Hmx1fl/fl;Wnt1-Cre mice. (C,D) Triple immunostaining for
-SMA, TH and PGP9.5 of Hmx1fl/fl (C) and Hmx1fl/fl;Wnt1-Cre (D) 19-days-old back
skin hair follicle. Arrowheads indicate sympathetic innervation of the arrector pili
muscle (C) and arrows point at the attachment point of the muscle just above the
bulge (C,D). Note PGP9.5+/TH+ fibers (green and blue, respectively) associated with
the -SMA+ arrector pili muscle (in red) in control but not in HMX1-deficient animals.
(E) Subcutaneous blood vessels in the dermis of Hmx1fl/fl and Hmx1fl/fl;Wnt1-Cre
mice stained for -SMA, PGP9.5 and TH on dorsal skin of 19-days-old mice.
Arrowheads point at PGP9.5+/TH+ fibers in subcutaneous vessels of control mice.
Note a deficit of PGP9.5+/TH+ fibers in the HMX1-deficient mice. An image of the hair
follicle (HF) from the same section is included to confirm the efficiency of the
antibodies used. Scale bar in (A,B), (C,D) and (E) represents 100, 50 and 50 m,
respectively.