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???displayArticle.abstract??? Heart induction in Xenopus has been thought to be dependent primarily on the interaction of the heart primordia with the Spemann organizer. We demonstrate, however, that signals derived from the deep dorsoanterior endoderm during early gastrulation are also essential for heart formation. The presence of deependoderm dramatically enhances heart formation in explants of heart primordia, both in the presence and absence of organizer. Likewise, extirpation of the entire endoderm can decrease the frequency of heart formation in embryos that retain organizer activity. Finally, we show that the combined presence of both endoderm and organizer is necessary and sufficient to induce heart in ventral mesoderm explants that would not otherwise form hearttissue. Xenopus heart induction, therefore, may be a multistep process requiring separate dorsalization and cardiogenic signalling events. This is the first demonstration of a heart-inducing role for the endoderm in Xenopus, indicating that the mechanism of heart formation may be similar in most vertebrates.
Figure 1. The deep dorsoanterior endoderm enhances heart formation in explants of dorsal marginal zone (DMZ). (A) Regions of the DMZ were explanted which encompassed the dorsal 150° of marginal zone, and included the Spemann organizer (30° to each side of the dorsal midline; stippled), lateralmesoderm including the two heartprimordia regions (30° to 75° lateral to the dorsal midline; hatched), and substantial amounts of the underlying deependoderm (shaded). The endoderm was either left associated with the explant or removed after explantation. A flap of dorsal ectoderm (depicted as transparent for clarity) was also included to promote healing. Explants were cultured for 7 days and scored for the formation of a beating heart. (B) The effect of deependoderm on heart formation in DMZ tissues explanted at stage 10 and stage 10.5 is presented as a percentage of total cases undergoing heart formation. The results from all trials were pooled to construct the histogram, since the data from individual experiments were judged to be not significantly different by a conditional exact test calculating the probabilities using the Turbo StatXact computer package (exact test: P≥ 0.05; see Materials and Methods). The number in parentheses, (n), represents the total number of explants. + and − refer to presence or absence of deependoderm, respectively.
Figure 2. The deependoderm can induce beating heart in the absence of organizer. (A) Bridged primordia explants were made by explanting regions of the DMZ as in Fig. 1A, and subsequently removing the organizer region (dorsal 60° of marginal zone) from the excised tissue, while leaving the two primordia connected by a narrow bridge of ectoderm. Explants were cultured with their associated endoderm or deprived of deependodermtissue. (B) The effect of deependoderm on the frequency of heart formation in bridged primordia was assessed in explant culture. The results of different trials were analyzed statistically, and some were found to be not significantly homogeneous (exact test: P<0.05; see Table 1). For consistency, the data from individual experiments have been pooled and represented here as a percentage of total explants with beating hearts as in Fig. 1B. Number in parentheses (n) represents the total number of explants. + and − indicate the presence or absence of deependoderm, respectively.
Figure 3. Beating hearts form in the absence of dorsoanterior structures. Stage 10 bridged primordia explant with endoderm, after 7 days in culture. Arrow points to location of beating heart.
Figure 4. Endoderm deficiency reduces the frequency of heart formation in whole embryos. (A) Stage 10 or stage 10.5 gastrulae were partially bisected along the ventral midline to access the endoderm-mesoderm boundary. The endodermtissue was gently scraped away from the two halves of marginal zone and discarded. Embryos then healed upside-down in round-bottom wells for 30 minutes in 0.75× MMR, during which time the ectoderm halves fused and the marginal zone invaginated into the cavity left by the operation. The embryos were then transferred to 0.1× MMR + gentamycin for culture, and assessed for the presence of beating hearttissue. (B) The effect of endoderm removal on heart formation in intact embryos is compared to partially bisected controls whose endoderm was not removed. Data from different trials is statistically homogeneous (exact test: P≥0.05), and presented as a percentage of total (n) explants with beating hearts.
Figure 5. Removal of endoderm at stage 10 can prevent heart formation in whole embryos. Embryos were cultured for 7 days after the operation. (A) Control bisected embryo (stage 10.5) whose endoderm was left intact. All controls had beating hearttissue and normal gut (arrowhead). (B) Embryo deprived of entire endoderm at stage 10. No beating hearttissue was evident in this explant. Asterisk indicates lack of gut development. (C) Embryo deprived of endoderm at stage 10.5. Location of beating heart is indicated by arrow. Gut development is absent (asterisk).
Figure 6. The presence of both organizer and endoderm is necessary for heart induction in ventral marginal zone (VMZ). (A) Small pieces of organizer (dorsal 30° of marginal zone) were explanted and used with or without their associated dorsoanterior deependoderm. Regions of the VMZ encompassing the ventral 150° (75° lateral to dorsal midline) of the embryo were simultaneously explanted with or without ventralendoderm. Three different types of recombinants were constructed by implanting the organizer fragment into the ventral midline of the VMZ explant, as in an in vivo organizer transplant graft. (B) The effect of no endoderm, ventralendoderm alone, or ventral and dorsal endoderm, on the frequency of heart formation in VMZ recombinants is expressed as a percentage of explants with beating hearts, since statistical tests indicated significant homogeneity between individual trials (exact test: P>0.05). The number in parentheses (n) refers to the total number of recombinants made
Figure 7. Ventralmesoderm is induced to form hearttissue in combination with both organizer and dorsoanterior endoderm. A VMZ recombinant was constructed as in Fig. 6A with an RLDx-labelled organizer segment which retained its associated dorsoanterior endoderm. Those with beating hearts after 7 days were sectioned, and the hearttissue was examined by epifluorescence illumination. Two examples are shown. (A,C) Phase micrograph of VMZ recombinant containing dorsal deependoderm, sectioned at level of hearttissue (arrow). Both myo-and endocardial layers are evident. (B,D) Epifluorescent micrograph of same sections as in A and C. Unlabelled VMZ cells contribute to approximately half of the hearttissue evident in B, whereas they give rise to most of the hearttissue in the explant shown in D. The scale bar in C represents 100 μm.