www.sciencemag.org/content//342/6164/1375/suppl/DC1
Supplementary Materials for
Progressive Specification Rather than Intercalation of Segments During Limb
Regeneration
Kathleen Roensch, Akira Tazaki, Osvaldo Chara, Elly M. Tanaka*
*Corresponding author. E-mail: [email protected]
Published 13 December 2013, Science 342, 1375 (2013)
DOI: 10.1126/science.1241796
This PDF file includes:
Materials and Methods
Figs. S1 to S7
References
Materials and Methods:
Axolotl surgery.
Ambystoma mexicanum were bred in our facility and were maintained at 17-20°C in Dresden tap water
and fed daily with artemia or fish pellets. For all surgeries, animals were anesthetized in 0.01% ethyl-paminobenzoate (Sigma).
The experiments described here were performed on 4.0-cm-long (from snout to tip of the tail) white
animals.
Cloning of Axolotl HoxA9, A11and A13.
To obtain full length sequence of axolotl HoxA9, 5’ RACE was performed using limb bud total
RNA with a primer 5’- GGCTGCTGTCGAAGGTGGTGCAGTCCCC -3’ by using Gene Racer
kit (Invitrogen) according to the manufacture’s instruction. The full coding region of HoxA9 was
PCR amplified from limb bud cDNA with primers 5’TTCGAATTCATATTTTTTCCTGGAGGTCCCGCT -3’ and 5’AGGCTCGAGTCACTCGTCCTTAGGCCGGTCC -3’ designed according to the sequence
obtained from the 5’RACE and according to the axolotl HoxA9 sequence from Genbank
(U20941.1), respectively. The PCR product was cloned into the EcoRI and XhoI site in the pCS2
vector.
For cloning full-length axolotl HoxA11, 5’ RACE was performed using limb bud total RNA with
a primer 5’- CCTGCCACCCACAGCTCTCCTCCGGGCC -3’ by using Gene Racer kit
(Invitrogen) according to the manufacture’s instruction. A long insert cDNA library was
screened with primers designed according to the sequence obtained from the 5’RACE, 5’TCCTCTTCCGGCAACAACGAGGAGAA -3’ and 5’AACACATATGTGCATTTGCCATCGAC -3’.
Full-length axolotl HoxA13 was cloned by screening the cDNA library (NT library, (37)) with
degenerate primers, 5’- MGIMGIGGIMGIAARAARMGIGT -3’ and 5’CATICKICKRTTYTGRAACCA -3’.
Accession numbers of the axolotl HoxA9, A11 and A13 are JX975067, JX975068, JX975069,
respectively.
HOXA antibody preparation.
HOXA9 and A13 antibodies were prepared as described previously(38). For HOXA11 antibody
production, a GST-HOXA11 fusion protein with amino acids
TAYGGPESGPADYAGDKGCEKGSPAVAGPPSAEACRGEADRRGAESSGGGGSSSSPESS
SGNNEEKASGSAPNG was used to immunize rabbits. Antibody was affinity purified using an
MBP fusion protein to the same sequence as previously described (38).
Immunohistochemistry
Limb buds and limb blastemas were collected from the level of the shoulder, fixed 4 h at 4°C in 1x
MEMFA (0.1M MOPS pH7.4, 2mM EGTA, 1mM MgSO4 and 3.7% formaldehyde) (Merck)). The
samples were washed in 1xPBS followed by 20% sucrose overnight at 4°C. The tissue was embedded in
tissue-tek (O.C.T. Compound, Sakura) and cut at 10 µm thickness (Microm HM 560). The
immunostaining was done by blocking (1xPBS, 1% goat serum, 0.3% Tween) the slides for 30 min at RT.
The primary rabbit anti-HOXA9 (1:5000), rabbit anti-HOXA11, rabbit anti-HOXA13 (1:1000) were
added to the slides and incubated overnight at 4°C. The antibodies were detected using the secondary
antibody Cy5 anti-rabbit IgG (1:200, Invitrogen). The cell nuclei were stained with HOECHST 33342
(1:500, Sigma). The slides were mounted with 50% glycerol. The regenerated limb blastemas as well as
limb buds were imaged using Zeiss Axiovert 200 microscope.
