Endomes QPCR

QPCR Data Relevant to Endomesoderm Network

May 18, 2010

Ub, ubiquitin mRNA control used for internal standardization; C_T, cycle no. at threshold; C, control embryo sample; Exp, sample from perturbed embryos. The quantity of transcript at any point in the reaction is I(1.94^C_T), where I is the initial amount in the reaction mixture. A positive number means the number of transcripts is increased by the perturbation; a negative number means the number of transcripts is decreased. Data are listed that are considered significant where significant means more than three-fold increase or decrease from control transcript levels (normalized C_T difference from control is <-1.6 or >+1.6). Smaller effects are shown as "NS", except where individual NS data are included together with other significant measurements to display scatter or inconsistencies amongst different batches of cDNA. Commas separate replicate measurements in the same cDNA batch; slashes indicate different batches of cDNA from independent experiments.

Note: Smaller effects are shown as "NS", except where individual NS data are included together with other significant measurements to display scatter or inconsistencies amongst different batches of cDNA. Commas separate replicate measurements in the same cDNA batch; slashes indicate different batches of cDNA from independent experiments, dashes indicate a missing data point

Perturbations

Perturbation:	Genes not affected or shown to be affected only indirectly (0 to 24h, unless otherwise noted)
Alx1 MASO	L1 (18, 24, 30 h), cyclophillin, delta, endo16, ets1, eve, gatac, gcm, pmar1, sm/pm27, snail, tbr, [erg, foxA, foxN2/3, foxO, hex, hnf6, jun, not, pks, soxC, tel, z48 (18-24 h)]
Blimp1b MASO	bra, hox11/13b, lim1, [dac, delta, hesC, msx, notch (18-27 h)]
Blimp1b-En	dec, dpt, endo16, foxa, gatae, gcm, not, nrl, otx a, pks, pmar1, soxb1, tbr
Bra MASO	capk, dec, delta, dpt, ephx, ficolin, msp130-like, nrl, pks, sm50, tbr, [blimp1b, dac, eve, foxN2/3, gcm, hesC, myc, notch, soxB1, soxC, z188 (18-27 h)]
Brn1/2/4 MASO	[Brn1/2/4, otxb1/2 (18, 30, 45 h)], [gatae, hox11/13b (45 h)]
Cad MOE	dec, elk, endo16, foxc, hnf6, nrl, soxb1, tbr, z12-1
Dac MASO	[blimp1b, bra, delta, eve, foxN2/3, gataC, gcm, hesC, myc, notch, soxB1, soxC, wnt8, z188 (18-27 h)]
DnN MOE	blimp1, bra, capk, dec, delta, e(spl)-1, elk, endo16, eve, foxa, foxb, gatac, hmx, hox11/13b, not, notch, nrl, otx b1/2, su(h), tbr, wnt8
Dri MASO	N, bra, delta, dri (in pmc's), e(s), ets1, eve, foxa, gatae, gcm, hnf6, hox11/13b, lim1, pmar1, tbr, wnt8, z12-1, [elk, otx a, su(h) (16-20 h)], [alx1, erg, foxN2/3, msp130L, pks, soxC, tel, z48 (18-24 h)], [not, nrl (16-24 h)]
Dri-En	delta, dri (in pmc's), ets1, hnf6, pmar1, tbr, z12-1 (16-20 h)
Elk-En	delta, elk, hnf6
Erg MASO	[foxN2/3, gcm, pmar1, soxC, tbr, z48 (18-24 h)]
Ets1 MASO	delta, endo16, eve, foxb, gcm, pmar1, sm30, tbr, [jun, krl, pks, z48 (18-24 h)]
Eve MASO	alx1, cyclophilin, dri, ets1, foxc, gatac, gatac, gsc, hmx, msp130, not, otx a, otx b1/2, otx b3, pmar1, sm30, soxb1, tbr, [foxG, msx, myc, notch, soxC (15-27 h)]
FoxA MASO	b1/2otx, hmx, not (0-48 h), nrl, otx a, pks, tbr, [brn1/2/4, delta, eve, hox11/13b, wnt8 (18-27, 32, 48 h)], [blimp1, foxc (18, 24, 32, 48 h)], [alx1, blimp1b, dac, elk, foxN2/3, foxP, hesC, hnf1, msx, myc, notch, otxb1/2, soxB1, soxC, tgif, unc4.1, z188 (18-27 h)]
FoxB MASO	cyclophillin, endo16, ets1, ficolin, foxa, gatae, hox11/13b, sm27, sm50, tbr, wnt8
