www.sciencemag.org/cgi/content/full/312/5777/1183/DC1

Supporting Online Material for

Regulatory Blueprint for a Chordate Embryo Kaoru S. Imai, Michael Levine, Nori Satoh, Yutaka Satou*

*To whom correspondence should be addressed.

E-mail: yutaka@ascidian.zool.kyoto-u.ac.jp

Published 26 May 2006, Science 312, 1183 (2006). DOI: 10.1126/science.1123404

The main PDF file includes

Materials and Methods SOM Text Figs. S1 to S9 Tables S1 to S7 References

Other Supporting Online Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/312/5777/1183/DC1)

Database S1 as a zipped archive: The Ciona Integrated Database, GHOST

1

Supporting Online Materials

Materials and Methods

Ascidian eggs and embryos

C. intestinalis adults, obtained from the Maizuru Fisheries Research Station of Kyoto

University, were maintained in aquaria in our laboratory under constant light to induce

oocyte maturation. Eggs and sperm were obtained surgically from gonoducts. Following

insemination, eggs were reared at 18°C in Millipore-filtered seawater (MFSW)

containing 50 μg/ml streptomycin sulfate.

cDNA clones and whole-mount in situ hybridization

Most cDNA clones were obtained from our EST collection (S1). These cDNAs were

basically the same as those examined in our previous study (S2). DIG-RNA probes for

whole-mount in situ hybridization (WMISH) were synthesized by in vitro transcription

with T7 RNA polymerase. The detailed procedure for WMISH has been described (S3).

Hierarchical clustering of blastomeres based on gene expression profiles

The expression of each gene in individual cells was converted into a binary code; if the

gene was expressed, a value of 1 was assigned, while if the gene was not expressed, 0

was assigned. Based on this binary matrix, correlation distance was used for hierarchical

clustering by the UPGMA method.

Gene knockdowns and reconstructing gene regulatory networks

Sequences of the morpholino oligonucleotides (Gene Tools, LLC) are shown in Table S2.

Microinjections were performed as described (S4). Relative quantification of mRNA

levels (qRT-PCR) was performed as described (S2). The majority of the primers used for

quantification were the same as those used previously (S2). Newly designed primers are

listed in Table S6.

The results of qRT-PCR examining the expression of SoxF, BMP3, and EphrinA-d were

not reproducible. Thus, these three genes were not listed in the study. In the qRT-PCR

analyses, genes with greater than a two-fold increase or decrease in mRNA quantity were

2

regarded as exhibiting significant changes in expression. These genes were determined to

be downstream genes in transcriptional networks. Genes with complex expression

patterns were also examined by WMISH (Table S4; Fig. S5; Fig. S6 for controls).

To present biochemical networks, several software packages have been developed. One

such program, Biotapestry, is designed to represent transcriptional networks (S5). Such

programs, however, are not intended for assays performed at the single-cell level.

Therefore, we have expressed our data in a novel fashion in a world-wide-web-based

database illustrating the expression patterns of genes and their regulative relationships at

a single cell level until the early gastrula stage and at a region/tissue level thereafter

(Database S1 or http://ghost.zool.kyoto-u.ac.jp/network/TFSTgenes.html).

3

Supporting Text

Regulatory codes define a pre-pattern in 16-cell embryo

As described in the main text, there are 5 distinct regulatory codes seen for the 8

different blastomeres (paired blastomeres are identical across the left-right axis) in

16-cell embryos (Fig. 1B): anterior animal blastomeres (a-line blastomeres), posterior

animal blastomeres (b-line blastomeres), anterior vegetal blastomeres (A-line

blastomeres; not identical to each other, but very similar), the posterior vegetal

blastomere (B5.1 blastomeres), and the posterior-most blastomere (B5.2). FoxA-a, FoxD,

Tbx6a and Tbx6b display restricted patterns of zygotic expression in 16-cell embryos (Fig.

S1). A signaling gene Fgf9/16/20 also displays a restricted pattern at this stage. These

restricted patterns depend on two localized maternal determinants, β-catenin and macho1,

which define vegetal and posterior fates by activating localized expression of these key

genes, respectively, (S6-S11). The combination of these two molecules could define four

distinct fates (antero-animal, antero-vegetal, postero-animal, and postero-vegetal). The

fifth regulatory code, seen for the posterior-most blastomere, is defined by the absence of

these factors at the 16-cell and 32-cell stages. This blastomere ultimately forms the germ

line, and the general absence of zygotic transcription might be mediated by a number of

localized maternal products encoded by the posterior-end-mark (pem) genes (S3, S12,

S13), or other components of the germ plasm. The regulatory codes seen in the 16-cell

embryo set the stage for inductive interactions that establish the major cell types of the

tadpole: gut, tail muscles, notochord, skin, and CNS. This was also supported by a

previous comprehensive screen showing that all or most localized maternal transcripts are

in the posterior pole like the pem gene transcripts (S13). Altogether, the regulatory codes

seen in the 16-cell embryo set the stage for inductive interactions that establish the major

cell types of the tadpole: gut, tail muscles, notochord, skin, and CNS.

Morpholino Oligonucleotide-based Gene Knockdowns

MOs can produce variable reductions in gene activity, sometimes resulting in

hypomorphic mutations rather than null phenotypes. Brachyury regulation has been

characterized in considerable detail, and thereby provides an opportunity to assess the

4

efficacy of Morpholino Oligonucleotide (MO)-mediated gene disruption. The MOs for

FoxA-a, FoxD, ZicL, and Fgf9/16/20, which are know activators of Brachyury (S6, S9,

S14), abolish Brachyury expression, suggesting a severe reduction in the activities of

these genes. Twist-like1 was also examined in detail in the previous study of a closely

related species, C. savignyi (S15), and the MO for Twist-like1 in C. intestinalis also gave

a similar effect. The Nodal MO we used were designed in the different position from the

MO used in the recent study (S16), and both of the MOs gave a similar result. This

“random” sampling of genes which were previously examined suggest that the MOs are

specific and effective in reducing or eliminating gene function.

We did not find any inconsistent phenotypes among two or more knockdowns

(inconsistent phenotypes mean that Gene A activates B, and B activates C but A

represses C, for example). This means that genes in a common pathway were found to be

related and consistent functions. This fact also supports the specificity of the MOs we

used.

Circuit diagrams for larval tissues

Endoderm (Fig. S8A)

FoxA-a is one of the earliest genes expressed in both the A-line and B-line lineages,

from which all endodermal cells are derived. MO-mediated suppression of FoxA-a

resulted in the complete loss of endoderm-specific alkaline phosphatase (AP) activity

(data not shown) and endodermal expression of GATA-a, SoxB1, and sFRP3/4-b (Fig.

S5A). The synthesis of the current study and earlier work suggests that FoxA-a

coordinates the expression of two regulatory pathways of endoderm differentiation,

through the regulation of Otx and Lhx3 (Fig. S8A). As it is required for AP activity (S17),

Lhx3 likely activates target genes essential for the differentiation of the larval endoderm,

including SoxB1 and sFRP3/4-b, which are downstream of FoxA-a (Fig. S5A) but not of

Otx (Fig. S5H). Lhx3 also works together with FoxA-a to activate Otx, which in turn

regulates GATA-a (Fig. S5A). It is possible that an ensuing cascade of GATA genes

controls the differentiation of the adult endoderm.

FoxA and GATA are used for endoderm differentiation in a broad spectrum of animals,

from C. elegans to vertebrates (S18). In addition, endoderm specification begins with a

5

localized source of β-catenin in nematodes, sea urchins and ascidians. The

Lhx3-dependent pathway seen in ascidian embryos is less well conserved, possibly due to

its specialized role in the differentiation of larval endoderm. Additional variations in the

Fox-GATA core network endow the endoderm with distinctive properties in different

systems. For example, Nodal signaling appears to be essential for initiating endoderm

specification in zebrafish and Xenopus, and possibly other vertebrates.

Tail Muscles (Fig. S8B)

The analysis of the 76 TF and ST genes and 27 MO-induced mutants suggests that

Ciona is the simplest metazoan to employ MyoD as a central myogenic agent, as seen in

vertebrate systems. Ciona has only one myogenic bHLH factor gene related to vertebrate

MyoD, Myf5, myogenin and MRF4 (S19, S20). In Ciona, MyoD coordinates the

expression of target genes required for the differentiation of the tail muscles, including

Otp, SYMD, Mox and a muscle actin gene (Fig. S8B). In contrast, the MyoD homologue

in Drosophila —nautilus—(S21), sea urchin —sum1—(S22) and Caenorhabditis

—hlh1—(S23) are not required for the differentiation of the major muscle groups. Thus,

the use of MyoD-like myogenic factors may be a chordate-specific innovation. There are

other localized regulatory factors present in the early muscle lineages (Fig. S1), but they

do not participate in the regulation of MyoD and myogenesis. For example, snail may be

activated prior to MyoD, but it functions as a transcriptional repressor that prevents the

differentiation of presumptive muscle cells into notochord (S24).

The anterior-most cells appear to develop at least partly in MyoD MO-injected embryos

(Fig. S5O). These cells appeared to arise from the B7.5 blastomeres, which also form the

cardiac mesoderm. The anterior lineage forms the heart primordium, or trunk ventral cells

(TVCs), while the posterior lineage forms the anterior-most tail muscles. It is conceivable

that myogenic agents controlling heart differentiation are able to mask the loss of MyoD

in the anterior tail muscles. Or, the MyoD MO we used might produce a hypomorphic

phenotype rather than a null phenotype.

Heart (Fig. S8C)

6

Cardiomyocyte differentiation is controlled by a highly conserved, interlocking cassette

of regulatory genes that include Nkx2.5 and Gata-4. The deployment of this network has

not been clearly elucidated in vertebrates. The analysis of early heart specification in

Ciona suggests that a bHLH regulatory gene, Mesp, plays an early role in the

specification of the heart field, prior to the onset of Nkx2.5 expression (Fig. S8C) (S25).

The function of Mesp is conserved in vertebrates (S26), but not in flies (S27), suggesting

that the establishment of the heart field may be different in flies and chordates.

B7.5 arises from the posterior-most blastomere, B6.3, of the 32-cell embryo. This cell is

unusual in that it is transcriptionally silent at the 16-cell and 32-cell stages when there is

intense zygotic expression of different patterning genes in all other blastomeres (Fig. S1).

After the division of B6.3 into B7.5 and B7.6, the B7.5 blastomere begins to express

Mesp at the time when it is specified to form cardiac mesoderm (or the heart field) and

the anterior-most tail muscles. Injection of a Mesp MO resulted in the loss of expression

of Nk4 (Nkx2.5), Hand, NoTrlc (Hand-like bHLH gene), FoxF, and tolloid in the TVCs

(Fig. S5U).

Knockdown of Fgf9/16/20 eliminated the expression of NoTrlc, FoxF, and Tolloid, and

caused diminished expression of Mesp (Fig. S5D). Fgf9/16/20 may be required to

maintain proper levels of Mesp, but not for initial activation. Indeed, there is evidence

that Tbx6b (and Tbx6c) are essential activators of Mesp expression (S28). It is possible

that the loss of NoTrlc, FoxF, and tolloid expression in FGF9/16/20 MO-injected

embryos might result from “sub-threshold” levels of the Mesp activator. This hypothesis

is supported by the fact that knockdown of β-catenin also reduced, but did not eliminate,

the expression of Mesp (S25), because Fgf9/16/20 is controlled by β-catenin.

Alternatively, Mesp and FGF9/16/20 work in parallel to co-regulate certain target genes

like FoxF and Tolloid.

Notochord

A-line (Fig. S8D): As described in the main text in detail, the prepattern seen in 16-cell

embryos establishes the presumptive notochord through the activation of Brachyury

expression.

7

B-line (Fig. S8E): Brachyury is also activated in the B8.6 blastomere, which generates the

secondary notochord cells located in posterior regions of the tail. This activation depends

on FoxA-a and FGF9/16/20 signaling (Fig. S5A, D), as seen for expression in A-line

blastomeres. However, ZicL is required only for activation in the A-line (S14, S29). It

appears that Notch signaling takes the place of the ZicL activator in B8.6 (S9, S30). This

blastomere arises from B7.3, which undergoes an asymmetric division to form B8.6 and

B8.5. B8.6 is in direct physical contact with the A7.6 mesenchyme cells. A7.6

specifically expresses a Delta-like Notch ligand gene, which might induce B8.6, but not

B8.5 (supporting online text), to express Brachyury through Suppressor of Hairless

{Su(H)} binding sites present in the Brachyury enhancer (S30).

