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Fig. 3. Comparative analysis of the expression of Runx and Cbfβ genes by whole-mount in situ hybridization. Runx3 is first detected at stage 20 in two domains, the developing primary sensory (Rohon-Beard) neurons and the trigeminal-profundal placode. At the same stage Cbfβ is also co-expressed in both domains, while Runx1 is restricted to Rohon-Beard neurons. At later stages, stage 25 and up, Runx3 expression appears to be largely confined to the head region and in a number of cranial nerves including the ganglion of the trigeminal, vagal and glossopharyngeal nerves. To confirm the tissue-specific expression of Runx genes, we also performed in situ hybridization for Pax3 and Islet1 in staged matched embryos. Pax3 and Islet1 share common expression domains with Runx genes in the profundal-trigeminal placode (Pax3 and Islet1), Rohon-Beard sensory neurons and cranial ganglia (Islet1). Stages are according to Nieuwkoop and Faber (1967). Stage 13, dorsal view anterior to top. Stage 20d, dorsal view anterior to the left. Stage 20f, frontal view dorsal to top. Stage 25–40, lateral view of the head region, anterior to the left and dorsal to top. ba, branchial arches region; bp, blood progenitors; hf, heart field; hg, hatching gland; gp, glossopharyngeal; nb, neural plate border; ol, olfactory placode; pr, profundal ganglia; rb, Rohon-Beard neurons; sa, statoacoustic ganglia; tg, trigeminal ganglia; pt, profundal-trigeminal placode; va, vagal nerve. The scale bars in the upper row represent 500 μm.
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Fig. 4. Comparative analysis of Runx3, Runx1 and Cbfβ in blood progenitors. (A) Whole-mount in situ hybridization of stage 31 embryos viewed from the ventral side, anterior to the left, highlights the expression of Runx3, Runx1 and Cbfβ in blood progenitors. In these cells Runx1 appears to be expressed at higher level than Runx3 and Cbfβ. The scale bar represents 500 μm. (B) In situ hybridization on adjacent sections of a stage 31 embryo shows that Runx3, Runx1 and Cbfβ expression domain overlap at least in the lateral most aspect of the lateral plate mesoderm (arrows). The scale bar represents 100 μm.
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Fig. 5. Comparative analysis of Runx3, Runx1, Cbfβ and Islet1 expression in the profundal-trigeminal placode and Rohon-Beard sensory neurons at stage 20. (A) Schematic representation of a stage 20 embryo view from the dorsal side, anterior to the left. Modified from Nieuwkoop and Faber (1967). (B) Schematic representation of a transverse section at the level of the diencephalon, corresponding to the line labeled “B†in panel (A). Dorsal to top. The blue areas indicate the position of the developing profundal-trigeminal placode. The boxed area labeled “C†is the region of the section shown in subsequent panels (C). (C) Runx3, Cbfβ and Islet1 are co-expressed in the profundal-trigeminal placode (blue arrows), while Runx1 is not detected in this tissue. (D) Transverse sections in the trunk region corresponding to the line labeled “D†in panel (A). All four genes are co-expressed in Rohon-Beard sensory neurons (red arrows). Islet1 is also expressed in the population of ventral interneurons (black arrows). The notochord is outlined with a solid line, while the position of the neural tube is underlined with dashed lines. The probes are indicated in the lower right corner of each panel. di, diencephalon; no, notochord; ov, optic vesicle. The scale bar in panel C represents 100 μm.
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Fig. 6. Comparative analysis of Runx3, Runx1, Runx2 and Cbfβ expression in the craniofacial cartilage at stage 40. (A) Schematic representation of the head of a stage 40 embryo viewed from the lateral side, dorsal to top, anterior to the left. Modified from Nieuwkoop and Faber (1967). (B and C) Schematic representations of the ventral (B) and dorsal (C) cranial cartilage elements. Modified from Sadaghiani and Thiebaud (1987). The cartilage elements are color-coded: ethmoid-trabecular (brown), quadrate (black), Meckel’s (blue), cerathoyal (green), basihyal (red) and branchial (yellow). In panels (A), (B) and (C) the lines labeled “Dâ€, “Eâ€, “Fâ€, “G†and “H†indicate the level of the transverse sections shown in the subsequent panels. cg, cement gland; he, heart; ph, pharynx; st, stomodeum. The scale bar in panel D represents 100 μm.
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Fig. 7. Comparative analysis of Runx3, Runx1, Runx2, Cbfβ and expression in neurogenic placode derivatives at stage 40. Transverse sections were performed at three levels along the antero-posterior axis; the pre-otic, the otic, and post-otic regions. Each column shows sections from approximately the same level. Trigeminal ganglion (red arrows), ganglion of anterodorsal lateral line nerve (black arrowheads), ganglia of facial and anteroventral lateral line nerves (blue arrowheads), ganglia of glossopharyngeal and middle lateral line nerves (blue arrows), statoacoustic ganglion (black arrows), ganglion of vagal nerve (green arrows), ganglion of posterior lateral line nerve (purple arrows). hb, hindbrain; no, notochord; ov, otic vesicle; The scale bar in the upper left panel represents 100 μm.
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Fig. 1. Sequence and structure comparison of Runx3 proteins across species. (A) The predicted amino acid sequences from Xenopus laevis, human, mouse and zebrafish Runx3 were aligned using ClustalW. Conserved amino acids in all four species or in at least two species are highlighted in black and grey, respectively. The runt domain, signature motif of this class of molecules, is underlined in red. (B) Phylogenetic tree analysis of Runx proteins from Xenopus laevis (Xl), human (Hs), mouse (Mm), chicken (Gg) and zebrafish (Dr). Accession numbers for the source sequences are indicated in materials and methods.
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Fig. 2. Temporal expression of Runx3, Runx1, Runx2 and Cbfβ during embryogenesis. (A) RT-PCR analysis of the developmental expression of Runx3, Runx1, Runx2 and Cbfβ. Stages are according to Nieuwkoop and Faber (1967). Ornithine decarboxylase (ODC) is shown as a loading control. G, onset of gastrulation; N, mid-neurula stage; T, tailbud stage; H, hatching stage. (B) Real-time RT-PCR analysis of Runx3, Runx1, Runx2 and Cbfβ. Each value has been normalized to the level of ODC.
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