In-situ hybridization on limb paraffin sections.
The limb blastemas and limb buds were fixed in 1x MEMFA (0.1M MOPS pH 7.4, 2mM EGTA,
1mM Mg SO4 x 7H2O and 3.7% formaldehyde) overnight at 4°C, washed in 1xPBS, dehydrate
by increasing ethanol series, washed 3x in Xylene, 3x in paraffin at 65°C and finally embedded
into paraffin. Paraffin-embedded tissues were sectioned at 10μm followed by drying at 37°C
incubator overnight. Before in-situ hybridization, the paraffin sections were dewaxed with 3x
Xylene, decreasing ethanol series and washed in 1xPBS/0.1% Tween (pre-treated with DEPC).
To synthesize the anti-sense RNA-probe for HoxA9, the plasmids were linearized with EcoRI.
The transcription was performed using T7 polymerase (Roche) for HoxA9, HoxA11 and HoxA13.
In situ hybridization was performed as described (39). Briefly, the sections were hybridized with
100ng/ml DIG-labeled probe in hybridization buffer (50% formamide, 10% dextran, 5x SSC,
0.1% Tween, 1mg/ml yeast RNA, 100μg/ml heparin, 1x Denhardt’s, 0.1% CHAPS, 5mM
EDTA) overnight at 70°C. Slides were washed 3x for 1 hour each and overnight at 70°C in 5x
SSC post-hybridization buffer (50% formamide, 5x SSC, 0.1% Tween) followed by 2x SSC
(50% formamide, 2x SSC, 0.1% Tween) twice for 1 hour each. Further the slides were washed
twice for 5 min each and once 20 min at RT in maleic acid buffer (100mM Maleic acid pH 7.5,
150mM NaCl, 0.1% Tween), blocked in maleic acid buffer and 1% blocking reagent (Roche) for
1 hour at RT. Anti-DIG Fab fragments conjugated with alkaline phosphatase (Roche) were
diluted 1:5000 in maleic acid buffer/1% blocking reagent and applied to the sections for 2 hours
at RT. Slides were washed 5x for 10 min each in maleic acid buffer and 2x for 10 min each in
alkaline phosphatase buffer (100mM Tris pH 9.5, 50mM MgCl2, 100mM NaCl, 0.1% Tween).
The slides were overlaid with BM purple (Roche) for 12-36 hours at 37°C and the reaction were
stopped with cold 1xPBS/1mM EDTA. The in-situ hybridized limbs were aquired on Zeiss
Axiovert microscope using the Axiovision software.
Retinoic Acid administration
To determine if retinoic acid (RA)-induced proximalization repressed onset of HOXA13
expression in the hand blastema, RA was dissolved in DMSO (100 µM) and injected
intraperitoneally at 100 µg/g body weight one day prior to limb amputation through the
metacarpals. Parallel control animals were injected with an equivalent volume of DMSO.
Immunostaining and imaging was performed as above, with all samples in parallel including a
10D upper arm blastema as a reference for positive signal.
Transplantation of GFP+ connective tissue blastema cells at different stages
Animals expressing GFP in connective tissue were generated by embryonic transplantations of
lateral plate mesoderm (12, 40). The transplanted embryos were bred in our facility up to a size
of 4.0 cm (from snouth to tip of the tail) and used as donors. The size of the host was about 4.0cm-long (from snouth to tip of the tail). 4 day or 8 day (proximal, distal or hand) blastemas were
transplanted into a 6 day mid-upper arm blastema of a white host. Briefly, limbs were amputated
in the middle of the upper arm. For donors, the upper arm blastema was removed into PBS with
Ca++Mg++ to prevent dissociation of the cells. The epidermis was peeled away. For 8 day
blastema transplants, the blastema was then split into a distal and a proximal piece. Up to two
pieces of blastema containing GFP+ cells were taken from each P or D piece for transplantation.
