Ft

d

Figure S1. Ethe length ofstaining of difference w

Extent of agf the ganglioacetylcholin

was observed

ganglionosisonated zone nesterase acd according t

s is similar i(in % of tota

ctivity. Datato a Student’

38 

in male andal colon lenga are presen’s t-test.

d female Holgth) in the conted as the

lTg/Tg animaolon of P20 mean ± SE

als. QuantificHolTg/Tg aniEM. No sig

cation of imals via gnificant

 

F

pn

v

Figure S2. C(A-C) Doubagainst βIIIpreparation neuronal-fatsquare in A.shown on tvibratome sl

Collagen VIble immunoI-Tubulin (gshowing thated eNCC in. Lateral viewthe right anlices compar

I is juxtaposofluorescencgreen) and at high leveln a HolTg/Tg ws of the z-and lower paring WT (B)

sed to eNCCce labelling

collagen Vs of collagenembryo. (A

axis along thanels, respec) and HolTg/T

39 

C in HolTg/Tg

of e15.5 smVI (red). (An VI are pre

A’) Zoom-in he x and y axctively. (B-C

Tg (C) tissues

g embryonicmall intestinA) Z-stack esent in a mu

view of thexes (at the leC) Single cs. Scale bar,

c intestines.nes performprojection

utually exclue region delevel indicateconfocal sec, 20µm (5µm

med with an

of a wholusive mannelimited by thd by white lctions of trm in A’).

ntibodies le-mount r around he white ines) are ansverse

 

F

p

i

no 

Figure S3. C(A) Single panels) and anti-βIII-Tubimmunofluosignificantlyneuronal-fatone-way AN

Collagen VIconfocal seHolTg/Tg (lo

bulin (greeorescence sigy increased oted eNCC (dNOVA). SI,

I protein levctions in th

ower panels)en) antibodgnals in canonly in regiodata are pressmall intesti

vels are incrhe plane of ) e11.5 inte

dies. Scale ndelas (cd) ons of HolTg

sented as theine.

40 

reased in e1the developstines doubl

bar, 20µmper µm2 sh

g/Tg embryone mean ± SE

11.5 HolTg/Tg

ping myentely labelled wm. (B) Qu

howing that nic intestineEM; n=6 inte

g intestines. eric plexus with anti-couantificationcollagen V

s that contaiestines per g

in wild-typeollagen VI (rn of colla

VI protein lein a high nugenotype; *p

e (upper red) and

agen VI evels are umber of p < 0.05;

 

 

F

p

ip

Figure S4. C(A) Single panels) and anti-βIII-Tubimmunofluoprotein leve(data are presmall intesti

Collagen VIconfocal seHolTg/Tg (lo

bulin (greeorescence sigels are signifesented as thine.

I protein levctions in th

ower panels)en) antibodgnals in candficantly incrhe mean ± SE

vels are incrhe plane of ) e15.5 inte

dies. Scale delas (cd) peeased in theEM; n=6 int

41 

reased in e1the developstines doubl

bar, 20µmer µm2 showe entire HolT

testines per g

15.5 HolTg/Tg

ping myentely labelled wm. (B) Qu

wing that, in Tg/Tg embryogenotype; *p

g intestines. eric plexus with anti-couantificationcomparison

onic intestinep < 0.05; one

in wild-typeollagen VI (rn of colla to WT, colles except the-way ANOV

 

e (upper red) and

agen VI lagen VI

he cecum VA). SI,

 

F

Figure S5. Hof post-nataSingle confo(green) and around and w

High levels al HolTg/Tg afocal section

collagen Vwithin myen

of collagen animals. ns of 3-weekVI (red). In cnteric ganglia

VI are aber

k old colonscontrast to wa from HolTg

42 

rrantly pres

s double labwild-type tisg/Tg mice. Sc

sent around

belled with ssues, abund

cale bar, 20µ

d and within

antibodies adant collage

µm.

n myenteric

against βIII-en VI is fou

c ganglia

-Tubulin und both

 

FP

W

Figure S6. CPolar histogeNCC at theWT and Hol

Cell migratigrams showine migration lTg/Tg eNCC

ion directiong the net trfront in e12(Student’s t

nality is unrajectories (2.5 embryos-test). 

