Falchuk KH et al. (2002), A role for biliverdin IXalpha in dorsal axis de...

XB-ART-7841

Proc Natl Acad Sci U S A 2002 Jan 08;991:251-6. doi: 10.1073/pnas.012616099.

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A role for biliverdin IXalpha in dorsal axis development of Xenopus laevis embryos.

Falchuk KH , Contin JM , Dziedzic TS , Feng Z , French TC , Heffron GJ , Montorzi M .

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The determinants of Xenopus laevis embryos that act before their first cell division are mandatory for the formation of mRNas required to establish the dorsal axis. Although their chemical identities are unknown, a number of their properties have long been recognized. One of the determinants is present in the cytoplasm and is sensitive to UV light. Thus, exposing stage 1 embryos to either standard 254-nm or, as shown here, to 366-nm UV light during the 0.3-0.4 time fraction of their first cycle inactivates the cytoplasmic determinant. As a consequence, both types of irradiated embryos fail to express dorsal markers, e.g., goosecoid and chordin, without affecting formation of ventral markers, e.g., Vent-1. The developmental outcome is dorsal axis-deficient morphology. We report here that biliverdin IXalpha, a normal constituent of cytoplasmic yolk platelets, is photo-transformed by irradiation with either 254- or 366-nm UV light and that the transformation triggers the dorsal axis deficiency. When the 254- or 366-nm UV-irradiated embryos, fated to dorsal axis deficiency, are incubated solely with microM amounts of biliverdin, they recover and form the axis. In contrast, incubation with either in vitro photo-transformed biliverdin or biliverdin IXalpha dimethyl ester does not induce recovery. The results define an approach to produce dorsal axis-deficient embryos by photo-transforming its biliverdin by irradiation with 366-nm UV light and identify an unsuspected role for biliverdin IXalpha in X. laevis embryogenesis.

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Species referenced: Xenopus laevis
Genes referenced: chrd gsc ventx1.2

