Hooker L et al. (2012), Microarray-based identification of Pitx3 target...

XB-ART-45704

Dev Dyn 2012 Sep 01;2419:1487-505. doi: 10.1002/dvdy.23836.

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Microarray-based identification of Pitx3 targets during Xenopus embryogenesis.

Hooker L , Smoczer C , KhosrowShahian F , Wolanski M , Crawford MJ .

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Unexpected phenotypes resulting from morpholino-mediated translational knockdown of Pitx3 in Xenopus laevis required further investigation regarding the genetic networks in which the gene might play a role. Microarray analysis was, therefore, used to assess global transcriptional changes downstream of Pitx3.From the large data set generated, selected candidate genes were confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization.We have identified four genes as likely direct targets of Pitx3 action: Pax6, β Crystallin-b1 (Crybb1), Hes7.1, and Hes4. Four others show equivocal promise worthy of consideration: Vent2, and Ripply2 (aka Ledgerline or Stripy), eFGF and RXRα. We also describe the expression pattern of additional and novel genes that are Pitx3-sensitive but that are unlikely to be direct targets.

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Species referenced: Xenopus laevis
Genes referenced: arsl baz2b bix1.3 cryba4 crybb1 crybb3 fgf4 gsc hes4 hes7.1 hes7.2 hoxa10 hoxa11 hoxa13 lgals1.1 lgals9 lhx1 lhx5 nrl obscn pax6 pitx3 ptgds rax rbp4l rdh10 rdh16 ripply2.1 ripply2.2 rxra sp5 sp5l tbx2 tbx4 tbx5 ttn ventx2.2 wnt1
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Phenotypes: Xla Wt + pitx3 MO (fig.10.a^1) [+]

