Martinelli and Fan Supplemental Figure 1

SphIEcoRIBamHI

SphI

tau-LacZ

tau-LacZ

PGK-neopA

XhoI

Gas1

recombinationconstruct PGK-tk

XhoI

SphI

SphI

SphI

SphI

Gas1LacZ

(Gas1-)

XhoI

XhoI PGK-neopA

SphI

A

e

22

+/- -/-+/- -/-e

+/- +/-

+/-

+/-+/+

E14.5 hl

B C D E

G Hn

ps

np

s

E14.5 head

dn

oe

dm

dn

fl

fl

hldn

s

o e

-/-

-/-

M c +/++/- -/-

Gas1LacZ

100

400

F

F igure S 1 . (A ) H om ologous recom b ination schem e to generate the G as1 LacZ (G as1 -) a lle le ; top , genom ic s tructu re o f G as1 locus; m idd le , recom b ina tion construct; bo ttom , recom bined a lle le . T he tau -LacZ -pA -P G K-neo casse tte is p laced a t the exact sam e loca tion as the PG K -neo casse tte in the p reviously pub lished G as1 neo a lle le (Lee e t a l. 2001b) and rep laces the entire cod ing reg ion o f G as1 . pA , po ly-A s igna l sequence ; P G K -tk , P G K -driven thym id ine k inase gene casse tte fo r nega tive se lection. R estric tion enzym e s ites (S ph I and Xho I) and 5 ’ and 3 ’ p robes used to screen fo r and confirm recom bination a re a lso the sam e as previous ly pub lished (Lee e t a l. 2001b); w h ite boxes, 5 ’ and 3 ’ U T R s of G as1 ; b lack box, cod ing reg ion o f G as1 ; a rrow heads, new P C R prim er se ts (see M ethods) fo r genotyp ing. (B ) P C R w ith the ind icated p rim er se ts can d is tingu ish w ild type (+ /+), he te rozygous (+ / -), and m utan t (-/-) em bryos; M , 100 bp ladder, 100 bp and 400 bp bands are labe led ; c , m ock con tro l P C R ; expected G as1 and LacZ bands a re labe led. (C ) X -ga l s ta in ing fo r LacZ activ ity (b lue ) in E7 .0 w ild type con tro l (le ft) and he te rozygous (righ t) em bryos. (D and F ) X -ga l s ta in ing pa tte rns o f E 9 .5 and E10 .5 he te rozygo tes (le ft) and m utan ts (righ t), respective ly. (E ) A transverse section o f E 9.5 a t the fo re lim b (fl) leve l. T hese sta in ing pa tte rns agree w ith the endogenous G as1 express ion ob ta ined by IS H (Lee and F an 2001). S -ga l (red ) s ta ined E 14 .5 he te rozygous (le ft) and m utan t (righ t) heads (G ) and h ind lim bs (h l) (H ) show tha t m utants have m icrophtha lm ia and sm a ll lim b /m a lfo rm ed d ig it 2 (w h ite a rrow head) pheno types as described fo r the G as1 neo m utant (L iu e t a l. 2002). U nder our experim enta l cond ition (37 °C , 2 h rs), w ild type em bryos fo r a ll s tages p resen ted here w ere nega tive fo r s ta in ing (not show n). O f no te , m utan ts have a sm a lle r head structu re than he te rozygo tes and w ild types observab le as early as E 9 .5 , w h ich w ill be described in de ta il in a separa te m anuscrip t. A bbrevia tions: np , neura l p la te ; ps, p rim itive s treak; n , node; s , som ite ; o , o tic ves ic le o r ea r; e , e ye ; dn , do rsa l neura l tube ; dm , derm om yotom e.

Martinelli and Fan Supplemental Figure 2

0

20

40

60

PAX7 PAX3 DBX1

% le

ngth

of N

.T.

Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/-

PAX7

PAX6

PAX3

GSH1/2

DB X1

NK X6.1

OL IG2

NK X2.2

HNF3β

S HH

Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/- Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/-

p=6.6E-5

p=.0076 p=1.6E-8

p=5.0E-7

p=2.2E-6

p=.0014

Gas1+/-;Shh+/- Gas1-/-;Shh+/+ Gas1-/-;Shh+/-

E12.5

E13.5

1 1 1

1 1 1

2 2

2 2

3 3 3

3 33

4

44

4

4

4

5 5 5

5 5 5

0

0.2

0.4

0.6

0.8

1

Forelimb Hindlimb

digi

t 2:4

leng

th ra

tio

Control Gas1 mutantA

B

Martinelli and Fan Supplemental Figure 3F igure S 2 . Q uan tita tion and add itiona l m a rker ana lys is fo r the neura l tube defect o f G as1 -/- and G as1 -/-;S hh +/- em bryos. Em bryos o f 24 -25 som ites w ere transverse ly sectioned a t the fo re lim b leve l and ana lyzed by IH C . G eno types a re labe led on top and m arkers on the le ft. In the le ft 3 co lum ns a re dorsa l m arkers, w h ile in the righ t 3 co lum ns a re ven tra l p rogen ito r m arkers. O f note , com prom ised floo rp la te induction w as a lso observed by the a lte ra tion o f N K X2.2 , H N F3β, and S H H express ion . A lso , in G as1 -/-;S hh +/- em bryos, O LIG 2 -positive ce lls occupy the m id line w ith little o r no S H H -positive ce lls . The resu lts a re consis ten t w ith reduced S H H s igna ling in the G as1 -/- neura l tube and a fu rthe r reduction in the G as1 -/-;S hh +/- neura l tube. D ashed line dem arks the boundaries o f express ion o f PA X7, P A X3, a nd D B X1. To quan tify the changes in the boundaries o f express ion fo r these 3 genes, the percen t o f the neura l tube w ith in the boundaries w as ca lcu la ted fo r G as1 +/-;S hh +/- (n = 5 ), G as1 -/- (n = 4 ), and G as1 -/-;S hh +/- (n = 3 ) em bryos. 2 sections w ere ana lyzed fo r each em bryo . A tw o-ta iled W elch ’s t-test w as perfo rm ed . For the boundaries o f a ll 3 genes , G as1 +/-; S hh +/- em bryos w ere s ta tis tica lly d iffe ren t from bo th G as1 -/- and G as1 -/-;S hh +/- em bryos (99% confidence leve l). P va lues a re ind icate d fo r each t-test and d isp layed on the bar graph . The express ion boundaries in G as1 -/- and G as1 -/-; S hh +/- em bryos w ere no t found to be s ta tis tica lly d iffe ren t.

F igu re S 3 . (A ) G as1 m u tant d ig it 2 is d isp roportiona lly sm a lle r than the o ther d ig its . T he lengths o f d ig its 2 and 4 w ere m easured on G as1 +/-;S hh +/- and G as1 -/- neona ta l ske le ta l p repara tions and the length ra tio ca lcu la ted . D ig it 4 w as used fo r com parison because it and d ig it 2 a re norm a lly o f s im ila r length . n fo r con tro l lim bs is 3 ; fo r G as1 -/- fo re lim bs, 19 ; fo r G as1 -/- h ind lim bs, 11 . E rro r ba rs = 1 s tandard devia tion . (B ) E xam ina tion o f fo re lim bs of E 12.5 and E 13 .5 m ice o f G as1 and S hh a lle lic com b ina tions (ind icated a t top ) revea led that d ig it 2 never fo rm s in the G as1 -/-;Shh +/- genotype . A rrow head ind ica tes la rge loca tion o f in te r -d ig ita l m esenchym e w here d ig it 2 no rm a lly form s. n = 3 fo r each geno type and stage . D ig it num bers are ind ica ted . S ca le bar = 1 m m . R ationa le fo r the determ ination o f m issing d ig it identity : D e te rm ina tion o f d ig it 1 w as stra igh tfo rw ard because it has one less pha lange . D ig its 4 and 5 w ere de te rm ined by m easuring the ir re la tive lengths as w e ll as exam ination o f m etacarpa l and m eta tarsa l shapes . A s p revious ly reported fo r G as1 m u tan ts, w e found tha t d ig its 4 and 5 a re no t a ffected in the G as1 -/-;Shh +/- (L iu e t a l. 2002). D is tingu ish ing the m issing d ig it as 2 as opposed to 3 is m ore com p lica ted. O ur lines o f a rgum ent tha t d ig it 2 is absen t in 4 -fingered lim bs a re severa l fo ld . 1 ) T he tim e course ana lys is p resen ted a bove show tha t in the G as1 -/-;S hh +/- geno type , d ig its 3 and 4 a re p resent in the ir no rm a l loca tions, w h ile the re is on ly la rge in te r-d ig ita l m esenchym e w here d ig it 2 no rm a lly fo rm s. 2 ) T he in te rm ed ia te pha lange of d ig it 2 in G as1 m u tan t fo re lim bs is freque n tly d isfigu red o r k inked (L iu e t a l. 2002). S uch a m a lfo rm ed d ig it is never observed in any 4 -fingered fo re lim bs, suggesting tha t none of the rem a in ing d ig its a re 2 . 3 ) W hen m etacarpa ls a re found to be fused in e ithe r geno type , it is a lw ays be tw een d ig its 2 and 3; w hen m eta ta rsa ls a re fused, it is a lw ays be tw een d ig its 1 and 2. N e ithe r o f these fus ions w ere ever observed in 4 -fingered lim bs. If the m iss ing d ig it w as actua lly 3 , then w e w ou ld s till expect to f ind fus ions be tw een the rem a in ing d ig its 1 and 2. 4 ) T he d isp roportiona lly reduced s ize o f d ig it 2 in the G as1 m utan t suggests tha t a m ore severe pheno type (as in the G as1 -/-;S hh +/- geno type) w ou ld cause a lack o f d ig it 2 to fo rm . E ven if the m iss ing d ig it is 3 o r adop ts a m ixed iden tity o f 2 and 3 , the conclus ions in the text a re s till app licab le because d ig it 3 a lso depends in part on d iffus ib le S H H (see re fe rences in text).

Martinelli and Fan Supplemental Figure 4

HNF3β GFP

GFP

GFP

GFP

OLIG2

NKX6.1 PAX6

F igure S 4 . G as1 express ion a lte rs the position o f neurona l p rogen ito rs in the neura l tube . C h ick trunk neura l tube w as e le ctropora ted w ith G as1N /A -IR E S -G FP (no t show n) o r G as1 -IR E S -G FP p lasm ids and ana lyzed fo r G FP , P A X6, N K X6.1 , O LIG 2, and H N F3β by doub le im m unofluo rescence (green fo r G FP , and red fo r PA X6, N K X6.1 , O L IG 2, o r H N F3β; excep t fo r O LIG 2, in w h ich the green s igna l is the au to fluo rescence from G FP ) on the sam e sections. Each pa ir o f im ages is from the sam e section; n ≥ 10 fo r each pa ir. A rrow s ind ica te ectop ic activa tion o f H H ta rge t genes; open a rrow s, ectop ic inh ib ition ; open a rrow heads, the ven tra l m id line.

Martinelli and Fan Supplemental Figure 5

Gas1/LacZ

Neural Plate

Neural Tube at PSM Level

Neural Tube at Soimte V

F igure S 5 . G as1 express ion w as de te rm ined by X -ga l s ta in ing E 9 .5 em bryos transverse ly sectioned a t va rious leve ls rang ing from poste rio r (le ft) to ante rio r (righ t). A x ia l leve ls a re ind ica ted on top , fo r com parison to F igure 7A .

Martinelli and Fan Supplemental Figure 6

Gas1-/-;Shh-/-Gas1+/+;Shh-/-Gas1-/-;Shh+/+Gas1+/-;Shh+/-

Ptc1

Gas1/LacZ

F igure S 6 . T he defective le ft/righ t asym m etry pa tte rn ing in the G as1 -/-;S hh -/- em bryo is associa ted w ith im pa ired H H s igna ling in the node as dete rm ined by P tc1 IS H in E8 .0 em bryos. G eno types a re ind ica ted on top . A rrow heads po in t to the m id line w here P tc1 express ion exis ts w hen S hh is p resen t. A rrow s po in t to the node . T he bo ttom p ic tu re is a X -ga l s ta ined G as1 -/-;S hh -/- em bryo revea ling LacZ express ion in the node fo r com parison to F ig . 7 E ,F . G iven the tw o cop ies o f the LacZ gene , the X -ga l s ta in ing w as tim e reduced accord ing ly (45 m in ) to m atch the leve ls o f p rim itive s treak s ta in ing in the LacZ hete rozygous em bryos (2 h r).