The host 6 day blastema was prepared by poking a hole in the epidermis with forceps at the midpoint of the blastema. Forceps were used to place the donor piece through the hole into the
middle of the blastema. The transplanted animals were kept in a covered dish for 3-4 h with
bencocaine soaked tissues. Afterwards they were transferred into tap water. The hosts were kept
for 30 days of regeneration followed by microscopy of the transplanted GFP+ connective tissue
cells using an Olympus SZX12 fluorescence stereomicroscope.
Calculating the spatial distribution of GFP+ cells between the forearm and the hand
We calculated the distribution of GFP+ cells along the proximo-distal axis of the regenerating
axolotl limbs using PIX, an open software developed in FORTRAN language. Images were
analyzed as follows: First, the proximo-distal axis was visually identified based on brightfield
images. Second, the fluorescence intensity from GFP+ cells was determined for each pixel after
background subtraction. At each coordinate along the proximo-distal axis, the summed
fluorescence intensity from all the pixels belonging to this coordinate (that is, all the pixels
located in the perpendicular direction at this coordinate of the proximo-distal axis) was
determined, resulting in a single line profile along the proximo-distal axis for each limb. In order
to compile all the individual limb data from one condition into one line profile, the individual
line profiles were normalized—the fluorescence intensity at each PD coordinate was normalized
by the total fluorescence intensity (the integral of the accumulated fluorescence intensity
calculated over the proximo-distal axis). The pixel positions along the PD axis were normalized
according to limb length. The average (see Figure 3j) and standard deviation (see Supplementary
Figure S5) of this line distribution was calculated for the four experimental conditions studied: 8
day hand blastema transplants (8D Hand BL), the distal portion of the 8 day upper arm blastema
(8D UA BL--dist), the proximal portion of the 8 day upper limb blastema (8D UA BL--prox) and
the early blastema transplants (4D UA BL).
References and Notes
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Supplementary Figure Legends
Figure S1. Multiple alignments for Vertebrate HOXA9, A11, A13.
Accession numbers used for multiple aliments are Hs_HOXA9 (NP_689952.1), Mm_HOXA9
(NP_034586.1), Gg_HOXA9 (XP_003640782.1), Xl_HOXA9 (NP_001091264.1),
Dr_HOXA9 (NP_571608.1), Hs_HOXA11 (NP_005514.1), Mm_HOXA11 (NP_034580.1)
Gg_HOXA11 (NP_989950.1), Xt_HOXA11 (XP_002933440.1), Dr_HOXA11
(NP_571222.1), Hs_HOXA13 (NP_000513.2, Mm_HOXA13 (NP_032290.1), Gg_HOXA13
(XP_003640757.1), Xt_HOXA13 (XP_002933431.1), Dr_HOXA13 (NP_001078963.1).
Figure S2. Nested HoxA9, HoxA11 and HoxA13 mRNA expression in the limb bud.
Serial sections of the developing forelimb bud at stage 36, 39, 40. Interestingly, HOXA11
transcript expression extends to the distal tip, suggesting post-transcriptional control of
HOXA11 protein expression.
A-C, HoxA9 expression.
D-F, HoxA11 expression.
G-I, Expression of HoxA13.
A,D,G, Early limb bud stage 36.
B,E,H, Mid bud stage 39.
C,F,I, Late limb bud stage 40. Scale bar, 100µm.
Figure S3. HOXA9 and HOXA11 expression are absent in the one day limb blastema.
Consecutive longitudinal limb sections to those shown in Figure 2A.
A, GFP-expressing blastema cell precursors (green) do not express HOXA9 (red). Images
taken at same exposure to those taken for 12 day blastema in Figure 2D.
B, Zoom of inset shown in A.
C, GFP-expressing blastema cell precursors (green) do not express HOXA11 (red). Images
taken at same exposure to those taken for 12 day blastema in Figure 2F.
D, Zoom of inset shown in C. Scale bar, 500 µm.
Figure S4. The first time point of detectable HOXA13 expression was day 8 postamputation.
Longitudinal section of 4 day and medium bud (8 day) forelimb blastemas amputated at the
level of the humerus.
