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Supplementary information for article:
Sea lamprey enlightens the origin of the coup
ling of retinoic acid signaling to vertebrate
hindbrain segmentation
Alice M. H. Bedois
1
, Hugo J. Parker
1
, Andrew J. Price
1
, Jason A. Morrison
1
, Marianne E. Bronner
2
and Robb Krumlauf
1,3,*
1
Stowers Institute for Medi
cal Research, Kansas Cit
y, Missouri 64110, USA
2
Division of Biology and Biological Engineering, Cali
fornia Institute of Technology, Pasadena, CA,
91125, USA
3
Department of Anatomy and Cell Biology
, Kansas University Medical Center, Kansas City, Kansas
66160, USA
*Corresponding author and contact details:
Robb Krumlauf, Stowers Institut
e for Medical Research, 1000 East
50th, Kansas City, MO 64110,
USA, Email: rek@stow
ers.org; Tel:1-816-926-4051
Supplementary Figure 1: Vertebrate Cyp26 protein alignments.
Protein alignments of vertebrate Cyp26A1 and
vertebrate Cyp26B1/C1. The yellow highlighting represents
the identity between sequences with a 95% threshold for
sequence identity. Conserved K, I helices and Heme domains
are indicated. Cutting site
s of the gRNAs used for the
CRISPR/Cas9 experiments are indicated with a t
eal arrow. Cc, Carcharodon carcharias; Eb,
Eptatretus burgeri
; Lc,
Latimeria chalumnae
; Mm,
Mus musculus
; Pm,
Petromyzon marinus
; Rt,
Rhincodon typus
.
Supplementary Figure 2: Vertebrate Aldh1a protein alignments.
Protein alignments of sea lamprey (Pm) Aldh1a1/a2a
and Aldh1a1/a2b with Aldh1a1 and Aldh1a2 from various jawe
d vertebrates. The yellow hi
ghlighting represents the
identity between sequences with a 95% ident
ity threshold. Conserved
Glutamic acid (Glu) and Cysteine (Cys) domains
are indicated. Cutting sites of the gRNAs used for the CRISPR
/Cas9 experiments are indicated with a blue arrow. Eb,
Eptatretus burgeri;
Lc,
Latimeria chalumnae
;
Lo
Lepisosteus oculatus;
Mm,
Mus musculus;
Pm,
Petromyzon marinus
; Rt,
Rhincodon
typus.
Supplementary Figure 3: Vertebrate
Aldh1a
Phylogeny.
Phylogenetic analysis of the vertebrate
Aldh1a
complement
with
Aldh1a8
used as an outgroup. Jawed Vertebrate (J.V.)
Aldh1a1/Aldh1a2/Aldh1a3
clades are indicated with black
lines. Trees were generated by Maximum Likelihood using the
WAG model with 500 iterations
for bootstrap testing, and
the resulting supporting value for each node is shown as
a percentage. A scale bar for
the evolutionary distance is
indicated below each tree. Species name abbrevia
tions are indicated on the top left of the panel.
Supplementary Figure 4: HCRv3 developm
ent and validation in the sea lamprey.
HCR panel representing a high-
power (40X) image of
krox20
(cyan),
kreisler
(yellow) and
hox
β
1
(magenta) with their merged expression. This image was
obtained after optimizing and adapting our HCRv3 protocol to
be able to visualize hindbrain segments. We then used this
protocol with this combination of genes to understand the ch
anges in hindbrain segmentation in Talarazole vs. DMSO
embryos (
Fig. 6a
).