Martinelli and Fan Supplemental Figure 1

SphIEcoRIBamHI

SphI

tau-LacZ

tau-LacZ

PGK-neopA

XhoI

Gas1

recombinationconstruct PGK-tk

XhoI

SphI

SphI

SphI

SphI

Gas1LacZ

(Gas1-)

XhoI

XhoI PGK-neopA

SphI

A

e

22

+/- -/-+/- -/-e

+/- +/-

+/-

+/-+/+

E14.5 hl

B C D E

G Hn

ps

np

s

E14.5 head

dn

oe

dm

dn

fl

fl

hldn

s

o e

-/-

-/-

M c +/++/- -/-

Gas1LacZ

100

400

F

F igure S 1 . (A ) H om ologous recom b ination schem e to generate the G as1 LacZ (G as1 -) a lle le ; top , genom ic s tructu re o f G as1 locus; m idd le , recom b ina tion construct; bo ttom , recom bined a lle le . T he tau -LacZ -pA -P G K-neo casse tte is p laced a t the exact sam e loca tion as the PG K -neo casse tte in the p reviously pub lished G as1 neo a lle le (Lee e t a l. 2001b) and rep laces the entire cod ing reg ion o f G as1 . pA , po ly-A s igna l sequence ; P G K -tk , P G K -driven thym id ine k inase gene casse tte fo r nega tive se lection. R estric tion enzym e s ites (S ph I and Xho I) and 5 ’ and 3 ’ p robes used to screen fo r and confirm recom bination a re a lso the sam e as previous ly pub lished (Lee e t a l. 2001b); w h ite boxes, 5 ’ and 3 ’ U T R s of G as1 ; b lack box, cod ing reg ion o f G as1 ; a rrow heads, new P C R prim er se ts (see M ethods) fo r genotyp ing. (B ) P C R w ith the ind icated p rim er se ts can d is tingu ish w ild type (+ /+), he te rozygous (+ / -), and m utan t (-/-) em bryos; M , 100 bp ladder, 100 bp and 400 bp bands are labe led ; c , m ock con tro l P C R ; expected G as1 and LacZ bands a re labe led. (C ) X -ga l s ta in ing fo r LacZ activ ity (b lue ) in E7 .0 w ild type con tro l (le ft) and he te rozygous (righ t) em bryos. (D and F ) X -ga l s ta in ing pa tte rns o f E 9 .5 and E10 .5 he te rozygo tes (le ft) and m utan ts (righ t), respective ly. (E ) A transverse section o f E 9.5 a t the fo re lim b (fl) leve l. T hese sta in ing pa tte rns agree w ith the endogenous G as1 express ion ob ta ined by IS H (Lee and F an 2001). S -ga l (red ) s ta ined E 14 .5 he te rozygous (le ft) and m utan t (righ t) heads (G ) and h ind lim bs (h l) (H ) show tha t m utants have m icrophtha lm ia and sm a ll lim b /m a lfo rm ed d ig it 2 (w h ite a rrow head) pheno types as described fo r the G as1 neo m utant (L iu e t a l. 2002). U nder our experim enta l cond ition (37 °C , 2 h rs), w ild type em bryos fo r a ll s tages p resen ted here w ere nega tive fo r s ta in ing (not show n). O f no te , m utan ts have a sm a lle r head structu re than he te rozygo tes and w ild types observab le as early as E 9 .5 , w h ich w ill be described in de ta il in a separa te m anuscrip t. A bbrevia tions: np , neura l p la te ; ps, p rim itive s treak; n , node; s , som ite ; o , o tic ves ic le o r ea r; e , e ye ; dn , do rsa l neura l tube ; dm , derm om yotom e.

Martinelli and Fan Supplemental Figure 2

0

20

40

60

PAX7 PAX3 DBX1

% le

ngth

of N

.T.

Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/-

PAX7

PAX6

PAX3

GSH1/2

DB X1

NK X6.1

OL IG2

NK X2.2

HNF3β

S HH

Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/- Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/-

p=6.6E-5

p=.0076 p=1.6E-8

p=5.0E-7

p=2.2E-6

p=.0014

Gas1+/-;Shh+/- Gas1-/-;Shh+/+ Gas1-/-;Shh+/-

E12.5

E13.5

1 1 1

1 1 1

2 2

2 2

3 3 3

3 33

4

44

4

4

4

5 5 5

5 5 5

0

0.2

0.4

0.6

0.8

1

Forelimb Hindlimb

digi

t 2:4

leng

th ra

tio

Control Gas1 mutantA

B

Martinelli and Fan Supplemental Figure 3F igure S 2 . Q uan tita tion and add itiona l m a rker ana lys is fo r the neura l tube defect o f G as1 -/- and G as1 -/-;S hh +/- em bryos. Em bryos o f 24 -25 som ites w ere transverse ly sectioned a t the fo re lim b leve l and ana lyzed by IH C . G eno types a re labe led on top and m arkers on the le ft. In the le ft 3 co lum ns a re dorsa l m arkers, w h ile in the righ t 3 co lum ns a re ven tra l p rogen ito r m arkers. O f note , com prom ised floo rp la te induction w as a lso observed by the a lte ra tion o f N K X2.2 , H N F3β, and S H H express ion . A lso , in G as1 -/-;S hh +/- em bryos, O LIG 2 -positive ce lls occupy the m id line w ith little o r no S H H -positive ce lls . The resu lts a re consis ten t w ith reduced S H H s igna ling in the G as1 -/- neura l tube and a fu rthe r reduction in the G as1 -/-;S hh +/- neura l tube. D ashed line dem arks the boundaries o f express ion o f PA X7, P A X3, a nd D B X1. To quan tify the changes in the boundaries o f express ion fo r these 3 genes, the percen t o f the neura l tube w ith in the boundaries w as ca lcu la ted fo r G as1 +/-;S hh +/- (n = 5 ), G as1 -/- (n = 4 ), and G as1 -/-;S hh +/- (n = 3 ) em bryos. 2 sections w ere ana lyzed fo r each em bryo . A tw o-ta iled W elch ’s t-test w as perfo rm ed . For the boundaries o f a ll 3 genes , G as1 +/-; S hh +/- em bryos w ere s ta tis tica lly d iffe ren t from bo th G as1 -/- and G as1 -/-;S hh +/- em bryos (99% confidence leve l). P va lues a re ind icate d fo r each t-test and d isp layed on the bar graph . The express ion boundaries in G as1 -/- and G as1 -/-; S hh +/- em bryos w ere no t found to be s ta tis tica lly d iffe ren t.

F igu re S 3 . (A ) G as1 m u tant d ig it 2 is d isp roportiona lly sm a lle r than the o ther d ig its . T he lengths o f d ig its 2 and 4 w ere m easured on G as1 +/-;S hh +/- and G as1 -/- neona ta l ske le ta l p repara tions and the length ra tio ca lcu la ted . D ig it 4 w as used fo r com parison because it and d ig it 2 a re norm a lly o f s im ila r length . n fo r con tro l lim bs is 3 ; fo r G as1 -/- fo re lim bs, 19 ; fo r G as1 -/- h ind lim bs, 11 . E rro r ba rs = 1 s tandard devia tion . (B ) E xam ina tion o f fo re lim bs of E 12.5 and E 13 .5 m ice o f G as1 and S hh a lle lic com b ina tions (ind icated a t top ) revea led that d ig it 2 never fo rm s in the G as1 -/-;Shh +/- genotype . A rrow head ind ica tes la rge loca tion o f in te r -d ig ita l m esenchym e w here d ig it 2 no rm a lly form s. n = 3 fo r each geno type and stage . D ig it num bers are ind ica ted . S ca le bar = 1 m m . R ationa le fo r the determ ination o f m issing d ig it identity : D e te rm ina tion o f d ig it 1 w as stra igh tfo rw ard because it has one less pha lange . D ig its 4 and 5 w ere de te rm ined by m easuring the ir re la tive lengths as w e ll as exam ination o f m etacarpa l and m eta tarsa l shapes . A s p revious ly reported fo r G as1 m u tan ts, w e found tha t d ig its 4 and 5 a re no t a ffected in the G as1 -/-;Shh +/- (L iu e t a l. 2002). D is tingu ish ing the m issing d ig it as 2 as opposed to 3 is m ore com p lica ted. O ur lines o f a rgum ent tha t d ig it 2 is absen t in 4 -fingered lim bs a re severa l fo ld . 1 ) T he tim e course ana lys is p resen ted a bove show tha t in the G as1 -/-;S hh +/- geno type , d ig its 3 and 4 a re p resent in the ir no rm a l loca tions, w h ile the re is on ly la rge in te r-d ig ita l m esenchym e w here d ig it 2 no rm a lly fo rm s. 2 ) T he in te rm ed ia te pha lange of d ig it 2 in G as1 m u tan t fo re lim bs is freque n tly d isfigu red o r k inked (L iu e t a l. 2002). S uch a m a lfo rm ed d ig it is never observed in any 4 -fingered fo re lim bs, suggesting tha t none of the rem a in ing d ig its a re 2 . 3 ) W hen m etacarpa ls a re found to be fused in e ithe r geno type , it is a lw ays be tw een d ig its 2 and 3; w hen m eta ta rsa ls a re fused, it is a lw ays be tw een d ig its 1 and 2. N e ithe r o f these fus ions w ere ever observed in 4 -fingered lim bs. If the m iss ing d ig it w as actua lly 3 , then w e w ou ld s till expect to f ind fus ions be tw een the rem a in ing d ig its 1 and 2. 4 ) T he d isp roportiona lly reduced s ize o f d ig it 2 in the G as1 m utan t suggests tha t a m ore severe pheno type (as in the G as1 -/-;S hh +/- geno type) w ou ld cause a lack o f d ig it 2 to fo rm . E ven if the m iss ing d ig it is 3 o r adop ts a m ixed iden tity o f 2 and 3 , the conclus ions in the text a re s till app licab le because d ig it 3 a lso depends in part on d iffus ib le S H H (see re fe rences in text).

Martinelli and Fan Supplemental Figure 4

HNF3β GFP

GFP

GFP

GFP

OLIG2

NKX6.1 PAX6

F igure S 4 . G as1 express ion a lte rs the position o f neurona l p rogen ito rs in the neura l tube . C h ick trunk neura l tube w as e le ctropora ted w ith G as1N /A -IR E S -G FP (no t show n) o r G as1 -IR E S -G FP p lasm ids and ana lyzed fo r G FP , P A X6, N K X6.1 , O LIG 2, and H N F3β by doub le im m unofluo rescence (green fo r G FP , and red fo r PA X6, N K X6.1 , O L IG 2, o r H N F3β; excep t fo r O LIG 2, in w h ich the green s igna l is the au to fluo rescence from G FP ) on the sam e sections. Each pa ir o f im ages is from the sam e section; n ≥ 10 fo r each pa ir. A rrow s ind ica te ectop ic activa tion o f H H ta rge t genes; open a rrow s, ectop ic inh ib ition ; open a rrow heads, the ven tra l m id line.

Martinelli and Fan Supplemental Figure 5

Gas1/LacZ

Neural Plate

Neural Tube at PSM Level

Neural Tube at Soimte V

F igure S 5 . G as1 express ion w as de te rm ined by X -ga l s ta in ing E 9 .5 em bryos transverse ly sectioned a t va rious leve ls rang ing from poste rio r (le ft) to ante rio r (righ t). A x ia l leve ls a re ind ica ted on top , fo r com parison to F igure 7A .

Martinelli and Fan Supplemental Figure 6

Gas1-/-;Shh-/-Gas1+/+;Shh-/-Gas1-/-;Shh+/+Gas1+/-;Shh+/-

Ptc1

Gas1/LacZ

F igure S 6 . T he defective le ft/righ t asym m etry pa tte rn ing in the G as1 -/-;S hh -/- em bryo is associa ted w ith im pa ired H H s igna ling in the node as dete rm ined by P tc1 IS H in E8 .0 em bryos. G eno types a re ind ica ted on top . A rrow heads po in t to the m id line w here P tc1 express ion exis ts w hen S hh is p resen t. A rrow s po in t to the node . T he bo ttom p ic tu re is a X -ga l s ta ined G as1 -/-;S hh -/- em bryo revea ling LacZ express ion in the node fo r com parison to F ig . 7 E ,F . G iven the tw o cop ies o f the LacZ gene , the X -ga l s ta in ing w as tim e reduced accord ing ly (45 m in ) to m atch the leve ls o f p rim itive s treak s ta in ing in the LacZ hete rozygous em bryos (2 h r).

