Schmidt JE et al. (1995), Localized BMP-4 mediates dorsal/ventral pattern...

XB-ART-19762

Dev Biol 1995 May 01;1691:37-50. doi: 10.1006/dbio.1995.1124.

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Localized BMP-4 mediates dorsal/ventral patterning in the early Xenopus embryo.

Schmidt JE , Suzuki A , Ueno N , Kimelman D .

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Mesoderm is initially induced in the amphibian embryo by events that occur in the early cleavage stages prior to the midblastula transition (MBT) and morphogenesis. These inductive interactions establish the mesoderm at the equator and create a distinction between the dorsal and the ventral regions. After the MBT, zygotic factors pattern the mesoderm and induce the neuroectoderm on the dorsal side of the embryo. Most previous studies have focused on the effects of signals originating in the dorsal mesoderm. We show that BMP-4 transcripts are localized to the ventral side of the gastrula embryo and provide evidence that localized expression of BMP-4 is important for regulating the expression of mesodermal and neural genes. We show that ectopic expression of BMP-4 inhibits the formation of dorsal and lateral mesoderm and reduces the size of the neural plate. Elimination of BMP-4 signaling with a dominant-negative BMP receptor expands the lateral mesoderm and neural plate without expanding the expression of genes along the dorsal midline. These results suggest that BMP-4 may act to oppose the action of dorsalizing signals and neural-inducing signals that originate in the dorsal organizer region. We suggest that BMP-4 may have an analogous role to the Drosophila gene, dpp, in dorsal/ventral pattern formation.

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Species referenced: Xenopus
Genes referenced: acta4 actc1 arsl bmp4 dspp evx1 gast gsc myod1 not tbxt wnt8a