A, GFP+ blastema precursors (green) at 4 dpa do not express HOXA13 (red). Images taken at
same exposure as 12 day blastema samples in Figure 2H.
B, Zoom of inset in A. Yellow arrowheads show that sparse signal in red, channel (D) is not
associated with GFP+ signal (E), and many spots are not associated with a Hoechst-positive
nucleus (blue, C).
F, Medium bud (8 day) upper arm blastema shows robust HOXA13 expression (red) at distal
tip (corresponding to transplanted cells in Figure 3 C,D).
G, Zoom of inset in F.
H. Single channel HOXA13 signal.
I. Single channel Hoechst channel.
Distal, left; proximal, right. Scale bar, 200µm.
Figure S5. Nested HoxA9, HoxA11 and HoxA13 mRNA expression during limb
regeneration visualized by section in-situ hybridization.
Longitudinal sections of an early bud (5 day), medium bud (8 day) and medium to late bud
(10 day) regenerating forelimb blastemas amputated at the level of the humerus. HoxA13
expression is absent from the 5 day limb blastema. By 8 days, the characteristic nested
expression pattern of HoxA9-A13 is observed.
A-C, Expression pattern of HoxA9 in the mesenchyme cells.
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D-F, Expression pattern of HoxA11 visualized in the mesenchyme. In the 8 day limb
blastema, the pattern of HoxA11 is more distal to HoxA9 expression.
G-I, Expression pattern of HoxA13 mRNA. No expression of HoxA13 was detected at 5
days post amputation (G). Onset of the HoxA13 expression domain by 8 days is restricted
distally to the blastema cells underneath the epidermis (H). Strong HoxA13 expression
domain detected in mesenchymal cells in the 10 day blastema (I).
Dashed line marks the plane of amputation. Scale bar, 500µm.
Figure S6. Early onset of HOXA13 in hand blastema, and its repression by the
proximalization factor, retinoic acid.
To confirm the association of HOXA13 expression with hand identity, Onset of HOXA13
expression in hand blastemas was investigated through a time course. HOXA13 expression
was seen in hand blastemas earlier than in upper arm blastemas.
A.Earliest time point of HOXA13 expression (red) is 4 days post amputation
C,D,E Zoomed inset of marked area in (A), in merge, HOXA13 and DAPI channels
respectively.
B. Administration of retinoic acid 1 day prior to amputation represses onset of HOXA13
(red) in 4 day blastema.
F,G,H. Zoomed inset of marked areas in (B) in merge, HOXA13 and DAPI channels
respectively.
I.Strong HOXA13 expression in hand blastema already by 6dpa.
K,L,M. Zoomed inset of marked area in (I), in merge, HOXA13 and DAPI channels
respectively.
J. Administration of retinoic acid 1 day prior to amputation represses onset of HOXA13 (red)
in 6 day blastema.
N,O,P. Zoomed inset of marked area in (J), in merge, HOXA13 and DAPI channels
respectively. Scale bar, 500 µm.
Figure S7. Standard deviation profile of the GFP fluorescence intensity data shown in
Figure 3J.
Standard deviation profile of the normalized GFP-fluorescence along the proximo-distal axis
for limbs in the different transplantation categories: 8 day hand blastema transplants (8D
Hand BL), the distal portion of the 8 day upper limb blastema (8D UA BL--dist), the
proximal portion of the 8 day upper arm blastema (8D UA BL--prox) and the early blastema
transplants (4D UA BL).
HOXA9
Hs_HOXA9
Mm_HOXA9
Gg_HOXA9
Am_HOXA9
Xl_HOXA9
Dr_HOXA9
MATTGALGNY
MATTGALGNY
MSAPGTLSNY
MSASGTLSNY
MSTSGTLSNY
MSTLGTLS.Y
YVDSFLLGAD
YVDSFLLGAD
YVDSFLVPE.
YVDSFLIHES
YVDSFLIHE.
YADSHLPHE.