Martinelli and Fan Supplemental Figure 1

SphIEcoRIBamHI

SphI

tau-LacZ

tau-LacZ

PGK-neopA

XhoI

Gas1

recombinationconstruct PGK-tk

XhoI

SphI

SphI

SphI

SphI

Gas1LacZ

(Gas1-)

XhoI

XhoI PGK-neopA

SphI

A

e

22

+/- -/-+/- -/-e

+/- +/-

+/-

+/-+/+

E14.5 hl

B C D E

G Hn

ps

np

s

E14.5 head

dn

oe

dm

dn

fl

fl

hldn

s

o e

-/-

-/-

M c +/++/- -/-

Gas1LacZ

100

400

F

F igure S 1 . (A ) H om ologous recom b ination schem e to generate the G as1 LacZ (G as1 -) a lle le ; top , genom ic s tructu re o f G as1 locus; m idd le , recom b ina tion construct; bo ttom , recom bined a lle le . T he tau -LacZ -pA -P G K-neo casse tte is p laced a t the exact sam e loca tion as the PG K -neo casse tte in the p reviously pub lished G as1 neo a lle le (Lee e t a l. 2001b) and rep laces the entire cod ing reg ion o f G as1 . pA , po ly-A s igna l sequence ; P G K -tk , P G K -driven thym id ine k inase gene casse tte fo r nega tive se lection. R estric tion enzym e s ites (S ph I and Xho I) and 5 ’ and 3 ’ p robes used to screen fo r and confirm recom bination a re a lso the sam e as previous ly pub lished (Lee e t a l. 2001b); w h ite boxes, 5 ’ and 3 ’ U T R s of G as1 ; b lack box, cod ing reg ion o f G as1 ; a rrow heads, new P C R prim er se ts (see M ethods) fo r genotyp ing. (B ) P C R w ith the ind icated p rim er se ts can d is tingu ish w ild type (+ /+), he te rozygous (+ / -), and m utan t (-/-) em bryos; M , 100 bp ladder, 100 bp and 400 bp bands are labe led ; c , m ock con tro l P C R ; expected G as1 and LacZ bands a re labe led. (C ) X -ga l s ta in ing fo r LacZ activ ity (b lue ) in E7 .0 w ild type con tro l (le ft) and he te rozygous (righ t) em bryos. (D and F ) X -ga l s ta in ing pa tte rns o f E 9 .5 and E10 .5 he te rozygo tes (le ft) and m utan ts (righ t), respective ly. (E ) A transverse section o f E 9.5 a t the fo re lim b (fl) leve l. T hese sta in ing pa tte rns agree w ith the endogenous G as1 express ion ob ta ined by IS H (Lee and F an 2001). S -ga l (red ) s ta ined E 14 .5 he te rozygous (le ft) and m utan t (righ t) heads (G ) and h ind lim bs (h l) (H ) show tha t m utants have m icrophtha lm ia and sm a ll lim b /m a lfo rm ed d ig it 2 (w h ite a rrow head) pheno types as described fo r the G as1 neo m utant (L iu e t a l. 2002). U nder our experim enta l cond ition (37 °C , 2 h rs), w ild type em bryos fo r a ll s tages p resen ted here w ere nega tive fo r s ta in ing (not show n). O f no te , m utan ts have a sm a lle r head structu re than he te rozygo tes and w ild types observab le as early as E 9 .5 , w h ich w ill be described in de ta il in a separa te m anuscrip t. A bbrevia tions: np , neura l p la te ; ps, p rim itive s treak; n , node; s , som ite ; o , o tic ves ic le o r ea r; e , e ye ; dn , do rsa l neura l tube ; dm , derm om yotom e.

Martinelli and Fan Supplemental Figure 2

0

20

40

60

PAX7 PAX3 DBX1

% le

ngth

of N

.T.

Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/-

PAX7

PAX6

PAX3

GSH1/2

DB X1

NK X6.1

OL IG2

NK X2.2

HNF3β

S HH

Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/- Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/-

p=6.6E-5

p=.0076 p=1.6E-8

p=5.0E-7

p=2.2E-6

p=.0014

Gas1+/-;Shh+/- Gas1-/-;Shh+/+ Gas1-/-;Shh+/-

E12.5

E13.5

1 1 1

1 1 1

2 2

2 2

3 3 3

3 33

4

44

4

4

4

5 5 5

5 5 5

0

0.2

0.4

0.6

0.8

1

Forelimb Hindlimb

digi

t 2:4

leng

th ra

tio

Control Gas1 mutantA

B

Martinelli and Fan Supplemental Figure 3F igure S 2 . Q uan tita tion and add itiona l m a rker ana lys is fo r the neura l tube defect o f G as1 -/- and G as1 -/-;S hh +/- em bryos. Em bryos o f 24 -25 som ites w ere transverse ly sectioned a t the fo re lim b leve l and ana lyzed by IH C . G eno types a re labe led on top and m arkers on the le ft. In the le ft 3 co lum ns a re dorsa l m arkers, w h ile in the righ t 3 co lum ns a re ven tra l p rogen ito r m arkers. O f note , com prom ised floo rp la te induction w as a lso observed by the a lte ra tion o f N K X2.2 , H N F3β, and S H H express ion . A lso , in G as1 -/-;S hh +/- em bryos, O LIG 2 -positive ce lls occupy the m id line w ith little o r no S H H -positive ce lls . The resu lts a re consis ten t w ith reduced S H H s igna ling in the G as1 -/- neura l tube and a fu rthe r reduction in the G as1 -/-;S hh +/- neura l tube. D ashed line dem arks the boundaries o f express ion o f PA X7, P A X3, a nd D B X1. To quan tify the changes in the boundaries o f express ion fo r these 3 genes, the percen t o f the neura l tube w ith in the boundaries w as ca lcu la ted fo r G as1 +/-;S hh +/- (n = 5 ), G as1 -/- (n = 4 ), and G as1 -/-;S hh +/- (n = 3 ) em bryos. 2 sections w ere ana lyzed fo r each em bryo . A tw o-ta iled W elch ’s t-test w as perfo rm ed . For the boundaries o f a ll 3 genes , G as1 +/-; S hh +/- em bryos w ere s ta tis tica lly d iffe ren t from bo th G as1 -/- and G as1 -/-;S hh +/- em bryos (99% confidence leve l). P va lues a re ind icate d fo r each t-test and d isp layed on the bar graph . The express ion boundaries in G as1 -/- and G as1 -/-; S hh +/- em bryos w ere no t found to be s ta tis tica lly d iffe ren t.

F igu re S 3 . (A ) G as1 m u tant d ig it 2 is d isp roportiona lly sm a lle r than the o ther d ig its . T he lengths o f d ig its 2 and 4 w ere m easured on G as1 +/-;S hh +/- and G as1 -/- neona ta l ske le ta l p repara tions and the length ra tio ca lcu la ted . D ig it 4 w as used fo r com parison because it and d ig it 2 a re norm a lly o f s im ila r length . n fo r con tro l lim bs is 3 ; fo r G as1 -/- fo re lim bs, 19 ; fo r G as1 -/- h ind lim bs, 11 . E rro r ba rs = 1 s tandard devia tion . (B ) E xam ina tion o f fo re lim bs of E 12.5 and E 13 .5 m ice o f G as1 and S hh a lle lic com b ina tions (ind icated a t top ) revea led that d ig it 2 never fo rm s in the G as1 -/-;Shh +/- genotype . A rrow head ind ica tes la rge loca tion o f in te r -d ig ita l m esenchym e w here d ig it 2 no rm a lly form s. n = 3 fo r each geno type and stage . D ig it num bers are ind ica ted . S ca le bar = 1 m m . R ationa le fo r the determ ination o f m issing d ig it identity : D e te rm ina tion o f d ig it 1 w as stra igh tfo rw ard because it has one less pha lange . D ig its 4 and 5 w ere de te rm ined by m easuring the ir re la tive lengths as w e ll as exam ination o f m etacarpa l and m eta tarsa l shapes . A s p revious ly reported fo r G as1 m u tan ts, w e found tha t d ig its 4 and 5 a re no t a ffected in the G as1 -/-;Shh +/- (L iu e t a l. 2002). D is tingu ish ing the m issing d ig it as 2 as opposed to 3 is m ore com p lica ted. O ur lines o f a rgum ent tha t d ig it 2 is absen t in 4 -fingered lim bs a re severa l fo ld . 1 ) T he tim e course ana lys is p resen ted a bove show tha t in the G as1 -/-;S hh +/- geno type , d ig its 3 and 4 a re p resent in the ir no rm a l loca tions, w h ile the re is on ly la rge in te r-d ig ita l m esenchym e w here d ig it 2 no rm a lly fo rm s. 2 ) T he in te rm ed ia te pha lange of d ig it 2 in G as1 m u tan t fo re lim bs is freque n tly d isfigu red o r k inked (L iu e t a l. 2002). S uch a m a lfo rm ed d ig it is never observed in any 4 -fingered fo re lim bs, suggesting tha t none of the rem a in ing d ig its a re 2 . 3 ) W hen m etacarpa ls a re found to be fused in e ithe r geno type , it is a lw ays be tw een d ig its 2 and 3; w hen m eta ta rsa ls a re fused, it is a lw ays be tw een d ig its 1 and 2. N e ithe r o f these fus ions w ere ever observed in 4 -fingered lim bs. If the m iss ing d ig it w as actua lly 3 , then w e w ou ld s till expect to f ind fus ions be tw een the rem a in ing d ig its 1 and 2. 4 ) T he d isp roportiona lly reduced s ize o f d ig it 2 in the G as1 m utan t suggests tha t a m ore severe pheno type (as in the G as1 -/-;S hh +/- geno type) w ou ld cause a lack o f d ig it 2 to fo rm . E ven if the m iss ing d ig it is 3 o r adop ts a m ixed iden tity o f 2 and 3 , the conclus ions in the text a re s till app licab le because d ig it 3 a lso depends in part on d iffus ib le S H H (see re fe rences in text).

Martinelli and Fan Supplemental Figure 4

HNF3β GFP

GFP

GFP

GFP

OLIG2

NKX6.1 PAX6

F igure S 4 . G as1 express ion a lte rs the position o f neurona l p rogen ito rs in the neura l tube . C h ick trunk neura l tube w as e le ctropora ted w ith G as1N /A -IR E S -G FP (no t show n) o r G as1 -IR E S -G FP p lasm ids and ana lyzed fo r G FP , P A X6, N K X6.1 , O LIG 2, and H N F3β by doub le im m unofluo rescence (green fo r G FP , and red fo r PA X6, N K X6.1 , O L IG 2, o r H N F3β; excep t fo r O LIG 2, in w h ich the green s igna l is the au to fluo rescence from G FP ) on the sam e sections. Each pa ir o f im ages is from the sam e section; n ≥ 10 fo r each pa ir. A rrow s ind ica te ectop ic activa tion o f H H ta rge t genes; open a rrow s, ectop ic inh ib ition ; open a rrow heads, the ven tra l m id line.

Martinelli and Fan Supplemental Figure 5

Gas1/LacZ

Neural Plate

Neural Tube at PSM Level

Neural Tube at Soimte V

F igure S 5 . G as1 express ion w as de te rm ined by X -ga l s ta in ing E 9 .5 em bryos transverse ly sectioned a t va rious leve ls rang ing from poste rio r (le ft) to ante rio r (righ t). A x ia l leve ls a re ind ica ted on top , fo r com parison to F igure 7A .

Martinelli and Fan Supplemental Figure 6

Gas1-/-;Shh-/-Gas1+/+;Shh-/-Gas1-/-;Shh+/+Gas1+/-;Shh+/-

Ptc1

Gas1/LacZ

F igure S 6 . T he defective le ft/righ t asym m etry pa tte rn ing in the G as1 -/-;S hh -/- em bryo is associa ted w ith im pa ired H H s igna ling in the node as dete rm ined by P tc1 IS H in E8 .0 em bryos. G eno types a re ind ica ted on top . A rrow heads po in t to the m id line w here P tc1 express ion exis ts w hen S hh is p resen t. A rrow s po in t to the node . T he bo ttom p ic tu re is a X -ga l s ta ined G as1 -/-;S hh -/- em bryo revea ling LacZ express ion in the node fo r com parison to F ig . 7 E ,F . G iven the tw o cop ies o f the LacZ gene , the X -ga l s ta in ing w as tim e reduced accord ing ly (45 m in ) to m atch the leve ls o f p rim itive s treak s ta in ing in the LacZ hete rozygous em bryos (2 h r).