43 

affected in Hin degrees)

s. No differe

HolTg/Tg eNCand traveled

ence in direc

CC. d distance (ictionality w

in µm) of inas detected

ndividual between

 

F

iH

NA 

 

 

 

Figure S7. N(A) Single identificatioHuC/D-) eNeNCC (in %Sox10+ cellsNo statisticANOVA.

Neuronal diconfocal sen and discr

NCC. Scale b%) within ths and HuC/D

cally signific

ifferentiatioections of erimination obar, 20µm. (Bhe whole eND+ cells (datcant differe

on of HolTg/T

e12.5 smallof neuronal-B) Quantitat

NCC populatta are presenences betwe

44 

Tg eNCC at intestines -fated (Sox1tive analysistion which inted as the meen group m

e12.5. double labe

10- HuC/D+

s of the relatiis representemean ± SEMmeans were

elled with ) and undifive abundaned by the c

M; n=5 intese noted acc

antibodies tfferentiated nce of neuronombined nu

stines per gecording to

to allow (Sox10+

nal-fated umber of enotype). one-way

 

F

tbnt 

 

Figure S8. N(A) Single cand discrimand undifferthe relative by the combn=6 intestinthe benefit o

Neuronal anconfocal sec

mination of nrentiated (Soabundance obined numbees per genot

of undifferen

nd glial diffctions of smneuronal-fateox10+ HuC/Dof each subser of Sox10type; *p < 0.ntiated proge

ferentiationmall intestine

ed (Sox10- HD- S100β-) eset (in %) w+ cells and 05; one-way

enitors is not

45 

of HolTg/Tg s triple labeHuC/D+ S10eNCC. Scale

within the whHuC/D+ cely ANOVA). ted in HolTg/

eNCC at e1elled with an00β-), glial-e bar, 20µmhole eNCC plls (data are A decreased

/Tg tissues.

15.5. ntibodies to -fated (Sox1

m. (B-D) Quapopulation wpresented a

d number of

allow ident0+ HuC/D-

antitative anwhich is repas the mean f glial-fated e

tification S100β+)

nalysis of presented

± SEM; eNCC to

 

F

iDvi

Figure S9. PCell proliferintestines ofDNA fragmvariation waintestines pe

Proliferatioration (A, af wild-type

mentation (Tas observed er genotype)

n and surviat e12.5 andand HolTg/T

UNEL), resaccording t. Scale bar, 5

ival of HolTg

d e15.5) andg embryos u

spectively. Cto one-way A50µm.

46 

g/Tg eNCC.d cell death using nucleiCounts wereANOVA (da

(B, at e12.ic markers fe restricted tata are prese

5) were evafor proliferato Sox10+ cented as the

aluated in thation (Ki67) cells. No sige mean ± SE

he small and for

gnificant EM; n=3

 

F

b

Figure S10.Single confo(blue), collabowels have

. Fibronectiocal sectionagen VI (rede no overt im

n remains us of e15.5 sd) and fibro

mpact on fibr

unaffected ismall intestinnectin (greeronectin leve

47 

n HolTg/Tg enes triple laen), showingels and organ

embryonic inabelled with g that highernization.

ntestines. antibodies a

r collagen Vagainst III-

VI levels in

-Tubulin HolTg/Tg

 

FRwdp

c

Figure S11.Representatiwith antiboddelineated bpatients (D) (d)) as well candelas (cd

. Tested humive single cdies against by green line

is indicatedas average

d) per µm2) a

man coloniconfocal sectβIII-Tubulin

es. Identificad in the uppe

level of perare indicated

c tissues nottions of trann (green; in tation number right cornei-ganglionic

d in the lowe

48 

t included innsverse cutsthe insets) ar of controls

er. Age at thec collagen Ver left corner

n Figure 7. s of human and collagen s (C), HSCRe time of tis

VI (n≥5 gangr. Scale bar,

colonic musVI (red). M

R patients (Hsue collectio

glia/child’s s50µm (75µm

scles doubleMyenteric ganH) and Downon (indicatedsample; exprm in insets).

e-labeled nglia are n-HSCR d in days ressed in

 

FEic

Figure S12.Evaluation insertion in constructs dactivity is re(data are prsignificant d

. The 153bpof transcripthe Holstei

driven by theeported in f

resented as tdifference wa

p deletion atptional activin genome. e minimal TKfold inductiothe mean ± as noted acc

t the Holsteivity for the Luciferase a

K promoter +on relative toSEM; n=9

cording to a S

49 

in locus is dsmall block

assays were+/- the 153bo the vectorindependentStudent’s t-t

devoid of conk of sequen

e performedbp fragment or only drivet experimentest.

nsistent regnces deletedin Neuro2a

of deleted sen by the TK

nts performe

gulatory actid by the traa cells with equences. LuK minimal pd in triplica

ivity. ansgenic reporter

uciferase promoter ate). No

50  

Table S1. Patients’ characteristics and full collagen VI quantification data for all tested human samples.