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	FIG. 1. Phenotypic comparison of dorsal and ventral injections of BMP·4 RNA. Four-cell embryos were injected into the two dorsal (A and C) or the two ventral (Band D) blastomeres with 2 ng (A and B) or 4 ng (C and D) of BMP-4 RNA. In each panel, the top three embryos were injected with BMP-4 RNA, and the bottom embryo is a control, uninjected embryo at the same stage (Stage 26). Complete ventralization can be achieved with different doses of BMP-4 RNA in embryos from different females; therefore, the embryos presented here are from the same experiment. Anterior is to the left in the control embryos and in embryos injected with 2 ng BMP-4 RNA. The closed blastopore is to the right in embryos injected with 4 ng BMP-4 RNA.
	FIG. 2. Gene expression in whole embryos injected with BMP-4 RNA. In all experiments, a percentage of injected and uninjected control embryos was allowed to develop until late tailbud or early tadpole stages to assure that the injected embryos were ventralized. (A) Vegetal view of gsc expression in an uninjected, stage 10.5 embryo. Dorsal is up, as is gsc expression. (B) Vegetal view of a stage 10.5 embryo injected dorsally with 4 ng BMP-4 RNA. gsc expression is eliminated from the dorsal side of the embryo. Dorsal is up. (C) Vegetal view of MyoD expression in an uninjected, stage 12 embryo. Dorsal is to the upper right. (D) Vegetal view of a stage 12 embryo injected dorsally wit h 2 ng BMP-4 RNA. MyoD expression is no longer cleared from the dorsal midline. The ventral expression is absent and the lateral expression is d iminished. Dorsal is up. (E) Vegetal view of Xwnt-8 expression in an uninjected, stage 12 embryo. Dorsal is up. (F) Vegetal view of a stage 12 embryo injected dorsally with 2 ng BMP-4 RNA. XY.mt-8 is no longer expressed on the ventral side of the embryo. Two lateral patches of expression remain and the dorsal clearing has narrowed. Dorsal is up. Note that these embryos are from the same batch of embryos as those stained for MyoD. (G) Lateral view of Xbra expression in an uninjected, stage 11 embryo. Xbra is expressed throughout t he marginal zone in the presumptive mesoderm. Dorsal is up. (H) Lateral view of a stage 11 embryo injected ventrally with 2 ng BMP·4 RNA. Xbra expression has expanded from the ventral marginal zone into the animal pole. Dorsal is up. (I) Dorsal view of Hairy II expression in an uninjected, stage 12.5 albino embryo. Anterior is up. Hairy II is expressed in a stripe that outlines the neural plate as well as within the dorsal midline. (J) Dorsal view of a stage 12.5 albino embryo injected with 2 ng BMP-4 RNA. Note that the embryo shown here was injected without dorsal or ventral distinction since pigmentation could not be used to differentiate dorsal from ventral. The neural plate as outlined by Hairy II has been greatly reduced in size. The results were identical to the results seen with dorsal and ventral injections ofBMP-4 RNA into pigmen ted embryos (data not shown). The blastopore is often larger in embryos injected with BMP-4 RNA than in control embryos; however, most injected embryos that are allowed to develop until tailbud or tadpole stages completed gast rulation. In B, D, and F, embryos injected dorsally with BMP-4 RNA are shown. Since the results did not differ significantly when embryos were injected ventrally, only dorsal-injected embryos are shown.
	FIG. 3. BMP-4 is cleared from the dorsal side of gastrula-stage embryos. Albino embryos at various stages were hybridized with an antisense BMP·4 RNA probe. (A) Lateral view of a stage 11 embryo, dorsal is up. The blastopore is to the upper right. BMP-4 expression is absent from the dorsal side of the embryo. Expression is strongest in the animal pole and appears to be at a lower level in the ventral marginal zone. An arrowhead indicates the ventral marginal zone. (B) Lateral/vegetal view of a stage 12 embryo. Staining is visible adjacent to the blastopore on the ventral side of the embryo. (C) Lateral view of the same embryo shown in B, anterior is to the left, dorsal is up. BMP-4 expression is absent from the dorsal side of the embryo in a region that approximates the future neural plate. (D) Future anterior view of the same embryo shown in B, the blastopore (arrowhead) and dorsal are to the right. A sharp boundary of expression is visible in this region. (E) Lateral view of a stage 22 en1bryo, anterior is to the left, dorsal is up. BMP-4 expression has resolved to two ventral regions, one anterior and one posterior. Additional expression can be seen above the eyes and along the future dorsal tail fin. (F) Lateral view of a stage 28 embryo, anterior is to the left, dorsal is up. The ventral posterior expression is reduced to a small narrow region; the ventral anterior expression is below the branchial arches. The expression above the eye is still present as well as expression along the tail fin. BMP-4 expression is now also visible in t he otic vesicle. No specific staining was detected with the sense probe (not shown).
	FrG. 4. Phenotypic effects of eliminating BMP-4 signaling in the early embryo. The top embryo was injected into the marginal zone of each cell at the four-cell stage with a total of 4 ng of the dominantnegative BMP receptor RNA. The head of this injected embryo is reduced in size, but appears relatively normal in structure compared to the cont rol embryo below. The body of the injected embryo has not elongated and is extremely reduced in size. Embryos are at stage 47, anterior is to the r ight, dorsal is up.