Martinelli and Fan Supplemental Figure 1

SphIEcoRIBamHI

SphI

tau-LacZ

tau-LacZ

PGK-neopA

XhoI

Gas1

recombinationconstruct PGK-tk

XhoI

SphI

SphI

SphI

SphI

Gas1LacZ

(Gas1-)

XhoI

XhoI PGK-neopA

SphI

A

e

22

+/- -/-+/- -/-e

+/- +/-

+/-

+/-+/+

E14.5 hl

B C D E

G Hn

ps

np

s

E14.5 head

dn

oe

dm

dn

fl

fl

hldn

s

o e

-/-

-/-

M c +/++/- -/-

Gas1LacZ

100

400

F

F igure S 1 . (A ) H om ologous recom b ination schem e to generate the G as1 LacZ (G as1 -) a lle le ; top , genom ic s tructu re o f G as1 locus; m idd le , recom b ina tion construct; bo ttom , recom bined a lle le . T he tau -LacZ -pA -P G K-neo casse tte is p laced a t the exact sam e loca tion as the PG K -neo casse tte in the p reviously pub lished G as1 neo a lle le (Lee e t a l. 2001b) and rep laces the entire cod ing reg ion o f G as1 . pA , po ly-A s igna l sequence ; P G K -tk , P G K -driven thym id ine k inase gene casse tte fo r nega tive se lection. R estric tion enzym e s ites (S ph I and Xho I) and 5 ’ and 3 ’ p robes used to screen fo r and confirm recom bination a re a lso the sam e as previous ly pub lished (Lee e t a l. 2001b); w h ite boxes, 5 ’ and 3 ’ U T R s of G as1 ; b lack box, cod ing reg ion o f G as1 ; a rrow heads, new P C R prim er se ts (see M ethods) fo r genotyp ing. (B ) P C R w ith the ind icated p rim er se ts can d is tingu ish w ild type (+ /+), he te rozygous (+ / -), and m utan t (-/-) em bryos; M , 100 bp ladder, 100 bp and 400 bp bands are labe led ; c , m ock con tro l P C R ; expected G as1 and LacZ bands a re labe led. (C ) X -ga l s ta in ing fo r LacZ activ ity (b lue ) in E7 .0 w ild type con tro l (le ft) and he te rozygous (righ t) em bryos. (D and F ) X -ga l s ta in ing pa tte rns o f E 9 .5 and E10 .5 he te rozygo tes (le ft) and m utan ts (righ t), respective ly. (E ) A transverse section o f E 9.5 a t the fo re lim b (fl) leve l. T hese sta in ing pa tte rns agree w ith the endogenous G as1 express ion ob ta ined by IS H (Lee and F an 2001). S -ga l (red ) s ta ined E 14 .5 he te rozygous (le ft) and m utan t (righ t) heads (G ) and h ind lim bs (h l) (H ) show tha t m utants have m icrophtha lm ia and sm a ll lim b /m a lfo rm ed d ig it 2 (w h ite a rrow head) pheno types as described fo r the G as1 neo m utant (L iu e t a l. 2002). U nder our experim enta l cond ition (37 °C , 2 h rs), w ild type em bryos fo r a ll s tages p resen ted here w ere nega tive fo r s ta in ing (not show n). O f no te , m utan ts have a sm a lle r head structu re than he te rozygo tes and w ild types observab le as early as E 9 .5 , w h ich w ill be described in de ta il in a separa te m anuscrip t. A bbrevia tions: np , neura l p la te ; ps, p rim itive s treak; n , node; s , som ite ; o , o tic ves ic le o r ea r; e , e ye ; dn , do rsa l neura l tube ; dm , derm om yotom e.

Martinelli and Fan Supplemental Figure 2

0

20

40

60

PAX7 PAX3 DBX1

% le

ngth

of N

.T.

Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/-

PAX7

PAX6

PAX3

GSH1/2

DB X1

NK X6.1

OL IG2

NK X2.2

HNF3β

S HH

Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/- Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/-

p=6.6E-5

p=.0076 p=1.6E-8

p=5.0E-7

p=2.2E-6

p=.0014

Gas1+/-;Shh+/- Gas1-/-;Shh+/+ Gas1-/-;Shh+/-

E12.5

E13.5

1 1 1

1 1 1

2 2

2 2

3 3 3

3 33

4

44

4

4

4

5 5 5

5 5 5

0

0.2

0.4

0.6

0.8

1

Forelimb Hindlimb

digi

t 2:4

leng

th ra

tioControl Gas1 mutantA

B

Martinelli and Fan Supplemental Figure 3F igure S 2 . Q uan tita tion and add itiona l m a rker ana lys is fo r the neura l tube defect o f G as1 -/- and G as1 -/-;S hh +/- em bryos. Em bryos o f 24 -25 som ites w ere transverse ly sectioned a t the fo re lim b leve l and ana lyzed by IH C . G eno types a re labe led on top and m arkers on the le ft. In the le ft 3 co lum ns a re dorsa l m arkers, w h ile in the righ t 3 co lum ns a re ven tra l p rogen ito r m arkers. O f note , com prom ised floo rp la te induction w as a lso observed by the a lte ra tion o f N K X2.2 , H N F3β, and S H H express ion . A lso , in G as1 -/-;S hh +/- em bryos, O LIG 2 -positive ce lls occupy the m id line w ith little o r no S H H -positive ce lls . The resu lts a re consis ten t w ith reduced S H H s igna ling in the G as1 -/- neura l tube and a fu rthe r reduction in the G as1 -/-;S hh +/- neura l tube. D ashed line dem arks the boundaries o f express ion o f PA X7, P A X3, a nd D B X1. To quan tify the changes in the boundaries o f express ion fo r these 3 genes, the percen t o f the neura l tube w ith in the boundaries w as ca lcu la ted fo r G as1 +/-;S hh +/- (n = 5 ), G as1 -/- (n = 4 ), and G as1 -/-;S hh +/- (n = 3 ) em bryos. 2 sections w ere ana lyzed fo r each em bryo . A tw o-ta iled W elch ’s t-test w as perfo rm ed . For the boundaries o f a ll 3 genes , G as1 +/-; S hh +/- em bryos w ere s ta tis tica lly d iffe ren t from bo th G as1 -/- and G as1 -/-;S hh +/- em bryos (99% confidence leve l). P va lues a re ind icate d fo r each t-test and d isp layed on the bar graph . The express ion boundaries in G as1 -/- and G as1 -/-; S hh +/- em bryos w ere no t found to be s ta tis tica lly d iffe ren t.

F igu re S 3 . (A ) G as1 m u tant d ig it 2 is d isp roportiona lly sm a lle r than the o ther d ig its . T he lengths o f d ig its 2 and 4 w ere m easured on G as1 +/-;S hh +/- and G as1 -/- neona ta l ske le ta l p repara tions and the length ra tio ca lcu la ted . D ig it 4 w as used fo r com parison because it and d ig it 2 a re norm a lly o f s im ila r length . n fo r con tro l lim bs is 3 ; fo r G as1 -/- fo re lim bs, 19 ; fo r G as1 -/- h ind lim bs, 11 . E rro r ba rs = 1 s tandard devia tion . (B ) E xam ina tion o f fo re lim bs of E 12.5 and E 13 .5 m ice o f G as1 and S hh a lle lic com b ina tions (ind icated a t top ) revea led that d ig it 2 never fo rm s in the G as1 -/-;Shh +/- genotype . A rrow head ind ica tes la rge loca tion o f in te r -d ig ita l m esenchym e w here d ig it 2 no rm a lly form s. n = 3 fo r each geno type and stage . D ig it num bers are ind ica ted . S ca le bar = 1 m m . R ationa le fo r the determ ination o f m issing d ig it identity : D e te rm ina tion o f d ig it 1 w as stra igh tfo rw ard because it has one less pha lange . D ig its 4 and 5 w ere de te rm ined by m easuring the ir re la tive lengths as w e ll as exam ination o f m etacarpa l and m eta tarsa l shapes . A s p revious ly reported fo r G as1 m u tan ts, w e found tha t d ig its 4 and 5 a re no t a ffected in the G as1 -/-;Shh +/- (L iu e t a l. 2002). D is tingu ish ing the m issing d ig it as 2 as opposed to 3 is m ore com p lica ted. O ur lines o f a rgum ent tha t d ig it 2 is absen t in 4 -fingered lim bs a re severa l fo ld . 1 ) T he tim e course ana lys is p resen ted a bove show tha t in the G as1 -/-;S hh +/- geno type , d ig its 3 and 4 a re p resent in the ir no rm a l loca tions, w h ile the re is on ly la rge in te r-d ig ita l m esenchym e w here d ig it 2 no rm a lly fo rm s. 2 ) T he in te rm ed ia te pha lange of d ig it 2 in G as1 m u tan t fo re lim bs is freque n tly d isfigu red o r k inked (L iu e t a l. 2002). S uch a m a lfo rm ed d ig it is never observed in any 4 -fingered fo re lim bs, suggesting tha t none of the rem a in ing d ig its a re 2 . 3 ) W hen m etacarpa ls a re found to be fused in e ithe r geno type , it is a lw ays be tw een d ig its 2 and 3; w hen m eta ta rsa ls a re fused, it is a lw ays be tw een d ig its 1 and 2. N e ithe r o f these fus ions w ere ever observed in 4 -fingered lim bs. If the m iss ing d ig it w as actua lly 3 , then w e w ou ld s till expect to f ind fus ions be tw een the rem a in ing d ig its 1 and 2. 4 ) T he d isp roportiona lly reduced s ize o f d ig it 2 in the G as1 m utan t suggests tha t a m ore severe pheno type (as in the G as1 -/-;S hh +/- geno type) w ou ld cause a lack o f d ig it 2 to fo rm . E ven if the m iss ing d ig it is 3 o r adop ts a m ixed iden tity o f 2 and 3 , the conclus ions in the text a re s till app licab le because d ig it 3 a lso depends in part on d iffus ib le S H H (see re fe rences in text).

Martinelli and Fan Supplemental Figure 4

HNF3β GFP

GFP

GFP

GFP

OLIG2

NKX6.1 PAX6

F igure S 4 . G as1 express ion a lte rs the position o f neurona l p rogen ito rs in the neura l tube . C h ick trunk neura l tube w as e le ctropora ted w ith G as1N /A -IR E S -G FP (no t show n) o r G as1 -IR E S -G FP p lasm ids and ana lyzed fo r G FP , P A X6, N K X6.1 , O LIG 2, and H N F3β by doub le im m unofluo rescence (green fo r G FP , and red fo r PA X6, N K X6.1 , O L IG 2, o r H N F3β; excep t fo r O LIG 2, in w h ich the green s igna l is the au to fluo rescence from G FP ) on the sam e sections. Each pa ir o f im ages is from the sam e section; n ≥ 10 fo r each pa ir. A rrow s ind ica te ectop ic activa tion o f H H ta rge t genes; open a rrow s, ectop ic inh ib ition ; open a rrow heads, the ven tra l m id line.

Martinelli and Fan Supplemental Figure 5

Gas1/LacZ

Neural Plate

Neural Tube at PSM Level

Neural Tube at Soimte V

F igure S 5 . G as1 express ion w as de te rm ined by X -ga l s ta in ing E 9 .5 em bryos transverse ly sectioned a t va rious leve ls rang ing from poste rio r (le ft) to ante rio r (righ t). A x ia l leve ls a re ind ica ted on top , fo r com parison to F igure 7A .

Martinelli and Fan Supplemental Figure 6

Gas1-/-;Shh-/-Gas1+/+;Shh-/-Gas1-/-;Shh+/+Gas1+/-;Shh+/-

Ptc1

Gas1/LacZ

F igure S 6 . T he defective le ft/righ t asym m etry pa tte rn ing in the G as1 -/-;S hh -/- em bryo is associa ted w ith im pa ired H H s igna ling in the node as dete rm ined by P tc1 IS H in E8 .0 em bryos. G eno types a re ind ica ted on top . A rrow heads po in t to the m id line w here P tc1 express ion exis ts w hen S hh is p resen t. A rrow s po in t to the node . T he bo ttom p ic tu re is a X -ga l s ta ined G as1 -/-;S hh -/- em bryo revea ling LacZ express ion in the node fo r com parison to F ig . 7 E ,F . G iven the tw o cop ies o f the LacZ gene , the X -ga l s ta in ing w as tim e reduced accord ing ly (45 m in ) to m atch the leve ls o f p rim itive s treak s ta in ing in the LacZ hete rozygous em bryos (2 h r).

