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 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 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 dysregulated both at E15.5 and P0 Tlx-/- retinas.

Gene E15.5 P0 Cluste Title Fold Fold r Name p-value p-value Change Change nuclear 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, 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 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 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 Biological Process with verification by PubMed search. 1. transcription factors and related . 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 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 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 5 (Drosophila) Nr2e3 -1.02 0.7998 -1.94 0.0003 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 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 /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 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 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 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 receptor Cd44 -2.14 0.1793 -2.85 0.0175 CD44 antigen Kit -1.05 0.5046 -2.70 0.0015 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 , 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 , 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 receptor Fgf15 1.02 0.7012 2.06 0.0017 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 () 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. Cell adhesion, cytoskeleton and matrix proteins

Crym -14.04 0.0001 -6.81 0.0004 crystallin, mu Omg -1.11 0.1688 -6.75 0.0012 oligodendrocyte myelin glycoprotein Sema5a -1.44 0.0025 -2.79 0.0051 semaphorin 5A Mcam -1.66 0.0186 -2.08 0.0029 melanoma cell adhesion molecule Espn -3.15 0.0006 -2.00 0.0011 espin Col2a1 1.12 0.3550 -1.95 0.0345 procollagen, type II, alpha 1 Pmp22 -1.73 0.0119 -1.80 0.0113 peripheral myelin protein Col9a3 -2.37 0.0968 -1.78 0.0269 procollagen, type IX, alpha 3 Spon1 -1.80 0.0069 -1.68 0.0310 spondin 1, (f-spondin) extracellular matrix protein Pcdh20 -1.62 0.0131 -1.07 0.8372 protocadherin 20 Tll -1.95 0.0216 -1.07 0.5491 tolloid-like Cdh1 -1.87 0.0020 1.22 0.1870 cadherin 1 Thbs1 -1.97 0.0074 1.39 0.1268 thrombospondin 1 Ramp3 1.90 0.0005 1.44 0.0024 receptor (calcitonin) activity modifying protein 3 Ntng1 1.08 0.6982 1.64 0.0020 netrin G1 Cntnap4 1.18 0.1442 1.70 0.0101 contactin associated protein 4 Lgals7 1.45 0.0077 1.74 0.0125 lectin, galactose binding, soluble 7 Cspg2 2.89 0.0059 1.87 0.0348 chondroitin sulfate proteoglycan 2 Spock1 1.35 0.1899 2.00 0.0010 sparc/osteonectin, cwcv and kazal-like proteoglycan 1 Itm2a 1.22 0.0049 2.26 0.0014 integral membrane protein 2A Ly86 1.53 0.0630 2.35 0.0048 lymphocyte antigen 86 Fst 1.71 0.0078 2.37 0.1274 follistatin Spp1 1.70 0.2962 2.52 0.1016 secreted phosphoprotein 1 Sepp1 1.53 0.0172 2.58 0.0167 selenoprotein P, plasma, 1 Efemp1 1.36 0.0049 2.74 0.0008 egf-containing fibulin-like extracellular matrix protein 1 Cdh4 2.02 0.0013 3.05 0.0013 cadherin 4 Cldn23 1.28 0.7475 4.22 0.0078 claudin 23

5. Metabolism and related protein

Cnp1 -1.80 0.0491 -5.16 0.0000 cyclic nucleotide phosphodiesterase 1 Fxyd1 -1.37 0.0879 -2.81 0.0301 FXYD domain-containing ion transport regulator 1 Popdc3 -1.26 0.7645 -2.28 0.0107 popeye domain containing 3 Ass1 -1.48 0.0757 -2.13 0.0004 argininosuccinate synthetase 1 Adam12 -1.02 0.7621 -2.09 0.0043 a disintegrin and metalloproteinase 12 (meltrin alpha) Car2 -1.26 0.0161 -1.77 0.0001 carbonic anhydrase 2 Rhbdl4 -1.64 0.0321 -1.73 0.0352 rhomboid, veinlet-like 4 (Drosophila) Cte1 1.66 0.0362 -1.61 0.0236 cytosolic acyl-CoA thioesterase 1 Scarb1 -2.79 0.0005 -1.54 0.0052 scavenger receptor class B, member 1 Cth -2.09 0.0004 -1.44 0.0891 cystathionase (cystathionine gamma-lyase) Cyp26a1 1.66 0.0009 1.40 0.0120 cytochrome P450, family 26, subfamily a, polypeptide 1 Galm 1.10 0.8142 1.70 0.0208 galactose mutarotase Scara3 1.60 0.0356 1.70 0.0002 scavenger receptor class A, member 3 Usp25 1.17 0.1211 1.78 0.0094 ubiquitin specific protease 25 Moxd1 -2.39 0.1471 2.09 0.0383 monooxygenase, DBH-like 1 Aldh1a3 1.40 0.0499 3.32 0.0000 aldehyde dehydrogenase family 1, subfamily A3 Cyp1b1 5.28 0.0002 3.46 0.0001 cytochrome P450, family 1, subfamily b, polypeptide 1 Cyp1b1 3.94 0.0001 3.62 0.0017 cytochrome P450, family 1, subfamily b, polypeptide 1 6. Channels, transporter and related proteins

