A 0.5 P = 0.05 0.4 0.3 q−value 0.2 0.1 0.0

0.0 0.2 0.4 0.6 0.8 1.0

p−value B 12000 8000 significant tests 4000 Significance cutoff 0

0.0 0.1 0.2 0.3 0.4 0.5

q−value cut−off

Supplemental Data Figure 1. Identification of temporally regulated genes using EDGE. Analysis of the p- value vs. q-value plot (A) and the q-value vs. no. of significant tests plot (B) clearly indicated that a p value cutoff of 0.05, corresponding to a q-value of 0.23, provided an ideal point to return the maximum number of significant genes with fewest false positives. A total of 1,170 distinct genes were returned with a P value <= 0.05. This list was filtered to remove any elements whose FC was below 1.2 between any two time points, giving a final list of 1,029 genes. Canonical Pathways Functions

Aryl Hydrocarbon Receptor Signaling Carbohydrate Metabolism

Endoplasmic Reticulum Stress Pathw ay Cell Death

ERK/MAPK Signaling Cellular Development Estrogen Receptor Signaling

G-Protein Coupled Receptor Signaling Cellular Movement

Hypoxia Signaling in the Cardiovascular System Embryonic Development

IGF-1 Signaling Endocrine System Development and Function IL-6 Signaling

Inositol Phosphate Metabolism Endocrine System Disorders

Insulin Receptor Signaling Gene Expression

Integrin Signaling Lipid Metabolism Leukocyte Extravasation Signaling Metabolic Disease PPAR Signaling

PPARa/RXRa Activation Molecular Transport

Protein Ubiquitination Pathw ay Nervous System Development and Function TGF-b Signaling Organ Development VEGF Signaling

Wnt/b-catenin Signaling Small Molecule Biochemistry

0 1 2 3 4 5 6 7 8 0 2 4 6 8 10 12 14 -log(p-value) -log(p-value)

Progenitors Descendants

Supplemental Data Figure 2. Comparison of canonical pathways and gene functions in endocrine progenitors and their descendants reveals striking functional differences. Pathway analysis was performed on genes that had increased expression in endocrine precursors (E13.5/E14.5 - red) and in those with increased expression in descendants of these cells (E16.5/E17.5 - green). A comparison analysis was performed using the Ingenuity Pathways Analysis application, and those pathways or functional groups with differences are highlighted. A Protein Family enzyme transcription regulator transporter kinase peptidase transmembrane receptor growth factor G-protein coupled receptor ligand-dependent nuclear receptor phosphatase translation regulator ion channel cytokine

B GO Biological Process

protein metabolism

cell organization and biogenesis

development

cell differentiation

protein biosynthesis

transcription from RNA Pol II promoter

chromosome organization and biogenesis

mRNA processing

cell fate commitment embryonic organ development

C GO Molecular Function nucleic acid binding signal transducer activity RNA binding structural constituent of ribosome actin binding growth factor activity protease inhibitor activity endopeptidase inhibitor activity calmodulin binding ligand-dependent nuclear receptor activity serine-type endopeptidase inhibitor activity hormone activity Supplementary Data Figure 3. Gene annotation enrichment analysis reveals a significant over- representation of transcriptional regulators in genes that are temporally regulated during development of the endocrine pancreas. Temporally regulated genes annotated with an Entrez Gene ID were mapped to protein families using the Ingenuity Pathways Knowledge Base (A). Using functional annotation for the Gene Ontology (GO) data base enriched terms within the biological process (B) and molecular function (C) categories were determined. IGFBP IGF-1 IGF-1 IGF-1 RE A, PP A, PP IGF-1 A, PP

IGF1R IGF1RIGF1R IGF1R IGF1R IGF1R A, P Cytoplasm SHC GRB2 SOS Ras GRB2 SHC A, P A, P P,P, PP PP PP PP A PP P, PP PP I, M PIP2 P P IRS1/2 I PP I GRB10 Nedd4 SHP2 Ras GAP A PP PI3K FAK Paxillin RE PI3K PP PP EC A c-Raf PIP3 IGF1R internalization A, P Crosstalk between and degradation A P P P integrin and IGF-1 signaling AKT Caspase 9 MEK1/2 PDK1 P I, P P P BAD A, P A, P A, P

RE P PKC p70 S6K PKA ERK1/2 (?,?) BAD TR Cell survival 14-3-3 Cell growth

TR

Nucleus P P AKT ERK1/2 JNK1 I, P CK2 P A, P P, PP FKHR P P Elk-1 SRF PP c-Jun c-Fos PP Cell survival T Supplementary Data Figure 4. Igf-1 Signalling. The network is displayed graphically as nodes (genes/gene products) and edges (the c-Fos biological relationships between the nodes). Nodes were colored to highlight those genes whose expression expression was significantly differentially expressed in either the temporal regulation analysis or static analysis. Color intensity indicates significance, from the most Cell growth and proliferation significant (dark red) to least significant (light pink) genes. Cytoplasm

Endoplasmic Reticulum Caspase 7 Stress induced by A, L Caspase Caspase Caspase hypoxia, glucose starvation 3 A, L 9 A, L 12 Apoptosis Unfolded proteins TRAF2PP PP JNK1 ASK1 A BiP A, P BiP IRE1 PP PP BiP A PP A, P PP PERK IRE1 ATF6

JIK TR Translation TR TR MBTPS initiation P IRE1 XBP1 M mRNA L EIF2A PERK A, P A ATF6 Selective I Translation XBP1 P58IPK