Martinelli and Fan Supplemental Figure 1

SphIEcoRIBamHI

SphI

tau-LacZ

tau-LacZ

PGK-neopA

XhoI

Gas1

recombinationconstruct PGK-tk

XhoI

SphI

SphI

SphI

SphI

Gas1LacZ

(Gas1-)

XhoI

XhoI PGK-neopA

SphI

A

e

22

+/- -/-+/- -/-e

+/- +/-

+/-

+/-+/+

E14.5 hl

B C D E

G Hn

ps

np

s

E14.5 head

dn

oe

dm

dn

fl

fl

hldn

s

o e

-/-

-/-

M c +/++/- -/-

Gas1LacZ

100

400

F

F igure S 1 . (A ) H om ologous recom b ination schem e to generate the G as1 LacZ (G as1 -) a lle le ; top , genom ic s tructu re o f G as1 locus; m idd le , recom b ina tion construct; bo ttom , recom bined a lle le . T he tau -LacZ -pA -P G K-neo casse tte is p laced a t the exact sam e loca tion as the PG K -neo casse tte in the p reviously pub lished G as1 neo a lle le (Lee e t a l. 2001b) and rep laces the entire cod ing reg ion o f G as1 . pA , po ly-A s igna l sequence ; P G K -tk , P G K -driven thym id ine k inase gene casse tte fo r nega tive se lection. R estric tion enzym e s ites (S ph I and Xho I) and 5 ’ and 3 ’ p robes used to screen fo r and confirm recom bination a re a lso the sam e as previous ly pub lished (Lee e t a l. 2001b); w h ite boxes, 5 ’ and 3 ’ U T R s of G as1 ; b lack box, cod ing reg ion o f G as1 ; a rrow heads, new P C R prim er se ts (see M ethods) fo r genotyp ing. (B ) P C R w ith the ind icated p rim er se ts can d is tingu ish w ild type (+ /+), he te rozygous (+ / -), and m utan t (-/-) em bryos; M , 100 bp ladder, 100 bp and 400 bp bands are labe led ; c , m ock con tro l P C R ; expected G as1 and LacZ bands a re labe led. (C ) X -ga l s ta in ing fo r LacZ activ ity (b lue ) in E7 .0 w ild type con tro l (le ft) and he te rozygous (righ t) em bryos. (D and F ) X -ga l s ta in ing pa tte rns o f E 9 .5 and E10 .5 he te rozygo tes (le ft) and m utan ts (righ t), respective ly. (E ) A transverse section o f E 9.5 a t the fo re lim b (fl) leve l. T hese sta in ing pa tte rns agree w ith the endogenous G as1 express ion ob ta ined by IS H (Lee and F an 2001). S -ga l (red ) s ta ined E 14 .5 he te rozygous (le ft) and m utan t (righ t) heads (G ) and h ind lim bs (h l) (H ) show tha t m utants have m icrophtha lm ia and sm a ll lim b /m a lfo rm ed d ig it 2 (w h ite a rrow head) pheno types as described fo r the G as1 neo m utant (L iu e t a l. 2002). U nder our experim enta l cond ition (37 °C , 2 h rs), w ild type em bryos fo r a ll s tages p resen ted here w ere nega tive fo r s ta in ing (not show n). O f no te , m utan ts have a sm a lle r head structu re than he te rozygo tes and w ild types observab le as early as E 9 .5 , w h ich w ill be described in de ta il in a separa te m anuscrip t. A bbrevia tions: np , neura l p la te ; ps, p rim itive s treak; n , node; s , som ite ; o , o tic ves ic le o r ea r; e , e ye ; dn , do rsa l neura l tube ; dm , derm om yotom e.

Martinelli and Fan Supplemental Figure 2

0

20

40

60

PAX7 PAX3 DBX1

% le

ngth

of N

.T.

Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/-

PAX7

PAX6

PAX3

GSH1/2

DB X1

NK X6.1

OL IG2

NK X2.2

HNF3β

S HH

Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/- Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/-

p=6.6E-5

p=.0076 p=1.6E-8

p=5.0E-7

p=2.2E-6

p=.0014

Gas1+/-;Shh+/- Gas1-/-;Shh+/+ Gas1-/-;Shh+/-

E12.5

E13.5

1 1 1

1 1 1

2 2

2 2

3 3 3

3 33

4

44

4

4

4

5 5 5

5 5 5

0

0.2

0.4

0.6

0.8

1

Forelimb Hindlimb

digi

t 2:4

leng

th ra

tio

Control Gas1 mutantA

B

Martinelli and Fan Supplemental Figure 3F igure S 2 . Q uan tita tion and add itiona l m a rker ana lys is fo r the neura l tube defect o f G as1 -/- and G as1 -/-;S hh +/- em bryos. Em bryos o f 24 -25 som ites w ere transverse ly sectioned a t the fo re lim b leve l and ana lyzed by IH C . G eno types a re labe led on top and m arkers on the le ft. In the le ft 3 co lum ns a re dorsa l m arkers, w h ile in the righ t 3 co lum ns a re ven tra l p rogen ito r m arkers. O f note , com prom ised floo rp la te induction w as a lso observed by the a lte ra tion o f N K X2.2 , H N F3β, and S H H express ion . A lso , in G as1 -/-;S hh +/- em bryos, O LIG 2 -positive ce lls occupy the m id line w ith little o r no S H H -positive ce lls . The resu lts a re consis ten t w ith reduced S H H s igna ling in the G as1 -/- neura l tube and a fu rthe r reduction in the G as1 -/-;S hh +/- neura l tube. D ashed line dem arks the boundaries o f express ion o f PA X7, P A X3, a nd D B X1. To quan tify the changes in the boundaries o f express ion fo r these 3 genes, the percen t o f the neura l tube w ith in the boundaries w as ca lcu la ted fo r G as1 +/-;S hh +/- (n = 5 ), G as1 -/- (n = 4 ), and G as1 -/-;S hh +/- (n = 3 ) em bryos. 2 sections w ere ana lyzed fo r each em bryo . A tw o-ta iled W elch ’s t-test w as perfo rm ed . For the boundaries o f a ll 3 genes , G as1 +/-; S hh +/- em bryos w ere s ta tis tica lly d iffe ren t from bo th G as1 -/- and G as1 -/-;S hh +/- em bryos (99% confidence leve l). P va lues a re ind icate d fo r each t-test and d isp layed on the bar graph . The express ion boundaries in G as1 -/- and G as1 -/-; S hh +/- em bryos w ere no t found to be s ta tis tica lly d iffe ren t.

F igu re S 3 . (A ) G as1 m u tant d ig it 2 is d isp roportiona lly sm a lle r than the o ther d ig its . T he lengths o f d ig its 2 and 4 w ere m easured on G as1 +/-;S hh +/- and G as1 -/- neona ta l ske le ta l p repara tions and the length ra tio ca lcu la ted . D ig it 4 w as used fo r com parison because it and d ig it 2 a re norm a lly o f s im ila r length . n fo r con tro l lim bs is 3 ; fo r G as1 -/- fo re lim bs, 19 ; fo r G as1 -/- h ind lim bs, 11 . E rro r ba rs = 1 s tandard devia tion . (B ) E xam ina tion o f fo re lim bs of E 12.5 and E 13 .5 m ice o f G as1 and S hh a lle lic com b ina tions (ind icated a t top ) revea led that d ig it 2 never fo rm s in the G as1 -/-;Shh +/- genotype . A rrow head ind ica tes la rge loca tion o f in te r -d ig ita l m esenchym e w here d ig it 2 no rm a lly form s. n = 3 fo r each geno type and stage . D ig it num bers are ind ica ted . S ca le bar = 1 m m . R ationa le fo r the determ ination o f m issing d ig it identity : D e te rm ina tion o f d ig it 1 w as stra igh tfo rw ard because it has one less pha lange . D ig its 4 and 5 w ere de te rm ined by m easuring the ir re la tive lengths as w e ll as exam ination o f m etacarpa l and m eta tarsa l shapes . A s p revious ly reported fo r G as1 m u tan ts, w e found tha t d ig its 4 and 5 a re no t a ffected in the G as1 -/-;Shh +/- (L iu e t a l. 2002). D is tingu ish ing the m issing d ig it as 2 as opposed to 3 is m ore com p lica ted. O ur lines o f a rgum ent tha t d ig it 2 is absen t in 4 -fingered lim bs a re severa l fo ld . 1 ) T he tim e course ana lys is p resen ted a bove show tha t in the G as1 -/-;S hh +/- geno type , d ig its 3 and 4 a re p resent in the ir no rm a l loca tions, w h ile the re is on ly la rge in te r-d ig ita l m esenchym e w here d ig it 2 no rm a lly fo rm s. 2 ) T he in te rm ed ia te pha lange of d ig it 2 in G as1 m u tan t fo re lim bs is freque n tly d isfigu red o r k inked (L iu e t a l. 2002). S uch a m a lfo rm ed d ig it is never observed in any 4 -fingered fo re lim bs, suggesting tha t none of the rem a in ing d ig its a re 2 . 3 ) W hen m etacarpa ls a re found to be fused in e ithe r geno type , it is a lw ays be tw een d ig its 2 and 3; w hen m eta ta rsa ls a re fused, it is a lw ays be tw een d ig its 1 and 2. N e ithe r o f these fus ions w ere ever observed in 4 -fingered lim bs. If the m iss ing d ig it w as actua lly 3 , then w e w ou ld s till expect to f ind fus ions be tw een the rem a in ing d ig its 1 and 2. 4 ) T he d isp roportiona lly reduced s ize o f d ig it 2 in the G as1 m utan t suggests tha t a m ore severe pheno type (as in the G as1 -/-;S hh +/- geno type) w ou ld cause a lack o f d ig it 2 to fo rm . E ven if the m iss ing d ig it is 3 o r adop ts a m ixed iden tity o f 2 and 3 , the conclus ions in the text a re s till app licab le because d ig it 3 a lso depends in part on d iffus ib le S H H (see re fe rences in text).

Martinelli and Fan Supplemental Figure 4

HNF3β GFP

GFP

GFP

GFP

OLIG2

NKX6.1 PAX6

F igure S 4 . G as1 express ion a lte rs the position o f neurona l p rogen ito rs in the neura l tube . C h ick trunk neura l tube w as e le ctropora ted w ith G as1N /A -IR E S -G FP (no t show n) o r G as1 -IR E S -G FP p lasm ids and ana lyzed fo r G FP , P A X6, N K X6.1 , O LIG 2, and H N F3β by doub le im m unofluo rescence (green fo r G FP , and red fo r PA X6, N K X6.1 , O L IG 2, o r H N F3β; excep t fo r O LIG 2, in w h ich the green s igna l is the au to fluo rescence from G FP ) on the sam e sections. Each pa ir o f im ages is from the sam e section; n ≥ 10 fo r each pa ir. A rrow s ind ica te ectop ic activa tion o f H H ta rge t genes; open a rrow s, ectop ic inh ib ition ; open a rrow heads, the ven tra l m id line.

Martinelli and Fan Supplemental Figure 5

Gas1/LacZ

Neural Plate

Neural Tube at PSM Level

Neural Tube at Soimte V

F igure S 5 . G as1 express ion w as de te rm ined by X -ga l s ta in ing E 9 .5 em bryos transverse ly sectioned a t va rious leve ls rang ing from poste rio r (le ft) to ante rio r (righ t). A x ia l leve ls a re ind ica ted on top , fo r com parison to F igure 7A .

Martinelli and Fan Supplemental Figure 6

Gas1-/-;Shh-/-Gas1+/+;Shh-/-Gas1-/-;Shh+/+Gas1+/-;Shh+/-

Ptc1

Gas1/LacZ

F igure S 6 . T he defective le ft/righ t asym m etry pa tte rn ing in the G as1 -/-;S hh -/- em bryo is associa ted w ith im pa ired H H s igna ling in the node as dete rm ined by P tc1 IS H in E8 .0 em bryos. G eno types a re ind ica ted on top . A rrow heads po in t to the m id line w here P tc1 express ion exis ts w hen S hh is p resen t. A rrow s po in t to the node . T he bo ttom p ic tu re is a X -ga l s ta ined G as1 -/-;S hh -/- em bryo revea ling LacZ express ion in the node fo r com parison to F ig . 7 E ,F . G iven the tw o cop ies o f the LacZ gene , the X -ga l s ta in ing w as tim e reduced accord ing ly (45 m in ) to m atch the leve ls o f p rim itive s treak s ta in ing in the LacZ hete rozygous em bryos (2 h r).

