Appendix identification of factors involved in the maintenance of embryonic stem cell self renewal and pluripotency

For this titration experiment, E14 mouse ES cells were treated with varying concentration of Activin A for 10 hours, and then harvested and assayed for Pitx2 transcript level using qPCR

Appendix

Appendix

I) Preparation of home-made alkaline phosphatase staining kit

Preparation was based on Sigma-Aldrich Procedure #86-Alkaline Phosphatase kit for leukocytes All reagents are available from Sigma-Aldrich

Sodium Nitrite 0.1M = 6.9g/L ml 1

Fast Red Violet LB Base mg 5

AMPD buffer pH 9.5

Napthol AS-BI phosphate mg 50

Prior to staining, solution A was mixed with solution B and water in 2:1:1 ratio This mixture was then used to stain the samples

II) Titration of Activin A

0

50

100

150

200

250

300

350

Concentration of Activin A added (ng/ml)

Figure 44 25ng/ml of Activin A was deemed to be the optimal concentration for use to upregulate Nodal signaling For this titration experiment, E14 mouse ES cells were treated with varying concentration of Activin A for 10 hours, and then harvested and assayed for Pitx2 transcript level using qPCR analysis The samples were normalized to endogenous -actin and mean values are plotted as percentages relative to non-treated ES cells harvested at start of experiment (100%) for qPCR measurement Experiments were performed in biological duplicates and duplicate Ct values were obtained for each of the biological replicates (n=2)

III) Titration of Lefty1

0

20

40

60

80

100

120

Concentration of Lefty1 (ng/ml)

Figure 45 50ng/ml of Lefty1was deemed to be the optimal concentration for use to lower Nodal signaling For this titration experiment, E14 mouse ES cells were treated with varying concentration of Activin A for 10 hours, and then harvested and assayed for Pitx2 transcript level using qPCR analysis The samples were normalized to endogenous -actin and mean values are plotted as percentages relative to non-treated ES cells harvested at start of experiment (100%) for qPCR measurement Experiments were performed in biological duplicates and duplicate Ct values were obtained for each of the biological replicates (n=2)

IV) Description of Ct method used for tabulation of qPCR data

For all the qPCR data presented in this thesis, the relative transcript levels for the gene of interest between test sample and control were tabulated using the Ct method -Actin was used as the housekeeping gene for normalization The difference between the mean test sample Ct values and the mean -Actin values ( Ctsample) were first tabulated

Ctsample = CtGene of interest – Ct -Actin

Similar tabulations were then made for the scrambled control sample ( Ctcontrol) Ct was then obtained by subtracting Ctcontrol from Ctsample

Ct method = Ctsample - Ctcontrol

The relative mRNA level of gene of interest expressed between sample and control, expressed as a percentage relative to control (Percent diff) were then tabulated with the following formula:

Percent diff = 2(- Ct) x 100%

V) qPCR primer sequences

Lefty2

Cadherin11 CGGCAAAGATTTCAGTAGAAGATGCc

Cerberus1 CGAAGAGGTCTCCCAGTGTACTTCG CCGTGACTCAGCCAGCAGAT Nodal CGGTTCTCATGCTCTACTCCAACCG GGCTTCTGTCTGGCAAATGATG E-Cadherin CGGATGGTCTTTGTTCTGGTTATCCG TGACGCAGCTCAAGAATCTCTCA Pdgfr CGGCCTCTGTTCTCTACACTGCCG CCCTCTGGGAGACCTTCATCAG Foxh1 CAGCACTAGCAGGGACTTGATGCTG GGTGGATGTGAGCCTGATTCC T-brachyury CGCTTGTTAGTTAGCTCCTTGAAGCG CACAGAGAGCGCAGGGAAGAG

Pthr1 CGGCATTAGGAAGTCTTGGAGCCG CCATTGGTGGTGGCAGGAAG Hnf4 CGTTGCCTGGAGGATTACATCAACG CATCTGCCAGGTGATGCTCTG

Mixl1 CGTCTATGGTCTGTCGGAAGACG CCACGCAGTGCTTTCCAAAC Fgf8 CGCTTTAGTTGAGGAACTCGAAGCG ACATGGCCTTTACCCGCAAG Goosecoid CGATTCTGTCCGAGTCCAAATCG GGAGACGACAGAAGCGATCCTC

Sox7 CGGTACGATTACCCCAACTACAAGTA

Pdgfr CGCTCCTTCTACCACCTCAGCG GCCGGATGGTCACTCTTTAGGA

Esx1 CTAACCCCAACCCCAACC TTCTTGACAGGTAATGCGTGA

NeuroD1 GCCTTTACCATGCACTACCC TGTTGTCTATGGGGATCTCG

Eomesodermin CCTGGTGGTGTTTTGTTGTG TTTAATAGCACCGGGCACTC E-Cadherin CGGATGGTCTTTGTTCTGGTTATCCG TGACGCAGCTCAAGAATCTCTCA BMP4 CGTGTTCACCTCCACCAGACACG CCTTCTGCGGGTCAAGGTATG

VI) A summary table documenting the qPCR analysis of germ layer markers for

experiments involving elevation of Nodal signaling

Gene expression level normalized to control (Mean values

expressed in %)

Gene

ALK4*

overexpression treatment Activin A overexpression Nodal Lefty2 RNAi

VII) A summary table documenting the qPCR analysis of germ layer markers for experiments involving inhibition of Nodal signaling

Gene expression level normalized to control

(Mean values expressed in %)

treatment (J1) treatment (E14) SB431542 treatment Lefty1

Nature/Function/Pathway(s)

Reduced OCT4

promoter activity when knockdown?

Perturbed Nanog

promoter activity when knockdown?

Lefty1 • Secreted protein

• Member of TGF- superfamily

• Involved in Nodal signaling

• Nodal antagonist

• High expression in ES cells and ICM

• Differential expression pattern between

ES cells and differentiated cells

• Co-expressed with ES transcription factor like Nanog during mouse pre-implantation development

• Oct4 and Nanog target in mouse Es cells

• LeftyB (human Lefty1) is regulated by Wnt pathway

Lefty2 • Secreted protein

• Member of TGF- superfamily

• Involved in Nodal signaling

• Nodal antagonist

• High expression in ES cells and ICM

• Differential expression pattern between

ES cells and differentiated cells

• Co-expressed with ES transcription factor like Oct4 during mouse pre-implantation development

• Oct4 , Sox2and Nanog target in human

ES cells

• LeftyA (human Lefty2) is regulated by Wnt pathway

Rex2 • Zinc finger protein • Expression detected in ES cells and F9

murine teratocarcinoma

• Differential expression pattern between

ES cells and differentiated cells

• Reduced expression during retinoic acid induced differentiation of F9 cells

Zfx • Zinc finger protein

• Transcription factor

• Link to canonical Wnt pathway

• High expression in ES cells

a-catenin • Subunit of Cadherin protein • Expression detected in ES cells

VIII) Candidate genes shortlisted

(Catna) complex

• Involved in Wnt signaling • Link to canonical Wnt pathway No Yes

-catenin

(Catnb)

• Subunit of Cadherin protein complex

• Intracellular mediator of canonical Wnt signaling

• Expression detected in ES cells

Dot1l • Non-SET domain protein

• Histone H3 methyltransferase • Expression detected in ES cells No No

Lef1 • Transcription factor

• Intracellular mediator of canonical Wnt signaling

• Expression detected in ES cells

Gata3 • Transcription factor • Co-expressed with ES transcription

factors like Nanog and Sox2 during