Legends for Supplemental Figures and Tables Figure S1. Expression of Tlx during retinogenesis. (A) Staged embryos were stained for β- galactosidase knocked into the Tlx locus to indicate Tlx expression. Tlx was expressed in the neural blast layer in the early phase of neural retina development (blue signal). (B) Expression of Tlx in neural retina was quantified using Q-PCR at multiple developmental stages. Figure S2. Expression of p27kip1 and cyclin D1 (Ccnd1) at various developmental stages in wild-type or Tlx-/- retinas. (A) Q-PCR analysis of p27kip1 mRNA expression. (B) Western blotting analysis of p27kip1 protein expression. (C) Q-PCR analysis of cyclin D1 mRNA expression. Figure S3. Q-PCR analysis of mRNA expression of Sf1 (A), Lrh1 (B), and Atn1 (C) in wild-type mouse retinas. RNAs from testis and liver were used as controls. Table S1. List of genes dysregulated both at E15.5 and P0 Tlx-/- retinas. Gene E15.5 P0 Cluste Gene Title Fold Fold r Name p-value p-value Change Change nuclear receptor subfamily 0, group B, Nr0b1 1.65 0.0024 2.99 0.0035 member 1 1 Pou4f3 1.91 0.0162 2.39 0.0031 POU domain, class 4, transcription factor 3 1 Tcfap2d 2.18 0.0000 2.37 0.0001 transcription factor AP-2, delta 1 Zic5 1.66 0.0002 2.02 0.0218 zinc finger protein of the cerebellum 5 1 Zfpm1 1.85 0.0030 1.88 0.0025 zinc finger protein, multitype 1 1 Pten 1.60 0.0155 1.82 0.0131 phospatase and tensin homolog 2 Itgb5 -1.85 0.0063 -1.85 0.0007 integrin beta 5 2 Gpr49 6.86 0.0001 15.16 0.0001 G protein-coupled receptor 49 3 Cmkor1 2.60 0.0007 2.72 0.0013 chemokine orphan receptor 1 3 Cacna1 d 2.81 0.0009 2.08 0.0017 L type calcium channel alpha 1D subunit 3 Edaradd 1.66 0.0037 1.62 0.0164 EDAR-associated death domain 3 Kdr -1.78 0.0014 -1.64 0.0008 kinase insert domain protein receptor 3 Rgs4 -2.33 0.0060 -1.69 0.0019 regulator of G-protein signaling 4 3 Pdlim4 -1.70 0.0000 -1.75 0.0042 PDZ and LIM domain 4 3 Cdh4 2.02 0.0013 3.05 0.0013 cadherin 4 4 Pmp22 -1.73 0.0119 -1.80 0.0113 peripheral myelin protein 4 Espn -3.15 0.0006 -2.00 0.0011 espin 4 Crym -14.04 0.0001 -6.81 0.0004 crystallin, mu 4 Cyp1b1 5.28 0.0002 3.62 0.0017 cytochrome P450, family 1, subfamily b1 5 Scara3 1.60 0.0356 1.70 0.0002 scavenger receptor class A, member 3 5 Cnp1 -1.80 0.0491 -5.16 0.0000 cyclic nucleotide phosphodiesterase 1 5 Accn1 2.85 0.0009 2.79 0.0001 degenerin 6 Crabp1 2.18 0.0068 1.68 0.0004 cellular retinoic acid binding protein I 6 FXYD domain-containing ion transport Fxyd7 -3.15 0.0661 -1.78 0.0133 regulator 7 6 Oact1 1.60 0.0225 2.00 0.0014 O-acyltransferase domain containing 1 7 transmembrane, prostate androgen induced Tmepai -1.68 0.0012 -1.58 0.0032 RNA 7 leucine rich repeat transmembrane Lrrtm1 -4.32 0.0088 -6.40 0.0000 neuronal 1 7 Shown are genes with p value ≤ 0.05 and fold change ≥1.6 at both E15.5 and P0; Unknown function genes and ESTs were not listed. Gene clustering was based on Gene Ontology Biological Process with verification by PubMed search. 1. transcription factors and related proteins. 2. cell cycle, cell proliferation and growth factors. 3. signal transduction. 4. cell adhesion, cytoskeleton and matrix proteins. 5. metabolism and related protein. 6. channels, transporter and related proteins. 