Nerve cord (Fig. S8F)

Nerve cord cells are specified dorsoventrally by the Nodal network as described in the

main text. Nodal activates TF and ST regulatory genes within the dorsal-most ependymal

cells, the roof cells, including Msxb, Pax3/7, snail and Chordin and also induces TF and

ST genes within lateral ependymal cells (A8.15 and A8.16 lineages), including Snail,

Delta-like, Neurogenin and E(spl)/Hairy-b (Fig. S5G). Among the latter genes,

Neurogenin and E(spl)/Hairy-b might require peak thresholds of Nodal signaling, and

consequently they are restricted to the A8.16 lineage due to sustained contact with the

Nodal-expressing b-line cells. Localized repressors help maintain distinct patterns of

gene expression in the floor plate and lateral ependymal cells (Fig. S8F). For example,

Snail keeps Mnx expression off in lateral ependymal cells (Fig. S5R), thereby restricting

expression to the floor plate. Conversely, FGF8/17/18, Cdx, and Pax6 are restricted to

lateral ependymal cells through the action of the FoxB repressor in the floor plate (Fig.

S5L).

Brain (Fig. S8F)

The brain, or cerebral vesicle, is located in a dorsal position of the larval trunk or head. It

receives sensory inputs from the associated otolith (gravity) and ocellus (light). Brain

specification begins with the localized activation of the Otx gene by FGF9/16/20

signaling at the 44-cell stage (S31). Otx participates in the activation of downstream TF

8

genes, together with DMRT1, which is independently activated by Fgf9/16/20 signaling

and FoxA-a. Otx and DMRT1 constitute a feed-forward regulatory circuit, leading to

localized expression of TF genes including Six1/2 and Six3/6 (Fig. S5H, J).

Mesenchyme

B-line (Fig. S8G): B-line mesenchyme cells are derived from B7.7 and B8.5 blastomeres

of the early gastrula. In these cells, Fgf9/16/20 induces expression of Twist-like1, which

triggers the specification of mesenchyme (S14). In concert with FGF signaling, Otx was

also required cell-autonomously for the activation of Twist-like1 (Figs S4 and S5H). Otx

is autonomously expressed in presumptive mesenchyme cells and/or their ancestral

blastomeres in a manner independent on FGF signaling (S31; Table S3). Although B7.7

and B8.5 exhibited very different TF-codes, both developed the expression of Otx; the

mechanism of Otx activation appeared to be similar in these cells. The expression of Otx

is under the control of β-catenin (S17) and macho1 (S32). Detailed analysis of the Otx

promoter (S31, S33) suggests the involvement of a T-box protein as well.

A-line (trunk lateral cells; TLCs) (Fig. S8H): Twist-like1 also plays a critical role in the

differentiation of the A-line mesenchyme. A previous study demonstrated that the

upstream regulatory mechanisms, however, are different from those functioning in B-line

mesenchyme (S15). In addition to FGF9/16/20 signaling and Otx activation, FoxD and

NoTrlc were both required for the activation of Twist-like1 in this lineage. The expression

of Otx and NoTrlc was under the regulation of FoxA-a. NoTrlc is also regulated by Nodal

(Fig S5G), which is likely the molecular foundation for an observation in Halocynthia

that TLC differentiation requires induction by cells in the animal hemisphere (S34). In

this cell lineage, the combination of Otx, NoTrlc, and FoxD appears to establish the

competence for response to FGF signaling by adopting the mesenchymal fate.

Fgf8/17/18 and MYTF were specifically expressed in A7.6 blastomeres at the 64-cell

and early gastrula stages. Expression of these genes is controlled by FoxD and

Neurogenin (Figs S4, S5B, S5Q). Delta-like is also expressed in these cells under the

control of FoxD and Neurogenin (Figs S4, S5B, S5Q), although this gene is also

expressed in A-line lateral nerve cord cells (A8.15 and A8.16) and b-line cells expressing

9

Nodal (b8.17, b8.18, b8.19 and b8.20). Expression of Delta-like in these cells was

regulated by Nodal (Figs S4, S5G), which may instruct neighboring B8.6 blastomeres to

become notochord.

Epidermis (Fig S8F)

Non-neuronal a- and b-line cells differentiated into the anterior and posterior epidermis,

respectively. Dll-B regulates the expression of most genes expressed in the epidermis,

although the upstream gene regulating Dll-B has not been identified. FoxA-a is important

in the determination of the antero-posterior boundary in the epidermis; MO knockdown

of FoxA-a resulted in the up-regulation of Emx, which is normally expressed in b-line. It

also causes and the down-regulation of sFRP1/5, which is normally expressed in a-line

epidermal cells. Epidermal cells are regionalized by distinct TF-codes into at least six

territories at the tailbud stage (Fig 2C). At least three of these territories, the

anterior-most epidermis surrounding the palp, the dorsal midline epidermis, and the

posterior ventral epidermis, contain developmentally distinct cell populations. Specific

peripheral neuronal cell types are derived from each territory. Differential gene

expression in the epidermal territories is first detected in 64-cell embryos, and later

stages.

The epidermis along the dorsal midline is derived from b8.18 and b8.20 blastomeres at

the early gastrula stage, and a part of these cells give rise to the dorsal epidermal tail

sensory neurons. Nodal activates Msxb within the dorsal midline (Fig S5G), and Msxb is

essential for the differentiation of the midline since MO disruption caused the

down-regulation of genes that are normally expressed in the dorsal midline (ash2,

CAGF9, Dll-C, and ZF-(C2H2)-24) and the up-regulation of SoxB2, which is not

normally expressed in these cells (Fig S5T).

The epidermis along the ventral midline is derived from a-line and b-line epidermal

cells of early embryos (compare Fig 1A with Fig 2B). A portion of the b-line ventral

epidermal cells also give rise to the ventral epidermal sensory neurons. ADMP is

expressed in a subset of B-line vegetal cells near the midline of early embryos and in the

a-line epidermal cells of later embryos. ADMP expressed in vegetal cells may induce the

differentiation of animal cells (at least in b-line) along the midline of early embryos.

10

Figure Legends

Fig. S1. Expression profiles of transcription factor genes in each blastomere at the 16-cell,

32-cell, 64-cell, and early gastrula stages. Expression of each gene is represented by a red

rectangle.

Fig. S2. Hierarchical clusterings of blastomeres based on regulatory-codes at (A) the

32-cell stage and (C) 64-cell stage. The developmental potentials of each cell are shown

with the same color codes as in Fig. 1.

Fig. S3. The expression pattern of transcription factor and signaling molecule genes (A)

at the late gastrula stage and (B) at the tailbud stage. In each panel, a photograph detailing

the results of the in situ hybridization is shown at the left. A schematic representation of

expression is shown at the right.

Fig. S4. Graphs representing the results of qRT-PCR analyses at the early gastrula.

Above each panel, the knocked-down genes are indicated. The bar in each graph

represents the change in the amount of gene transcript with a logarithm assuming two as

the bottom. Values exhibiting a greater than two-fold change, either an increase or

decrease, are shown in orange and light blue bars, respectively. Values exhibiting a

greater than four-fold change, either an increase or decrease, are shown as four-fold

change for simplicity and in red and blue bars, respectively. Each bar in each panel

represents transcription factor and signaling molecule genes as shown in the top.

Fig. S5. Effects of gene knockdown. Knocked-down genes are indicated at the left of

each panel. The genes examined by WMISH are designated above each photograph. The

amount of expression lost in each experimental embryo is shown by blue arrowheads,

while ectopic expression is indicated by red arrows. The examined developmental stages

are shown at the top right of each photograph; 64, 64-cell stage; eG, early gastrula stage;

lG, late gastrula stage; TB, tailbud stage; AR, arrested at the 110-cell stage with

cytochalasin B and fixed at a point equivalent to the late gastrula stage. The orientation of

11

each embryo is shown in the left bottom of each photograph; vg, vegetal view; an, animal

view; dor, dorsal view; lat, lateral view; vent, ventral view.

Fig. S6. Controls for whole-mount in situ hybridization experiments. The examined

developmental stages are shown at the right top of each photograph; 64, 64-cell stage; eG,

early gastrula stage; lG, late gastrula stage; TB, tailbud stage; AR, arrested at the 110-cell

stage with cytochalasin B and fixed at a point equivalent to the late gastrula stage. The

orientation of each embryo is shown at the bottom left of each photograph; vg, vegetal

view; an, animal view; dor, dorsal view; lat, lateral view; vent, ventral view.

Fig. S7. Graphs representing the results of qRT-PCR analyses at the late gastrula. Above

each panel, the knocked-down genes are indicated. The bar in each graph represents the

change in the amount of gene transcript with a logarithm assuming two as the bottom.

Values exhibiting a greater than two-fold change, either an increase or decrease, are

shown in orange and light blue bars, respectively. Values exhibiting a greater than

four-fold change, either an increase or decrease, are shown in red and blue bars,

respectively and as four-fold change for simplicity. Each bar in each panel represents

transcription factor and signaling molecule genes as shown in the top.

Fig. S8. Simplified schematic representations of epistatic relationships among

transcription factor and signaling molecule genes in differentiation of Ciona larval tissues.

Genes are represented in the order of their expressions from the left (eggs) to the right

(tailbud embryos). Arrows indicate transcriptionally regulatory interactions. Black and

light blue arrows indicate regulatory interactions by transcription factor and signaling

molecule genes. The flat-head arrows indicate repression and the dotted arrows indicate

possible interactions that have not yet been proven definitely. Genes enclosed by red lines

are expressed in the differentiated tissues, and genes other than transcription factor and

signaling molecule genes are included among these genes enclosed by red lines. In (A),

(C) and (F), gene regulatory networks in other animals indicated are shown in grey boxes

for comparison. Yellow lines in (F) show cell boundaries. Note that these representations

12

are simplified for better understanding and include results previously published. See the

text for the details.

Fig. S9. Expression patterns of (A) Delta-like and (B) Pax3/7 during embryogenesis. (A)

Delta-like was not expressed maternally. Expressed began in b7.9 and b7.10 at the 64-cell

stage. At the early gastrula stage, Delta-like was expressed in b8.17, b8.19, A7.6, A8.15,

and A8.16 blastomeres. This gene continues to be expressed in the nerve cord lineage at

the late gastrula, neurula, and tailbud stages. (B) Pax3/7-like is first expressed in

decendents of b8.17, b8.18, b8.19, and b8.20 at the late gastrula stage. This gene is also

expressed in A9.32 and a9.49 blastomeres. Neural expression continues until the tailbud

stage.

Database S1. Unzip the archive file named 1123404DatabaseS1.zip and place the

unzipped files in your local hard disk. Open the file named as ‘TFSTgenes.html’ using a

web browser. This database is best browsed with Netscape ver6 or later or Firefox but

can be browsed with Internet explorer. The same database can be browsed in

http://ghost.zool.kyoto-u.ac.jp/network/TFSTgenes.html.

