Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract??? Tail bud formation in Xenopus depends on interaction between a dorsal domain (dorsal roof) expressing lunatic fringe and Notch, and a ventral domain (posterior wall) expressing the Notch ligand Delta. Ectopic expression of an activated form of Notch, Notch ICD, by means of an animal cap graft into the posterior neural plate, results in the formation of an ectopic tail-like structure containing a neural tube and fin. However, somites are never formed in these tails. Here, we show that BMP signaling is activated in the posterior wall of the tail bud and is involved in the formation of tailsomites from this region. Grafts into the posterior neural plate, in which BMP signaling is activated, will form tail-like outgrowths. Unlike the Notch ICD tails, the BMP tails contain well-organized somites as well as neural tube and fin, with the graft contributing to both somites and neural tube. Through a variety of epistasis-type experiments, we show that the most likely model involves a requirement for BMP signaling upstream of Notch activation, resulting in formation of the secondary neural tube, as well as a Notch-independent pathway leading to the formation of tailsomites from the posterior wall.
FIG. 1. Endogenous BMP activity in the tail bud. (A) Embryos bisected along the anteriorposterior axis and stained using an antibody that recognizes the activated form of Smad. Strong anti-phospho-Smad1/5/8 staining is seen in the region immediately ventral to the future tail bud (black arrows), correlating to the expression of BMP4 mRNA. Nuclear phosphorylated BMP Smads are also present at lower levels in the tail-forming region itself from stage 17 on. (A) stage 12, (B) stage 15, (C) stage 17, (D) stage 20, (E) stage 25. All embryos are oriented with anterior to the left and dorsal uppermost. (F) In situ hybridization to BMP4 to show sites of endogenous transcription. (F) BMP4 expression just anterior to the ventral and lateral lips of the blastopore (black arrow). Embryo viewed from the vegetal side with dorsal uppermost. (G) Expression in embryos bisected after in situ hybridization; arrow points to strong expression in the region immediately ventral to the tail bud. Orientation is anterior to the left and dorsal uppermost. (G) stage 14, (H) stage 20, (I) stage 25. ftb, position of future tail bud; tb, tail bud; bp, blastopore.
FIG. 2. Ventralizing activity of BMP pathway components. Embryos were injected into each of 4 cells at the 4-cell stage and analyzed at stage 40 using the DAI index of Kao and Hopwood (1988), where a score of 5 indicates a normal embryo and a score of 0 indicates a completely ventralized embryo. (A) Control (uninjected) embryos at stage 40 score 5 on the DAI scale (N = 62). (B) Embryos injected with 2 ng Smad5 mRNA have defects in ventralfin and proctodeum. (C) Embryos injected with 250 pg of BMP4 mRNA are ventralized with an average DAI score of 1.8 (N = 44). (D) Embryos injected with 2 ng Smad5*, an activated form lacking the last two amino acids, are partially ventralized with an average DAI score of 2.6 (N = 46). (E) Embryos injected with 2 ng mRNA encoding the hormone-inducible fusion protein Smad5-GR develop normally with an average DAI score of 4.9 (N = 35). (F) Embryos injected with the same dose of Smad5-GR mRNA but treated with 10 mM dexamethasone from stage 6 are partially ventralized with an average DAI score of 2.2 (N = 29). (G) Embryos injected with 500 pg of the constitutively active BMP receptor Alk3 are extremely ventralized with an average DAI score of 1.2 (N = 62). (H) Embryos injected with 100 pg mRNA encoding dominant negative Smad5-sbn are dorsalized with an average DAI score of 6.2 (N = 44).
FIG. 3. Effects of injecting BMP pathway components into animal caps. Animal caps were dissected at stage 8.5 and cultured in NAM/2
until control stage 30. Uninduced caps form compact wrinkled balls, those producing ventral-type mesoderm form translucent vesicles, and
those producing dorsal-type mesoderm elongate to a sausage shape. (A) Untreated animal caps do not form mesoderm. (B) Animal caps from
embryos injected with 250 pg BMP4 mRNA form ventralmesoderm. (C) Animal caps from embryos injected with 500 pg of the constitutive
BMP receptor Alk3 form ventralmesoderm. (D) Animal caps from embryos injected with 2 ng Smad5 mRNA do not form mesoderm. (E)
Animal caps from embryos injected with 2 ng of active Smad5* mRNA form ventralmesoderm. (F) Animal caps from embryos expressing
2 ng of Smad5-GR mRNA do not form mesoderm. (G) Animal caps from embryos injected with 2 ng of Smad5-GR mRNA and treated with
10 mM dexamethasone form ventralmesoderm. (H) Animal caps injected with 250 pg BMP4 and 100 pg Smad5-sbn mRNA elongate, the
formation of dorsal mesoderm indicating that both endogenous and exogenous BMP4 are inhibited.
FIG. 4. RT-PCR and luciferase reporter assays to demonstrate activity of Smad constructs. (A) RT-PCR to show the nature of inductions
in animal caps using markers for ventralmesoderm (Xhox3, Globin), axial mesoderm (cardiac actin), neural tissue (Nrp-1), and the loading
control gene ODC. A total of 102 animal caps at stage 30 were pooled for each sample and positive and negative control RNA was made
from 5 pooled WT embryos at the same stage and reverse-transcribed with (Embryo) or without reverse transcriptase enzyme (-RT).