Ctrl_C1H
GGAGCGTCCTCTACAGCATCCGCGACACGCACGAGACGGCGCCGGCCTTCAGCTCGCCGC 60
Cr_C1H_1
GGAGCGTCCTCTACAGCATCCGCGACACGCACGAGACGGCGCCGGCCTTCAGCTCGCCGC 60
Cr_C1H_2
GGAGCGTCCTGTACAGCATCCGCGACACGCACGAGACGGCGCCGGCCTTCAGCTCGCCGC 60
Cr_C1H_3
GGAGCGTCCTATACAGCATCCGCGACACGCACGAGACGGCGCCGGCCTTCAGCTCGCCGC 60
Cr_C1H_4
GGAGCGTCCTCTACAGCATCCGCGACACGCACGAGACGGCGCCGGCCTTCAGCTCGCCGC 60
Cr_C1H_5
GGAGCGTCCTCTACAGCATCCGCGACACGCACGAGACGGCGCCGGCCTTCAGCTCGCCGC 60
********** *************************************************
Ctrl_C1H
TTGACTTCGACCCCGACCGCTTCGACGCGACGCGCGCCGAGGACTCGAAGGAGCGC
TTCA
120
Cr_C1H_1
TTGACTTCGACCCCGACCGCTTAGACGCGACGCGCGCCGAGGACTCGAAGGAGCGC
TTCA
120
Cr_C1H_2
TTGACTTCGACCCCGACCGCTTCGACGCGACGCGCGCCGAGGACTCGAAGGAGCGC
TTCA
120
Cr_C1H_3
TTGACTTCGACCCCGACCGCTTCGACGCGACGCGCGCCGAGGACTCGAAGGAGCGC
TTCA
120
Cr_C1H_4
TTGACTTCGACCCCGACCGCTTCGACGCGACGCGCGCCGAGGACTCGAAGGAGCGC
TTCA
120
Cr_C1H_5
TTGACTTCGACCCCGACCGCTTCG------------------------------------ 69
************************
Ctrl_C1H
GCTACCTGCCGTTCGG
CGG
CGGCGTGCGCAGCTGCCTGGGCAAGGAGCTCGCCAAGCTCA 180
Cr_C1H_1
GCTACCTGC
C----GG
CGG
CGGCGTGCGCAGCTGCCTGGGCAAGGAGCTCGCCAAGCTCA 176
Cr_C1H_2
GCTACCTGC
----CGG
CGG
CGGCGTGCGCAGCTGCCTGGGCAAGGAGCTCGCCAAGCTCA 176
Cr_C1H_3
GCTACCTGC
----CGG
CGG
CGGCGTGCGCAGCTGCCTGGGCAAGGAGCTCGCCAAGCTCA 176
Cr_C1H_4
GCTACCTGCC
----GG
CGG
CGGCGTGCGCAGCTGCCTGGGCAAGGAGCTCGCCAAGCTCA 176
Cr_C1H_5
---------------G
CGG
CGGCGTGCGCAGCTGCCTGGGCAAGGAGCTCGCCAAGCTCA 129
*********************************************
Ctrl_A1H
CCCGACCGCTGGATGACCGGCGGCGACGCCGGCGGCGGCAACGGAGACGCGGCATCGTCG 120
Cr_A1H_1
CCCGACCGCTGGATGACCGGCGGCGACGCCGGCGGCGGCAACGGAGACGCGGCATCGTC- 119
Cr_A1H_2
CCCGACCGCTGGATGACCGGCGGCGACGCCGGCGGCGGCAACGGAGACGCGGCATCGTCG 120
Cr_A1H_3
CCCGCCCGCGGGATGACCGGCGGCGCGGCCTGCGGCGGCATCGGAGACGTGGCATTGTCG 120
Cr_A1H_4
CCCGACCGCTGGATGACCGGCGGCGACGCCGGCGGCGGCAACGGAGACGCGGCATCGTC- 119
**** **** *************** *** ********* ******** ***** ***
Ctrl_A1H
CGCTTCACCTACATCCCGGGGG
CGG
CTCGCGCAGCTGCGTGGGCAAGGAGCTGGCGCGAC 180
Cr_A1H_1
----------------------------GCGCAGCTGCGTGGGCAAGGAGCTGGCGCGAC 151
Cr_A1H_2
CGCTTCACCTCG--------------------CGCTGCGTGGGCAAGGAGCTGGCGCGAC 160
Cr_A1H_3
CGCGTCTC--------------------CTCCCGCTGCGTGGGCAAGGAGCTGGCGCGAC 160
Cr_A1H_4
----------------------------GCGCAGCTGCGTGGGCAAGGAGCTGGCGCGAC 151