7. miscellaneous genes. Fold changes represented the geometric mean after pair-wise comparison between values from TLX KO retinas to that from wild type controls. Numbers with negative sign indicated decreased expression in TLX KO retinas. Table S2. Global gene expression changes in Tlx-/- retinas at E15.5 and P0. Gene E15.5 P0 Gene Title Fold Fold Name p-value p-value Change Change 1. Transcription factors and related proteins Etv4 1.05 0.9308 -7.64 0.0200 ets variant gene 4 (E1AF) Foxd1 -1.45 0.0026 -2.21 0.0003 forkhead box D1 Etv5 -1.58 0.0054 -2.09 0.0004 ets variant gene 5 Hey2 -1.23 0.2040 -2.02 0.0026 hairy/enhancer-of-split related with YRPW motif 2 Irx5 1.10 0.4787 -1.95 0.0028 Iroquois related homeobox 5 (Drosophila) Nr2e3 -1.02 0.7998 -1.94 0.0003 nuclear receptor subfamily 2, group E, member 3 Heyl -1.29 0.0646 -1.91 0.0038 hairy/enhancer-of-split related with YRPW motif-like Sox9 -1.21 0.0505 -1.87 0.0017 SRY-box containing gene 9 Klf3 1.07 0.6294 -1.75 0.0272 Kruppel-like factor 3 (basic) Olig2 -1.48 0.0084 -1.73 0.0139 oligodendrocyte transcription factor 2 Nrl -1.08 0.6075 -1.66 0.0065 neural retina leucine zipper gene Nr1d2 -1.07 0.9164 -1.65 0.0007 nuclear receptor subfamily 1, group D, member 2 Hes2 -1.95 0.0089 -1.60 0.0459 hairy and enhancer of split 2 (Drosophila) Ascl1 -1.73 0.0288 -1.58 0.0769 achaete-scute complex homolog-like 1 (Drosophila) Rora -1.74 0.0012 -1.33 0.0016 RAR-related orphan receptor alpha Hod -1.84 0.0454 -1.23 0.0644 homeobox only domain Tcfap2c -1.97 0.0011 -1.17 0.0171 transcription factor AP-2, gamma Max -2.09 0.0120 -1.11 0.4365 Max protein Eomes -1.70 0.0062 -1.06 0.3584 eomesodermin homolog (Xenopus laevis) Nfib 1.65 0.0075 -1.04 0.3667 nuclear factor I/B Mllt2h 2.05 0.0047 1.05 0.7032 homolog of human MLLT2 unidentified gene Gfi1 -2.14 0.0004 1.48 0.0106 growth factor independent 1 Klf7 1.18 0.0039 1.64 0.0060 Kruppel-like factor 7 (ubiquitous) Isl2 -1.38 0.0234 1.66 0.0013 insulin related protein 2 (islet 2) Bhlhb4 3.43 0.0096 1.70 0.4360 basic helix-loop-helix domain containing, class B4 Ptx3 1.87 0.0073 1.70 0.0945 pentaxin related gene Ebf1 1.06 0.5342 1.71 0.0085 early B-cell factor 1 Zfpm1 1.85 0.0030 1.88 0.0025 zinc finger protein, multitype 1 Ebf3 1.49 0.0093 1.90 0.0009 early B-cell factor 3 Eya2 1.77 0.0006 1.98 0.3813 eyes absent 2 homolog (Drosophila) Zic5 1.64 0.0006 2.02 0.0218 zinc finger protein of the cerebellum 5 Ebf2 1.39 0.0615 2.11 0.0072 early B-cell factor 2 Pou3f1 1.11 0.6620 2.26 0.0048 POU domain, class 3, transcription factor 1 Foxg1 1.41 0.0122 2.28 0.0196 forkhead box G1 Tcfap2d 2.18 0.0000 2.37 0.0001 transcription factor AP-2, delta Pou4f3 1.91 0.0162 2.39 0.0031 POU domain, class 4, transcription factor 3 Pthr1 1.23 0.0761 2.41 0.0049 parathyroid hormone receptor 1 Atoh7 1.21 0.0027 2.79 0.0070 atonal homolog 7 (Drosophila) Nr0b1 1.65 0.0024 2.99 0.0035 nuclear receptor subfamily 0, group B, member 1 Fosl2 1.40 0.5734 4.32 0.0026 fos-like antigen 2 2. Cell cycle, cell proliferation and growth factors Ngfr -1.28 0.1547 -3.79 0.0034 nerve growth factor receptor Cd44 -2.14 0.1793 -2.85 0.0175 CD44 antigen Kit -1.05 0.5046 -2.70 0.0015 kit oncogene Septin8 -1.21 