AP-2

-like

2Br

achy

ury

Dll-

BD

MRT

1E(

spl)/

hair

y-a

E(sp

l)/ha

iry-

bE1

2/E4

7EL

KEm

cEt

s/po

inte

d2Fl

i/ERG

1Fl

i/ERG

3Fo

sFo

xA-a

FoxB

FoxC

FoxD

-a/b

FoxH

-bFo

xPG

ATA-

bH

NF4

Irx-

BJu

nLh

x3M

esp

Mnx

msx

bM

yTF

Myo

DN

euro

geni

nN

oTrl

cO

rpha

n Fo

x-2

Otp

Otx

PPAR

RAR

ROR

SYM

D1

Snai

lSo

xB1

SoxB

2So

xCSo

xFTb

x2/3

Tbx6

aTb

x6b/

c/d

TTF1

TWIS

T-lik

e-1a

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cL

Zinc

Fin

ger (

C2H

2)-2

5Zi

nc F

inge

r (C2

H2)

-2

Zinc

Fin

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C2H

2)-3

4Zi

nc F

inge

r (C

3H)

A5.1A5.2a5.3a5.4B5.1B5.2b5.3b5.4

A6.1A6.2A6.3A6.4a6.5a6.6a6.7a6.8B6.1B6.2B6.3B6.4b6.5b6.6b6.7b6.8

A7.1A7.2A7.3A7.4A7.5A7.6A7.7A7.8a7.9a7.10a7.11a7.12a7.13a7.14a7.15a7.16B7.1B7.2B7.3B7.4B7.5B7.6B7.7B7.8b7.9b7.10b7.11b7.12b7.13b7.14b7.15b7.16

16-c

ell s

tage

32-c

ell s

tage

64-c

ell s

tage

A7.1A7.2A8.5A8.6A8.7A8.8A7.5A7.6A8.13A8.14A8.15A8.16a8.17a8.18a8.19a8.20a8.21a8.22a8.23a8.24a8.25a8.26a8.27a8.28a8.29a8.30a8.31a8.32B7.1B7.2B8.5B8.6B8.7B8.8B7.5B7.6B7.7B8.15B8.16b8.17b8.18b8.19b8.20b8.21b8.22b8.23b8.24b8.25b8.26b8.27b8.28b8.29b8.30b8.31b8.32

early

gas

trula

stag

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Figure S1

B7.6

A7.5A7.1A7.2B7.1B7.2A7.3A7.7A7.4A7.8B7.3B7.7B7.4B7.8B7.5

b7.9b7.10

A7.6a7.13a7.9a7.10

a7.14a7.16a7.15a7.11a7.12b7.14b7.11b7.13b7.16b7.12b7.15

B6.1A6.1A6.3A6.2A6.4B6.3b6.8b6.7b6.6b6.5a6.8a6.7a6.5a6.6B6.2B6.4

AB

32-cell stage64-cell stage

Figure S2

endoderm

notochordmuscle

mesenchyme

trunk ventral cell

neural cellsepidermis

nerve cord

MYTF

Fgf8/17/18

FoxC

Lmx

Pax6

Cdx

Neurogenin

COE

Snail

Mnx

FoxB

ZF-C2H2-33

A

ZicL

Pax3/7

Emc

Msxb

Chordin

NK4 (lateral view)

SoxB2 (dorsal view)

ZF(C2H2)-2 (lateral view)Ach2 (dorsal view)

Irx-C (ventral view) ZF(C2H2)-24 (dorsal view)

(ventral view)

msxb (ventral view)

(dorsal view)

CAGF9 (dorsal view)

(ventral view) Dll-C (dorsal view)

B

FoxH-a (dorsal view)TB

dor

Figure S3

FoxA

-aAD

MP

AP-2

-like

2Br

achy

ury

dick

kopf

Dll-

BD

MRT

1et

s/po

inte

d2Fg

f9/1

6/20

Figure S4-1

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

AD

MP

AP

-2-li

ke2

BM

P2/

4B

rach

yury

chor

din

Del

ta-li

kedi

ckko

pfD

ll-B

DM

RT1

DU

SP

1.2.

4.5

E12

/E47

E(s

pl)/h

airy

-aE

(spl

)/hai

ry-b

ELK

Em

cE

ph1

Eph

rinA

-cet

s/po

inte

d2FG

F8/1

7/18

FGF9

/16/

20Fl

i/ER

G1

Fli/E

RG

3Fo

sFo

xA-a

FoxB

FoxD

FoxH

-bFo

xPFz

4G

ATA

-bH

edge

hog1

HN

F4Irx

-Ble

fty/a

ntiv

inLh

x3M

nxm

sxb

Myo

DM

YTF

Neu

roge

nin

noda

lno

ggin

NoT

rlcO

rpha

n Fo

x-2

Orp

han

Wnt

eO

tpO

txP

PAR

RA

RR

OR

SM

YD

1sF

RP

1/5

Sna

ilS

OC

S1/

2/3/

CIS

Sox

B1

Sox

B2

Sox

CTb

x2/3

Tbx6

aTb

x6b/

c/d

TGFβ

not

ass

igne

d 1

Tollo

idTT

F1TW

IST-

like-

1a/b

Wnt

5ZF

(C2H

2)-2

ZF (C

2H2)

-25

ZF (C

2H2)

-34

ZF (C

3H)

ZicL

snai

lFo

xBFo

xD-a

/bLe

fty/a

ntiv

inM

sxb

Myo

DN

euro

geni

nN

odal

Otx

NoT

rlc

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

Figure S4-2

SoxC

Tbx6

b/c/

dTb

x2/3

Twis

t-lik

e1a/

bw

nt5

ZicL

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

-2

-1

0

1

2

Figure S4-3

Brachyury Chordin DMRT1 Eph1 FoxB Lhx3 Mnx

NoTrlc Nodal

Brachyury Chordin DMRT1Dll-B Fgf8/17/18 FoxB Mnx

NoTrlc

Chordin Lhx3 MyoDMnx

Snail

Cdx Lmx

Brachyury Chordin DMRT1 FoxB Mnx MyoD Nodal ZicLMsxb MYTF

NoTrlc

Otx TTF1 ZicL

Emx MYTF

Twist-like1 Twist-like2sFRP1/5

GATA-a SoxB1 sFRP3/4-b

Nodal Wnt5MYTF Neurogenin Twist-like1 ZicL

MYTF Pax6

ZF(C2H2)-33 Meis Six1/2 Six3/6

NodalCdx FoxCFgf8/17/18 MYTF Neurogenin Pax6 Snail

AP2-

like2

MO

SoxC

MO

Fgf9

/16/

20 M

OZi

cL M

OFo

xD-a

/b M

OFo

xA-a

MO

emc

eG 64

64

64 6464

64

64

64

64 64

64eG

eGeG

eG eG

eGeG

eG lG lG lG

lG lGlG lG lG lG lG

lG

eG eG eG eG eG eG

eG

eG

eG

eG

eG

eG

lG

eG

eG

eG

TB

TB

TB

TB TB

TB TB

eG 64 64

eG eG lG

lG

lG

lG lG lG

lG lG

lG

lG

AR

AR

vg vg an vg

vg vg vgvg vg vg

vg

vg

vg

vg vg vg vg vg

vg vg

vg

vg

vg vg vg

vg vg an vg vg vg vg vg vgvg vg

anvg vg vg vg vg vg vg

vg

vg vg vg

vg

vg vgvgvg

vg vg vg

vg vg vgvgvgvg

vgan

vgvgvg

Delta-like

Delta-like Fgf9/16/20

Delta-like

eG

eG

64

eG

AR

an

an

eG

vg

vg vg

Figure S5-1

A

E

F

D

C

B

Msxb

Nod

al M

O eG

vg

MYTFlG

vg

NeurogeninlG

vg

SnaillG

vg

Pax6lG

vg

emclG

vg

LmxlG

vg

FoxClG

vg

NoTrlceG

vg

Delta-likeeG

vg

G

TB

dor

Mnx

Twis

t-lik

e1 M

O

Snai

l MO

FoxB

MO

NoT

rlc

MO

Myo

D M

O

Tbx6

b M

O

Neu

roge

nin

MO

ADM

P M

OM

sxb

MO

Mes

p M

O

FoxC

MO

Ets/

Poin

ted2

MO

DM

RT1

MO

Dll-

B M

OO

tx M

O

NoTrlc TolloidFoxF ZF(C2H2)-2

MYTF

FoxClG

vg

Lhx364

vg

MYTFlG

vg

Twist-like1eG

vg

SMYD1eG

vg

MyoDeG

vg

eG

vg

MYTFeG

vg

Fgf8/17/18eG

vg

Nodal64

vg

Mnx64

vg

FoxClG

vg

FoxClG

vg

lG

vg

MnxlG

vg

Fgf8/17/18lG

vg

Pax6lG

vg

CdxlG

vg

Twist-like1AR

vg

TTF1eG

vg

FoxH-aTB

Six3/6TB

ZF(C2H2)-24TB

MYTFlG

vg

HlxTB

Hox4TB

TBTBTB TB

ZF(C2H2)-24TB

vent

IrxCTB

vent

dor

SoxB2TB

dor

Nk4TB

vent

TB

vent

Dll-CCAGF9TB

vent

COETB

dor

MoxTB

lat

Muscle actinTB

lat

TB

lat

Ash2TB

dor

Dll-CTB

dor

CAGF9TB

dor

ZF(C2H2)-24TB

lat

lat latlatlat

Six3/6TB

lat

Six1/2TB

dor

MeisTB

dor

Six1/2TB

dor

Six1/2TB

dor

MeisTB

dor

Six3/6TB

dor

GATA-aTB

lat

SoxB1TB

lat

sFRP3/4-bTB

lat

eG

vg

Delta-like

Twist-like1eG

vg

Figure S5-2

K

VU

T

S

RQ

PON

ML

J

I

H

Brachyury Dll-B DMRT1

Eph1 Fgf8/17/18 Lhx3 Mnx MyoD

Nodal Otx

Twist-like2 Wnt5

Emx

sFRP1/5

FoxB

NoTrlcMYTF

ZicL

Chordin Delta-like

Fgf9/16/20

Snail SYMD TTF1

Twist-like1

eG

vg

64

vg

64

vg

64

vg

64

vg

64

vg

64

vg

64

vg

64 64

vg vg

64

an

eG

vg

eG

vg

eG

vg

eG

vg

eG

vg

eG

vg

eG

vg

eG

vg

lG

lG

an

eG

vg

eG

vg

eG

vg

eG

vg

eG

vg

eG

vg

eG

vg

eG

vg

eG

vg

eG

vg

eG eG

vgvg

eG

an

vg vg

AR AR

Six3/6 Six1/2 Meis COE Mox Muscle actin

FoxF Tolloid NoTrlc GATA-a SoxB1 sFRP3/4-b

Hox4 Hlx

TB

lat

TB

lat

TB

lat

TB

lat

TB

lat

TB

lat

TB

lat

TB

lat lat

TB

lat

TB

lat

HexTB

lat

TB

dor

TB

dor dor

TB

TB

A

B C

D E

F

Figure S6

vg

-2-1012

-2-1012

-2-1012

-2-1012

Figure S7-1AD

MP

AP-2

-like

2Br

achy

ury

dick

kopf

Dll-

BD

MRT

1et

s/po

inte

d2Fo

xA-a

Fgf9

/16/

20

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

AD

MP

AP

-2-li

ke2

BM

P2/

4B

MP

5/7-

like

Cdx

chor

din

CO

ED

elta

-like

dick

kopf

Dll-

BD

MR

T1E

mc

Em

xFG

F8/1

7/18

FGF9

/16/

20Fl

i/ER

G1

Fos

FoxA

-aFo

xBFo

xCFo

xD-a

/bG

ATA

-bH

exJu

nLA

G1-

like

5M

esp

Mis

tM

nxm

sxb

Myo

DM

YTF

Neu

roge

nin

noda

lN

oTrlc

Otp

Otx

Pax

3/7

Pax

6R

AR

SM

YD

1sF

RP

1/5

Sna

ilS

OC

S1/

2/3/

CIS

Sox

CTb

x6a

Tbx6

b/c/

dTW

IST-

like-

1a/b

TWIS

T-lik

e-2

Wnt

5ZF

(C2H

2)-3

3Zi

cL

FoxB

FoxD

-a/b

FoxC

Lefty

/ant

ivin

Mes

pM

sxb

Myo

DN

euro

geni

nN

odal

NoT

rlc

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

Figure S7-2

SoxC

Tbx6

b/c/

dTb

x2/3

Twis

t-lik

e1a/

bw

nt5

ZicL

Otx

snai

l

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

-2-1012

Figure S7-3

Figure S8

G

β-catenin

macho1

T-box gene (not identified)