Concentrations of injected mRNAs were as stated in Fig. 2. (B) Schematic of Xvent2b promoteruciferase reporter construct. The BMP
responsive element (BRE) is contained within the 2275 to 2152 portion of the promoter. The TATA box lies between 232 and 134. (C)
Results of luciferase assays performed on protein extracts from embryos injected at the 4-cell stage and harvested at stage 10. These results
are representative of three repeat experiments and measured in duplicate to generate error bars (standard errors). (C) Dorsal injections reveal
that Smad5 (1 ng per embryo) does not stimulate luciferase activity over background, whereas Smad5* (1 ng per embryo) increases
production of luciferase threefold. BMP4 (250 pg per embryo), included as a positive control, increases luciferase production ninefold. (D)
On the ventral side, injections of reporter DNA alone produce high levels of luciferase as a result of the endogenous factors present on the
ventral side. Smad5-sbn (200 pg per embryo) reduces this activity to less than 1/3, showing that it is capable of interfering with the
endogenous BMP pathway.
FIG. 5. Activation of the BMP pathway in animal cap grafts to the posterior neural plate results in the formation of ectopic tails expressing tail bud markers. (A) Results of grafts into the posterior neural plate. Arrows point to ectopic tails, if formed. (A) Grafts from animal caps injected with 2 ng Smad5 mRNA do not form ectopic tails. (B) Grafts from animal caps injected with 2 ng Smad5-GR mRNA do not form ectopic tails. (C) Ectopic tails are formed from grafts of animal caps injected with 250 pg BMP4 mRNA. (D) Ectopic tails are formed from grafts of animal caps injected with 2 ng Smad5* mRNA. (E) Ectopic tails are formed from grafts of animal caps injected with Smad5-GR mRNA and treated with dexamethasone from stage 6. (F) Ectopic tails are formed from grafts of animal caps injected with 500 pg Alk3 mRNA. (G) Comparison of tissue-specific markers (dark blue) in the host tail (black arrow) and ectopic tail formed by Smad5* graft (white arrow). (G) The tail bud marker Xbra is expressed in both host- and graft-derived tail buds. (H) FGF-8 is expressed in both host- and graft-derived tail buds. (I) Xhox3 is expressed in both host- and graft-derived tail buds.
FIG. 6. Activation of the BMP pathway in animal cap grafts to the posterior neural plate leads to the formation of ectopic tails (white arrow)
containing somites, neural tube, and fin. (A and B) Two examples of grafts labeled with nuclear b-galactosidase (blue) to show contribution of the
graft to the ectopic tail by stage 36. The fin and epidermis of the secondary tail are not labeled and are therefore induced from the host by the
graft. (C) Sagittal section through posterior of a grafted embryo at stage 36. Graft cells are labeled with nuclear b-galactosidase and are found in
the somites and neural tube of the ectopic tail but not in the fin, finmesenchyme, or epidermis. (D) Drawing of the section in C, showing the
identity of the tissues contributing to the host and graft tails. nt, host neural tube; nt9, graft-derived ectopic neural tube; s, host somites; s9, graft
derived ectopic tailsomites; nc, notochord (host); fin, tailfin; fin9, tailfin of ectopic tail (host-derived). (E) 12/101 antibody staining to show
somites (dark blue) in both host- and graft-derived tails. Black arrows denote host tails.
FIG. 7. BMP signaling is required for tail bud outgrowth and acts upstream of Notch activation and cleavage. (A) Animal cap grafts
from embryos injected with 2 ng of Smad5* and 400 pg mRNA encoding the antimorphic Xhox3VP16 fusion fail to form ectopic tails
(white arrow), suggesting a requirement for Xhox3 downstream of BMP signaling. (B) Animal cap grafts from embryos injected with
400 pg Smad5-sbn and 400 pg Xhox3EnR mRNA do form ectopic tails, indicating that Xhox3 is required downstream of Smad5. (C)
Animal cap grafts from embryos injected with 400 pg Xhox3EnR mRNA and 50 pg Noggin-CSKA DNA form ectopic tails. (D) Animal
cap grafts from embryos injected with 250 pg BMP4 and 400 pg Smad5-sbn do not form ectopic tails. (E) Coinjection of 400 pg
Smad5-sbn mRNA with the same dose of Notch ICD does not block ectopic tail formation, suggesting that Notch signaling is
downstream of Smad 5 activity. (F) Coinjection of 50 pg Noggin-CSKA DNA with 400 pg Notch ICD mRNA does not block formation
of ectopic tails from the graft. (G) Calpeptin treatment of embryos grafted with Notch ICD expressing animal caps does not affect the
formation of ectopic tails. (H) Treatment of embryos grafted with animal caps expressing a membrane-tethered constitutively cleaved
form of Notch, mNotchDE, with calpeptin prevents formation of ectopic tails. (I) Treatment with calpeptin also prevents the
formation of ectopic tails from grafts expressing BMP4.