***************************
Ctrl_A1H
TCATCCTGAGGATCGTGGTGGTGGAGCTGTGCCGGCGCTGCGAGTGGGAGCTGCCCAACG 240
Cr_A1H_1
TCATCCTGAGGATCGTGGTGGTGGAGCTGTGCCGGCGCTGCGAGTGGGAGCTGCCCAACG 211
Cr_A1H_2
TCATCCTGAGGATCGTGGTGGTGGAGCTGTGCCGGCGCTGCGAGTGGGAGCTGCCCAACG 220
Cr_A1H_3
TCATCCTGAGGATCGTGGTGGTGGAGCTGTGCCGGCGCTGCGAGTGGGAGCTGCCCAACG 220
Cr_A1H_4
TCATCCTGAGGATCGTGGTGGTGGAGCTGTGCCGGCGCTGCGAGTGGGAGCTGCCCAACG 211
************************************************************
Ctrl_A1I
CCGCTCACCACTCCCGCCCCCTCCAGGACCTGAAGGAGTCG
GCCACGGAGCTCCTGTTCG
420
Cr_A1I
CCGCTCACCACTCCCGCCCCCTCCAGGACCTGAAGGAGTCG
GCCACGGAGCTCCTGT
--
G
419
********************************************************* *
Ctrl_A1I
G
GGG
ACACGAGACGACGGCCAGCGCCGCCACGTCACTCGTCATGCACCTGGCAATCCACC 480
Cr_A1I
G
GGG
ACACGAGACGACGGCCAGCGCCGCCACGTCGCTCGTCATGCACCTGGCAATCCACC 478
********************************** *************************
Cyp26A1_H
Cyp26A1_I
Cyp26B1/C1a_H
Mutant
CRISPR
loci
4/7
1/12
5/6
Control
CRISPR
loci
5/5
6/6
3/3
Supplementary Figure 5
:
Genotyping strategy and sequencing results for
Cyp26
CRISPR embryos
Cartoon illustrating the
design of primers for amplifying each site targeted by the
gRNAs
in the
Cyp26
loci. The primer
binding sites are indicated in bold purple. For each site, we sh
ow an alignment of mutant loci (Cr_) sequences with the
sequence of a control locus (Ctrl), the site targeted by the gRNA
is indicated in bold and the PAM in red. A table indicating
the proportion of mutant CRISPR or contro
l CRISPR loci for each site is shown.
Ctrl_Glu
CCCTTACGTAACGCAGGTGGGG
AAGTTGATCCAGGAGGAGGC
CGG
CAAGAGTAACCTGAA 179
Cr_Glu_1
CCTTTACGTAACGCAGGTGGGG
AAGTTGATCCAGGA
--------------GTAACGTGAA 73
Cr_Glu_2
CCCTTACGTAACGCAGGTGGGG
AAGTTGATCCAGGA
--------------GTAACCTGAA 165
Cr_Glu_3
CCCTTACGTAACGCAGGTGGGG
AAGTTGATCCAGGA
--------------GTAACCTGAA 166
Cr_Glu_4
CCCTTACGTAACGCAGGTGGGG
AAGTTGATCCAGGA
GTAACCTGAAGC------------ 165
Cr_Glu_5
CCCTTACGTAACGCAGGTGGGG
AAGTTGATCCAGGA
--------------GTAACCTGAA 165
** *********************************
Ctrl_Glu
GCGCGTGACGCTGGAGCTTGGCGGGAAGAGCCCCATCATCGTCTTCGCCGACGCCGACCG 239
Cr_Glu_1
GCGAGTGACGCTGAAGCTTGGCGGGAAGAGCCCCATCATCGTATTCGCCGAA-CCGACCG 132
Cr_Glu_2
GCGCGTGACGCTGGAGCTTGGCGGGAAGAGCCCCATCATCGTCTTCGCCGACGCCGACCG 225
Cr_Glu_3