Fgf9/16/20

Otx

Twist-like1

Twist-like2HexMistFli/Erg1HlxFosHox4LAG1-like5

B-line mesenchyme

H

β-catenin

Fgf9/16/20

Otx

Twist-like1

Twist-like2

Hex

Fli/Erg1

A-line mesenchyme

FoxA

FoxD NoTrlc

Nodal

Dll-B

Dll-B

SoxB2

EmxFoxH-aSOCS1/2/3GATA-b

ADMP

ZF(C2H2)-24Dll-CCAGFAsh2

Irx-CNK4

ventral midlinetail epidermis

ventral midlinetrunk epidermis

lateral part of epidermis

FoxC

Otx

DMRT1

ZF(C2H2)-2

Six1/2Six3/6

Meis

palps

Brain

ZicL

MYTF

COE

ZicL

Fgf9/16/20

Delta-like

Chordin

Fgf8/17/18

LmxNeurogenin

Snail

SoxC

Mnx

A8.15-A8.16-line (lateral) nerve cord

A8.7-A8.8-line (ventral) nerve cord(floor plate)

Pax6

Pax6

FoxA-a

FoxA-a

FoxDFoxB

Fgf8/17/18

Mnx

Cdx

Cdx

FoxC, MYTF

Nodal

Nodal

SnailPax3/7

Nodal

b-line (dorsal) nerve cord (roof plate)

Delta-like Chordin

dorsal midline epidermis

ZF(C2H2)-24Dll-CCAGFAsh2

Msxb

ZicL

SoxB2

β-catenin

F

SoxC

muscle

macho-1 Tbx6b

ZicL

MyoD

mox

SYMD

otpsnail

Brachyury

B

β-catenin FoxA-a Otx GATA-a

Lhx3 Alkaline phosphataseSoxB1sFRP3/4-b

larval endoderm

adult endoderm?A

TVCs

β-catenin

heartmacho1 Tbx6b

Fgf9/16/20

Mesp

Hand

NoTrlc/Hand-like

NK4

Tolloid

FoxF

C

FoxAβ-catenin

Fgf9/16/20

FoxD

BrachyuryZicL

tropomyosin -likeprickle

netrin..........

A-line notochordD

Nodal

FoxAβ-catenin Fgf9/16/20

FoxD

Brachyury

delta-like

tropomyosin -likeprickle

netrin..........

B-line notochordE

Figure S9

tailbud(dosal view)

tailbud(lateral view)

lategastrula

earlygastrula

64-cellstage

32-cellstageA

B

Del

ta-li

kePa

x3/7

27

Table S1. Genes examined in this study

* No primer pairs specific enough for qRT-PCR experiments were obtained for these

genes.

Gene Name Number of genes

Genes expressed at and before the early gastrula stage Genes encoding transcription factors

AP-2-like2, Brachyury, Dll-B, DMRT1, E(spl)/hairy-a, E(spl)/hairy-b, E12/E47, ELK, Emc, ets/pointed2, Fli/ERG1, Fli/ERG3, Fos, FoxA-a, FoxB, FoxC, FoxD-a/b, FoxH-b, FoxP, GATA-b, HNF4, Irx-B, Jun, Lhx3, Mesp, Mnx, msxb, MyTF, MyoD, Neurogenin, NoTrlc, Orphan Fox-2, Otp, Otx, PPAR, RAR, ROR, SYMD1, Snail, SoxB1, SoxB2, SoxC, SoxF*, Tbx2/3, Tbx6a, Tbx6b/c/d, TTF1, TWIST-like-1a/b, ZicL, ZF(C2H2)-2, ZF(C2H2)-25, ZF(C2H2)-34, ZF(C3H)

53

Genes encoding signal transduction molecules ADMP, BMP2/4, BMP3*, chordin, Delta-like, dickkopf, DUSP1.2.4.5, Eph1, EphrinA-c, EphrinA-d*, FGF8/17/18, FGF9/16/20, Fz4, Hedgehog1, lefty/antivin, nodal, noggin, Orphan Wnt e, sFRP1/5, SOCS1/2/3/CIS, TGFβ not assigned 1, Tolloid, Wnt5

23

Genes expressed at the late gastrula stage Genes encoding transcription factors

AP-2-like2, Cdx, COE, Dll-B, DMRT1, Emc, Emx, Fli/ERG1, Fos, FoxA-a, FoxB, FoxC, FoxD-a/b, GATA-b, Hex, Jun, LAG1-like5, Mesp, Mist, Mnx, msxb, MyoD, MYTF, Neurogenin, NoTrlc, Otp, Otx, Pax3/7, Pax6, RAR, SYMD1, Snail, SoxC, Tbx6a, Tbx6b/c/d, TWIST-like-1a/b, TWIST-like-2, ZF (C2H2)-33, ZicL

39

Genes encoding signal transduction molecules ADMP, BMP2/4, BMP5/7-like, chordin, Delta-like, dickkopf, FGF8/17/18, FGF9/16/20, nodal, sFRP1/5, SOCS1/2/3/CIS, Wnt5 12

Genes expressed at the tailbud stage Genes encoding transcription factors

Achaete-Scute a-like2, CAGF9, Dll-C, FoxH-a, Hlx, Hox4, Irx-C, Msxb, NK4, SoxB2, ZF(C2H2)-2, ZF(C2H2)-24, Six1/2, Six3/6, Twist-like2, Meis, COE, Mox, FoxF, NoTrlc, GATA-a, SoxB1

22

Genes encoding signal transduction molecules Tolloid, sFRP3/4-b 2

28

Table S2. Morpholino oligonucleotides used in this study.

Gene Sequence ADMP TATCGTGTAGTTTGCTTTCTATATA AP-2-like2 CAACGCTGCCTGCCTTGATCCTCGG BMP2/4 AGAAAACCGCTGGATTTTATTGTGA BMP3 TTAAGGTTTTACTGAGTCTCATTAT Brachyury ACGTCATTGTAGGTTTGTAACTCGC dickkopf CAGCAACATCGCGCTTCAACTACAG Dll-B TCGGAGATTCAACGACGCTTGACAT DMRT1 CTGTTTGCTATAATTTCTGTAACTC DUSP1.2.4.5 CAGGAGATAGGTTGGCTCACCATTT E(spl)/hairy-a TTCTTCGTTCAACAGGCATGATTGT E(spl)/hairy-b TTAAGAAGCAGCAGAAAAGTAATTC E12/E47 AACTGTTACATTAAAAACCTTAAAC ELK CATCATTTCCAACAGATAAGCAATA Emc CAACTTTAACCATTTTGCTGATTCT Eph1 TAATCCATCGTAACAAATATACAGA EphrinA-c GAGCGGATCATGGTGTAGGTTTCAA EphrinA-d TTGAGTTGCCATTCTTCGTTTTAAT ets/pointed2 TACCATGTTTCTGAAGCGATGCTTT FGF8/17/18 CATTTTCGTATGTAATCCAAGAGAA FGF9/16/20 CATAGACATTTTCAGTATGGAAGGC Fli/ERG1 TTAACGCTGCTCAACATATTCCTGG Fli/ERG3 ACAGTTTGCGTCATGGCGAATCGGC Fos GATACATTCCTCCTAGAGTTTTGCA FoxA-a ATCCGATTTCAAAAGCTTTCTCAGA FoxB GTCCTGGTCGTGGCATTTTTAATTC FoxC CATTGTCATTATAGAGAATCAAACC FoxD-a/b GCACACAACACTGCACTGTCATCAT FoxH-b TCGGCGGCATCGGAGGGACGAACTC FoxP TTCGTGGACAGCCTTGCGCATTCTC Fz4 GTCTAATCTCCATGTCCAATTATAC Hedgehog1 TTTCAGCCTTATACATTGATATCTA HNF4 CATTCCGTAGGTGAAGGCGCCGATG Irx-B TTAGATAGACCGTGTCTTGATTCCG Jun AGAGGCGTGAGACAGATAGTAGGTA lefty/antivin CATTATTATTGAATGTTCTAGATTT Lhx3 GCTTTAGTATCGAGAATCATTTTAA Mesp CATAATACAAGTTTCAAATCAACCT Mnx CATTTTAAATCTTAAATATCAAAGG

29

msxb ATTCGTTTACTGTCATTTTTAATTT MyoD GCTCCTCTAGAGAGATACACGTCAT Neurogenin AAATCCAACATTTTGTAGCAAGAGC nodal TCGAATATTTTGTAACTTCCGGTTT noggin CATTTCGAATAATTTTAACCGAATT NoTrlc ACTGTTGTCATGTTAAGCTACTGTG Orphan Fox-2 GTAGTTGGAGTCTTACCTAATACAT Orphan Wnt e CATTCCAAATCCTTATCCTCTTTAA Otx CATGTTAGGAATTGAACCCGTGGTA PPAR AAAATAGATATGTAGTAATAAACTA RAR CAACCGTTTCCATTTTCAAAACCAA ROR CATGTTTTAATACAGGTGGAAATGA sFRP1/5 TTTATACATGAGAACTAAACCAAGA Snail GTCATGATGTAATCACAGTAATATA SOCS1/2/3/CIS CATTTCTCATTTATTTCCTTACAAC SoxB1 AACATGAAGTCGTTCTGAGATGGCT SoxB2 CATAATAACGTGTTAGAATATCTTG SoxC TTGATGTACTTGCCATAGCTGAACT SoxF CATTTTCGATTCTACAGTGACGCCT SYMD1 CAACTCCAGGATAAGACATTGTAAT Tbx2/3 GAGGTCCACACCAACACTTTAACAT Tbx6a CATTTGTAATTCCACTTCAGTGTTC Tbx6b/c/d TTACAATTTCCTCTCTCTTTCGATT TGFβ not assigned 1 CATCTTTAACCCAACACTTTCAACG TTF1 CATCTCACAGCAAAGTCTCCAGTGT Twist-like1a/b AAAGTAAGTGACGTTTGGTTTGCTA Wnt5 CATATTTCCGGCAACGATTCAAACT ZF (C2H2)-2 CTATCAAGTCCTATATTAACCTCTA ZF (C2H2)-25 GGATATGTAATTGTACATTGCTTGT ZF (C2H2)-34 CATTTCTTTATCTCGTGCGCGGTTT ZF (C3H) GAGATCATAGTTGAAGACATAGTGG ZicL GATCAACCATTACATTAGAATACAT

Table S3. Relative changes of mRNA amounts meseaured by qRT-PCR at the early gastrula stage by gene knockdowns with specific morpholino oligonucleotidesGenes knocked-down