0.0565 -1.85 0.0046 septin 8 Itgb5 -1.85 0.0063 -1.85 0.0007 integrin beta 5 Pdgfra -1.73 0.0211 -1.80 0.0199 platelet derived growth factor receptor, alpha polypeptide Catnbip1 -1.49 0.0617 -1.78 0.0333 catenin beta interacting protein 1 Gfra1 -1.26 0.2860 -1.74 0.0250 glial cell derived neurotrophic factor family receptor alpha 1 Lama1 -1.36 0.1942 -1.68 0.0004 laminin, alpha 1 Spag5 1.03 0.5708 -1.65 0.0023 sperm associated antigen 5 Slit2 1.38 0.0085 1.59 0.0030 slit homolog 2 (Drosophila) Csf1r 1.15 0.3609 1.73 0.0031 colony stimulating factor 1 receptor Dapk1 1.24 0.1820 1.74 0.0003 death associated protein kinase 1 Acvr1c -1.05 0.5461 1.78 0.0044 activin A receptor, type IC Fign 1.18 0.1606 1.81 0.0301 fidgetin Pten 1.60 0.0155 1.82 0.0131 phospatase and tensin homolog Egfr 1.06 0.9274 1.84 0.0015 epidermal growth factor receptor Fgf15 1.02 0.7012 2.06 0.0017 fibroblast growth factor 15 Igfbpl1 1.59 0.0063 2.18 0.0004 insulin-like growth factor binding protein-like 1 3. Signal transduction Cxcl16 -1.03 0.9686 -8.19 0.0027 chemokine (C-X-C motif) ligand 16 Sag -1.48 0.1862 -3.30 0.0134 retinal S-antigen Pde6b 1.34 0.7509 -3.15 0.0247 phosphodiesterase 6B, cGMP, beta polypeptide Gng1 -1.16 0.6379 -2.05 0.0009 guanine nucleotide binding protein (G protein), gamma 1 Dusp16 -1.03 0.6973 -1.90 0.0006 dual specificity phosphatase 16 Pdlim4 -1.70 0.0000 -1.75 0.0042 PDZ and LIM domain 4 Cxcr4 -1.24 0.0054 -1.69 0.0043 chemokine (C-X-C motif) receptor 4 Rgs4 -2.33 0.0060 -1.69 0.0019 regulator of G-protein signaling 4 Baiap1 -1.35 0.0017 -1.65 0.0105 BAI1-associated protein 1 Kdr -1.78 0.0014 -1.64 0.0008 kinase insert domain protein receptor Epha3 -1.81 0.0044 -1.43 0.1592 Eph receptor A3 Hpca -1.90 0.0014 -1.32 0.0717 hippocalcin Rpgrip1 -2.37 0.0007 -1.11 0.1504 retinitis pigmentosa GTPase regulator interacting protein 1 Pkib -2.26 0.0027 -1.10 0.5017 protein kinase inhibitor beta, cAMP dependent Apeg1 -1.80 0.0182 1.11 0.3094 aortic preferentially expressed gene 1 Ptprk 1.68 0.0072 1.16 0.2927 protein tyrosine phosphatase, receptor type, K Cnr1 1.95 0.0133 1.18 0.3726 cannabinoid receptor 1 (brain) Plce1 2.76 0.0001 1.29 0.2795 phospholipase C, epsilon 1 Cacng5 1.88 0.0004 1.32 0.2263 calcium channel, voltage-dependent, gamma subunit 5 Rragd 1.73 0.0031 1.53 0.0275 Ras-related GTP binding D Cx3cr1 1.33 0.1226 1.60 0.0399 chemokine (C-X3-C) receptor 1 Adrb1 -1.27 0.1341 1.61 0.0644 adrenergic receptor, beta 1 Edaradd 1.66 0.0037 1.62 0.0164 EDAR-associated death domain Pde9a 1.03 0.2034 1.71 0.0041 phosphodiesterase 9A Hck 1.15 0.5010 1.74 0.0164 hemopoietic cell kinase Tm4sf12 1.24 0.6936 1.98 0.0038 transmembrane 4 superfamily member 12 Rassf3 1.54 0.0311 2.02 0.0110 Ras association (RalGDS/AF-6) domain family 3 Adcyap1 1.44 0.0197 2.02 0.0027 adenylate cyclase activating polypeptide 1 Cacna1d 2.81 0.0009 2.08 0.0017 L type calcium channel alpha 1D subunit Snf1lk 1.11 0.8149 2.09 0.0084 SNF1-like kinase Edg1 1.41 0.0058 2.11 0.0007 endothelial differentiation G-protein-coupled receptor 1 Sstr2 1.24 0.0142 2.30 0.0012 somatostatin receptor 2 Cmkor1 2.60 0.0007 2.72 0.0013 chemokine orphan receptor 1 Gpr49 6.86 0.0001 15.16 0.0001 G protein-coupled receptor 49 4.