GCGCGTGACGCTGGAGCTTGGCGGGAAGAGCCCCATCATCGTCTTCGCCGACGCCGACCG 226
Cr_Glu_4
---GCGGACGCTGGAGCGTGGCGGGAAGAGCCCCATCATCGTTTTGACCGACGCCGACCG 222
Cr_Glu_5
GCGCGTGACGCTGGAGCTTGGCGGGAAGAGCCCCATCATCGTCTTCGCCGACGCCGACCG 225
******* *** ************************ ** **** *******
Ctrl_Cys
CCCCTGCTGTGACACTGCTGACTCGGCGTGGTTCCCACCCCTCCACCCCCCCCCTCCCAC 240
Cr_Cys_1
CCCCTGCTGTGACACTGCTGACTCGGCGTGGTTCCCACCCCTCCACCCCCCCCCTCCCAC 240
Cr_Cys_2
CCCCTGCTGTGACACTGCTGACTCGGCGTGGTTCCCACCCCTCCACCCCCCCCCTCCCAC 240
Cr_Cys_3
CCCCTGCTGTGACACTGCTGACTCGGCGTGGTTCCCACCCCTCCACCCCCCCC-TCCCAC 240
Cr_Cys_4
CCCCTGCTGTGACACTGCTGACTCGGCGTGGTTCCCACCCCTCCACCCCCCCCCTCCCAC 238
Cr_Cys_5
CCCCTGCTGTGACACTGCTGACTCGGCGTGGTTCCCACCCCTCCACCC------------ 238
Cr_Cys_6
CCCCTGCTGTGACACTGCTGACTCGGCGTGGTTCCCACCCCTCCACCC------------ 238
Cr_Cys_7
CCCCTGCTGTGACACTGCTGACTCGGCGTGGTTCCCACCCCTCCACCC------------ 240
************************************************
Ctrl_Cys
TACGAGCAGTGGACGCGGCGGTGGAGCAGGCGCA
CCAGGGGGTGTTCTGGAACC
AGG
GCC 300
Cr_Cys_1
-------------------------------------------------------
GG
GCC 258
Cr_Cys_2
-------------------------------------------------------
GG
GCC 258
Cr_Cys_3
-------------------------------------------------------
GG
GCC 249
Cr_Cys_4
-------------------------------------------------------
GG
GCC 260
Cr_Cys_5
------------------------------------------------------
AGG
GCC 250
Cr_Cys_6
------------------------------------------------------
AGG
GCC 250
Cr_Cys_7
------------------------------------------------------
AGG
GCC 244
*****
Aldh1a1/a2a_Glu
Aldh1a1/a2a_Cys
Mutant
CRISPR
loci
5/5
7/11
Control
CRISPR
loci
5/5
3/3
Supplementary Figure 6
:
Genotyping strategy and sequencing results for
Aldh1a1/a2a
CRISPR embryos.
Cartoon illustrating the
design of primers for amplifyi
ng each site targeted by the
gRNAs
in the
Aldh1a1/a2a
gene loci.
Primer binding sites are indicated in bold purple. For each site
, we show an alignment of mut
ant loci (Cr_) sequences with
the sequence of a control locus (Ctrl), the site targeted by
the gRNA is indicated in bold and the PAM in red. A table
indicating the proportion of mutant CRISPR or c
ontrol CRISPR loci for each site is shown.
Supplementary Figure 7
:
Effect of DEAB concentration and timi
ng of treatment on hindbrain patterning.