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	Figure 1. Microarray data represented according to putative gene function. The 100 most up- and down-regulated transcripts affected by Pitx3-morpholino-mediated knockdown were categorized by sequence analysis for stages 19 and 27 of X. laevis embryonic development. Colors correspond to functional groups in the legend (right).Download figure to PowerPoint
	Figure 2. In situ hybridization analysis for putative targets of Pitx3 involved in eye development. Visual comparisons of gene expression patterns between right-side injected control-morpholino (Cmo) or Pitx3-morpholino (Pmo) embryos and their untreated contralateral control. A–E: Pitx3 expression patterns are presented for comparison (adapted from Khosrowshahian et al., 2005; Smoczer et al., In Press). A: demonstrates faint but detectable signal throughout the ectoderm and in agreement with reverse transcriptase-polymerase chain reaction (RT-PCR) results. B: Expression is detectable throughout neural ridge, while at stage 22, the gene is expressed in a cleared specimen where an arrow indicates presomitic mesoderm. By stage 27 (D), Pitx3 is detectable throughout much of the head ectoderm, as well as in branchial arches and somites. This pattern restricts later to somites, otic vesicle, lens, and brain (D). F–G′: Vent2 expression is reduced in the developing eye field at stage 19 for the Pitx3-morpholino (Pmo) injected side (A′ white arrow) and at stage 27 (B′ black arrow), when compared with control-morpholino (Cmo) injected embryos (A,B). H–I′: Pax6 shows reduced expression in eye field on Pmo side of embryos at stage 19 (C′ black arrow) and 27 (D′ white arrow). J–J′: Crybb1 shows drastic loss of expression in the eye vesicle on the Pmo-treated side of stage 27 embryo (E′) and no difference caused by Cmo treatment (E). Dotted line represents the midline of the embryo, separating injected right side from contralateral left side control.Download figure to PowerPoint
	Fig. 3. Characterization of a novel transcript, Rbp4l, in X. laevis. A: Protein alignment showing distinct groups between retinol binding proteins and purpurin family members. B: Temporal expression of Rbp4l throughout embryonic stages of development, showing slight detection at stages 17 and 24, and an increase in expression at stages 31 and 35. Confirmation of microarray predictions by means of reverse transcriptase-polymer- ase chain reaction (RT-PCR), showing an increase in Rbp4l expression in response to Pitx3-morpholino (Pmo) at stages 19 and 27, when com- pared with wild-type (WT) and control-morpholino (Cmo) treatments. C: In situ hybridization with antisense riboprobe against Rbp4l transcript shows expression at stages 27 (C), 31 (D), and 35 (E) concentrated in the developing lens (white arrows, D and E) and at the dorsal midline of the developing midbrain region. F: An embryo injected unilaterally with Pitx3 morpholino on its right side (left of the dotted line) displayed enhanced and general expression in the craniofacial region. G: A schematic diagram of Rbp4l protein depicting a secretory signal at the N-terminus (red) and three characteristic lipocalin motifs (blue) that classify this protein as a member of the kernel subfamily of lipocalins. GenBank accession num- bers used to generate phylogenetic tree (A) are as follows: xRbp4l CD362061 (X. laevis), rRbp4 plasma BC167099 (rat), mRbp4 BC031809 (mouse), hRBP4 plasma AL356214 (human), cRbp4 precursor NM_205238 (chick), xlRbp4 precursor NM_001087726 (X. laevis), xlRb4 plasma NM_001086998 (X. laevis), xtRbp4 plasma NM_001015748 (X. tropicalis), zRbp4 NM_130920 (zebrafish), zpurpurin AB242211 (zebrafish), spurpurin NP_001135080 (salmon), ccpurpurin NP_001187969 (channel catfish), gpurpurin BAD42450 (goldfish), bcpurpurin AD028302 (blue catfish), cpur- purin P08938 (chick).
	Fig. 4. Characterization of a novel transcript Galectin IX in X. laevis. A: Protein alignment showing amino acid similarities between Xenopus Galectin family members. B: Temporal expres- sion of Galectin IX throughout embryonic stages of development, shows expression beginning at gastrulation (stage 10), decreasing at stage 12, and expressing consistently at stages 17 through 35. Confirmation of microarray predictions by means of reverse transcriptase-polymer- ase chain reaction (RT-PCR), detect an increase in expression at stage 19 and a decrease at stage 27 for Pitx3-morpholino (Pmo) -treated samples, compared with wild-type (WT) and con- trol-morpholino (Cmo). C: Galectin IX transcript expresses at stages 24 (C), 27 (D), and 31 (E) concentrated in the developing eye (white arrows) and presumptive pronephros, persisting in the nephric tubules and ducts. GenBank accession numbers used to generate phylogenetic tree (A) are as follows: xGalectinIa AB056478, xGalectinIb AB060969, xGalectinIIa AB060970, xGalecti- nIIb AB080016, xGalectinIIIa AB060971, xGalectinIIIb AB080017, xGalectinIVa AB060972, xGa- lectinVa M88105, xGalectinVb AB080018, xGalectinVIa AB080019, xGalectinVIIa AB080020, xGalectinVIIIa AB080021, xGalectinIX BJ056659.
	Fig. 5. Characterization of a novel transcript, Rdh16, in X. laevis. A: Protein alignment showing amino acid similarities between Xenopus retinol dehydrogenase (rdh) family members. B: Tem- poral expression of Rdh16 throughout embryonic stages of development shows faint expression beginning at stage 24 and 27, then increasing at stages 31 and 35. We were unable to confirm the microarray predictions by means of reverse transcriptase-polymerase chain reaction (RT- PCR), as no change in expression was detected between wild-type (WT) control-morpholino (Cmo), or Pitx3-morpholino (Pmo) embryos. C: In situ hybridization with antisense riboprobe against Rdh16 transcript, shows expression at stages 27 (C), 31 (D), and 35 (E) concentrated in the eyecup, branchial arches, and otic vesicle, as well as along the lateral plate mesoderm, with a focus on the posterior half (D), and on in the developing myotomes. GenBank accession num- bers used to generate phylogenetic tree (A) are as follows: xRdh16 NP_001083356, xRdh7 NP_001079189, xRdh13 NP_001085680, xRdh5 NP_001086194, xRdh9 NP_001090337, xRdh10 ACN32204.
