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Figure 1. Cardiac formation during Xenopus early embryogenesis. A: Precardiac mesoderm (PCM, blue) is formed on the prospective dorsal side of the early gastrula (stage 10). B-G: The region of PCM formation is defined by the expression of Nkx2.5 (arrowhead; Tonissen et al.,[1994]). B: By the end of the gastrula (stage 13), the PCM is fused and lies at the anterior end of the embryo. C: At the neurula (stage 18), the PCM is located on the ventral side behind the cement gland. D: At the early tail bud (stage 23), the PCM is separated. At the mid-tail bud (stage 28), the PCM is re-fused. F: At the late tail bud (stage 34), a tubular and beating heart is formed. G: At this point, the tadpole heart separates into the atrium and ventricle. A: Vegetal view, with dorsal upward. B-G: Ventral view, with anterior toward the left.
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Figure 2. Cardiac induction from animal caps and in vivo transplantation. A: Animal caps excised from blastulae were placed in physiologic saline that lacked calcium, to loosen the cell adhesions. The culture medium was replaced with 100 ng/ml activin dissolved in physiologic saline that contained calcium, and the cells were dissociated by pipetting. Individual cells were then reaggregated, and the reaggregates began to beat approximately 2 days later. Reaggregates that had been cultured for 1 day were broken into appropriate sizes and transplanted through an incision that had already been made anterior to the cloaca in the neurula. B: XcTnI, a marker gene for myocardial differentiation, is expressed specifically in the myocardium of a normal 3-day-old embryo (left). The dissociated animal cap cells form spherical reaggregates, regardless of whether they have been treated with activin. XcTnI signals are detected exclusively in the activin-treated reaggregates (right). C: The internal anatomy of a 1-year-old frog with a well-developed ectopic heart. The ectopic heart adjacent to the host's intestine is incorporated into the host's vascular system. The blood from the host's mesenteric artery (black arrows) flows into the host's anterior abdominal vein (white arrows) by means of the ectopic heart (h). D: Histologic section of an ectopic heart. The heart can be divided into multiple chambers: a ventricle-like chamber (v) that comprises a thick and deeply penetrating layer of myocardium; and an atrium-like chamber (a) that is surrounded by a thin layer of myocardium.
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Figure 3. Developmental expression profiles and knockdown analysis of XHAPLN3 and Xhas2. A-D: Whole-mount in situ hybridization of Nkx2.5 (A), XHAPLN3 (B), Xversican (C), and Xhas2 (D). Lateral views are shown, with the anterior toward the left and dorsal side upward. The arrowhead indicates the precardiac mesoderm (PCM). E-G: The effects of depleting XHAPLN3 and Xhas2 are validated by the pattern of XcTnI expression. Normal tadpole heart structure (E, arrow). F,G: However, both XHAPLN3-morphant (F) and Xhas2-morphant (G) show no XcTnI expression. Ventral views are shown, with anterior upward.
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hapln3 (hyaluronan and proteoglycan link protein 3 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anterior left, dorsal up.
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vcan (versican) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anterior left, dorsal up.
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Figure 4. Already-known genes that were identified using the microarray. Gene names are indicated in red and arranged by embryonic stage. Nkx2.5 and GATA4/5/6 are among the earliest heart-related transcription factors (Tonissen et al.,1994, Afouda et al.,2005). Moreover, Nkx2.5 expression is regulated by both XHMGA2 and Smads (Monzen et al.,2008). The RNA-binding protein hermes also regulates Nkx2.5 expression (Gerber et al.,2002). Tbx5, Tbx20, myocardin, and islet1 are transcription factors that interact with Nkx2.5 and GATAs and regulate gene expression in the precardiac region (Stennard et al.,2003, Small et al.,2005, Brade et al.,2007). Both the Notch signal (Notch1, Serrate1, and Delta1) and hyaluronan matrix (Xhas2, Xversican, and XHAPLN3) genes are essential for the correct specification of myocardial cell fates (Rones et al.,2000, Ito et al.,2008). Finally, terminal differentiation marker genes (cardiac troponin I/C/T, MHCa/b, MLC2, cardiac actin, and ANF), which encode principally structural proteins, are expressed (Gurdon et al.,1989, Drysdale et al.,1994, Small and Krieg,2003). Moreover, cardiogenesis is maintained by several secreted factors (Eroshkin et al.,2006, Zhu et al.,2008).Download figure to PowerPoint
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Figure 5. Temporal expression patterns of already-known genes that are related to blood vessel differentiation in Xenopus. Dashed arrows indicate the temporal expression of each gene in prospective and forming regions of the blood vessel, as follows: DLP, dorsal lateral plate; AA, aortic arches; PCV, posterior cardinal vein; ISV, intersomitic vein. The differentiation processes of blood vessel formation are indicated in the middle. The timing of definitive differentiation of the hemangioblast is not well understood, but it is known that the hemangioblasts located in the DLP differentiate into adult blood cells and vascular endothelial cells. Fli1 is known to be a marker of hemangioblasts (Liu et al.,2008). The start of vasculogenesis and angiogenesis is clearly observed as the expression of Xmsr in the AA or PCV and the expression of Xtie2 in the sprouting ISV, as determined by whole-mount in situ hybridization. We identify two novel potential marker genes, Ami and XRASGRP2, for the late differentiation of blood vessels, angiogenesis and/or maturation.Download figure to PowerPoint
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Figure 6. In vitro induction of pronephros or pancreas from animal caps treated with activin and retinoic acid (RA). Animal caps were excised and treated with activin and/or RA in Steinberg's solution (SS) supplemented with 0.1% bovine serum albumin (BSA). Explants were washed with SS and cultured in SS with 0.1% BSA. All the treatments and culturing were performed at 20°C. A: When animal caps were excised and treated with 10 ng/ml activin and 104 M RA simultaneously for 3 hr, washed and cultured for 4 days. The explants mainly differentiated into pronephric tissues. After this explant was transplanted to a pronephrectomized Xenopus embryo of stage 20–25, the transplanted embryo developed normally. In contrast, the pronephrectomized Xenopus embryo showed edema at the tadpole stages. B: When animal caps were treated with 100 ng/ml activin for 1 hr, washed and cultured for 3–5 hr in SS with 0.1% BSA, and then treated with 104 M RA for 1 hr, the explants differentiated into pancreatic tissues at a high frequency.Download figure to PowerPoint
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Figure 7. Schematic representation of pronephros development in X. laevis. The expression of molecular signals is accompanied by the differentiation of the tubule, duct, and glomus. The expression of Lim-1 and Pax-8 in the pronephric mesoderm determines the analgen of pronephros at stage 12.5. At stage 20, the anlagen of the pronephros thickens and becomes morphologically discernible, and the expression of Pax-2, Wnt-4, WT1, and other molecules begins at pronephros. By stage 25, the genes involved in the patterning and morphogenesis of the pronephros are activated. The pronephros is separated from the lateral plate, and further differentiation commences at the late tail-bud stage. The genes shown in blue, green, and red letters represent genes that are involved in the development of the tubule, duct, and glomus.Download figure to PowerPoint
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Figure 8. List of genes implicated in brain development. Representative genes involved in brain formation are indicated for each developmental stage. During the gastrula stage, neural tissue is induced and the anterior–posterior (AP) axis is determined, resulting in fundamental neural tissue patterning. At the neurula stage, the anterior neural plate is subdivided into the telencephalon (red), diencephalon (yellow), mesencephalon (green), and rhombencephalon by the restricted expression of several brain-specific genes. At the tadpole stage, the brain region assumes a three-dimensional structure.Download figure to PowerPoint
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