	FIG. 5. The dominant·negative BMP receptor blocks BMP-4 signaling, but not activin signaling. {A) Animal caps were isolated at stage 9 from uninjected, BMP-4 RNA- or lillMPR RNA-injected embryos or from embryos injected with both BMP-4 and ~BMPR RNAs. In some cases, activin was added to the animal caps at stage 9. In all cases, RNA was harvested at stage 11. The levels of Jl'bra and the ubiquitously expressed gene, EF-lc:, were determined by RNase protection analysis. Lane 1, control caps. Lane 2, activin-treated caps. Lane 3, animal caps from embryos injected with 2 ng BMP-4 RNA. Lane 4, animal caps from embryos injected with 2 ng BMP-4 RNA and 2 ng ~MPR RNA. Lane 5, a nimal caps from embryos injected with 2 ng .J.BMPR RNA. Lane 6, animal caps from embryos injected with 2 ng .J.BMPR RNA and treated with activin. (B) Embryos injected with BMP·4 RNA and/or ~BMPR RNA from the same experiment shown in A were allowed to develop until stage 31. RNA was isolated and analyzed for muscle actin expression by RNase protection analysis. Lane 1, uninjected embryos. Lane 2, embyros injected with 2 ng BMP· 4 RNA. Lane 3. embryos injected with 2 ng ~MPR RNA. Lane 4, embryos injected with 2 ng BMP-4 RNA and 2 ng .J.BMPR RNA. EF-la was used as a control for loading.
	FIG. 6. Gene expression in gastrula and neurula embryos injected with dominant-negative BMP receptor RNA. (A) Vegetal view of gsc expression in an uninjected stage 10 embryo. Dorsal is up. (B) Vegetal view of gsc expression in a stage 10 embryo injected with ~BMPR RNA. gsc. expression is unchanged. Dorsal is llp (C) Dorsoposterior view of Xnot expression in an uninjected, stage 12.5 embryo. Anterior is at the upper left corner. (D) Dorsoposterior view of Xnot expression in a stage 12.5 embryo injected with aBMPR RNA. Xnat expression closely resembles that in uninjected embryos, although the region of expression may not extend as far anteriorly. Anterior is at the upper left corner. (E) Vegetal view of MyoD expression in an uninjected stage 11.5 embyro. Dorsal is up. MyoD is expressed in the lateral and ventral marginal zone. (F) Vegetal view of MyoD expression in embryos injected with ~BMPR RNA. MvoD expression in the latt:ral regions appears unchanged; however, the ventral expression appears stronger than in control embryos. Dorsal is up. (G) Vegetal view of Xwnt-Sexpression in an uninjected stage 11.5 embryo. Dorsal is up. X1~mt-8 is expressed in the lateral and ventral marginal zone distant from the dorsal midline. (H) Vegetal view of Xwnt-8 expression in a stage 11.5 embryo i njeeted with 6BMPR RNA. Xumt-8 expression is nearly eliminated. Dorsal is up. Embryos in D, E, F, and G are from the same experiment. (I) Vegetal view of Xbra expression in an uninjected, stage 11 embryo. Dorsal is up. (J) Veg(~tal view of Xfrra expression in a stage 11 embryo injected with aBMPR RNA. Dorsal is up. Xbra. expression is unchanged in injected embryos as compared to controls. (K) Dorsolateral view of Hairy II expression in an uninjected, stage 13 embryo. Hairy II expression is in a stripe along the border of the neural plate and does not extend to the blastopore (at right). In addition to this stripe of expression, Hai1y II is expressed along the dorsal midline (arrowhead). Anterior is to the left. (L) Dorsolateral view of Hairy II expression in a stage 13 embryo injected with 11BMPR RNA. The stripe of Hairy II expr~sion is thicker and continues around to the ventral side of the embryo. Expression along the dorsal midline (arrowhead) is shortened. Anterior is to the left. (M) Ventral view of H(l;iry II expression in an uninjected, stage 13 embryo. Hairy II is not expressed on the ventral side. Anterior is to the left. (N) Ventral view of Hairy II expression in a stage 13 embryo injected with LlBMPR RNA. The stripe of Hairy II expression bordering the neural plate extends around the entire ventral side of the embryo. Anterior is to the left. Embryos in K-N are albino embryos. (0) Dorsal view of MyoDexpression in stage 15 embryos, anterior is up. The embryo on the left was injected with .J.BMPR RNA, the embryo on the right is an uninjected control embryo. MyoD expression is cleared from the dorsal midline in both injected and uninjected embryos. The posterior region is reduced in injected embryos compared to control embryos. (P) Lateral view of the embryos shown in 0 . The top embryo was injected with 6BMPR RNA and the bottom embryo is an uninjected control embryo. No MyoD expression is present on the ventral side of the control embryo; however, MyoD expression extends around the entire ventroposterior region of the injected embryo. Morphological changes in the embryo are already evident at this stage. Anterior is to the left, do:rsal is up. (Q) View of the blastopore (arrowhead) in the same embryo as in 0 and P. The dorsal paraxial expression of MyoD extends around the entire \'entral side of the embryo. Dorsal is up. (R) Dorsoposterior view of the blastopore (arrowhead) of the uninjected, stage 15 control embryo shown in 0 and P. No ventral expression is apparent. Anterior is up. Photographs for Q and R were taken such that t he view was centered on the blastopore. Due to the abnormal shape of the injected embryos, the blastopore is found directly at one end of the embryo; thus, t he view looks down the anterior/ posterior axis. The blastopore of uninjected embryos at stage 15 is on the dorsoposterior surface of the embryo; therefore, the view is dorsoposterior.
	FIG. 7. The transcript levels of the ventroposterior gene, Xh.ox:J, are reduced, whereas the levels of the donal paraxial gene MyoD are increased in embryos injected with J.BMPR RNA. Embryos were injected into the marginal zone of all four blastomeres at the four-cell stage with 4 ng of .J.BMPR RNA. RNA was isolated at stage 13 and analyzed by RN ase protection using Xhox3 (A), MyoD (B), and EF-la probes. The same sample of RNA was used to detect the levels of MyoD and Xho:c3. In both panels, RNA from uninjected embryos is in lane 1 and RNA from ~BMPR-injected embryos is in lane 2. EF-la was used as a control for loading.