ID Age

(in days) Gender (M or F)

Mean peri-ganglionic collagen VI levels (in candelas/µm2)

Mean intra-ganglionic collagen VI levels (in candelas/µm2)

C1 1 M 780 390

C2 1 M 389 346

C3 60 M 401 195

C4 178 M 727 409

C5 191 M 123 211

C6 217 F 528 278

C7 278 M 281 149

C8 452 M 321 211

H1 17 M 1200 374

H2 20 M 1200 646

H3 35 F 1005 622

H4 40 M 1148 413

H5 42 M 1048 419

H6 51 M 773 194

H7 63 M 1140 514

H8 75 F 992 436

H9 96 M 578 293

H10 222 M 969 421

H11 321 M 978 363

H12 454 M 633 341

D1 49 F 1358 389

D2 97 M 1201 647

D3 182 M 1049 537

D4 336 M 1659 640

Note: Age is at time of tissue collection.

51  

Table S2. Details of the oligonucleotide primers used for RT-PCR and genotyping

ID Sense primer Antisense primer

Wdr82 5’-CAGTATGATAGGACCTGTGAGTGG 5’-AGCCACTTTTATACCACTCTCTCC

Glyctk 5’-CTATCCTGCTCAGGTGATAAGCC 5’-TTGAGAATATGCAGGCAGTCTTGC

Col6a4 5’-AAGAGGATTTTCAGGAGAGAAGGG 5’-AGATTATCAATTCCAGGATCCCCC

Col6a5 5’-GTGACTCAGTACAGGGAAGGG 5’-GTGGTCCCCCACTGACTCATC

Col6a6 5’-TCAGCATCTGGCCTGTTAGG 5’-CGTACTCGGGGCCAGAATCTT

Genotyping F 5’-GTGGTGGACCTAACCTTACAAGGA n/a

Genotyping R1 n/a 5’-CAGGGCTAAGTCTTGGCTTACTTG

Genotyping R2 n/a 5’-CACAGCTTGCTGTATCAGAGCCAT

Table S3. Characteristics of the primary and secondary antibodies used in this study

Antibody Host species

RRID number

Source

Anti-βIII-Tubulin Mouse AB_2256751 Abcam, ab78078

Anti-collagen VI Rabbit AB_305585 Abcam, ab6588

Anti-Sox10 Goat AB_2195374 Santa Cruz Biotechnology, sc-17342

Anti-S100β Rabbit AB_10013383 DakoCytomation, Z0311

Anti-HuC/D Mouse AB_2314656 Molecular Probes, A-21271

Anti-Ki67 Rabbit AB_443209 Abcam, ab15580

Anti-Goat Alexa Fluor 488 Bovine AB_2340883 Jackson ImmunoResearch, 805-545-180

Anti-Rabbit Alexa Fluor 594 Donkey AB_2340621 Jackson ImmunoResearch, 711-585-152

Anti-Mouse Alexa Fluor 647 Donkey AB_2340862 Jackson ImmunoResearch, 715-605-150

52  

Supporting information

Supplemental Acknowledgments: The following French Departments of Pediatric Surgery from the

Ente-Hirsch study group are thanked for participating to this work: Etienne Suply, Stephan de Napoli,

Marc-David Leclair (University Hospital of Nantes); Guillaume Levard, Véronique Couvrat-Carcauzon

(University Hospital of Poitiers); Philine de Vries (University Hospital of Brest); Guillaume Podevin

(University Hospital of Angers); Sabine Sarnacki, Erik Hervieux (Necker Hospital, Hospital for Sick

Children, Paris).

Movie S1: Example of eNCC migration in an e13.5 control embryo.

Movie S2: Example of eNCC migration in an e13.5 HolTg/Tg embryo.

Supplemental Dataset 1: Full rRNA-depleted transcriptome of e12.5 WT::G4-RFP and

HolTg/Tg::G4-RFP eNCC