AADELSVGRY
AADELGAGRY
.GDELAAPRY
EELVQSRYAA
.GDDLVQPRF
.NDDHLAPRF
APGTLGQPPR
APGTLGQPPR
APAPLGPPPR
AAGPLAGAGR
APGSLAQPAR
SSGPVVQQQS
QAATLAEHPD
QAAALAEHPD
..PALAEHPE
Q.PGMGEHPE
Q.AASAEHPE
RELTLLEYSE
FSPCSFQSKA
FSPCSFQSKA
LTPCSFQPKA
FTPCSFQSKS
FTPCSFQSKS
QEPYTFQAKS
TVFGASWNPV
AVFGASWNPV
PVFGPPWSPA
PVFGSSWSPV
SVFSSPWNPV
SIFGASWSPV
HAAGANAVPA
HAAGANAVPA
HPAGASGVPA
HPQGAAGGSV
HPQTANNVSS
QPTGAS...I
AVYHHHHHHP
AVYHHHHHP.
VYHPYAHHQ.
PAVYHHHHP.
VYHPYVHHQ.
AYHPYIHHP.
YVHPQAPVAA
YVHPQAPVAA
.......APV
..YVHHQAPG
.......ASM
.......CST
Hs_HOXA9
Mm_HOXA9
Gg_HOXA9
Am_HOXA9
Xl_HOXA9
Dr_HOXA9
AAPDGRYMRS
AAPDGRYMRS
APPDGRYMRS
APEASRYMRS
SASDCRYMRS
GDSDGASVRP
WLEPTPG.AL
WLEPTPG.AL
WLEPVPG.SL
WLEPMPG.AL
WLDPMATGSL
WALEPLP.AL
SFAGLPSSRP
SFAGLPSSRP
SFPGLPTSRH
SFPGLPAARH
PFPAFPSSRS
PFTGLSTDTH
YGIKPEPLS.
YGIKPEPLS.
YGIKPEPLA.
YGIKPEPLPP
YGIKPEPVAT
QDIKLEPLVG
.ARRGDCPTL
.ARRGDCPTL
.ARRGDCTTF
GTRRGDCTTF
..RRGDCSTY
...SGECTTH
DT.HTLSLTD
DT.HTLSLTD
DT.HTLSLSD
DSSHTLSLSD
DS.RSLPVSD
TLLVAETDNN
YACGSPPVDR
YACGSPPVDR
YACGSPPVDR
YGSSP....A
YGCDSP.VDR
TTQTERKVPD
EKQPSEGAFS
EKQPSEGAFS
DKQSHEGAFS
DKQSSEGAFP
DKPPGDPPFA
DAVSNGSHDE
ENNAENESGG
ENNAENESGG
ENNGESEANG
EAPAETEASG
VTNEENLSNG
KIPAETKLDL
DKPPIDPNNP
DKPPIDPNNP
EKPHIDPNNP
DKPAIDPNNP
DKAPIDPNNP
DPSKCNQDNP
Hs_HOXA9
Mm_HOXA9
Gg_HOXA9
Am_HOXA9
Xl_HOXA9
Dr_HOXA9
AANWLHARST
AANWLHARST
AANWLHARST
AANWLHARST
AANWLHARST
LSNWLHAKST
RKKRCPYTKH
RKKRCPYTKH
RKKRCPYTKH
RKKRCPYTKH
RKKRCPYTKH
RKKRCPYTKH
QTLELEKEFL
QTLELEKEFL
QTLELEKEFL
QTLELEKEFL
QTLELEKEFL
QTLELEKEFL
FNMYLTRDRR
FNMYLTRDRR
FNMYLTRDRR
FNMYLTRDRR
FNMYLTRDRR
FNMYLSRDRR
YEVARLLNLT
YEVARLLNLT
YEVARLLNLT
YEVARLLNLT
YEVARLLNLT
YEVARLLNLT
ERQVKIWFQN
ERQVKIWFQN
ERQVKIWFQN
ERQVKIWFQN
ERQVKIWFQN
ERQVKIWFQN
RRMKMKKINK
RRMKMKKINK
RRMKMKKINK
RRMKMKKINK
RRMKMKKINK
RRMKMKKCNK
DRAKDE
DRAKDE
DRAKDE
DRPKDE
DRSKDE
DRPKDI
HOXA11
Hs_HoxA11
Mm_HoxA11
Gg_HoxA11
Am_HoxA11
Xl_HoxA11
Dr_HoxA11
..........