Genes examined ADMP AP-2-like2 brachyury dkk Dll-B DMRT1 ets/pointed2 FGF9/16/20 FoxA-a FoxB FoxD-a/b lefty/antivin msxb MyoD neurogenin nodal noTrlc Otx Snail SoxC Tbx6b/c/d tbx2/3 TWIST-like-1a/b wnt5 ZicLADMP 1.36 1.18 0.91 0.75 1.02 1.13 0.99 0.90 1.17 0.98 0.85 0.95 0.96 1.06 0.84 0.86 0.78 1.12 1.21 1.00 0.74 0.93 0.82 0.59 0.75AP-2-like2 1.32 0.85 0.59 0.74 0.92 1.07 1.04 1.22 1.13 0.93 1.09 0.89 1.02 0.93 0.94 0.96 0.97 1.01 1.01 0.80 0.99 0.61 1.59 0.83 1.14BMP2/4 0.88 1.02 0.76 1.31 0.73 1.21 1.89 1.19 0.80 1.14 1.15 0.88 0.91 1.13 1.05 1.08 1.13 1.04 1.01 1.01 0.72 0.62 0.53 0.95 0.52Brachyury 1.13 1.03 0.92 0.97 1.32 0.57 0.32 0.09 0.00 0.99 0.01 0.73 0.93 1.04 0.93 1.18 1.16 0.97 1.26 0.50 0.79 1.11 1.13 0.59 0.01chordin 1.48 1.47 0.53 0.70 1.08 0.90 0.64 0.31 0.94 0.76 0.94 1.22 0.87 1.21 1.04 0.68 1.43 1.43 1.48 0.90 1.13 1.18 0.55 0.57 0.43Delta-like 1.49 1.30 0.88 1.13 1.69 0.96 1.07 0.22 0.95 0.98 0.60 1.02 0.88 1.09 0.96 0.04 1.97 0.58 1.29 1.48 1.36 1.41 1.19 0.85 0.58dickkopf 0.92 1.06 0.63 0.74 1.30 0.93 0.79 0.64 0.72 1.15 1.10 1.04 0.97 1.03 0.91 0.90 1.68 0.92 1.23 0.84 1.13 0.79 1.01 0.93 1.09Dll-B 1.95 1.24 0.77 1.55 1.30 0.89 1.01 0.91 1.07 0.99 1.40 1.15 1.02 1.08 0.93 1.01 1.34 1.11 1.22 0.85 0.94 0.88 1.06 0.93 0.62DMRT1 1.16 1.09 0.62 1.31 1.16 0.57 0.35 0.02 0.16 1.01 1.87 0.98 0.91 1.39 1.04 1.88 1.40 1.16 1.02 0.41 1.83 1.06 1.62 1.06 0.91DUSP1.2.4.5 0.97 1.28 0.83 1.16 1.77 0.86 0.37 0.19 0.46 0.84 1.12 0.94 0.84 1.67 1.14 1.49 1.35 0.74 1.17 0.64 1.46 0.93 1.23 1.59 0.66E12/E47 1.18 1.20 0.91 1.06 1.07 0.90 0.93 0.96 0.94 0.96 0.95 0.77 1.01 1.13 0.91 1.02 1.16 0.99 0.74 0.86 0.97 1.03 0.92 0.97 0.98E(spl)/hairy-a 1.19 1.23 1.73 0.74 1.16 0.92 0.88 1.13 1.31 0.97 1.57 1.11 1.46 1.58 0.86 0.57 1.08 1.66 1.55 0.89 1.19 0.88 1.35 1.32 0.86E(spl)/hairy-b 0.90 0.81 0.62 0.78 1.03 1.01 0.77 0.63 1.35 1.01 0.52 1.02 1.45 1.06 0.76 1.02 0.85 0.97 1.01 0.98 0.86 0.58 0.65 0.88 1.01ELK 1.09 1.07 0.77 0.99 1.39 0.81 0.25 0.15 0.98 1.06 0.95 0.75 0.97 0.98 1.04 1.04 1.06 1.23 1.19 0.93 1.01 1.06 0.70 0.97 0.75Emc 0.96 2.01 0.88 1.06 1.72 1.12 1.00 0.80 1.21 1.25 1.67 0.94 1.12 1.42 1.03 0.14 1.03 1.25 1.08 0.88 1.65 0.97 1.53 0.90 0.66Eph1 1.35 1.06 0.56 0.80 1.68 0.95 0.84 0.65 0.49 0.57 0.81 0.92 0.93 1.24 0.78 1.02 0.82 1.21 1.09 0.66 1.55 1.92 1.17 0.80 0.71EphrinA-c 1.37 1.84 1.06 1.02 1.14 0.82 0.41 0.39 0.54 1.20 1.21 1.14 1.07 1.43 0.86 1.10 0.95 1.06 1.44 0.89 1.40 0.77 1.92 1.21 0.86ets/pointed2 1.00 1.04 0.57 1.16 0.88 0.77 1.18 0.89 0.95 1.08 0.97 0.96 0.94 1.04 0.87 0.83 1.01 0.82 1.07 0.92 1.09 0.89 1.09 0.96 0.82FGF8/17/18 0.71 1.04 0.86 0.91 0.81 0.75 1.78 0.74 1.45 1.46 0.43 0.90 1.04 0.57 0.12 1.52 1.20 0.55 1.68 0.61 1.23 0.84 0.61 1.40 0.66FGF9/16/20 1.38 0.80 1.01 0.86 0.93 0.77 1.12 2.75 0.75 1.11 0.61 0.80 1.08 1.37 0.84 1.29 1.71 0.80 1.06 0.85 1.02 0.62 1.13 0.77 0.53Fli/ERG1 1.23 0.90 0.56 0.91 0.59 1.06 0.01 0.03 0.93 1.15 1.43 1.06 0.97 1.29 0.78 0.81 1.07 0.11 0.95 0.72 1.33 1.61 0.34 0.59 0.55Fli/ERG3 1.57 0.98 0.63 1.05 1.22 0.88 0.68 0.57 1.17 1.31 1.10 1.16 0.90 1.11 0.78 1.16 0.97 0.70 1.15 0.87 1.27 1.26 1.22 0.74 1.09Fos 1.57 0.97 0.57 0.84 0.94 0.81 0.45 0.38 1.25 1.02 1.02 0.62 1.06 0.95 0.96 1.26 0.79 0.73 0.93 0.57 0.84 0.58 0.35 0.77 0.71FoxA-a 1.46 1.09 0.72 1.04 1.56 0.85 0.81 0.88 1.29 1.21 0.67 1.00 1.08 1.39 1.06 1.30 1.62 1.16 1.27 1.64 1.08 0.93 1.34 1.34 0.84FoxB 0.74 0.69 1.37 0.75 0.81 0.95 1.41 1.58 0.22 5.39 0.35 0.84 1.43 0.58 0.65 1.91 1.26 0.90 1.11 0.69 0.87 0.79 1.79 1.33 0.53FoxD-a/b 1.38 1.39 0.57 0.91 1.53 1.06 1.00 1.39 1.08 1.32 9.58 1.17 0.95 1.40 1.73 1.55 1.30 0.79 1.16 0.86 1.89 1.00 1.52 1.53 1.39FoxH-b 1.74 1.25 0.86 0.85 0.68 0.97 1.13 1.12 0.74 1.21 1.07 1.26 0.86 1.16 0.92 0.96 1.09 1.13 1.26 0.93 1.13 1.24 0.72 0.99 0.93FoxP 0.90 1.13 1.09 1.04 1.09 0.90 1.13 0.65 1.09 0.83 0.97 1.02 0.71 1.10 1.34 0.91 0.93 0.93 1.07 0.74 1.21 0.93 1.16 1.28 1.10Fz4 0.95 0.85 0.52 1.25 0.75 0.76 1.18 0.56 0.38 0.90 0.55 0.79 0.96 1.37 0.72 0.57 0.57 0.63 0.89 0.66 0.81 0.97 0.75 1.01 0.81GATA-b 1.06 0.92 0.70 1.14 0.48 1.52 1.32 1.82 0.02 1.21 1.02 1.24 1.06 0.95 0.92 1.80 1.42 1.33 1.41 0.51 0.64 0.68 1.03 0.81 1.19Hedgehog1 1.45 1.27 0.53 1.31 1.21 1.13 1.51 1.35 1.73 1.53 1.20 1.19 1.64 1.16 1.25 1.32 0.63 1.01 1.42 1.28 1.41 1.31 1.02 1.68 1.64HNF4 1.14 0.88 0.68 1.02 0.97 0.95 1.11 0.97 1.13 0.85 0.95 0.90 0.97 1.14 1.03 0.99 0.93 0.97 1.01 0.95 1.18 0.98 1.06 1.08 0.78Irx-B 1.82 0.91 0.78 0.97 0.90 0.99 0.64 0.95 1.79 1.27 0.99 1.13 1.14 1.78 1.16 1.34 1.29 1.27 0.74 1.01 1.01 1.56 1.16 0.74 0.76lefty/antivin 0.95 1.10 1.14 0.75 1.18 1.13 0.93 1.09 1.06 1.09 0.95 1.13 1.09 1.39 1.04 0.64 1.39 1.37 1.02 1.09 0.84 0.80 0.69 0.50 0.59Lhx3 1.22 1.10 0.60 0.93 1.72 1.03 0.91 0.56 0.41 1.30 0.60 1.02 1.35 1.23 0.90 1.17 0.79 1.09 1.42 0.95 0.99 0.89 1.42 1.66 1.14Mnx 1.78 0.94 0.71 0.89 1.47 0.61 0.03 0.13 0.42 0.95 0.07 1.09 0.78 0.77 1.02 1.30 1.34 0.84 1.49 1.71 0.67 0.66 1.92 1.29 0.06msxb 0.88 1.29 0.82 1.27 0.89 0.85 0.06 0.04 2.04 1.18 1.20 0.89 0.56 1.14 0.97 0.01 1.67 0.65 1.38 1.77 1.16 1.13 0.60 0.69 0.50MyoD 1.57 1.09 0.65 0.97 0.51 0.85 1.05 1.41 0.94 0.70 0.85 0.84 0.89 1.91 0.93 1.15 0.99 0.66 1.01 0.64 0.99 0.59 0.76 0.72 0.26MYTF 0.56 0.52 0.55 0.65 0.73 0.81 1.95 0.66 1.18 1.01 0.20 0.97 0.79 0.97 0.18 0.66 0.61 0.55 1.40 0.73 0.90 0.75 0.54 0.62 0.72Neurogenin 0.88 0.93 1.16 0.75 0.73 0.93 1.47 0.98 0.50 1.23 0.59 1.02 1.05 1.07 1.57 0.56 0.90 0.83 0.91 1.00 0.56 0.87 0.77 0.60 0.60nodal 0.90 1.76 1.63 1.20 1.80 0.98 0.62 0.38 10.20 0.63 2.99 0.84 0.93 1.80 1.59 1.20 1.27 1.56 1.84 2.20 1.89 1.97 1.88 1.36 0.86noggin 1.16 0.95 0.52 1.42 0.95 0.79 0.65 0.47 0.64 1.09 0.96 0.84 0.76 1.14 0.85 0.95 1.53 0.75 0.98 0.69 0.59 0.83 0.74 0.51 1.01NoTrlc 1.12 1.01 0.66 0.86 1.06 1.30 1.31 0.63 0.04 0.90 0.45 1.58 1.14 1.47 0.79 0.24 1.21 0.52 1.30 0.69 1.13 1.05 0.56 0.67 1.43Orphan Fox-2 1.07 1.33 1.20 0.74 1.27 1.37 0.95 1.07 1.14 1.16 1.21 1.31 1.09 1.16 1.04 1.19 0.99 1.41 0.97 0.97 1.11 0.93 1.10 0.80 0.79Orphan Wnt e 0.91 1.18 0.70 0.93 0.76 1.13 0.80 1.85 0.72 0.92 1.03 0.97 0.85 1.27 0.98 1.01 0.70 0.63 0.89 0.98 0.61 0.74 0.63 1.05 0.90Otp 0.82 0.79 0.88 1.17 0.65 1.18 1.06 1.34 1.59 1.31 0.64 1.15 0.88 0.47 0.73 1.16 1.21 0.75 0.91 0.81 0.63 0.56 0.66 0.86 0.25Otx 1.18 0.99 0.66 0.75 1.22 0.77 0.82 0.50 0.63 0.88 1.04 0.72 1.00 1.39 0.78 0.65 1.41 0.71 1.18 0.70 1.12 0.72 0.70 0.75 0.61PPAR 1.15 0.98 1.41 1.17 1.49 1.21 1.10 0.76 1.10 1.09 0.78 0.92 1.01 0.82 1.09 1.13 1.48 1.06 1.08 0.90 0.96 1.11 1.06 1.19 0.86RAR 1.91 1.18 0.78 1.24 0.84 0.93 0.99 1.03 1.39 0.88 1.06 1.14 0.94 1.06 1.12 0.91 0.80 1.41 1.38 0.90 1.06 1.31 1.36 0.78 0.99ROR 1.28 1.18 0.90 0.81 1.09 0.96 0.81 0.91 0.78 0.85 0.79 1.11 0.79 1.12 0.95 0.99 0.99 0.90 1.06 0.69 1.15 0.95 0.80 1.02 0.85SYMD1 1.84 1.35 0.98 1.09 0.80 0.96 1.09 1.40 0.84 0.80 0.81 0.68 0.96 0.42 0.80 0.87 0.86 0.56 1.27 0.85 0.64 0.69 0.69 0.67 0.71sFRP1/5 1.85 1.36 0.95 1.09 0.91 0.88 0.99 1.01 0.50 1.07 1.18 0.74 0.85 1.21 0.99 1.13 1.17 1.20 0.82 0.53 1.14 0.81 0.60 0.87 0.83Snail 1.43 1.16 1.29 0.96 0.99 1.48 0.63 1.02 0.79 1.12 0.83 1.05 0.93 1.19 0.92 0.78 1.28 0.93 1.39 1.16 0.86 0.95 0.60 0.77 0.56SOCS1/2/3/CIS 1.80 0.77 0.50 0.78 1.00 0.87 1.12 0.94 0.69 1.36 0.53 0.91 1.17 1.23 1.14 0.95 1.53 1.09 1.01 0.80 1.33 1.09 0.67 0.60 0.67SoxB1 1.08 1.12 1.04 0.71 0.93 0.54 1.24 1.04 1.23 0.84 1.00 0.55 0.88 1.21 0.88 0.85 1.09 1.13 1.11 0.81 1.19 0.57 1.30 0.60 0.61SoxB2 1.39 0.63 0.91 0.75 0.57 0.59 0.72 1.12 1.39 0.85 0.86 1.09 0.84 1.11 0.99 1.46 0.88 1.19 1.13 0.81 0.90 0.57 0.66 0.68 0.84SoxC 1.36 0.94 0.64 1.08 1.19 0.91 0.63 0.78 0.93 1.16 1.13 1.03 0.82 1.12 1.35 1.19 0.80 0.51 1.04 0.82 1.06 0.84 0.65 1.39 0.86Tbx2/3 0.51 1.24 1.15 0.61 0.85 1.41 1.18 0.62 1.06 1.31 1.21 1.01 0.72 1.45 0.94 0.70 1.91 0.69 1.68 0.67 0.92 0.72 0.69 0.62 1.59Tbx6a 1.29 1.20 0.63 0.95 1.05 1.13 0.73 0.90 1.38 0.95 1.67 1.08 0.90 1.11 0.93 1.40 1.58 0.90 1.22 0.87 0.82 0.78 0.65 0.74 1.08Tbx6b/c/d 1.43 1.13 0.85 0.72 0.84 0.56 1.37 1.27 0.77 1.36 0.75 0.75 1.04 0.91 0.84 1.12 1.24 0.62 1.21 0.95 1.09 0.60 0.77 0.92 0.53TGFβ not assigned 1 1.68 1.29 0.90 1.01 0.97 1.00 1.00 1.09 0.76 1.23 1.12 0.68 1.16 1.26 0.86 1.10 1.21 0.99 0.85 0.93 1.07 0.88 0.99 0.84 0.72Tolloid 1.12 1.16 0.72 1.05 1.20 0.90 0.59 0.63 0.90 1.11 0.99 1.23 0.91 1.09 0.81 0.92 1.29 0.87 1.21 0.75 1.00 1.06 0.76 1.07 0.92TTF1 1.21 1.16 0.67 1.21 1.45 0.95 0.52 0.32 0.47 0.67 0.55 0.85 1.20 1.41 0.84 0.91 1.03 1.14 1.03 0.94 1.19 0.84 1.27 0.91 0.71TWIST-like-1a/b 1.46 0.97 0.50 1.01 0.79 0.58 0.03 0.07 0.67 0.88 1.35 0.55 0.95 1.06 0.99 0.71 1.40 0.31 0.97 0.52 1.18 0.76 0.70 0.62 0.54Wnt5 1.44 1.07 1.82 0.95 0.82 1.23 1.30 1.51 1.32 1.08 1.04 0.99 1.06 1.21 0.98 1.57 1.16 1.01 1.05 1.07 0.85 0.98 0.93 1.53 0.98ZF (C2H2)-2 1.32 1.16 1.13 0.72 1.09 0.86 1.27 1.14 1.16 0.88 1.09 0.73 1.39 1.06 0.97 1.10 0.95 0.97 1.01 0.89 1.18 0.76 1.88 1.13 0.83ZF (C2H2)-25 1.09 1.31 0.67 0.97 1.09 0.70 0.96 0.91 1.02 0.88 0.90 0.74 0.97 1.01 0.78 1.06 1.18 1.03 0.87 0.70 0.99 0.84 1.08 0.85 0.55ZF (C2H2)-34 0.75 1.81 1.13 0.84 1.37 0.85 1.13 1.04 0.90 0.91 1.31 1.21 1.08 0.88 1.23 0.95 0.95 1.06 1.04 0.84 1.57 0.91 1.14 1.22 1.06ZF (C3H) 1.51 0.85 1.09 1.17 1.13 0.60 0.40 0.20 0.73 1.78 1.16 0.73 0.75 1.02 1.01 1.14 1.35 1.04 1.49 0.58 1.25 0.99 1.22 0.58 0.67ZicL 1.21 1.21 0.91 0.89 0.83 1.04 1.06 1.06 0.39 0.93 0.30 1.07 1.04 1.15 0.89 0.91 1.42 0.87 1.21 0.90 0.89 0.74 1.11 0.96 1.56