FoxN2/3 MASO	[cyclophillin, dri, foxA, foxN2/3, foxb, gatac, gcm, hex, hnf6, jun, krl, msp130, msp130L, not, pm27, pmar1, sm50, snail, soxC, tbr, tgif, z48 (18-24 h)]
GataC MASO	bra, capk, delta, dpt, elk, eve, gatae, gcm, not, pks
GataE MASO	??????, blimp1, capk, dec, dri, elk, ets1, eve, ferritin, gatae, gcm, hmx, nk1, otx a, [alx1, dac, delta, foxN2/3, hesC, soxB1, soxC (18-27 h)]
Gcm MASO	capk, dec, delta, elk, gatac, gatae, hmx, not, nrl
Gsc MASO	alx1, blimp1, bra, decorin, delta (in pmc's), dri, ets1, eve, foxb, fvmo, gatac, gcm, gsc, hmx, hnf6, krl, pks, pmar1, tbr, wnt8, z12-1 (17, 23 h)
Hex MASO	[dri, foxA, gcm, gsc, jun, krl, pmar1, tbr, z48 (18-24 h)]
Hnf1 MASO	[Brn1/2/4, alx1, blimp1b, bra, dac, delta, elk, endo16, eve, foxG, foxN2/3, foxP, foxa, foxb, gatac, gatae, gcm, hh, hox11/13b, krl, msx, myc, notch, otxb1/2, soxC, soxb1, tgif, unc4.1, wnt8, z188 (24-27 h)]
Hnf6 MASO	alx1, blimp1, bra, cyclophillin, delta, dri (in pmc's), elk, ets1, eve, foxa, gatae, gcm, hnf6 (in pmc's), otx a, pmar1, tbr, wnt8, [foxb, krl, z12-1 (19-20 h)]
Hox11/13b MASO	blimp1, otx a, otx b, soxb1, wnt8, [Brn1/2/4, alx1, dac, delta, elk, hnf6, otxb1/2, soxC, z188 (18-27 h)]
Krl MASO	alx1, blimp1, bra, dec, delta (12, 18-27 h), dri, ets1, eve, eve, gatae, gcm, gsc, hmx, krl, lim1, nrl, otx b1/2, pm27, pmar1, sm50, tbr, wnt8, z12-1, [dac, foxN2/3, foxP, gataC, hesC, msx, myc, notch, soxC, z188 (18-27 h)]
MutantSu(H) MOE	[endo16, sm50 (18, 24, 30 h)]
Myc MASO	[blimp1b, bra, dac, delta, endo16, eve, foxG, foxN2/3, foxP, gataC, gataE, gcm, hesC, hnf6, notch, soxB1, soxC, wnt8, z188 (18-27 h)]
N MASO	[foxa, foxb (18-27, 29, 41 h)], [bra, eve (21, 24-27, 29, 41 h)], [espl1, gatae, hmx, nrl, su (21, 29, 41 h)], [brn1/2/4, dac, foxN2/3, foxP, hesC, hh, myc, soxB1, soxC, wnt8 (18-27 h)]
Otx-En	dec, delta, elk, eve, gcm, otx a, pks, tbr
OtxHD MASO	[alx1, blimp1b, bra, dac, delta, elk, eve, foxN2/3, foxP, gatae, gcm, hnf6, hox11/13b, krl, myc, notch, otxb1/2, soxC, soxb1, unc4.1, wnt8, z188 (18-27 h)]
Otxa MASO	[alx1, blimp1b, bra, endo16, eve, foxG, foxP, foxa, foxb, gatac, gatae, gcm, hox11/13b, myc, soxC, soxb1, tgif, unc4.1, z188 (18-27 h)]
Pmar1 MOE	blimp1, elk, foxa, hnf6, krl, sm30, wnt8
Pmar1-En	blimp1, elk, foxa, hnf6, sm30, wnt8
SoxC MASO	[cyclophillin, dri, erg, ets1, foxO, hex, jun, msp130L, not, nrl, pm27, pmar1, sm50, snail, tbr, tel, vegfrII, z48 (18-24 h)], [alx1, blimp1b, bra, dac, delta, elk, endo16, eve, foxA, foxG, foxN2/3, foxP, foxb, gataC, gatae, gcm, hh, hnf1, hnf6, hox11/13b, krl, msx, myc, notch, otxb1/2, soxb1, unc4.1, wnt8, z188 (18-27 h)]
Soxb1 MASO	blimp1, bra, dec, dpt, dri, elk, endo16, foxa, foxb, foxc, gatac, krl, nrl, pmar1, wnt8, z12-1, [delta, gatae, gcm, hox11/13b, tbr (0-16 h)]
Soxb1 MOE	blimp1, dri, endo16, hnf6, tbr
Soxb1-En	alx1 (12 h), [bra, delta, endo16, ets-1, eve, foxa, foxb, gatac, gcm, hox11/13b, krl, msp130, pm27, sm50, wnt8 (18, 24 h)]
Tbr MASO	alx1, cyclophilin, delta, endo16, ets1, ets1, eve, ficolin, gatae, gcm, msp130, msp130L, pm27, [cyclophillin, foxA, foxN2/3, foxO, gatac, hex, jun, not, pks, pmar1, soxC, tel, vegfrII, z48 (18-24 h)]
Tel MASO	[cyclophillin, dri, erg, ets1, ficolin, foxA, foxB, foxN2/3, foxO, gcm, hex, hnf6, jun, krl, msp130, msp130L, not, pks, pmar1, pmar1, soxC, tbr, vegfrII, z48 (18-24 h)]
Tgif MASO	[cyclophillin, ets1, jun, pmar1, tbr, tel (18-24 h)], [dac, delta, elk, endo16, eve, gatac, hesC, hox11/13b, msx, myc, notch, soxC, wnt8, z188 (18-27 h)]