	Fig. 6. In situ hybridization analysis for putative Pitx3 target genes Obscnl and Baz2b. Visual comparisons of gene expression patterns between control-morpholino (Cmo) and Pitx3-morpho- lino (Pmo) right side-injected embryos. A: Obscnl shows a loss of expression in the branchial arches (black arrow), otic vesicle, and retina when treated at stage 27 with Pmo (A0) versus Cmo (A). B: Baz2b is substantially reduced in response to Pmo (B0) in the retinal layer of the optic protuberance (white arrow), as well as in the pronephros and in the anterior region of the dorsal axis, when compared with Cmo (B).
	Fig. 7. Characterization of a novel transcript, Baz2b, in X. laevis. A: Protein alignment showing amino acid similarities between Baz2B homologs across organisms. B: Temporal expression of Baz2b throughout embryonic stages of development show expression as a maternal transcript in the egg E and throughout development to tailbud stage, with slight reductions in transcript level at stages 10 and 19. B Confirmation of microarray predictions by means of reverse tran- scriptase-polymerase chain reaction (RT-PCR) show abolished expression at stage 27 in response to Pitx3-morpholino (Pmo) when compared with control-morpholino (Cmo) and wild- type (WT) embryos. C: Baz2b expression at stages 21 (C), 31 (D), and 35 (E) is concentrated in the developing eye, as well as the branchial arches and otic vesicle. Dark expression is seen in the pronephros, persisting in the tubules (E). F: A schematic diagram of Baz2b protein depict- ing various domains characteristic of Baz2B: methyl-CpG binding domain (MBD), DNA binding domain (DDT), zinc finger domain (Z), adjacent to the bromodomain (BR). GenBank accession numbers used to generate phylogenetic tree (A) are as follows: xBaz2b BQ400337 (X. laevis), mBaz2b BC150814 (mouse), rBaz2b NM_001108260 (rat), hBAZ2B NM_013450 (human), cBaz2b NM_204677 (chick), xtBaz2b BC166361 (X. tropicalis).
	Fig. 8. In situ hybridization analysis for putative brain targets of Pitx3. Comparisons of gene expression patterns between right-side injected control-morpholino (Cmo) or Pitx3-morpholino (Pmo) embryos and their untreated contralateral control. A0: Hes7.1 at stage 19 shows decreased expression in the midbrain hindbrain boundary or isthmus (black arrow) in response to Pmo (A0) versus Cmo (A) and again at stage 27 Pmo (B0) (black arrow) versus Cmo (B). C0: Spr1 stained embryos show increased expression (black arrow) at stage 19 when treated with Pmo (C0), where no change in expression is observed with Cmo (C). At stage 27, Spr1 expression in the isthmus is abolished on the Pmo side (D0) (white arrow). Dotted line represents the midline of the embryo, separating injected right-side from contralateral left-side control.
	Fig. 9. In situ hybridization analysis for putative segmentation targets of Pitx3. Visual comparisons of gene expression patterns between right- side injected control-morpholino (Cmo) or Pitx3-morpholino (Pmo) embryos and contralateral control. A0: Ripply2 expression, showing as two stripes in the presomitic mesoderm, shows an anterior shift (black arrow) in expression at stage 19 when treated with Pmo (A0) instead of Cmo (A). At stage 27, Ripply2 expression pattern loses its distinct shape and becomes unrestricted in response to Pmo (B0), whereas with Cmo treatment, precise patterning of this gene expression remains intact (B). C0: Hes4 expression becomes blurred in Pmo-treated embryos at stage 19 (C0) and at stage 27 (D0) Hes4 expression is absent in the presomitic mesoderm (black arrow) and pronephros areas, compared with Cmo-treated embryos (D). E0: Hes7 no longer expresses in the most anterior stripe (black arrow), and the remaining two stripes are shifted anteriorly in comparison to the contralateral control (E0). At stage 27, on the Pmo side of the embryo (F0), Hes7 shows increased expression in the presomitic mesoderm (white arrow) and again an anterior shift of the striped pattern (black arrow). Dotted line represents the midline of the embryo, separating injected right-side from contralateral left-side control.
	Fig. 10.In situ hybridization analysis for putative tailbud targets of Pitx3. A0: HoxA11 shows decreased posterior expression in the tailbud region (white arrows) of Pmo embryos at stages 19 (A0) and 27 (B0); C0 :Spr2 displays a broader and larger domain of expression (black arrows) when treated with Pmo, both at stage 19 (C0) and 27 (D0), compared with Cmo-treated embryos (C, D). E0: Lim1 expression disappears from paraxial mesoderm (red arrow) and is up-regulated in lateral mesoderm (black arrow) at stage 19 when treated with Pmo (E0). At stage 27 (F0), Pmo reduces Lim1 expression in the developing pronephros (black arrow) and in the head mesenchyme and along the dorsal axis.
	bazb2 (bromodomain adjacent to zinc finger domain, 2B) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 21, lateral view, anterior left, dorsal up.
	bazb2 (bromodomain adjacent to zinc finger domain, 2B) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 31, lateral view, anterior left, dorsal up.
	obscn (obscurin, cytoskeletal calmodulin and titin-interacting RhoGEF ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 27, lateral view, anterior right, dorsal up.