31

Table S4. Effects of gene knockdowns examined by whole-mount in situ hybridization

Genes supressed Genes examined

Stage examined*1 Figure

Details of effects of gene suppression

FoxA-a Brachyury eG Figure S5A down-regulated in A-line and B-line notochord

Chordin 64 Figure S5A down-regulated in A-line notochord

Delta-like 64 Figure S5A

ectopically expressed in a-line neural cells; not affected in TLCs and b-line neural cells.

DMRT1 eG Figure S5A down-regulated in a-line neural cells

Emx lG Figure S5A ectopically expressed in a-line epidermal cells

Eph1 eG Figure S5A down-regulated in the endodemal cells

FoxB 64 Figure S5A down-regulated in A-line nerve cord cells and TLCs

Lhx3 64 Figure S5A

most of the expression in vegetal cells were down-regulated but not all of the expression was completely lost

Mnx 64 Figure S5A down-regulated in A-line and B-line notochord

MYTF lG Figure S5A

down-regulated in a-line neural cells and A-line nerve cord cells

nodal eG Figure S5A ectopically expressed in a-line neural cells

noTrlc eG Figure S5A down-regulated in TLC

32

Otx 64 Figure S5A

down-regulated in endodermal cells; not affected in B7.3 and B7.7

sFRP1/5 lG Figure S5A down-regulated in a-line epidermal cells

TTF1 eG Figure S5A down-regulated in endodermal cells

Twist-like1 ARR Figure S5A

down-regulated in TLCs; not affected in B-line mesenchyme

Twist-like2 ARR Figure S5A

down-regulated in TLCs; not affected in B-line mesenchyme

ZicL eG Figure S5A

down-regulated in A-line nerve cord cells and a-line neural cells; not affected in b-line neural cells and B-line muscle cells

GATA-a TB Figure S5A down-regulated in endodermal cells

SoxB1 TB Figure S5A down-regulated in endodermal cells

sFRP3/4-b TB Figure S5A down-regulated in endodermal cells

Alkaline Phosphatase activity TB data not shown

Alkaline phosphatase activities were not detected

FoxD-a/b Brachyury eG Figure S5B down-regulated in A-line and B-line notochord

Chordin 64 Figure S5B down-regulated in A-line notochord

Chordin eG Figure S5B down-regulated in A-line notochord

Delta-like eG Figure S5B

down-regulated in A-line nerve cord cells and TLCs; not affected in b-line

33

neural cells.

Dll-B eG Figure S5B ectopically expressed in A-line nerve cord cells

DMRT1 eG Figure S5B ectopically expressed in A-line nerve cord cells

FGF8/17/18 eG Figure S5B down-regulated in TLC

FGF9/16/20 eG Figure S5B down-regulated in A-line nerve cord cells

FoxB 64 Figure S5B down-regulated in A-line nerve cord cells and TLCs

Mnx 64 Figure S5B down-regulated in A-line and B-line notochord

MYTF eG Figure S5B down-regulated in TLC

MYTF lG Figure S5B

down-regulated in a-line neural cells and A-line nerve cord cells

neurogenin lG Figure S5B down-regulated in a-line neural cells

nodal eG Figure S5B ectopically expressed in A-line nerve cord cells

noTrlc eG Figure S5B not affected

Twist-like1 ARR Figure S5B

down-regulated in TLCs; ectopically expressed in B-line notochord

Wnt5 64 Figure S5B down-regulated in A-line nerve cord cells

ZicL 64 Figure S5B

down-regulated in A-line nerve cord cells and B-line notochord

ZicL lG Figure S5B down-regulated in a-line neural cells and A-line nerve

34

cord cells

ZicL Chordin eG Figure S5C down-regulated in A-line notochord

Cdx lG Figure S5C down-regulated in A-line nerve cord cells

Delta-like eG Figure S5C

down-regulated in A-line nerve cord cells; not affected in TLCs and b-line neural cells.

Lhx3 64 Figure S5C

down-regulated in A-line notochord; not affected in endodermal cells

Lmx lG Figure S5C down-regulated in A-line nerve cord cells

Meis TB Figure S5C down-regulated in a-line neural cells

Mnx 64 Figure S5C down-regulated in A-line and B-line notochord

Mnx lG Figure S5C down-regulated in A-line nerve cord cells

MyoD eG Figure S5C down-regulated in B8.7 and B8.8 muscle cells

MYTF lG Figure S5C down-regulated in A-line nerve cord cells

Pax6 lG Figure S5C down-regulated in A-line nerve cord cells

Six1/2 TB Figure S5C down-regulated in a-line neural cells

Six3/6 TB Figure S5C down-regulated in a-line neural cells

Snail eG Figure S5C s

Snail lG Figure S5C down-regulated in a-line neural cells ,b-line neural

35

cells and A-line nerve cord cells

ZF(C2H2)-33 lG Figure S5C down-regulated in A-line nerve cord cells

FGF9/16/20 Brachyury eG Figure S5D down-regulated in A-line and B-line notochord

Chordin 64 Figure S5D

down-regulated in A-line notochord and b-line neural cells

DMRT1 eG Figure S5D down-regulated in a-line neural cells

FoxB eG Figure S5D ectopically expressed in A-line notochord

Mnx eG Figure S5D down-regulated in A-line and B-line notochord

msxb lG Figure S5D down-regulated in b-line neural cells

MyoD eG Figure S5D ectopically expressed in B-line mesenchyme

MYTF lG Figure S5D down-regulated in a-line neural cells

Nodal eG Figure S5D down-regulated in b-line neural cells

ZicL eG Figure S5D

down-regulated in a-line neural cells and b-line neural cells; not affected in other blastomeres

ZicL lG Figure S5D

down-regulated in a-line neural cells and b-line neural cells

NoTrlc TB Figure S5D down-regulated in TVCs

SoxC Nodal 64 Figure S5E ectopically expressed in a-line

36

neuronal cells

Cdx lG Figure S5E

ectopically expressed in medial part of A-line nerve cord cells

Fgf8/17/18 lG Figure S5E

ectopically expressed in medial part of A-line nerve cord cells

FoxC lG Figure S5E down-regulated in a-line neural cells

MYTF lG Figure S5E

down-regulated in a-line neural cells; not affected in a-line neural cells

neurogenin lG Figure S5E

ectopically expressed in medial part of A-line nerve cord cells

Pax6 lG Figure S5E

ectopically expressed in medial part of A-line nerve cord cells

Snail lG Figure S5E

ectopically expressed in medial part of A-line nerve cord and a-line neural cells

AP-2-like2 Emc lG Figure S5F ectopically expressed in epidermal cells

Nodal Delta-like eG Figure S5G

down-regulated in TLCs, A-line and b-line nerve cord cells

Emc lG Figure S5G

down-regulated in a-line neural cells and A-line nerve cord cells

37

FoxC lG Figure S5G ectopically expressed in b-line neural cells

Lmx lG Figure S5G down-regulated in A-line nerve cord cells

msxb lG Figure S5G down-regulated in b-line neural cells

MYTF lG Figure S5G

ectopically expressed in b-line neural cells; not affected in A-line nerve cord cells and a-line neural cells

Neurogenin lG Figure S5G down-regulated in A-line nerve cord cells

NoTrlc eG Figure S5G down-regulated in TLCs

Pax6 lG Figure S5G down-regulated in A-line nerve cord cells

Snail lG Figure S5G

down-regulated in a-line neural cells ,b-line neural cells and A-line nerve cord cells