Martinelli and Fan Supplemental Figure 1

SphIEcoRIBamHI

SphI

tau-LacZ

tau-LacZ

PGK-neopA

XhoI

Gas1

recombinationconstruct PGK-tk

XhoI

SphI

SphI

SphI

SphI

Gas1LacZ

(Gas1-)

XhoI

XhoI PGK-neopA

SphI

A

e

22

+/- -/-+/- -/-e

+/- +/-

+/-

+/-+/+

E14.5 hl

B C D E

G Hn

ps

np

s

E14.5 head

dn

oe

dm

dn

fl

fl

hldn

s

o e

-/-

-/-

M c +/++/- -/-

Gas1LacZ

100

400

F

F igure S 1 . (A ) H om ologous recom b ination schem e to generate the G as1 LacZ (G as1 -) a lle le ; top , genom ic s tructu re o f G as1 locus; m idd le , recom b ina tion construct; bo ttom , recom bined a lle le . T he tau -LacZ -pA -P G K-neo casse tte is p laced a t the exact sam e loca tion as the PG K -neo casse tte in the p reviously pub lished G as1 neo a lle le (Lee e t a l. 2001b) and rep laces the entire cod ing reg ion o f G as1 . pA , po ly-A s igna l sequence ; P G K -tk , P G K -driven thym id ine k inase gene casse tte fo r nega tive se lection. R estric tion enzym e s ites (S ph I and Xho I) and 5 ’ and 3 ’ p robes used to screen fo r and confirm recom bination a re a lso the sam e as previous ly pub lished (Lee e t a l. 2001b); w h ite boxes, 5 ’ and 3 ’ U T R s of G as1 ; b lack box, cod ing reg ion o f G as1 ; a rrow heads, new P C R prim er se ts (see M ethods) fo r genotyp ing. (B ) P C R w ith the ind icated p rim er se ts can d is tingu ish w ild type (+ /+), he te rozygous (+ / -), and m utan t (-/-) em bryos; M , 100 bp ladder, 100 bp and 400 bp bands are labe led ; c , m ock con tro l P C R ; expected G as1 and LacZ bands a re labe led. (C ) X -ga l s ta in ing fo r LacZ activ ity (b lue ) in E7 .0 w ild type con tro l (le ft) and he te rozygous (righ t) em bryos. (D and F ) X -ga l s ta in ing pa tte rns o f E 9 .5 and E10 .5 he te rozygo tes (le ft) and m utan ts (righ t), respective ly. (E ) A transverse section o f E 9.5 a t the fo re lim b (fl) leve l. T hese sta in ing pa tte rns agree w ith the endogenous G as1 express ion ob ta ined by IS H (Lee and F an 2001). S -ga l (red ) s ta ined E 14 .5 he te rozygous (le ft) and m utan t (righ t) heads (G ) and h ind lim bs (h l) (H ) show tha t m utants have m icrophtha lm ia and sm a ll lim b /m a lfo rm ed d ig it 2 (w h ite a rrow head) pheno types as described fo r the G as1 neo m utant (L iu e t a l. 2002). U nder our experim enta l cond ition (37 °C , 2 h rs), w ild type em bryos fo r a ll s tages p resen ted here w ere nega tive fo r s ta in ing (not show n). O f no te , m utan ts have a sm a lle r head structu re than he te rozygo tes and w ild types observab le as early as E 9 .5 , w h ich w ill be described in de ta il in a separa te m anuscrip t. A bbrevia tions: np , neura l p la te ; ps, p rim itive s treak; n , node; s , som ite ; o , o tic ves ic le o r ea r; e , e ye ; dn , do rsa l neura l tube ; dm , derm om yotom e.

Martinelli and Fan Supplemental Figure 2

0

20

40

60

PAX7 PAX3 DBX1

% le

ngth

of N

.T.

Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/-

PAX7

PAX6

PAX3

GSH1/2

DB X1

NK X6.1

OL IG2

NK X2.2

HNF3β

S HH

Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/- Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/-

p=6.6E-5

p=.0076 p=1.6E-8

p=5.0E-7

p=2.2E-6

p=.0014

Gas1+/-;Shh+/- Gas1-/-;Shh+/+ Gas1-/-;Shh+/-

E12.5

E13.5

1 1 1

1 1 1

2 2

2 2

3 3 3

3 33

4

44

4

4

4

5 5 5

5 5 5

0

0.2

0.4

0.6

0.8

1

Forelimb Hindlimb

digi

t 2:4

leng

th ra

tio

Control Gas1 mutantA

B

Martinelli and Fan Supplemental Figure 3F igure S 2 . Q uan tita tion and add itiona l m a rker ana lys is fo r the neura l tube defect o f G as1 -/- and G as1 -/-;S hh +/- em bryos. Em bryos o f 24 -25 som ites w ere transverse ly sectioned a t the fo re lim b leve l and ana lyzed by IH C . G eno types a re labe led on top and m arkers on the le ft. In the le ft 3 co lum ns a re dorsa l m arkers, w h ile in the righ t 3 co lum ns a re ven tra l p rogen ito r m arkers. O f note , com prom ised floo rp la te induction w as a lso observed by the a lte ra tion o f N K X2.2 , H N F3β, and S H H express ion . A lso , in G as1 -/-;S hh +/- em bryos, O LIG 2 -positive ce lls occupy the m id line w ith little o r no S H H -positive ce lls . The resu lts a re consis ten t w ith reduced S H H s igna ling in the G as1 -/- neura l tube and a fu rthe r reduction in the G as1 -/-;S hh +/- neura l tube. D ashed line dem arks the boundaries o f express ion o f PA X7, P A X3, a nd D B X1. To quan tify the changes in the boundaries o f express ion fo r these 3 genes, the percen t o f the neura l tube w ith in the boundaries w as ca lcu la ted fo r G as1 +/-;S hh +/- (n = 5 ), G as1 -/- (n = 4 ), and G as1 -/-;S hh +/- (n = 3 ) em bryos. 2 sections w ere ana lyzed fo r each em bryo . A tw o-ta iled W elch ’s t-test w as perfo rm ed . For the boundaries o f a ll 3 genes , G as1 +/-; S hh +/- em bryos w ere s ta tis tica lly d iffe ren t from bo th G as1 -/- and G as1 -/-;S hh +/- em bryos (99% confidence leve l). P va lues a re ind icate d fo r each t-test and d isp layed on the bar graph . The express ion boundaries in G as1 -/- and G as1 -/-; S hh +/- em bryos w ere no t found to be s ta tis tica lly d iffe ren t.

F igu re S 3 . (A ) G as1 m u tant d ig it 2 is d isp roportiona lly sm a lle r than the o ther d ig its . T he lengths o f d ig its 2 and 4 w ere m easured on G as1 +/-;S hh +/- and G as1 -/- neona ta l ske le ta l p repara tions and the length ra tio ca lcu la ted . D ig it 4 w as used fo r com parison because it and d ig it 2 a re norm a lly o f s im ila r length . n fo r con tro l lim bs is 3 ; fo r G as1 -/- fo re lim bs, 19 ; fo r G as1 -/- h ind lim bs, 11 . E rro r ba rs = 1 s tandard devia tion . (B ) E xam ina tion o f fo re lim bs of E 12.5 and E 13 .5 m ice o f G as1 and S hh a lle lic com b ina tions (ind icated a t top ) revea led that d ig it 2 never fo rm s in the G as1 -/-;Shh +/- genotype . A rrow head ind ica tes la rge loca tion o f in te r -d ig ita l m esenchym e w here d ig it 2 no rm a lly form s. n = 3 fo r each geno type and stage . D ig it num bers are ind ica ted . S ca le bar = 1 m m . R ationa le fo r the determ ination o f m issing d ig it identity : D e te rm ina tion o f d ig it 1 w as stra igh tfo rw ard because it has one less pha lange . D ig its 4 and 5 w ere de te rm ined by m easuring the ir re la tive lengths as w e ll as exam ination o f m etacarpa l and m eta tarsa l shapes . A s p revious ly reported fo r G as1 m u tan ts, w e found tha t d ig its 4 and 5 a re no t a ffected in the G as1 -/-;Shh +/- (L iu e t a l. 2002). D is tingu ish ing the m issing d ig it as 2 as opposed to 3 is m ore com p lica ted. O ur lines o f a rgum ent tha t d ig it 2 is absen t in 4 -fingered lim bs a re severa l fo ld . 1 ) T he tim e course ana lys is p resen ted a bove show tha t in the G as1 -/-;S hh +/- geno type , d ig its 3 and 4 a re p resent in the ir no rm a l loca tions, w h ile the re is on ly la rge in te r-d ig ita l m esenchym e w here d ig it 2 no rm a lly fo rm s. 2 ) T he in te rm ed ia te pha lange of d ig it 2 in G as1 m u tan t fo re lim bs is freque n tly d isfigu red o r k inked (L iu e t a l. 2002). S uch a m a lfo rm ed d ig it is never observed in any 4 -fingered fo re lim bs, suggesting tha t none of the rem a in ing d ig its a re 2 . 3 ) W hen m etacarpa ls a re found to be fused in e ithe r geno type , it is a lw ays be tw een d ig its 2 and 3; w hen m eta ta rsa ls a re fused, it is a lw ays be tw een d ig its 1 and 2. N e ithe r o f these fus ions w ere ever observed in 4 -fingered lim bs. If the m iss ing d ig it w as actua lly 3 , then w e w ou ld s till expect to f ind fus ions be tw een the rem a in ing d ig its 1 and 2. 4 ) T he d isp roportiona lly reduced s ize o f d ig it 2 in the G as1 m utan t suggests tha t a m ore severe pheno type (as in the G as1 -/-;S hh +/- geno type) w ou ld cause a lack o f d ig it 2 to fo rm . E ven if the m iss ing d ig it is 3 o r adop ts a m ixed iden tity o f 2 and 3 , the conclus ions in the text a re s till app licab le because d ig it 3 a lso depends in part on d iffus ib le S H H (see re fe rences in text).

Martinelli and Fan Supplemental Figure 4

HNF3β GFP

GFP

GFP

GFP

OLIG2

NKX6.1 PAX6

F igure S 4 . G as1 express ion a lte rs the position o f neurona l p rogen ito rs in the neura l tube . C h ick trunk neura l tube w as e le ctropora ted w ith G as1N /A -IR E S -G FP (no t show n) o r G as1 -IR E S -G FP p lasm ids and ana lyzed fo r G FP , P A X6, N K X6.1 , O LIG 2, and H N F3β by doub le im m unofluo rescence (green fo r G FP , and red fo r PA X6, N K X6.1 , O L IG 2, o r H N F3β; excep t fo r O LIG 2, in w h ich the green s igna l is the au to fluo rescence from G FP ) on the sam e sections. Each pa ir o f im ages is from the sam e section; n ≥ 10 fo r each pa ir. A rrow s ind ica te ectop ic activa tion o f H H ta rge t genes; open a rrow s, ectop ic inh ib ition ; open a rrow heads, the ven tra l m id line.

Martinelli and Fan Supplemental Figure 5

Gas1/LacZ

Neural Plate

Neural Tube at PSM Level

Neural Tube at Soimte V

F igure S 5 . G as1 express ion w as de te rm ined by X -ga l s ta in ing E 9 .5 em bryos transverse ly sectioned a t va rious leve ls rang ing from poste rio r (le ft) to ante rio r (righ t). A x ia l leve ls a re ind ica ted on top , fo r com parison to F igure 7A .

Martinelli and Fan Supplemental Figure 6

Gas1-/-;Shh-/-Gas1+/+;Shh-/-Gas1-/-;Shh+/+Gas1+/-;Shh+/-

Ptc1

Gas1/LacZ

F igure S 6 . T he defective le ft/righ t asym m etry pa tte rn ing in the G as1 -/-;S hh -/- em bryo is associa ted w ith im pa ired H H s igna ling in the node as dete rm ined by P tc1 IS H in E8 .0 em bryos. G eno types a re ind ica ted on top . A rrow heads po in t to the m id line w here P tc1 express ion exis ts w hen S hh is p resen t. A rrow s po in t to the node . T he bo ttom p ic tu re is a X -ga l s ta ined G as1 -/-;S hh -/- em bryo revea ling LacZ express ion in the node fo r com parison to F ig . 7 E ,F . G iven the tw o cop ies o f the LacZ gene , the X -ga l s ta in ing w as tim e reduced accord ing ly (45 m in ) to m atch the leve ls o f p rim itive s treak s ta in ing in the LacZ hete rozygous em bryos (2 h r).