.........M
.........M
MRTARGSPSM
MRTARGS.LM
.........M
MDFDERGPCS
MDFDERGPCS
MDFDERVPCS
MDFDERVPCS
MDFDERVPCS
MDFDERVPVG
SNMYLPSCTY
SNMYLPSCTY
SNMYLPSCTY
SNMYLPSCTY
SNMYLPSCTY
SNMYLPGCTY
YVSGPDFSSL
YVSGPDFSSL
YVSGPDFSSL
YVSGPDFSSL
YVSGPDFSSL
YVSGTDFSSL
PSFLPQTPSS
PSFLPQTPSS
PSFLPQTPSS
PSFLPQNPAS
PSFLPQTPSS
PPFLPQTPSS
RPMTYSYS.S
RPMTYSYS.S
RPMTYSYS.S
RPMPYSYS.S
RPMTYSYS.S
CPMTYSYSTS
NLPQVQPVRE
NLPQVQPVRE
NLPQVQPVRE
NLPQVQPVRE
NLPQVQPVRE
SLPQVQSVRE
VTFREYAIEP
VTFREYAIEP
VTFREYAIDP
VTFREYAIDP
VTFREYAIDT
VSFRDYAIDT
ATKWHPRGNL
ATKWHPRGNL
SSKWHPRNNL
ASKWHPRSNL
SSKWHHRNNL
SSKWHSRGNL
AHCYSAEELV
AHCYSAEELV
PHCYSAEEIM
AHCYSAEELM
PHCYSAEEIM
PHCYATEDMV
Hs_HoxA11
Mm_HoxA11
Gg_HoxA11
Am_HoxA11
Xl_HoxA11
Dr_HoxA11
HRDCLQAPSA
HRDCLQAPSA
HRDCLPSTTT
HRDCGPNPNA
HRDCLPASNT
HRECLSNP..
AGVPGDVLAK
AGVPGDVLAK
ASM.GEVFGK
GGM.GDVFGK
ASV.GEMFAK
.GTLGDMLSK
SSANVYHHPT
SSANVYHHPT
STANVYHHPS
GAPSPYHHPA
NPTNVYH.PN
NNSVLYH.SN
PAVSSNFYST
PAVSSNFYST
ANVSSNFYST
PGASSNFYST
ANVSSNFYST
SSHTSNVYGS
VGRNGVLPQA
VGRNGVLPQA
VGRNGVLPQA
VGRNGVLPQA
VGRNGVLPQA
VGRNGVLPQA
FDQFFETAYG
FDQFFETAYG
FDQFFETAYG
FDQFFETAYG
FDQFFETAYG
FDQFFETAYG
TPENLASSDY
TPENLASSDY
TAENPSSADY
GPES.GPADY
TTES.QPSDY
NVEN.QPTEH
PGDKSAEKGP
PGDKNAEKGP
PPDKSGEKAP
AGDKGCEKGS
SVDKSCDKVA
PVDRATSKAP
PAATATSAAA
QAAAATSAAA
..........
..........
..........
..........
.AAAATGAPA
VAAAATGAPA
..AAAGATAA
..PAVAGPPS
..AAAATTSS
....PPAESG
Hs_HoxA11
Mm_HoxA11
Gg_HoxA11
Am_HoxA11
Xl_HoxA11
Dr_HoxA11
TSSSDSGGGG
TSSSDGGGGG
TSSSEGGCGG
AEACRGEADR
SEACREPEEK
SDSCR.....
GCRETAAAAE
GCQE..AAAE
.....AAAAA
.........R
........ER
..........