Footnotes

¹Must be affected indirectly via intercellular signaling because Bra is not present in cells where these genes are active.

²At 30 h affected indirectly via cell signaling, since at 30 h Dri and Gsc are expressed only in OE.

³Thought to be indirect input, via GataE.

⁴Probably via GataC.

⁵Probably via Bra.

⁶Probably in OE where both Elk and Lim1 are expressed then.

⁷Probably indirect via Gcm.

⁸Probably indirect via gcm.

⁹The inputs inferred from effects on endo16.

¹⁰Effect on endo16 is via Ui since no necessary GataE site in endo16.

¹¹Probably via ets1; based on ECRA.

¹²Indirect, because rescued by blimp1 MOE.

¹⁴Probably indirect via elk.

¹⁵Weak effect 18 h only, probably not significant, since wnt8 expression is disappearing in gatae domain at 18 h. Effect seen is probably via Otx which represses wnt8 in this period.

¹⁷Likely indirect via gatae.

¹⁸Late Krl effects likely to be indirect results of effects of Krl loss on endomesoderm specification (Howard et al., 2000).

¹⁹Obviously indirect via pmar1.

²⁰Only when dri is expressed in OE (>30 h).

²¹Possibly indirect via, because if there were an Otx site in gatac gene effect would have been seen earlier than 24 h.

²²Must be indirect because direct sm50 inputs are known.

²³Normalized to different standards in same run, i.e., ubiquitin and 18S rRNA.

²⁴ alx1 expressed specifically in pmc's; discovered by Ettensohn et al.

²⁶In collaboration with R. and L. Angerer who provided the samples measured.

²⁷Likely an artifact of expressing Blimp1-En MOE pmc's, since foxb is expressed only in pmc's at this stage (i.e., there could be a later interaction of Blimp1 and Soxb in hindgut where they are both expressed after gastrulation).

²⁸Weak effect likely indirect since gatac is expressed in smc's at 27 h and foxb in pmc's, later hindgut on oral side (plus oral ectoderm).

³⁰Not indirect via GataE because N MASO does not affect gatac though dnN does.

³¹Indirect effects on gatac does not turn on until after Tcf input is faded out, but all other inputs depend on endomesoderm specification.

³²Effect on initial phase of gatac expression, before it becomes exclusive with gcm (oral vs aboral and lateral).

³³Could be indirect via Ets1 effect on tbr; effect of Ets1 on foxb is stronger than tbr effect on foxb.

³⁴At least early (<24 h) effects on the three fvmo genes probably via gcm.

³⁵Via dri (see Amore et al., 2002).

³⁷Implies late blastula pmc domain to mesoderm indirect effect on oral side, where gatac is then expressed in mesoderm: not yet understood.

³⁸Since Gsc is a repressor (Angerer et al.) implies existence of mid-late blastula repressor of skeletogenic function controlled by gsc: perhaps a timing device.

⁴⁰Very possibly indirect via dri.

⁴¹Unlikely a direct transcriptional effect, since eve expression is not affected by Soxb1-Eng MOE.

⁴²Possibly indirect.

⁴³Likely via dri.

⁴⁵Probably in OE because most gsc expression at 24 h is there.

⁴⁷Double perturbation results show both inputs, Alx1 and Ets1, are required when indicated.

⁴⁸Data for mesenchyme blastula stage of Lytechinus variegatus

⁴⁹Probably Indirect

⁵⁰Unc4.1 is expressed in the aboral veg1 ectoderm, starting at 24h; effect must be indirect