Martinelli and Fan Supplemental Figure 1

SphIEcoRIBamHI

SphI

tau-LacZ

tau-LacZ

PGK-neopA

XhoI

Gas1

recombinationconstruct PGK-tk

XhoI

SphI

SphI

SphI

SphI

Gas1LacZ

(Gas1-)

XhoI

XhoI PGK-neopA

SphI

A

e

22

+/- -/-+/- -/-e

+/- +/-

+/-

+/-+/+

E14.5 hl

B C D E

G Hn

ps

np

s

E14.5 head

dn

oe

dm

dn

fl

fl

hldn

s

o e

-/-

-/-

M c +/++/- -/-

Gas1LacZ

100

400

F

F igure S 1 . (A ) H om ologous recom b ination schem e to generate the G as1 LacZ (G as1 -) a lle le ; top , genom ic s tructu re o f G as1 locus; m idd le , recom b ina tion construct; bo ttom , recom bined a lle le . T he tau -LacZ -pA -P G K-neo casse tte is p laced a t the exact sam e loca tion as the PG K -neo casse tte in the p reviously pub lished G as1 neo a lle le (Lee e t a l. 2001b) and rep laces the entire cod ing reg ion o f G as1 . pA , po ly-A s igna l sequence ; P G K -tk , P G K -driven thym id ine k inase gene casse tte fo r nega tive se lection. R estric tion enzym e s ites (S ph I and Xho I) and 5 ’ and 3 ’ p robes used to screen fo r and confirm recom bination a re a lso the sam e as previous ly pub lished (Lee e t a l. 2001b); w h ite boxes, 5 ’ and 3 ’ U T R s of G as1 ; b lack box, cod ing reg ion o f G as1 ; a rrow heads, new P C R prim er se ts (see M ethods) fo r genotyp ing. (B ) P C R w ith the ind icated p rim er se ts can d is tingu ish w ild type (+ /+), he te rozygous (+ / -), and m utan t (-/-) em bryos; M , 100 bp ladder, 100 bp and 400 bp bands are labe led ; c , m ock con tro l P C R ; expected G as1 and LacZ bands a re labe led. (C ) X -ga l s ta in ing fo r LacZ activ ity (b lue ) in E7 .0 w ild type con tro l (le ft) and he te rozygous (righ t) em bryos. (D and F ) X -ga l s ta in ing pa tte rns o f E 9 .5 and E10 .5 he te rozygo tes (le ft) and m utan ts (righ t), respective ly. (E ) A transverse section o f E 9.5 a t the fo re lim b (fl) leve l. T hese sta in ing pa tte rns agree w ith the endogenous G as1 express ion ob ta ined by IS H (Lee and F an 2001). S -ga l (red ) s ta ined E 14 .5 he te rozygous (le ft) and m utan t (righ t) heads (G ) and h ind lim bs (h l) (H ) show tha t m utants have m icrophtha lm ia and sm a ll lim b /m a lfo rm ed d ig it 2 (w h ite a rrow head) pheno types as described fo r the G as1 neo m utant (L iu e t a l. 2002). U nder our experim enta l cond ition (37 °C , 2 h rs), w ild type em bryos fo r a ll s tages p resen ted here w ere nega tive fo r s ta in ing (not show n). O f no te , m utan ts have a sm a lle r head structu re than he te rozygo tes and w ild types observab le as early as E 9 .5 , w h ich w ill be described in de ta il in a separa te m anuscrip t. A bbrevia tions: np , neura l p la te ; ps, p rim itive s treak; n , node; s , som ite ; o , o tic ves ic le o r ea r; e , e ye ; dn , do rsa l neura l tube ; dm , derm om yotom e.

Martinelli and Fan Supplemental Figure 2

0

20

40

60

PAX7 PAX3 DBX1

% le

ngth

of N

.T.

Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/-

PAX7

PAX6

PAX3

GSH1/2

DB X1

NK X6.1

OL IG2

NK X2.2

HNF3β

S HH

Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/- Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/-

p=6.6E-5

p=.0076 p=1.6E-8

p=5.0E-7

p=2.2E-6

p=.0014

Gas1+/-;Shh+/- Gas1-/-;Shh+/+ Gas1-/-;Shh+/-

E12.5

E13.5

1 1 1

1 1 1

2 2

2 2

3 3 3

3 33

4

44

4

4

4

5 5 5

5 5 5

0

0.2

0.4

0.6

0.8

1

Forelimb Hindlimb

digi

t 2:4

leng

th ra

tio

Control Gas1 mutantA

B

Martinelli and Fan Supplemental Figure 3F igure S 2 . Q uan tita tion and add itiona l m a rker ana lys is fo r the neura l tube defect o f G as1 -/- and G as1 -/-;S hh +/- em bryos. Em bryos o f 24 -25 som ites w ere transverse ly sectioned a t the fo re lim b leve l and ana lyzed by IH C . G eno types a re labe led on top and m arkers on the le ft. In the le ft 3 co lum ns a re dorsa l m arkers, w h ile in the righ t 3 co lum ns a re ven tra l p rogen ito r m arkers. O f note , com prom ised floo rp la te induction w as a lso observed by the a lte ra tion o f N K X2.2 , H N F3β, and S H H express ion . A lso , in G as1 -/-;S hh +/- em bryos, O LIG 2 -positive ce lls occupy the m id line w ith little o r no S H H -positive ce lls . The resu lts a re consis ten t w ith reduced S H H s igna ling in the G as1 -/- neura l tube and a fu rthe r reduction in the G as1 -/-;S hh +/- neura l tube. D ashed line dem arks the boundaries o f express ion o f PA X7, P A X3, a nd D B X1. To quan tify the changes in the boundaries o f express ion fo r these 3 genes, the percen t o f the neura l tube w ith in the boundaries w as ca lcu la ted fo r G as1 +/-;S hh +/- (n = 5 ), G as1 -/- (n = 4 ), and G as1 -/-;S hh +/- (n = 3 ) em bryos. 2 sections w ere ana lyzed fo r each em bryo . A tw o-ta iled W elch ’s t-test w as perfo rm ed . For the boundaries o f a ll 3 genes , G as1 +/-; S hh +/- em bryos w ere s ta tis tica lly d iffe ren t from bo th G as1 -/- and G as1 -/-;S hh +/- em bryos (99% confidence leve l). P va lues a re ind icate d fo r each t-test and d isp layed on the bar graph . The express ion boundaries in G as1 -/- and G as1 -/-; S hh +/- em bryos w ere no t found to be s ta tis tica lly d iffe ren t.

F igu re S 3 . (A ) G as1 m u tant d ig it 2 is d isp roportiona lly sm a lle r than the o ther d ig its . T he lengths o f d ig its 2 and 4 w ere m easured on G as1 +/-;S hh +/- and G as1 -/- neona ta l ske le ta l p repara tions and the length ra tio ca lcu la ted . D ig it 4 w as used fo r com parison because it and d ig it 2 a re norm a lly o f s im ila r length . n fo r con tro l lim bs is 3 ; fo r G as1 -/- fo re lim bs, 19 ; fo r G as1 -/- h ind lim bs, 11 . E rro r ba rs = 1 s tandard devia tion . (B ) E xam ina tion o f fo re lim bs of E 12.5 and E 13 .5 m ice o f G as1 and S hh a lle lic com b ina tions (ind icated a t top ) revea led that d ig it 2 never fo rm s in the G as1 -/-;Shh +/- genotype . A rrow head ind ica tes la rge loca tion o f in te r -d ig ita l m esenchym e w here d ig it 2 no rm a lly form s. n = 3 fo r each geno type and stage . D ig it num bers are ind ica ted . S ca le bar = 1 m m . R ationa le fo r the determ ination o f m issing d ig it identity : D e te rm ina tion o f d ig it 1 w as stra igh tfo rw ard because it has one less pha lange . D ig its 4 and 5 w ere de te rm ined by m easuring the ir re la tive lengths as w e ll as exam ination o f m etacarpa l and m eta tarsa l shapes . A s p revious ly reported fo r G as1 m u tan ts, w e found tha t d ig its 4 and 5 a re no t a ffected in the G as1 -/-;Shh +/- (L iu e t a l. 2002). D is tingu ish ing the m issing d ig it as 2 as opposed to 3 is m ore com p lica ted. O ur lines o f a rgum ent tha t d ig it 2 is absen t in 4 -fingered lim bs a re severa l fo ld . 1 ) T he tim e course ana lys is p resen ted a bove show tha t in the G as1 -/-;S hh +/- geno type , d ig its 3 and 4 a re p resent in the ir no rm a l loca tions, w h ile the re is on ly la rge in te r-d ig ita l m esenchym e w here d ig it 2 no rm a lly fo rm s. 2 ) T he in te rm ed ia te pha lange of d ig it 2 in G as1 m u tan t fo re lim bs is freque n tly d isfigu red o r k inked (L iu e t a l. 2002). S uch a m a lfo rm ed d ig it is never observed in any 4 -fingered fo re lim bs, suggesting tha t none of the rem a in ing d ig its a re 2 . 3 ) W hen m etacarpa ls a re found to be fused in e ithe r geno type , it is a lw ays be tw een d ig its 2 and 3; w hen m eta ta rsa ls a re fused, it is a lw ays be tw een d ig its 1 and 2. N e ithe r o f these fus ions w ere ever observed in 4 -fingered lim bs. If the m iss ing d ig it w as actua lly 3 , then w e w ou ld s till expect to f ind fus ions be tw een the rem a in ing d ig its 1 and 2. 4 ) T he d isp roportiona lly reduced s ize o f d ig it 2 in the G as1 m utan t suggests tha t a m ore severe pheno type (as in the G as1 -/-;S hh +/- geno type) w ou ld cause a lack o f d ig it 2 to fo rm . E ven if the m iss ing d ig it is 3 o r adop ts a m ixed iden tity o f 2 and 3 , the conclus ions in the text a re s till app licab le because d ig it 3 a lso depends in part on d iffus ib le S H H (see re fe rences in text).

Martinelli and Fan Supplemental Figure 4

HNF3β GFP

GFP

GFP

GFP

OLIG2

NKX6.1 PAX6

F igure S 4 . G as1 express ion a lte rs the position o f neurona l p rogen ito rs in the neura l tube . C h ick trunk neura l tube w as e le ctropora ted w ith G as1N /A -IR E S -G FP (no t show n) o r G as1 -IR E S -G FP p lasm ids and ana lyzed fo r G FP , P A X6, N K X6.1 , O LIG 2, and H N F3β by doub le im m unofluo rescence (green fo r G FP , and red fo r PA X6, N K X6.1 , O L IG 2, o r H N F3β; excep t fo r O LIG 2, in w h ich the green s igna l is the au to fluo rescence from G FP ) on the sam e sections. Each pa ir o f im ages is from the sam e section; n ≥ 10 fo r each pa ir. A rrow s ind ica te ectop ic activa tion o f H H ta rge t genes; open a rrow s, ectop ic inh ib ition ; open a rrow heads, the ven tra l m id line.

Martinelli and Fan Supplemental Figure 5

Gas1/LacZ

Neural Plate

Neural Tube at PSM Level

Neural Tube at Soimte V

F igure S 5 . G as1 express ion w as de te rm ined by X -ga l s ta in ing E 9 .5 em bryos transverse ly sectioned a t va rious leve ls rang ing from poste rio r (le ft) to ante rio r (righ t). A x ia l leve ls a re ind ica ted on top , fo r com parison to F igure 7A .

Martinelli and Fan Supplemental Figure 6

Gas1-/-;Shh-/-Gas1+/+;Shh-/-Gas1-/-;Shh+/+Gas1+/-;Shh+/-

Ptc1

Gas1/LacZ

F igure S 6 . T he defective le ft/righ t asym m etry pa tte rn ing in the G as1 -/-;S hh -/- em bryo is associa ted w ith im pa ired H H s igna ling in the node as dete rm ined by P tc1 IS H in E8 .0 em bryos. G eno types a re ind ica ted on top . A rrow heads po in t to the m id line w here P tc1 express ion exis ts w hen S hh is p resen t. A rrow s po in t to the node . T he bo ttom p ic tu re is a X -ga l s ta ined G as1 -/-;S hh -/- em bryo revea ling LacZ express ion in the node fo r com parison to F ig . 7 E ,F . G iven the tw o cop ies o f the LacZ gene , the X -ga l s ta in ing w as tim e reduced accord ing ly (45 m in ) to m atch the leve ls o f p rim itive s treak s ta in ing in the LacZ hete rozygous em bryos (2 h r).