TR ATF4 TR Golgi apparatus

TR Transcription of: ATF4 XBP1 ATF6 BiP CHOP UPRE GRP94 Nucleus Calreticulin

Supplementary Data Figure 5. Endoplasmic Reticulum Stress Pathway. The network is displayed graphically as nodes (genes/gene products) and edges (the biological relationships between the nodes). Nodes were colored to highlight those genes whose expression expression was significantly differentially expressed in either the temporal regulation analysis or static analysis. Color intensity indicates significance, from the most significant (dark red) to least significant (light pink) genes. Wnt TGF-β WIF1 EC DKK EC A,RE PP A, PP SFRP PP PP EC Extracellular Cadherin (E,N,P,VE) KREMENLRP1/5/6 Frizzled TGFBR

A, PP Cytoplasm PPRE P P PP CK1/2 A EC Gaq/o P Src β-catenin P GBP Dsh A, P PP A, PPAR-1 HPK1 ILK TR CK1 AKT TR PIN1 A I, P I, P P P P EC APC β-catenin GSK3 GBP Dsh TAK1 TAB1 PPRE P I PP A, PP PP PP Axin P, PP β-TrCP MDM2 A I, PP PP EC p14ARF RE RE I, M, PP PP2A Ub RE NLK EC p53

Ub Proteasomal β-catenin β-catenin RE Degradation

TR Nucleus RA PP TR SOX RAR

I Supplementary Data Figure 6. Wnt/β- I, PP Catenin Signalling. The network is Groucho HDAC1 CBP displayed graphically as nodes (genes/gene PP Bcl-9 PP A NLK A LEF/TCF β-cateninLEF/TCF I, P products) and edges (the biological relation- PP ships between the nodes). Nodes were colored to highlight those genes whose

T T T T T expression expression was significantly T T T T differentially expressed in either the tempo- Cyclin c-Myc TCF1 MMP7 Axin-2 CD44 ral regulation analysis or static analysis. D1 TCF4 PPARδ CX43

T Color intensity indicates significance, from the most significant (dark red) to least p14ARF significant (light pink) genes. Insulin Glucose A, PP

GLUT4Syntaxin Lipid raft Insulin receptor TRRE PP PP LAR Cytoplasm Cbl I VAMP2 Synip C3G A, P A, P PP A, P A, P PP I A GAB1 CIP4/2 CRK PIP2 I PTP1B PP PTEN A, P TR TC10 SHC A, P Glucose RE EC RE PI3K IRS PP SOCS3 TR EC A SHIP EC GRB2 PIP3 I SHP2 NCK JAK1/2 A PP GLUT4 PP IRS1 VAMP2 PKC PDK1 A, P A, P A, P SOS PP CRK I, P A, P SGK JNK1 A Fyn ATP TSC1 TSC2 AKT Raptor PP I, P, PP I, P A, PPA A, P A, P I, P BAD I Ras mTOR PDE3B A, P

A, P ENaC A GSK3 PP1 EC cAMP AMP 4E-BP1 RE c-Raf GRB10 A, P A A I, P I, P I, P A I, PP A ATP A TR p70 S6K eIF4E eIF2B citrate GYS PKA MEK1/2 lyase TR A, P A Sodium Hormone- Transport ERK1/2 Stress Protein sensitive Fatty Acid lipase Synthesis Synthesis Apoptosis Lipolysis TR

Nucleus AKT SGK ERK1/2 I, P I, P I, P Transcription AFX I, P FKHR FKHRL1

Cell growth Transcription Apoptosis Transcription Cell Growth

Supplementary Data Figure 7. Insulin Receptor Signalling. The network is displayed graphically as nodes (genes/gene products) and edges (the biological relationships between the nodes). Nodes were colored to highlight those genes whose expression expression was significantly differentially expressed in either the temporal regulation analysis or static analysis. Color intensity indicates significance, from the most significant (dark red) to least significant (light pink) genes. ONECUT1

A, E, PD, PP, RB, T M, PP PP A, E EP300 E FOXA2 A E, RB A, PP, RB CREBBP

A PD PD, PP, T GCG PP A, PP, T TNDM A, PP

A, PP TCF1

PP PP E E PP

NEUROG3 E OLIG1 E E E FGF8 E E TITF1 NKX2-4 (includes EG:4823) E E E E E NHLH1 SNAI2 E E NEUROG1 E E E DRD1 E E E E E E PP LHX4 NTRK2 CD63 RB E E CDH8 E E E DLX6 E E PSEN1 E HLXB9 NR2F1 E CD24 E E E E HES6 A, P, RB PPA E EN1 E E EFNB2 GSH-2 E SEMA3CE CDH2 E E E E ERBB3 E E NRP1 NSBP1 E LHX6 ROBO1 NTRK1 NEUROD2 PD E PP, RB E PSEN2 E E EBF1 RORB E SEMA3A

CTSL2 PD, T E E XRCC1 RHOB NUMBL E LO RARB DLL4 SYP E PCSK1 NOTCH2 CHGA E E A, L, PP E E ABCC8 DLL1 E IGF2R

NKX6-1 MAFA PD ISL1 PAX6 NKX2-2 E E

NEUROD1 EIF3S10 PD PD PD PD E, PD PDX1

PD L PAX4 PP IAPP RB SST GCK PPY E, LOPP

INS

Supplementary Data Figure 8. Genes interacting with Neurog3 that are documented in the Ingenuity Pathway Analysis Knowledge Base. The pathway analysis database documents 68 genes with litterature defining their interac- tion with Neurog3. 47 of these genes were identified in a study where microarray analysis demonstrated increased expression of these genes in an in vitro study where pancreatic duct cell lines were infected with a Neurog3- expressing adenovirus (Gasa et al., 2004). Of the genes they identified, less than 20% of them were also identified in the present study, and were restricted to those genes whose interaction with Neurog3 has been reported elsewhere.