Otx FoxC lG Figure S5H down-regulated in a-line neural cells

Lhx3 64 Figure S5H not affected

MYTF lG Figure S5H

down-regulated in a-line neural cells; not affected in A-line nerve cord cells

TTF1 eG Figure S5H not affected

Twist-like1 eG Figure S5H down-regulated in TLCs and B-line mesenchyme cells

GATA-a TB Figure S5H down-regulated in endodermal cells

Meis TB Figure S5H down-regulated in a-line neural cells

38

sFRP3/4-b TB Figure S5H not affected

Six1/2 TB Figure S5H down-regulated in a-line neural cells

Six3/6 TB Figure S5H down-regulated in a-line neural cells

SoxB1 TB Figure S5H not affected

Dll-B MYTF lG Figure S5I

down-regulated in a-line neural cells; not affected in A-line nerve cord cells

FoxH-a TB Figure S5I down-regulated in epidermal cells

Six1/2 TB Figure S5I down-regulated in a-line neural cells

Six3/6 TB Figure S5I down-regulated in a-line neural cells

ZF(C2H2)-24 TB Figure S5I down-regulated in epidermis around the papila

DMRT1 FoxC lG Figure S5J down-regulated in a-line neural cells

Meis TB Figure S5J down-regulated in a-line neural cells

Six1/2 TB Figure S5J down-regulated in a-line neural cells

Six3/6 TB Figure S5J down-regulated in a-line neural cells

Ets/Pointed2 FoxC lG Figure S5K down-regulated in a-line neural cells

Mnx eG Figure S5K down-regulated in A-line and B-line notochord

Nodal eG Figure S5K down-regulated in b-line neural cells

Twist-like1-a/b eG Figure S5K down-regulated in and B-line mesenchyme

FoxB Cdx lG Figure S5L ectopically expressed in medial part of

39

A-line nerve cord cells

FGF8/17/18 lG Figure S5L

ectopically expressed in medial part of A-line nerve cord cells

Mnx lG Figure S5L down-regulated in A-line nerve cord cells

Pax6 lG Figure S5L

ectopically expressed in medial part of A-line nerve cord cells

Twist-like1 Hlx TB Figure S5M down-regulated in mesenchyme

Hox4 TB Figure S5M down-regulated in mesenchyme

NoTrlc Twist-like1-a/b ARR Figure S5N down-regulated in TLCs

MyoD SYMD1 eG Figure S5O down-regulated in B8.7 and B8.8 muscle cells

Mox TB Figure S5O down-regulated in muscle cells

muscle actin TB Figure S5O

down-regulated in muscle cells; not affected in small region in the anterior part of muscle

Tbx6b/c/d MyoD eG Figure S5P down-regulated in B8.7 and B8.8 muscle cells

Neurogenin Delta-like eG Figure S5Q

down-regulated in TLCs; not affected in A-line nerve cord cells and b-line neural cells.

40

FGF8/17/18 eG Figure S5Q down-regulated in TLC

MYTF eG Figure S5Q down-regulated in TLC

COE TB Figure S5Q down-regulated in neural cells

Snail Mnx lG Figure S5R

ectopically expressed in lateral part of A-line nerve cord cells

MYTF eG Figure S5R

ectopically expressed in lateral part of B7.7 mesenchyme

ADMP NK4 TB Figure S5S

down-regulated in ventral part of trunk epidermal cells

CAGF9 TB Figure S5S

down-regulated in ventral part of tail epidermal cells; not affected in dorsal midline epidermis

Dll-C TB Figure S5S

down-regulated in ventral part of tail epidermal cells; not affected in dorsal midline epidermis

IrxC TB Figure S5S

down-regulated in ventral part of trunk epidermal cells

ZF(C2H2)-24 TB Figure S5S

down-regulated in ventral part of tail epidermal cells; not affected in dorsal midline epidermis

Msxb Achaete-Scute a-like2 TB Figure S5T

down-regulated in dorsal midline epidermal cells; not affected in ventral part of tail epidermal cells

41

CAGF9 TB Figure S5T

down-regulated in dorsal midline epidermal cells; not affected in ventral part of tail epidermal cells

Dll-C TB Figure S5T

down-regulated in dorsal midline epidermal cells; not affected in ventral part of tail epidermal cells

SoxB2 TB Figure S5T

ectopically expressed in dorsal midline epidermal cells

ZF(C2H2)-24 TB Figure S5T

down-regulated in dorsal midline epidermal cells; not affected in ventral part of tail epidermal cells

Mesp FoxF TB Figure S5U

down-regulated in TVCs; not affected in trunk epidermal cells

NoTrlc TB Figure S5U down-regulated in TVCs

Tolloid TB Figure S5U down-regulated in TVCs

FoxC ZF(C2H2)-2 TB Figure S5V down-regulated in papilas

*1 64, 64-cell stage; eG, early gastrula stage; lG, late gastrula stage; tailbud, tailbud stage; ARR, embryos arrested with cytochalasinB at the 110-cell stage and incubated to the stage equivalent to the late gastrula stage

Table S5. Relative changes of mRNA amounts meseaured by qRT-PCR at the late gastrula stage by gene knockdowns with specific morpholino oligonucleotidesMorpholino