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	Figure 1 Northern blots of RNA from UV-irradiated stage 10.5 embryos. The dorsal markers goosecoid (gsc) and chordin (ch) and ventral one (Vent 1) are present in control RNA. The two dorsal mRNAs are absent, whereas the ventral marker, Vent 1, is present in both 254- and 366-nm irradiated embryos.
	Figure 2 Morphology of embryos irradiated with UV light. (A) Control embryos after reaching stages 30–35. (B) Adorsal embryos formed after exposure to 254-nm UV light. (C) Adorsal embryos formed after irradiation with 366-nm UV light. (D) Embryos irradiated with either long or short wavelength UV light after addition of frog-biliverdin IXα or (E) commercial biliverdin IXα. (Magnifications: A, ×7; B–D, ×12; E, ×10.)
	Figure 3 Biliverdin IXα rescues UV-irradiated embryos from dorsal axis deficiency. (A) Average DAI score of embryos irradiated with 254-nm UV light (gray) is 0.35. About 51% of embryos treated with biliverdin (black) are scored with a DAI between 5 and 4 with an average DAI score of 2.72. (B) Average DAI score of embryos irradiated with 366-nm UV light (gray) is 0.72. Nearly 55% of embryos incubated with biliverdin (black) recover and are scored with 4–5 with an average DAI of 3.08 for the total population. The recoveries are statistically significant (paired t with P < 0.001). (C) The extent of embryo recovery from 254 nm (□) or 366-nm (●) UV light irradiation depends on biliverdin concentration. Recovery was determined with the equation, Ri = (x − uv)/(c − uv), where Ri = recovery index, x = average DAI for embryos incubated with biliverdin, uv = average DAI for embryos exposed to UV, and c = average DAI for control embryos.
	Figure 4 HPLC elution profile of an extract of eggs or stage 1 embryos. (A) The chromatographic profile exhibits a number of peaks with distinctive characteristics. The peak pertinent to this report elutes with a retention time of 23.3 min (arrow). (B) Comparison of peak areas of the HPLC profiles of extracts from control (black) and 366-nm UV light irradiated embryos (gray) demonstrate that the 23.3-min peak is the only one that is markedly reduced.
	Figure 5 Rechromatography of purified 23.3-min fraction before and after UV light irradiation. (A) Before irradiation. (B) Chromatography of the same 23.3-min fraction after 366-nm UV light irradiation in vitro. The 23.3-min fraction is virtually abolished, and a major new species is generated. The photo-transformation product differs in its elution time (41.6 min) and spectral properties.
	Figure 6 Physical chemical features of the HPLC fraction 23.3 min identifies it as biliverdin IXα. The numbers in the structure indicate the position of the protons and the carbons described in Fig. 7. (A) The 23.3-min HPLC fraction has a unique UV-Vis absorption spectrum with characteristic peaks at 375 and 665 nm in ethanol. (B) MS positive ion mode analyses of the 23.3-min HPLC fraction yields a single high abundance peak with a mass-to-charge ratio of (1+) 583.2553. The molecule is predicted to have 19 double-bond equivalents, including all rings and carboxyl groups. There are five exchangeable protons ascertained by the mass increase in the presence of deuterium. These characteristics are identical to those of (1+) biliverdin. This identification was reinforced by the identical TLC Rf values (0.85 in a 3:1 chloroform/methanol mixture), cochromatographic behavior of a commercial biliverdin sample and the yolk platelet material purified on C18 with HPLC, and superposition of both absorption and NMR spectra (see Fig. 7).
	Figure 7 NMR one-dimensional 1H spectrum and total correlation spectroscopy (TOCSY) spectra of the 23.3-min HPLC fraction identified it as biliverdin IXα. (A) The one-dimensional 1H spectrum of the pure 23.3-min HPLC fraction is identical to that of commercial biliverdin IXα. Chemical shifts are relative to trimethylsilyl propionate at 0.00 ppm: 1H NMR (methanol-d4) δ 6.54(m, 1H, H-21), 5.41(d, 1H, H-22), 6.05(d, 1H, H-22′), 6.22(s, 1H, H-5), 7.11(s, 1H, H-10), 6.19(s, 1H, H-15), 6.75(m, 1H, H-171), 5.72(d, 1H, H-172), 5.68(m, 1H, H-172′), 2.47(t, 4H, H-82, H-122), 2.96(t, 4H, H-81, H-121), 2.09, 2.02, 2.00, 1.73(s, 12H, H-31, H-71, H-131, H-181); 13C NMR (methanol-d4) δ 173.3(C-1, C-19), 127.1(C-21), 119.9(C-22), 100.0(C-5, C-15), 117.1(C-10), 127.6(C-171), 123.1(C-172), 21.5(C-81, C-121), 38.2(C-82, C-122) 177.8(C-83, C-123), 139.5(C-8, C-12). (B) Plot of the vinyl region from TOCSY spectrum of the oocyte molecule. The chemical shifts and coupling patterns are identical to commercial biliverdin IXα. Additionally, the coupling between the carbonyl carbon and the α and β methylene protons of the propionic acid side chains were verified from the distortionless enhancement by polarization transfer-heteronuclear multiple quantum correlation and heteronuclear multiple bond correlation spectra (data not shown).
	Figure 8 Molecular switch for induction of dorsal axis. Biliverdin is proposed to interact with a cortical factor to trigger or switch on the downstream activation of genes. When the chemical switch is turned on, the Nieuwkoop center and the Spemann–Mangold organizer are sequentially formed. The UV irradiation of biliverdin renders an ineffective photo-product, the chemical switch remains off, and the dorsalizing gene products that participate in the configuration of the dorsal axis are not formed. The result is adorsal teratology. MBT, midblastula transition.

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Brannon, Activation of Siamois by the Wnt pathway. 1996, Pubmed, Xenbase