Martinelli and Fan Supplemental Figure 1

SphIEcoRIBamHI

SphI

tau-LacZ

tau-LacZ

PGK-neopA

XhoI

Gas1

recombinationconstruct PGK-tk

XhoI

SphI

SphI

SphI

SphI

Gas1LacZ

(Gas1-)

XhoI

XhoI PGK-neopA

SphI

A

e

22

+/- -/-+/- -/-e

+/- +/-

+/-

+/-+/+

E14.5 hl

B C D E

G Hn

ps

np

s

E14.5 head

dn

oe

dm

dn

fl

fl

hldn

s

o e

-/-

-/-

M c +/++/- -/-

Gas1LacZ

100

400

F

F igure S 1 . (A ) H om ologous recom b ination schem e to generate the G as1 LacZ (G as1 -) a lle le ; top , genom ic s tructu re o f G as1 locus; m idd le , recom b ina tion construct; bo ttom , recom bined a lle le . T he tau -LacZ -pA -P G K-neo casse tte is p laced a t the exact sam e loca tion as the PG K -neo casse tte in the p reviously pub lished G as1 neo a lle le (Lee e t a l. 2001b) and rep laces the entire cod ing reg ion o f G as1 . pA , po ly-A s igna l sequence ; P G K -tk , P G K -driven thym id ine k inase gene casse tte fo r nega tive se lection. R estric tion enzym e s ites (S ph I and Xho I) and 5 ’ and 3 ’ p robes used to screen fo r and confirm recom bination a re a lso the sam e as previous ly pub lished (Lee e t a l. 2001b); w h ite boxes, 5 ’ and 3 ’ U T R s of G as1 ; b lack box, cod ing reg ion o f G as1 ; a rrow heads, new P C R prim er se ts (see M ethods) fo r genotyp ing. (B ) P C R w ith the ind icated p rim er se ts can d is tingu ish w ild type (+ /+), he te rozygous (+ / -), and m utan t (-/-) em bryos; M , 100 bp ladder, 100 bp and 400 bp bands are labe led ; c , m ock con tro l P C R ; expected G as1 and LacZ bands a re labe led. (C ) X -ga l s ta in ing fo r LacZ activ ity (b lue ) in E7 .0 w ild type con tro l (le ft) and he te rozygous (righ t) em bryos. (D and F ) X -ga l s ta in ing pa tte rns o f E 9 .5 and E10 .5 he te rozygo tes (le ft) and m utan ts (righ t), respective ly. (E ) A transverse section o f E 9.5 a t the fo re lim b (fl) leve l. T hese sta in ing pa tte rns agree w ith the endogenous G as1 express ion ob ta ined by IS H (Lee and F an 2001). S -ga l (red ) s ta ined E 14 .5 he te rozygous (le ft) and m utan t (righ t) heads (G ) and h ind lim bs (h l) (H ) show tha t m utants have m icrophtha lm ia and sm a ll lim b /m a lfo rm ed d ig it 2 (w h ite a rrow head) pheno types as described fo r the G as1 neo m utant (L iu e t a l. 2002). U nder our experim enta l cond ition (37 °C , 2 h rs), w ild type em bryos fo r a ll s tages p resen ted here w ere nega tive fo r s ta in ing (not show n). O f no te , m utan ts have a sm a lle r head structu re than he te rozygo tes and w ild types observab le as early as E 9 .5 , w h ich w ill be described in de ta il in a separa te m anuscrip t. A bbrevia tions: np , neura l p la te ; ps, p rim itive s treak; n , node; s , som ite ; o , o tic ves ic le o r ea r; e , e ye ; dn , do rsa l neura l tube ; dm , derm om yotom e.

Martinelli and Fan Supplemental Figure 2

0

20

40

60

PAX7 PAX3 DBX1

% le

ngth

of N

.T.

Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/-

PAX7

PAX6

PAX3

GSH1/2

DB X1

NK X6.1

OL IG2

NK X2.2

HNF3β

S HH

Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/- Gas1+/-;Shh+/- Gas1-/- Gas1-/-;Shh+/-

p=6.6E-5

p=.0076 p=1.6E-8

p=5.0E-7

p=2.2E-6

p=.0014

Gas1+/-;Shh+/- Gas1-/-;Shh+/+ Gas1-/-;Shh+/-

E12.5

E13.5

1 1 1

1 1 1

2 2

2 2

3 3 3

3 33

4

44

4

4

4

5 5 5

5 5 5

0

0.2

0.4

0.6

0.8

1

Forelimb Hindlimb

digi

t 2:4

leng

th ra

tio

Control Gas1 mutantA

B

Martinelli and Fan Supplemental Figure 3F igure S 2 . Q uan tita tion and add itiona l m a rker ana lys is fo r the neura l tube defect o f G as1 -/- and G as1 -/-;S hh +/- em bryos. Em bryos o f 24 -25 som ites w ere transverse ly sectioned a t the fo re lim b leve l and ana lyzed by IH C . G eno types a re labe led on top and m arkers on the le ft. In the le ft 3 co lum ns a re dorsa l m arkers, w h ile in the righ t 3 co lum ns a re ven tra l p rogen ito r m arkers. O f note , com prom ised floo rp la te induction w as a lso observed by the a lte ra tion o f N K X2.2 , H N F3β, and S H H express ion . A lso , in G as1 -/-;S hh +/- em bryos, O LIG 2 -positive ce lls occupy the m id line w ith little o r no S H H -positive ce lls . The resu lts a re consis ten t w ith reduced S H H s igna ling in the G as1 -/- neura l tube and a fu rthe r reduction in the G as1 -/-;S hh +/- neura l tube. D ashed line dem arks the boundaries o f express ion o f PA X7, P A X3, a nd D B X1. To quan tify the changes in the boundaries o f express ion fo r these 3 genes, the percen t o f the neura l tube w ith in the boundaries w as ca lcu la ted fo r G as1 +/-;S hh +/- (n = 5 ), G as1 -/- (n = 4 ), and G as1 -/-;S hh +/- (n = 3 ) em bryos. 2 sections w ere ana lyzed fo r each em bryo . A tw o-ta iled W elch ’s t-test w as perfo rm ed . For the boundaries o f a ll 3 genes , G as1 +/-; S hh +/- em bryos w ere s ta tis tica lly d iffe ren t from bo th G as1 -/- and G as1 -/-;S hh +/- em bryos (99% confidence leve l). P va lues a re ind icate d fo r each t-test and d isp layed on the bar graph . The express ion boundaries in G as1 -/- and G as1 -/-; S hh +/- em bryos w ere no t found to be s ta tis tica lly d iffe ren t.

F igu re S 3 . (A ) G as1 m u tant d ig it 2 is d isp roportiona lly sm a lle r than the o ther d ig its . T he lengths o f d ig its 2 and 4 w ere m easured on G as1 +/-;S hh +/- and G as1 -/- neona ta l ske le ta l p repara tions and the length ra tio ca lcu la ted . D ig it 4 w as used fo r com parison because it and d ig it 2 a re norm a lly o f s im ila r length . n fo r con tro l lim bs is 3 ; fo r G as1 -/- fo re lim bs, 19 ; fo r G as1 -/- h ind lim bs, 11 . E rro r ba rs = 1 s tandard devia tion . (B ) E xam ina tion o f fo re lim bs of E 12.5 and E 13 .5 m ice o f G as1 and S hh a lle lic com b ina tions (ind icated a t top ) revea led that d ig it 2 never fo rm s in the G as1 -/-;Shh +/- genotype . A rrow head ind ica tes la rge loca tion o f in te r -d ig ita l m esenchym e w here d ig it 2 no rm a lly form s. n = 3 fo r each geno type and stage . D ig it num bers are ind ica ted . S ca le bar = 1 m m . R ationa le fo r the determ ination o f m issing d ig it identity : D e te rm ina tion o f d ig it 1 w as stra igh tfo rw ard because it has one less pha lange . D ig its 4 and 5 w ere de te rm ined by m easuring the ir re la tive lengths as w e ll as exam ination o f m etacarpa l and m eta tarsa l shapes . A s p revious ly reported fo r G as1 m u tan ts, w e found tha t d ig its 4 and 5 a re no t a ffected in the G as1 -/-;Shh +/- (L iu e t a l. 2002). D is tingu ish ing the m issing d ig it as 2 as opposed to 3 is m ore com p lica ted. O ur lines o f a rgum ent tha t d ig it 2 is absen t in 4 -fingered lim bs a re severa l fo ld . 1 ) T he tim e course ana lys is p resen ted a bove show tha t in the G as1 -/-;S hh +/- geno type , d ig its 3 and 4 a re p resent in the ir no rm a l loca tions, w h ile the re is on ly la rge in te r-d ig ita l m esenchym e w here d ig it 2 no rm a lly fo rm s. 2 ) T he in te rm ed ia te pha lange of d ig it 2 in G as1 m u tan t fo re lim bs is freque n tly d isfigu red o r k inked (L iu e t a l. 2002). S uch a m a lfo rm ed d ig it is never observed in any 4 -fingered fo re lim bs, suggesting tha t none of the rem a in ing d ig its a re 2 . 3 ) W hen m etacarpa ls a re found to be fused in e ithe r geno type , it is a lw ays be tw een d ig its 2 and 3; w hen m eta ta rsa ls a re fused, it is a lw ays be tw een d ig its 1 and 2. N e ithe r o f these fus ions w ere ever observed in 4 -fingered lim bs. If the m iss ing d ig it w as actua lly 3 , then w e w ou ld s till expect to f ind fus ions be tw een the rem a in ing d ig its 1 and 2. 4 ) T he d isp roportiona lly reduced s ize o f d ig it 2 in the G as1 m utan t suggests tha t a m ore severe pheno type (as in the G as1 -/-;S hh +/- geno type) w ou ld cause a lack o f d ig it 2 to fo rm . E ven if the m iss ing d ig it is 3 o r adop ts a m ixed iden tity o f 2 and 3 , the conclus ions in the text a re s till app licab le because d ig it 3 a lso depends in part on d iffus ib le S H H (see re fe rences in text).

Martinelli and Fan Supplemental Figure 4

HNF3β GFP

GFP

GFP

GFP

OLIG2

NKX6.1 PAX6

F igure S 4 . G as1 express ion a lte rs the position o f neurona l p rogen ito rs in the neura l tube . C h ick trunk neura l tube w as e le ctropora ted w ith G as1N /A -IR E S -G FP (no t show n) o r G as1 -IR E S -G FP p lasm ids and ana lyzed fo r G FP , P A X6, N K X6.1 , O LIG 2, and H N F3β by doub le im m unofluo rescence (green fo r G FP , and red fo r PA X6, N K X6.1 , O L IG 2, o r H N F3β; excep t fo r O LIG 2, in w h ich the green s igna l is the au to fluo rescence from G FP ) on the sam e sections. Each pa ir o f im ages is from the sam e section; n ≥ 10 fo r each pa ir. A rrow s ind ica te ectop ic activa tion o f H H ta rge t genes; open a rrow s, ectop ic inh ib ition ; open a rrow heads, the ven tra l m id line.

Martinelli and Fan Supplemental Figure 5

Gas1/LacZ

Neural Plate

Neural Tube at PSM Level

Neural Tube at Soimte V

F igure S 5 . G as1 express ion w as de te rm ined by X -ga l s ta in ing E 9 .5 em bryos transverse ly sectioned a t va rious leve ls rang ing from poste rio r (le ft) to ante rio r (righ t). A x ia l leve ls a re ind ica ted on top , fo r com parison to F igure 7A .

Martinelli and Fan Supplemental Figure 6

Gas1-/-;Shh-/-Gas1+/+;Shh-/-Gas1-/-;Shh+/+Gas1+/-;Shh+/-

Ptc1

Gas1/LacZ

F igure S 6 . T he defective le ft/righ t asym m etry pa tte rn ing in the G as1 -/-;S hh -/- em bryo is associa ted w ith im pa ired H H s igna ling in the node as dete rm ined by P tc1 IS H in E8 .0 em bryos. G eno types a re ind ica ted on top . A rrow heads po in t to the m id line w here P tc1 express ion exis ts w hen S hh is p resen t. A rrow s po in t to the node . T he bo ttom p ic tu re is a X -ga l s ta ined G as1 -/-;S hh -/- em bryo revea ling LacZ express ion in the node fo r com parison to F ig . 7 E ,F . G iven the tw o cop ies o f the LacZ gene , the X -ga l s ta in ing w as tim e reduced accord ing ly (45 m in ) to m atch the leve ls o f p rim itive s treak s ta in ing in the LacZ hete rozygous em bryos (2 h r).