EKERRRRPES
EKERRRRPES
GKERRRRPES
GAESSGGGGS
RAESSGRS.S
GTDETERCEE
SSSPESSSGH
SSSPESSSGH
GSSPESSSGN
SSSPESSSGN
SSSSQSSSGN
TSSPEPSSGN
TEDKAGGSS.
TEDKAGGSG.
NEEKSGSSS.
NEEKASGSAP
NEDKANSSS.
NEDKFSGSS.
.GQRTRKKRC
.GQRTRKKRC
.GQRTRKKRC
NGQRTRKKRC
.GQRTRKKRC
NGQKTRKKRC
PYTKYQIREL
PYTKYQIREL
PYTKYQIREL
PYTKYQIREL
PYTKYQIREL
PYTKYQIREL
EREFFFSVYI
EREFFFSVYI
EREFFFSVYI
EREFFFSVYI
EREFFFSVYI
EREFFFSVYI
NKEKRLQLSR
NKEKRLQLSR
NKEKRLQLSR
NKEKRLQLSR
NKEKRLQLSR
NKEKRLQLSR
MLNLTDRQVK
MLNLTDRQVK
MLNLTDRQVK
MLNLTDRQVK
MLNLTDRQVK
MLNLTDRQVK
Hs_HoxA11
Mm_HoxA11
Gg_HoxA11
Am_HoxA11
Xl_HoxA11
Dr_HoxA11
IWFQNRRMKE
IWFQNRRMKE
IWFQNRRMKE
IWFQNRRMKE
IWFQNRRMKE
IWFQNRRMKE
KKINRDRLQY
KKINRDRLQY
KKINRDRLQY
KKINRDRLQY
KKINRDRLQY
KKLNRDRLQY
YSANPLL
YSANPLL
YSANPLL
YSANPLL
YSANPLQ
YTTNPLL
Hs_HOXA13
Mm_HOXA13
Gg_HOXA13
Am_HOXA13
Xt_HOXA13
Dr_HOXA13
MTASVLLHPR
MTASVLLHPR
MTASVLLHPR
MTASVLLRPR
MTASVLLHPR
MTTSLLLRPR
WIEPTVMFLY
WIEPTVMFLY
WIEP.VMFLY
WIEP.VMFLY
WAEP.VMFLY
WIDP.VMFLY
DNGGGLVADE
DNGGGLVADE
DNS....LDE
DNS....LDE
DNS....LEE
DNGGG..LDD
LNKNMEGAAA
LNKNMEGAAA
INKNMDG...
INKNMDG...
MNKNMDG...
TSKNMEG...
AAAAAAAAAA
AAAAAAAAAA
..........
..........
..........
..........
AGAGGGGFPH
AGAGGGGFPH
..........
..........
..........
..........
PAAAAAGGNF
PAAAAAGGNF
...FHAGSNF
....FPGSSF
....FPVSSF
....FTGGNF
SVAAAAAAAA
SVAAAAAAAA
........AA
..........
..........
..........
AAAANQCRNL
AAAANQCRNL
AAAANPCRNL
.AAANQCRNL
..AANQCRNL
..SPSPCRNL
MAHPAPLAPG
MAHPAPLAPG
MAHPAPLAAP
MAHPAPLGPP
IGHHAPLAP.
MSHPASLAP.
Hs_HOXA13
Mm_HOXA13
Gg_HOXA13
Am_HOXA13
Xt_HOXA13
Dr_HOXA13
AASAYSSAPG
AAAAYSSAPG
SAAAYTSS..
GAAYSAPGGS
.SSAYPPS..
.SATYPSS..
EAPPSAAAAA
EAPPSAAAAA
EAPAAGMAEP
EGPAE.....
EVPVSAIAEP
EVAAAAAGDS
AAAAAAAAAA
AAAAAAAAAA
..........
..........
..........
..........
AAASSSGGPG
AAASSSGGPG
..........
..........
..........
..........