(a)
cISH
of key patterning hindbrain markers in em
bryos treated with 10 μM of DEAB at gastr
ulation (st13), 10 μM of DEAB at early
neurulation (st17) or 50 μM of DEAB at early neurulation
(st17). For each gene, the most
representative phenotype is
shown, and numbers of experimental replicates are indicat
ed. These molecular phenotypes obtained for embryos treated
with 10 μM of DEAB at gastrulation (st13)
are less severe than the
ones obtained when treating the embryos with 50 μM
DEAB (
Fig. 5d
) and are reminiscent of the phenotypes obtained in the Cr
Aldh1a1/a2a
mutants (
Fig. 8c
).
(b)
Cartoon
summarizing the effects of differ
ent concentrations and timing of
treatments on hindbrain patterning.
Mouse (
Mus musculus
): Aldh1a1: NP_001348432.1; Aldh1a2: NP_033048.2; Aldh8a1:
NP_848828.1;
Cyp26A1: XP_017173542.1; Cyp26B
1: NP_001171184.1; Cyp26C1:
XP_017173748.1; Cyp51: NP_064394.2
Spotted gar (
Lepisosteus oculatus
): Aldh1a1: XP_015222024.1; Aldh1a2:
XP_006628784.1; Aldh8a1: XP_006625886.1;
Cyp26A1-LG5: XP_015202021.1; Cyp26B1-LG5: XP_015
202022.1; Cyp26B1-LG4: XP_006629157.1;
Cyp51: XP_006636075.1
West indian coelacanth (
Latimeria chalumnae
): Aldh1a1: XP_006001725.2; Aldh1a2: XP_005998797.2;
Aldh8a1: XP_014352052.1; Cyp26A1: XP_005991616.1; Cyp26B1: XP_005997719.1;
Cyp26C1: XP_014341348.1; Cyp51: XP_014350652.1
Whale shark (
Rhincodon typus
): Aldh1a1-like: XP_015222024.1; Aldh1a2: XP_020369553.1;
Aldh8a1: XP_048452951.1; Cyp26A1: XP_020382659.1; Cyp26B1: XP_020378695.1;
Cyp26C1: XP_020367038.1; Cyp51: XP_020371022.1
Inshore hagfish (
Eptatretus burgeri
): Aldh1a1: ENSEBUG0
0000010361; Aldh8a1:
ENSEBUG00000011145;
Cyp26C1 (10538): ENSEBUG0
0000010538; Cyp26C1 (069
09): ENSEBUG00000006909;
Cyp51: ENSEBUG00000006843
Sea lamprey (
Petromyzon marinus
): Aldh1a1/a2a (Chr1): XP_032805864.1;
Aldh1a1/a2b (Ch40): XP_032824682.1; Aldh8a1: XP_032810842.1; Cyp26A1: XP_032808019.1-2;
Cyp26B1/C1a (Chr11): XP_032808017.1; Cyp26B1/
C1b (Chr9): XP_032806163.1; Cyp51: XP_032816590.1
Thorny Skate (
Amblyraja radiata
): Aldh1a1-like: XP_032873441.1; Aldh1a2: XP_032906516.1;
Cyp26A1: XP_032889480.1; Cyp26B1: XP
_032885953.1; Cyp26C1: XP_032889482.1;
Chicken (
Gallus gallus
): Aldh1a1: NP_989908.2; Aldh1a2: NP_001384737.1;
Cyp26A1: NP_001001129.2; Cyp26B1: XP
_015141554.1; Cyp26C1: XP_421678.6;
Chinese soft-shelled turtle (
Pelodiscus sinensis
): Aldh1a1: XP_006137885.1; Al
dh1a2: XP_006112531.1; Cyp26A1:
XP_006113245.1; Cyp26B1: XP_025042348.1;
Cyp26C1: XP_025035575.1;
West African lungfish (
Protopterus annectens
): Aldh1a1: XP_043916765.1; Al
dh1a2: XP_043934839.1; Cyp26A1:
XP_043912621.1; Cyp26B1:
XP_043919907.1; Cyp26C1:
XP_043912622.1;
Reedfish (
Erpetoichthys calabaricus
): Aldh1a1: XP_028658993.1; Al
dh1a2: XP_028678887.1; Cyp26A1:
XP_028651199.1; Cyp26B1:
XP_028657295.1; Cyp26C1:
XP_028651196.1
Amphioxus (
Branchiostoma floridae
): BfAldh1a1/a2a: XP_035683184.1;
BfAldh1a1/a2b: XP_035666435.1;
BfAldh1a1/a2c: XP_035683791.1; BfAldh1a1/a2d: XP_035
683789.1; BfAldh1a1/a2e: XP_035659507.1; BfAldh1a1/a2f:
XP_035659462.1;
Bf
Cyp26-1: LOC118413434; BfCyp26-2: XP_03
5672703.1; BfCyp26-3: XP_035672700.1;
Supplementary Figure 8: Protein accession
numbers used for phylogeny analysis.