Rlbp1 -1.33 0.0082 -2.87 0.0018 retinaldehyde binding protein 1 Slc16a2 -1.04 0.2045 -2.30 0.0006 solute carrier family 16, member 2 Fxyd7 -3.15 0.0661 -1.78 0.0133 FXYD domain-containing ion transport regulator 7 Slc16a3 -1.18 0.5866 -1.69 0.0003 solute carrier family 16, member 3 Smc4l1 -1.77 0.0095 -1.31 0.0128 SMC4 structural maintenance of 4-like 1 Synpr -1.71 0.0411 -1.03 0.9471 synaptoporin Viaat -1.68 0.0506 1.17 0.0676 vesicular inhibitory amino acid transporter Snag1 1.75 0.0016 1.20 0.2893 sorting nexin associated golgi protein 1 Arf6 -1.84 0.0255 1.23 0.2675 ADP-ribosylation factor 6 Slc7a3 -2.02 0.0044 1.29 0.3712 solute carrier family 7, member 3 Cftr 2.26 0.0035 1.35 0.4252 cystic fibrosis transmembrane regulator homolog Chrna3 1.20 0.2300 1.62 0.0033 cholinergic receptor, nicotinic, alpha polypeptide 3 Crabp1 2.18 0.0068 1.68 0.0004 cellular retinoic acid binding protein I Kctd12 1.08 0.1937 1.84 0.0001 potassium channel tetramerisation domain containing 12 Cacna1d 2.62 0.0001 2.08 0.0017 L type calcium channel, voltage-dependent, alpha 1D Accn1 2.85 0.0009 2.79 0.0001 amiloride-sensitive cation channel 1, neuronal (degenerin)

7. Miscellaneous genes

Lrrtm1 -4.32 0.0088 -6.40 0.0000 leucine rich repeat transmembrane neuronal 1 Fbxo2 -1.62 0.4859 -4.09 0.0195 F-box only protein 2 Ttyh1 -1.43 0.1883 -2.08 0.0107 tweety homolog 1 (Drosophila) Lrrfip1 -1.38 0.0133 -2.03 0.0008 leucine rich repeat (in FLII) interacting protein 1 Ttyh1 -1.27 0.1623 -1.90 0.0245 tweety homolog 1 (Drosophila) Plod2 -1.31 0.0151 -1.66 0.0555 procollagen lysine, 2-oxoglutarate 5-dioxygenase 2 Slc25a13 -1.44 0.0125 -1.61 0.0589 solute carrier family 25, member 13 Crb1 -1.56 0.0037 -1.60 0.0073 crumbs homolog 1 (Drosophila) Epb4.1l2 -1.68 0.0006 -1.60 0.0013 erythrocyte protein band 4.1-like 2 Tmepai -1.68 0.0012 -1.58 0.0032 transmembrane, prostate androgen induced RNA Plekhb1 -2.03 0.0017 -1.45 0.0794 pleckstrin homology domain containing, family B, member 1 Pabpc1 3.17 0.0018 -1.32 0.1431 poly A binding protein, cytoplasmic 1 Deadc1 -2.60 0.0003 -1.32 0.5962 deaminase domain containing 1 Ndrg1 -1.77 0.0009 -1.24 0.0216 N- downstream regulated gene 1 Raly -1.90 0.0063 -1.19 0.4118 hnRNP-associated with lethal yellow Cotl1 -2.30 0.0052 -1.15 0.4698 coactosin-like 1 (Dictyostelium) Ubxd4 2.00 0.0039 -1.11 0.4184 UBX domain containing 4 Bcl7a 2.44 0.0582 1.01 0.8559 B-cell CLL/lymphoma 7A Siat7e -1.94 0.0180 1.01 0.9647 sialyltransferase 7 E Kif5c 1.65 0.0223 1.22 0.4855 kinesin family member 5C Otub1 1.70 0.0015 1.26 0.1306 OTU domain, ubiquitin aldehyde binding 1 Dlgh4 -1.84 0.0086 1.33 0.1936 discs, large homolog 4 (Drosophila) Hyou1 1.77 0.0148 1.40 0.2001 hypoxia up-regulated 1 Fgd6 1.25 0.0041 1.64 0.0189 FYVE, RhoGEF and PH domain containing 6 C1qb 1.39 0.1223 1.65 0.0350 complement component 1, q subcomponent, beta Elavl4 1.38 0.0074 1.66 0.0006 ELAV (embryonic lethal, abnormal vision, Drosophila)-like 4 Ltb4dh 1.02 0.1709 1.68 0.0152 leukotriene B4 12-hydroxydehydrogenase Olfm1 -1.18 0.4153 1.73 0.0071 olfactomedin 1 Serpine2 1.30 0.0490 1.77 0.0010 serine (or cysteine) proteinase inhibitor, clade E, member 2 Tyrobp 1.24 0.1930 1.98 0.0519 TYRO protein binding protein Oact1 1.60 0.0225 2.00 0.0014 O-acyltransferase (membrane bound) domain containing 1 Epb4.1l4 1.38 0.0019 2.02 0.0001 a erythrocyte protein band 4.1-like 4a C1qa 1.48 0.1358 2.06 0.0242 complement component 1, q subcomponent, alpha Lasp1 -1.10 0.6341 2.08 0.0434 LIM and SH3 protein 1 C4 -1.18 0.9059 2.18 0.0008 complement component 4 (within H-2S) Lrpb7 1.23 0.0063 2.26 0.0065 leucine rich protein, B7 gene Shown are genes with p value ≤ 0.05 and fold change ³1.6 at either E15.5 or P0; Gene clustering was based on Gene Ontology Biological Process with verification by PubMed search. Unknown function genes and ESTs were not listed. 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.