Genes examined ADMP AP-2-like2 brachyury dkk Dll-B DMRT1 ets/pointed2 FGF9/16/20 FoxA-a FoxB FoxC FoxD-a/b lefty/antivin Mesp msxb MyoD neurogenin nodal noTrlc Otx Snail SoxC Tbx6b/c/d tbx2/3 TWIST-like-1a/b wnt5 ZicLADMP 2.89 1.26 0.96 1.19 0.62 1.21 0.99 1.31 0.81 1.17 1.73 1.46 1.03 0.96 1.25 0.85 1.23 1.09 1.12 1.08 0.98 1.27 1.15 1.30 0.86 0.75 1.01AP-2-like2 1.12 1.17 1.42 0.88 1.80 1.00 1.20 0.95 1.16 0.88 1.68 1.67 0.72 1.02 0.98 1.16 1.14 0.99 1.68 1.16 0.74 1.06 1.31 1.06 1.15 0.80 1.07BMP2/4 0.81 1.12 1.11 1.23 0.68 0.69 1.74 1.30 1.16 1.16 0.96 1.35 1.19 1.49 1.79 0.84 1.12 1.21 0.93 1.04 1.11 1.93 0.66 0.74 1.57 0.77 1.16BMP5/7-like 1.10 0.63 0.80 0.98 0.77 0.78 0.82 0.79 0.70 1.21 0.89 0.84 0.86 0.99 1.05 0.83 1.19 0.88 0.93 0.99 0.83 1.11 0.77 0.81 1.34 0.95 0.81Cdx 0.58 0.72 1.06 1.28 0.60 0.98 0.87 1.08 1.59 0.89 0.63 0.84 1.18 0.86 0.80 0.67 0.98 0.61 1.06 0.65 0.68 0.90 0.91 0.74 0.60 0.64 1.20chordin 0.64 1.09 1.13 1.05 1.35 1.05 0.23 0.26 1.88 0.56 0.83 1.71 1.00 0.92 1.04 1.17 0.97 0.23 0.90 1.13 1.46 1.34 1.13 0.93 0.55 0.58 0.99COE 0.76 0.58 1.48 0.59 1.10 0.63 0.97 0.12 1.07 0.78 1.10 0.05 0.70 0.91 0.91 0.81 0.04 0.48 1.13 0.69 0.91 1.35 0.52 0.70 0.95 0.85 0.23Delta-like 1.49 0.86 0.92 0.91 1.13 1.22 1.11 0.57 1.05 0.81 0.97 0.99 1.46 1.69 1.64 1.21 1.25 0.50 1.55 1.13 1.33 2.99 1.30 0.88 1.17 1.67 1.01dickkopf 0.85 1.13 1.33 3.05 1.99 0.84 0.86 0.64 0.90 0.80 1.24 0.88 1.00 1.04 1.08 1.60 0.91 0.56 0.99 0.75 0.92 0.91 0.95 0.81 1.23 1.39 0.77Dll-B 0.87 1.62 0.91 1.36 4.82 0.92 1.09 1.27 1.09 1.69 1.33 1.19 0.68 0.89 1.15 0.95 1.32 1.21 1.21 0.68 0.90 1.38 0.84 1.16 1.06 0.66 1.01DMRT1 1.16 1.75 1.42 1.38 1.40 3.05 1.06 0.12 0.49 0.66 1.40 2.36 0.95 0.82 1.01 1.15 0.97 1.58 0.97 0.90 1.72 0.60 1.61 0.81 1.62 0.87 1.56Emc 0.90 6.87 1.23 0.50 1.91 0.99 1.21 0.29 1.12 0.70 0.97 1.65 1.56 0.84 1.16 1.79 1.34 0.28 1.22 1.27 1.71 1.58 1.40 0.63 1.53 1.46 1.34Emx 0.86 1.91 1.00 1.92 0.31 1.35 1.45 0.84 2.20 0.61 1.10 0.65 1.47 0.74 1.39 1.13 0.72 1.01 1.05 1.04 1.04 1.33 0.77 0.81 1.10 1.26 0.85FGF8/17/18 0.49 1.16 0.95 1.49 1.01 0.56 0.65 0.31 1.00 1.27 1.82 0.29 1.07 1.22 0.78 1.31 0.85 0.69 0.58 0.75 1.57 1.35 1.36 0.66 0.72 1.41 0.75FGF9/16/20 0.69 0.93 0.71 1.10 0.59 0.86 1.02 3.58 0.68 0.65 0.72 1.06 0.79 0.63 0.97 1.04 0.96 1.01 1.34 0.67 1.13 0.71 1.09 0.96 0.60 0.82 0.58Fli/ERG1 1.06 1.09 0.80 1.07 1.27 1.00 0.36 0.07 0.52 1.01 0.61 0.98 0.92 0.97 0.88 0.61 0.91 0.69 0.63 0.02 0.93 0.70 1.26 1.03 0.36 1.27 0.72Fos 0.65 0.93 0.96 0.90 1.54 0.90 0.49 0.13 0.68 0.63 1.05 0.67 0.74 0.83 0.73 0.84 0.82 0.95 0.81 0.48 0.93 0.66 0.97 0.75 0.35 0.90 0.43FoxA-a 1.16 0.73 1.07 1.13 1.45 1.03 0.90 0.82 0.73 0.86 1.69 0.71 1.15 0.80 1.74 1.19 1.11 1.09 0.63 0.88 0.98 1.05 1.00 0.92 1.14 1.12 0.91FoxB 0.81 0.58 0.86 0.83 0.50 0.95 1.02 2.20 0.74 3.32 0.97 0.53 1.40 0.91 1.39 0.82 0.81 0.86 0.95 0.62 0.97 0.69 1.09 0.57 1.79 1.10 0.59FoxC 0.58 0.77 1.62 1.85 0.06 0.17 0.18 0.03 0.01 0.76 2.66 0.63 0.83 1.21 0.83 1.39 1.09 1.85 0.61 0.12 1.09 0.05 1.06 1.65 0.62 0.95 1.09FoxD-a/b 0.97 1.49 1.00 1.39 0.79 0.80 1.43 1.99 0.81 0.81 1.42 4.89 1.06 0.74 0.89 1.05 1.38 0.98 1.66 0.33 0.81 1.35 1.95 0.67 0.45 1.33 1.51GATA-b 1.53 1.60 0.82 1.85 0.01 1.61 1.23 1.51 0.16 1.22 1.59 0.83 0.91 1.56 1.27 1.18 0.66 1.21 0.91 1.35 0.89 0.47 0.95 0.76 1.07 1.91 0.77Hex 1.05 0.81 0.99 0.71 1.21 0.98 0.57 0.09 0.69 0.54 0.81 0.74 0.86 0.93 0.81 0.66 0.80 0.75 0.99 0.10 0.99 0.55 1.06 0.93 0.15 0.88 0.61Jun 0.77 0.88 0.96 1.24 1.06 0.89 0.67 0.22 1.21 0.57 0.59 0.59 0.82 0.84 1.13 0.95 1.00 1.39 0.78 0.39 0.75 1.13 0.95 0.79 0.65 0.59 0.52LAG1-like 5 0.94 0.73 0.91 0.80 0.70 0.80 0.58 0.15 0.58 0.98 0.75 0.65 0.89 0.85 0.93 0.63 0.93 0.62 0.52 0.07 0.88 0.54 0.64 0.53 0.21 0.98 0.63Mesp 0.81 1.18 1.09 0.95 0.93 0.79 0.89 0.58 1.47 0.72 1.63 0.96 1.59 4.08 0.81 1.84 1.06 0.75 0.56 1.37 1.32 0.67 0.63 0.85 0.99 0.90 0.75Mist 0.62 0.80 1.03 1.04 1.01 1.14 0.08 0.00 0.52 0.78 0.75 0.72 0.91 0.98 1.09 0.91 0.86 0.87 1.17 0.03 0.80 0.54 0.70 0.78 0.09 0.68 0.78Mnx 1.16 0.76 1.07 0.95 1.06 1.06 1.45 1.35 0.87 0.57 0.94 0.45 1.20 0.97 1.21 0.94 1.18 1.29 1.29 0.90 0.54 1.79 0.16 0.81 1.92 1.08 0.33msxb 0.72 0.82 1.06 1.19 0.60 0.84 0.37 0.25 1.39 1.33 0.51 0.99 0.99 0.85 0.57 1.11 0.93 0.02 1.39 0.67 0.87 1.16 1.16 0.66 0.77 1.08 0.53MyoD 1.05 0.75 1.10 0.70 1.17 0.94 1.21 1.95 0.69 0.56 1.18 0.69 0.97 0.95 1.10 4.41 0.67 1.04 1.39 1.01 0.57 1.14 0.27 0.90 0.57 1.14 0.31MYTF 0.56 0.70 0.90 1.13 0.50 0.81 0.50 0.37 0.13 0.66 0.71 0.10 0.90 0.77 0.88 0.77 0.91 1.61 0.82 0.34 1.46 0.65 0.89 0.93 0.77 0.62 0.24Neurogenin 1.17 1.05 0.95 1.21 1.46 0.66 0.84 0.16 0.77 0.96 1.41 0.29 1.10 1.07 1.06 0.71 5.46 0.48 1.45 0.88 1.06 1.30 1.09 0.81 0.69 1.01 0.30nodal 0.57 0.96 0.52 1.20 1.82 1.34 0.48 0.87 7.26 0.68 1.23 3.07 1.93 0.64 0.93 1.47 0.95 14.83 0.67 1.60 1.71 2.89 1.44 0.74 1.99 1.36 1.39NoTrlc 0.81 0.96 1.21 1.13 1.30 0.85 0.80 0.74 0.16 0.62 1.11 0.63 0.95 0.66 1.09 1.21 0.81 0.38 2.19 0.51 1.29 1.11 0.84 0.95 0.79 1.11 1.09Otp 0.82 1.13 0.93 0.93 1.27 0.92 1.74 1.46 1.06 0.53 1.71 0.51 1.14 0.66 1.00 0.22 0.91 1.26 0.98 0.76 0.80 1.06 0.31 1.10 0.76 0.92 0.69Otx 0.99 0.84 1.40 1.15 1.03 0.68 0.70 0.83 0.15 0.51 1.09 0.66 1.04 0.95 1.04 1.04 1.01 1.16 0.88 2.17 1.06 0.57 1.25 0.84 0.68 0.82 0.73Pax3/7 0.55 0.99 0.92 1.13 0.57 1.14 1.08 0.02 2.62 0.77 0.80 1.99 1.12 0.82 0.82 1.01 1.07 0.02 0.87 1.21 1.16 1.67 1.43 0.78 1.16 1.05 1.41Pax6 0.72 1.33 0.86 1.16 1.60 0.56 0.79 0.33 0.31 0.76 0.80 0.05 1.25 1.87 1.84 0.80 0.71 0.33 0.77 0.61 0.77 0.88 1.06 0.84 0.64 0.59 0.07RAR 0.83 0.78 0.97 1.18 0.93 0.97 1.05 1.35 0.90 0.76 0.79 1.42 1.08 1.09 1.09 0.79 1.13 0.89 0.60 1.13 1.01 1.02 0.87 1.16 1.19 1.44 1.30SYMD1 0.62 0.78 1.08 0.85 0.97 0.77 0.43 1.36 0.85 0.63 1.01 0.58 1.02 0.95 0.96 0.30 0.88 1.01 1.39 0.78 0.79 0.88 0.24 0.70 0.60 0.51 0.50sFRP1/5 0.69 1.02 0.99 0.99 0.80 1.16 0.92 0.99 0.17 0.79 0.97 1.34 1.02 0.76 1.03 0.98 0.95 0.89 0.74 0.88 0.90 0.65 1.02 1.01 0.69 1.04 1.47Snail 1.21 0.88 1.13 0.95 1.02 0.88 0.98 1.64 1.11 0.91 1.57 0.60 0.90 0.95 1.16 0.80 1.06 0.84 0.52 1.16 4.96 2.41 0.18 0.90 0.90 1.13 0.34SOCS1/2/3/CIS 0.67 0.73 0.98 1.18 0.34 0.99 0.62 1.05 1.79 0.83 0.84 0.81 1.13 0.95 1.09 1.02 1.32 0.21 1.06 1.29 0.94 1.03 0.76 0.71 0.97 0.60 0.70SoxC 0.67 0.62 0.85 1.14 1.51 0.81 0.62 0.62 1.25 0.63 1.26 0.82 1.13 0.95 0.91 1.28 1.01 1.17 0.70 0.50 0.95 0.86 1.40 0.72 0.86 1.47 0.76Tbx6a 1.14 1.33 1.22 0.90 1.14 0.86 0.56 0.70 0.88 0.82 1.33 0.60 0.99 0.93 1.01 1.23 0.95 1.21 0.81 0.95 1.29 1.04 0.62 0.87 0.91 0.80 0.47Tbx6b/c/d 1.27 0.97 1.31 1.07 1.04 1.06 1.29 0.95 0.72 0.85 1.55 0.54 1.04 0.86 1.65 0.90 0.91 0.90 0.95 0.81 0.51 1.84 5.50 1.21 0.69 0.85 0.08TWIST-like-1a/b 0.97 0.61 0.59 1.25 1.09 0.87 0.36 0.03 0.35 0.53 1.18 0.62 0.87 0.88 1.11 0.85 0.89 0.67 0.84 0.13 0.95 0.80 0.83 0.76 0.61 0.72 0.36TWIST-like-2 0.91 0.74 0.96 0.94 1.12 0.80 0.13 0.12 0.60 1.01 1.31 0.93 0.92 0.86 0.89 0.88 0.86 0.70 1.06 0.16 0.84 0.69 0.61 0.86 0.13 1.23 0.72Wnt5 0.86 1.06 0.79 1.43 1.13 1.04 0.70 0.68 0.93 0.87 0.91 0.65 1.01 0.94 1.06 1.33 1.07 1.31 0.66 1.01 0.99 1.30 0.50 1.04 0.91 1.93 0.48ZF (C2H2)-33 0.70 1.09 0.94 0.98 0.80 0.97 0.82 1.42 0.18 0.75 1.03 0.18 0.92 0.93 0.81 0.92 0.97 1.10 1.13 0.62 1.66 0.62 1.24 0.77 0.69 0.90 0.22ZicL 1.95 1.21 0.99 0.97 0.72 1.10 0.60 0.53 0.27 0.83 1.81 0.15 1.20 0.84 1.36 1.59 0.91 1.12 0.77 1.07 1.19 1.52 0.87 1.07 0.88 1.13 5.86

43

Table S6. Primers for qRT-PCRs designed in this study. Gene Forward primer Reverse primer

BMP2/4 AGCAACGATGTCGAAGAATGA GGCGTCATTTCTGGCTGTTA BMP5/7-like CATCGGTTCCAGTGATCTTAT TCAGGCATCGGCGCATTGT Brachyury ACGTCACCGCCTTAGGATT AGGACCACGTGGTCGTGAA Cdx CAGCGCGATATTGCAATAGT TTTAACGCCTCGTGCGAACA COE GATGGCGACAGAGAACATTTT AATGGATTGGAACTCGTCATCT Delta-like GAAAACCCGTCATGCAAGCT TCCTGGTTTTGGTTGGCATT Emx GCAAGCGTCTATGACGTAATA TGGAACGAACTACTACAGCAA EphrinA-c AATCCACCTCCACGCAACAT GCGAAAGGCCAATTGCATT ets/pointed2 TCTATACAGGCCGACAGTTTT CTGCGAGCGAAATGTCTCTT FGF9/16/20 CTTTCCGACAAGGAAGCTAT GCGTTCGTCTGCTGTGAAA Fli/ERG3 GCGTATCACAAACCTCTAGAA GTATGTGGAGAAGCTCATGAA Fos CCGCACACACATTCCCATT ACGAAATAGCGACAGTAGTAGA FoxA-a CACCATCAGTGAATGACACTA GAGACGGAGTGGACACTGA FoxC GCGACACAAAGTATTCAGCAA TCTCCCACGAGGCACAGTT FoxD-a/b GCCTTGTTTTGGCAGAACTTA GGTTTCACAGCTGCCACTT GATA-b GAGTTCTCCAGGTACGTTTA TGGTTCCCCCTTCCTCTGT Hex TGGTTGAACGACCAAGAAACTA TCATGGTGGTAAGCAGGTAGT Jun TGCCGGAAACTGTGACGTA TAAGCAAGGCAACTCAGCTA LAG1-like 5 TGCCATCGTGAAAAAAGTCCTT ATTCACCTTCAACGTCACTTCT Lhx3 CCACGTGACACATTTCCAATA CGGCATAGAAATCACAGTGAA Mist AGTGCAAGTCTGCATTTGCA TTTGTGTGCTGGCACACCAT MyoD CAGAAACCAAACCAGAAACGA CTGACGTCATCGGACTGATA MYTF AGTGCTGTAGCGACCTTTCT GAACTGCTTGTTTCACTGCAT Neurogenin CTCGTTGTTGGCTAGAAGAAA TCCCAGAAAATGGAGGTGAAA NoTrlc GTCAAGAAAACCCCGTCACA CTCCAGTCAGTTTTTCTTCTCT Pax3/7 GACCCGTTTATGGAACACTA CCTTTTAGAAGCGCACTGTGA Pax6 GGCCATGTTTGAGTTAACCTA GACATCATCGCATGACCATT PPAR TGAGATTGTGGAGACAGCTT ACAATGTTGGTTGCATGAAGGA SMYD AATTATTCCGCTGTGGTTGCT TTATTTCGCAGAGGCCCATTT SOCS1/2/3/CIS CACCGCAGAACTCCCACTTTA CCAACCTGCAAACCTCGTTT Tbx2/3 CCGCTGTGCAGGAATTAAGAA TCCTCGGAATGACGTCTTGTT Tbx6a ATACCCCAACAGCACCACAT TGCCCACTAAACAAGTTTCTCA Tbx6b/c/d AACGACCACATCTCCGTTTTA AGTGCCGCATGCAGTAGTAA TWIST-like-1a/b CACGAGATTGGAAGGAAAGAA AGCAGCGGCTTGATCGATA TWIST-like-2 CGTTATCAAACCCCTCAACTT CGTTTGTTGCGTCATAACCAT ZF (C2H2)-25 TACCACACGGTCAGGTACATT GAAGCACTCAAAAGCTTGAAGA ZF (C2H2)-33 TGCGCAGATTTTGTTGCAGAA ATCGAGAGATCCGCAAGGAA ZicL TTGCGACCGAAGCTACACT TGTTGTCGATTACGTCACCAT

44

Table S7. Genes regulated by auto-regulatory loops or negative feedback loops and the relative changes in the amount of mRNA, which were measured by qRT-PCR using tailbud embryos.

Gene Name ADMP 2.89 Brachyury 4.17 dickkopf 3.05 Dll-B 4.82 DMRT1 3.05 ets/pointed2 2.14 FGF9/16/20 3.58 FoxB 3.32 FoxC 2.66 FoxD-a/b 4.89 lefty/antivin 2.73 Mesp 4.08 MyoD 4.41 Neurogenin 5.46 nodal 14.83 NoTrlc 2.19 Otx 2.17 Snail 2.17 Tbx2/3 2.46 Tbx6b/c/d 5.50 Wnt5 1.93 ZicL 5.86

45

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