Jin Y , Weinstein DC .

R03 HD077015 NICHD NIH HHS , R15 GM124577 NIGMS NIH HHS

             cement gland development

	Fig. 1. Knockdown ofpitx1andpitx2cinhibits endogenous cement gland formation andXag1expression. (A) 0% of uninjected embryos (n = 56) lack an observable cement gland. (B) 40% of tadpole stage embryos (n = 46) lack an observable cement gland following injection of Pitx1-MO and Pitx2c-MO. (C) 0% embryos (n = 36) lack an observable cement gland following injection of Pitx1–5MM and Pitx2c-5MM. (D) Cement gland in Pitx1-MO and Pitx2c-MO-injected embryo is rescued by injection of pitx1 RNA: 6% of embryos (n = 35) lack an observable cement gland. (E) 73% of uninjected neurula stage embryos (n = 22) exhibit strong Xag1 staining. (F) 36% of embryos (n = 28) display strong Xag1 staining following injection of Pitx1-MO and Pitx2c-MO. (G) 80% of embryos (n = 20) exhibit strong Xag1 staining following injection of Pitx1–5MM and Pitx2c-5MM. Embryos in (A-D) are lateral views, anterior to left; embryos in (E-G) are ventral views, anterior to top. Morpholino oligos were injected into the animal pole of early cleavage stages embryos. Black arrows indicate actual or expected sites of the cement gland; black arrowheads indicate Xag1 staining.
	Fig. 2. Pitx1 transcriptionally activates cement gland differentiation genes both directly and indirectly. (A) Injection of VP16-pitx1 RNA, or wild-type pitx1 RNA, leads to induction of cement gland markers. Indicated doses of pitx1, VP16-pitx1, and EnR-pitx1 RNA were injected at early cleavage stages. RT-PCR analysis was performed on animal caps collected at mid-neurula stages after dissection at late blastula stages. (B) nkx3.1, TGFβ1, sim2, crx, epha4 and traf4, but not Xag1, Xcg, agr2 or xa-1, are strongly induced by Pitx1-GR in both the presence and absence of cycloheximide. 400 pg pitx1-GR RNA was injected into early cleavage stages embryos. RT-PCR analysis was performed on animal caps dissected at late blastula stages and cultured with or without dexamethasone and/or cycloheximide until mid-neurula stages. Dexamethasone (10 µM) was added 10 min after cycloheximide (10 µg/ml) addition at stage 12, as listed. Dexamethasone and/or cycloheximide treatments were for approximately 5 h. EF1α is used as a loading control (Krieg et al., 1989). The -RT lane contains all reagents except reverse transcriptase, and is used as a negative control.
	Fig. 3. follistatinexpression in the cement gland is dependent on Pitx1. (A) Injection of pitx1 RNA results in the induction of follistatin. 400 pg pitx1 RNA was injected at early cleavage stages. RT-PCR analysis was performed on animal caps harvested at mid-neurula stages after dissection at late blastula stages. (B) Cycloheximide treatment results in strong expression of follistatin. (C) follistatin expression is observed in anterior notochord at early neurula stages. (D) follistatin expression is observed in the hindbrain (arrow) and pronephros (arrowheads) at late neurula stages. (E) 66% of uninjected late neurula stage embryos (n = 29) have detectable follistatin staining in the presumptive cement gland region. (F) 36% of embryos (n = 33) have detectable follistatin staining in the cement gland primordium following injection of Pitx1-MO and Pitx2c-MO; (G) 77% of embryos (n = 31) have expanded follistatin staining following injection of pitx1 RNA. Morpholino oligos or pitx1 RNA were injected into the animal pole of early cleavage stages embryos. Embryos in (C, D) are dorsal views, anterior to left; embryos in (E-G) are ventral views, anterior to left. Black arrows and arrowheads indicate actual or expected follistatin staining.
	Fig. 4. Pitx1 may function as a transcriptional activator to inhibit BMP signaling. RT-PCR analysis (A, C and D) and Western blot analysis (B) were performed on animal caps harvested at mid-neurula stages after dissection at late blastula stages. (A) Injection of pitx1 RNA inhibits BMP targets in a dose dependent manner. 10–400 pg pitx1 RNA was injected at early cleavage stages, as listed. Ornithine decarboxylase (ODC) is used as a loading control (Bassez et al., 1990). (B) Injection of pitx1 RNA results in decreased C-terminal phosphorylation of Smad1/5; total levels of Smad1 are not significantly affected. Embryos were injected with 20 pg, 100 pg and 400 pg pitx1 RNA at early cleavage stages, as listed. β-tubulin is used as a loading control. (C) Injection of VP16-pitx1 RNA leads to down-regulation of BMP target genes, similar to that seen with wild-type pitx1; injection of EnR-pitx1 RNA does not alter expression of BMP-responsive genes. Indicated doses of pitx1, VP16-pitx1, and EnR-pitx1 RNA were injected at early cleavage stages. (D) Pitx1-mediated inhibition of the BMP target gene sizzled is partially rescued by co-injection of Follistatin-MO. 200 pg pitx1 RNA was co-injected with 192 ng Follistatin-MO or scrambled control morpholino oligo at early cleavage stages.
	Fig. 5. Inhibition of BMP signaling is required for the expression ofpitxgenes, while BMP/Smad1 signaling is required for cement gland development. (A) Pitx1-mediated inhibition of the BMP target gene sizzled is rescued by co-expression of either Smad1 or CA-BMPRIa. Pitx1-mediated induction of two pitx2 transcript variants, pitx2b and pitx2c, as well as endogenous pitx1, are down-regulated following enhancement of BMP signaling. 50 pg pitx1 RNA was either injected alone or co-injected with 1.6 ng smad1 or CA-BMPRIa RNA at early cleavage stages. RT-PCR analysis was performed on animal caps harvested at mid-neurula stages after dissection at late blastula stages. (B) 90% of neurula stage control embryos (n = 20) display strong pitx1 staining. Water was injected into the animal pole of 4-cell stage Xenopus embryos. (C) 88% of embryos (n = 16) exhibit weak and spatially restricted pitx1 staining following injection of 500 pg CA-BMPRIa RNA at early cleavage stages. Embryo views are anterior, ventral to bottom. (D) Injection of noggin RNA inhibits Pitx1-mediated induction of cement gland differentiation genes. 400 pg pitx1-GR RNA and/or 20 pg noggin RNA were injected into Xenopus embryos. RT-PCR analysis was performed on animal caps dissected at late blastula stages and cultured with or without dexamethasone until mid-neurula stages. Dexamethasone (10 µM) was added to animal caps at stage 12. (E) Pitx1 binds to Smad1. 2 ng pitx1-myc RNA was injected at early cleavage stages. Pull-down of native Smad1 from injected embryos leads to co-immunoprecipitation of exogenous Pitx1. Normal rabbit IgG antibodies was used in parallel studies as a negative control.

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