PAGPAGAEAA
PAGPAGAEAA
........AV
........AG
.........A
.........G
KQCSPCSAAA
KQCSPCSAAA
KQCSPCSAAV
KQCSPCS..A
KQCNPCS.AV
KQCSPCS.AV
QSSSGPAALP
QSSSGPAALP
QSSSG.AALP
QGSSG.AALP
QSTPN.ASLP
QGSAS.ASIS
YGYFGSG.YY
YGYFGSG.YY
YGYFGSG.YY
YGYFGSG.YY
YGYFGSG.YY
YGYFGGGGYY
PCARMGPHPN
PCARMGPHPN
PCRMT..HHN
PCRVG..HHG
PCRMS..HHN
PCRMSHHHGS
..AIKSCAQP
..AIKSCAQP
..AIKSCAQP
..GIKSCAQP
..TIKSCSQP
GGGVKTCAQS
Hs_HOXA13
Mm_HOXA13
Gg_HOXA13
Am_HOXA13
Xt_HOXA13
Dr_HOXA13
ASAAAAAAFA
AS..AAAAFA
AS.....TFA
SS......FA
SS......FA
PAS..GSPYG
DKYMDTAGPA
DKYMDTAGPA
DKYMDTS.VS
DKYMDTSGSA
EKYMDTSGSA
EKYMDTSAST
AEEFSS.RAK
AEEFSS.RAK
GEEFTS.RAK
GEEFTS.RAK
GEDFPS.RPK
GEDYTSSRAK
EFAFYHQGYA
EFAFYHQGYA
EFAFY.QGYA
EFAFY.QGYA
EFAFY.QSYP
EFALY.SSYA
AGPYHHHQPM
AGPYHHHQPV
AGPY...QPV
AGPY...QPV
PGPY...QPV
SSPY...QPV
PGYLDMP.VV
PGYLDMP.VV
PGYLDVP.VV
PGYLDMPTMV
PSYLDMP.VV
PSYLDVP.VV
PGLGGPGESR
PGLGGPGESR
PTIGGPGEPR
PTVGGPGEPR
STIGTAGEPR
QAISGPSEPR
HEPLGLPMES
HEPLGLPMES
HDSL.LPMDS
HEPL.LPMEP
HEPL.LPMDG
HESL.LPMES
YQPWALPN.G
YQPWALPN.G
YQPWAITN.G
YQPWALTN.G
YQAWPITN.G
YQPWAITTSG
WNGQMYCPKE
WNGQMYCPKE
WNGQVYCPKE
WNGQVYCSKE
WNGQVYCAKD
WNGQVYCTKE
Hs_HOXA13
Mm_HOXA13
Gg_HOXA13
Am_HOXA13
Xt_HOXA13
Dr_HOXA13
QAQPPHLWKS
QTQPPHLWKS
QSQPPHLWKS
QGQPPHLWKS
QAQPTHLWKS
QQQTGNVWKS
TLPDVVSHP.
TLPDVVSHP.
TLPDVVSHP.
SLPDVVSHP.
SLPDVV.HQ.
SIPESVSHGG
SDASSYRRGR
SDASSYRRGR
SDANSYRRGR
SDANSYRRGR
SDSSSYRRGR
ADGSSFRRGR
KKRVPYTKVQ
KKRVPYTKVQ
KKRVPYTKVQ
KKRVPYTKVQ
KKRVPYTKVQ
KKRVPYTKVQ
LKELEREYAT
LKELEREYAT
LKELEREYAT
LKELEREYAT
LKELEREYAT
LKELEREYAT
NKFITKDKRR
NKFITKDKRR
NKFITKDKRR
NKFITKDKRR
NKFITKDKRR
NKFITKDKRR
RISATTNLSE
RISATTNLSE
RISATTNLSE
RISATTNLSE
RISATTNLSE
RISAQTNLSE
RQVTIWFQNR
RQVTIWFQNR
RQVTIWFQNR
RQVTIWFQNR
RQVTIWFQNR
RQVTIWFQNR
RVKEKKVINK
RVKEKKVINK
RVKEKKVINK
RVKEKKVINK
RVKEKKVINK
RVKEKKVVNK
LKTTS
LKTTS
LKTTS
LKTTS
LKSTS
LKSSS
HOXA13
FigureS1