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PLoS One
2013 Jul 01;87:e69866. doi: 10.1371/journal.pone.0069866.
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Expression and functional characterization of Xhmg-at-hook genes in Xenopus laevis.
Macrì S
,
Sgarra R
,
Ros G
,
Maurizio E
,
Zammitti S
,
Milani O
,
Onorati M
,
Vignali R
,
Manfioletti G
.
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High Mobility Group A proteins (HMGA1 and HMGA2) are architectural nuclear factors involved in development, cell differentiation, and cancer formation and progression. Here we report the cloning, developmental expression and functional analysis of a new multi-AT-hook factor in Xenopus laevis (XHMG-AT-hook) that exists in three different isoforms. Xhmg-at-hook1 and 3 isoforms, but not isoform 2, are expressed throughout the entire development of Xenopus, both in the maternal and zygotic phase. Localized transcripts are present in the animal pole in the early maternal phase; during the zygotic phase, mRNA can be detected in the developing central nervous system (CNS), including the eye, and in the neural crest. We show evidence that XHMG-AT-hook proteins differ from typical HMGA proteins in terms of their properties in DNA binding and in protein/protein interaction. Finally, we provide evidence that they are involved in early CNS development and in neural crest differentiation.
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23936116
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Figure 2. Xhmg-at-hook1-3 expression analyses.(A) RT-PCR analysis of Xhmg-at-hook and ODC transcription during Xenopus laevis development. Numbers refer to embryo stages. (B–G) Results of WISH on Xenopus laevis embryos. (B–B’) Stage 2: Xhmg-at-hook maternal transcripts are localised in the animal pole (ap). (C) Stage 22: faint staining is detectable in both the developing eye (white arrowhead) and CNS (black arrowhead). (D) Stage 25: Xhmg-at-hook expression is in the anterior half of the embryo around branchial pouches (black arrows). (E, F, G) At tailbud stages 31, 35–36, 39 respectively, labelling is present in the brain, eye, neural tube (nt), somites (som) and branchial region (f, forebrain; m, midbrain; h, hindbrain; ov, otic vesicles; cg, cement gland; vp, vegetal pole). (H) Transversal section of a stage 28 hybridised embryo showing Xhmg-at-hook mRNA presence in the brain region, eye vesicles (white arrowhead) and NCC derived-mesenchyme around the pharynx (arrows) (H). (I) Horizontal section of a stage 33 hybridised embryo showing Xhmg-at-hook mRNA presence in the NCC derived pharyngeal arches (arrows).
Figure 3. Results of combined antisense MoXat1 and MoXat3 injections in Xenopus embryos. Reduction of Xotx2 (A–C or J–L, respectively for strong or slight reduction), nrp-1 (D–F, strong; M–O, slight) and Twist (G–H, strong; P–Q, slight) expression is observed on the injected side of embryos (inj), compared to uninjected side (un). Strong or weak reduction (I, R respectively) of pharyngeal skeleton is observed on the injected side of antisense MO treated swimming tadpoles compared to control side. Beta-gal red staining traces injected side of embryos.
Figure 4. XLHMGA2 and XHMG-AT-hook1 DNA-binding properties.(A) Electrophoretic mobility shift assay performed with in vitro transcribed and translated (IVT) HA-tagged XLHMGA2βa (HA-XLA2Ãa) and XHMG-AT-hook1 (HAâXATH1) proteins. Two different DNA probes were used: upper panel, E3 (0.1 pmoles); lower panel HCRII (0.1 pmoles); EMSAs were performed incubating 2, 4, and 6 µL of IVT proteins. (B) Western blot analysis of IVT proteins is shown (red ponceau stained membrane (left) and α-HA antibody recognition (right) to assess the production of the XLHMGA2βa and XHMG-AT-hook1 proteins.
Figure 5. XLHMGA2βa, but not XHMG-AT-hook1, interacts with the same molecular partners of mammalian HMGA.(A) Blue coomassie stained analysis of different HMGA molecular partners produced as GST-fused protein and of GST alone. PR: pocket region; CT: C-terminal region; ZnF: Zinc finger region. (B) GST-pull down assays performed with the GST-fused HMGA molecular partners shown in panel A and IVT and [35S]-methionine radiolabeled XHMG-AT-hook1 (HAâXATH1), hHMGA2 (HAâhA2), and XLHMGA2βa (HAâXLA2βa). For each IVT protein used input is shown in lanes 1, 6, and 10 (10% of the amount used in GST-pull down experiments). GST alone is used as a negative control.
Figure 1. XHMG-AT-hook proteins and organization of their transcripts and loci. (A) ClustalW alignment of XHMG-AT-hook protein isoforms. The amino acid sequences of the three different XHMG-AT-hook1-3 protein sequences (XATH1â3) found in X. laevis and of the one (XATH3) found in X. tropicalis are shown. The conserved AT-hooks are shown in bold; internal repeats are boxed in different shades of yellow or brown respectively. The C-terminal region is boxed in orange. (B) Genomic organization of the Xhmg-at-hook locus in Xenopus tropicalis. The exon/intron organization is indicated together with the proposed mechanisms of generation of the different Xhmg-at-hook1-3 (XATH1-3) transcripts in Xenopus laevis, based on homology with the genomic sequences of Xenopus tropicalis (see also description in the text). doi:10.1371/journal.pone.0069866.g001
Figure S2. Results of antisense morpholino MoXat1 (A-I) or MoXat3 (J-R) injections in Xenopus embryos. Injections of 4 ng of single MO does not produce reduction in the expression of Xotx2 (A-C, J-L), nrp-1 (D-F; M-O) or Twist (G-H, P-Q) on the injected (inj) side of treated embryos compared to control (un) side. No reduction of pharyngeal skeleton is observed on the injected side in swimming larvae (I, R) compared to uninjected side. β-gal red staining traces injected side of embryos.
Figure S3. Results of standard control MO injections in Xenopus embryos. Injections of 8 ng of MO does not produce reduction in the expression of Xotx2 (A-C), nrp-1 (D-F) or Twist (G-H) on the injected (inj) side of embryos compared to control (un) side. No reduction of pharyngeal skeleton is observed on the injected side in swimming larvae (I) compared to uninjected side. β-gal red staining traces injected side of embryos.
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