Protein sequences
corresponding to each gene were retrieved using both EN
SEMBL and NCBI databases, focusing on the latest available
version of the genome and/or the most complete available genome annotation. Amphioxus
Cyp26
and
Aldh1
gene
families were used as outg
roups for the analyses of
Cyp26
and
Aldh1a
complements.
Mouse (
Mus musculus
):
Aldh1a1
: NM_001361503.1;
Aldh1a2
: NM_009022.4;
Cyp26A1
: XM_017318053.2;
Cyp26B1
: NM_001177713.1;
Cyp26C1
: XM_017318259.3
Grey short-tailed opossum (
Monodelphis domestica
):
Aldh1a1
: XM_001373091.4;
Aldh1a2
: XM_007479682.2;
Cyp26A1
: XM_001375255.4;
Cyp26C1
: XM_007478798.1
Platypus (
Ornithorhynchus anatinus
):
Aldh1a1
: XM_007667019.3;
Aldh1a2
: XM_029070050.1;
Cyp26B1
: XM_029046562.2;
Cyp26C1
: XM_029060242.1
Chicken (
Gallus gallus
):
Aldh1a1
: NM_204577.5;
Aldh1a2
: NM_001397808.1;
Cyp26A1
: XM_015288598.4;
Cyp26B1
: XM_015286068.4;
Cyp26C1
: XM_421678.8
Spotted gar (
Lepisosteus oculatus
):
Aldh1a1
: XM_015366538.1;
Aldh1a2
: XM_006628721.2;
Cyp26A1LG5
: XM_015346535.1;
Cyp26B1-LG5
: XM_015346536.1;
Cyp26B1-LG4
: XM_006629094.2
West indian coelacanth (
Latimeria chalumnae
):
Aldh1a1
: XM_006001663.2;
Aldh1a2
: XM_005998735.2;
Cyp26A1
: XM_005991554.1;
Cyp26B1
: XM_005997657.2;
Cyp26C1
: XM_014485862.1
Whale shark (
Rhincodon typus
):
Aldh1a1-like
: XM_020527364.2;
Aldh1a2
: XM_020513964.2;
Cyp26A1
: XM_048608684.1;
Cyp26B1
: XM_020523106.1;
Cyp26C1
: XM_020511449.1
Sea lamprey (
Petromyzon marinus
):
Aldh1a1/a2a
(Chr1): XM_032949973.1;
Aldh1a1/a2b
(Chr40): XM_032968791.1;
Cyp26A1
: XM_032952128.1;
Cyp26B1/C1a
(Chr11): XM_032952126.1;
Cyp26B1/C1b
(Chr9): XM_032950272.1
Supplementary Figure 9: mRNA accession
numbers used for synteny analysis.
mRNA accession numbers of
Cyp26
and
Aldh1a
used to conduct the synteny analyses in different vertebrate models.
Supplementary Figure 10: comparison of
Cyp26
expression developmental dynamics in different vertebrates.
Schematic representation of the dynamic changes in
Cyp26
expression during early processes of development
(gastrulation-neurulation) and dur
ing hindbrain segmentation in
mouse, zebrafish and
lamprey, illustrating
both similarities
and differences between orthologues.