Beck CW and Slack JM (1999), A developmental pathway controlling outgrowth o...

XB-ART-13365

Development 1999 Apr 01;1268:1611-20. doi: 10.1242/dev.126.8.1611.

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A developmental pathway controlling outgrowth of the Xenopus tail bud.

Beck CW , Slack JM .

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We have developed a new assay to identify factors promoting formation and outgrowth of the tail bud. A piece of animal cap filled with the test mRNAs is grafted into the posterior region of the neural plate of a host embryo. With this assay we show that expression of a constitutively active Notch (Notch ICD) in the posterior neural plate is sufficient to produce an ectopic tail consisting of neural tube and fin. The ectopic tails express the evenskipped homologue Xhox3, a marker for the distal tail tip. Xhox3 will also induce formation of an ectopic tail in our assay. We show that an antimorphic version of Xhox3, Xhox3VP16, will prevent tail formation by Notch ICD, showing that Xhox3 is downstream of Notch signalling. An inducible version of this reagent, Xhox3VP16GR, specifically blocks tail formation when induced in tailbud stage embryos, comfirming the importance of Xhox3 for tail bud outgrowth in normal development. Grafts containing Notch ICD will only form tails if placed in the posterior part of the neural plate. However, if Xwnt3a is also present in the grafts they can form tails at any anteroposterior level. Since Xwnt3a expression is localised appropriately in the posterior at the time of tail bud formation it is likely to be responsible for restricting tail forming competence to the posterior neural plate in our assay. Combined expression of Xwnt3a and active Notch in animal cap explants is sufficient to induce Xhox3, provoke elongation and form neural tubes. Conservation of gene expression in the tail bud of other vertebrates suggests that this pathway may describe a general mechanism controlling tail outgrowth and secondary neurulation.

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Species referenced: Xenopus laevis
Genes referenced: cdx4 dll1 evx1 fgf8 gal.1 lfng mt-tr ncam1 notch1 notch4 notch4.2 nr3c1 shh tbxt trna wnt3a

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	Fig. 1. Constructs used in this study. VP16 or EnR effector domains are substituted for the putative repressor domain of the wild-type Xhox3. In the inducible version, a glucocorticoid receptor ligand-binding domain is added at the 3¢ end.
	Fig. 2. Ectopic tails formed by grafts expressing Notch ICD. (A) Assay for outgrowth-forming ability. Test mRNA was injected quadrilaterally into 4-cell-stage embryos, which were then allowed to develop to stage 9. Animal caps were removed and trimmed into strips of 100´600 mm. A slit 600 mm long was made in the host neural plate 100-200 mm anterior to the slit blastopore, and the neural plate pushed forward to create an opening for the graft. Grafted embryos were analysed for ectopic tail formation at stage 34-36. (B,C) Embryos grafted with control animal cap. There is no pattern disruption, and visualisation of the graft by X-gal staining (cyan) shows incorporation of the graft into the host neural tube. (D,E) Embryos grafted with Notch ICD expressing animal cap develop an ectopic tail. The X-gal stain shows that grafted tissue which ends up more anteriorly is incorporated into the host neural tube, while the more posterior graft tissue is incorporated into the ectopic tail bud. (F,G) Section though a Notch ICD grafted embryo at stage 35 to show the lack of somites and notochord in the ectopic tail formed from the graft. (H,I) Section through a Notch ICD graft onto a host labelled with FDA to show that the ectopic tail epidermis is composed of host cells (fluorescent in H, green in I), whereas the neural tube of the ectopic tail is composed primarily of nonfluorescent, graft-derived cells. The composition of the fin mesenchyme is mixed. fin, host fin; fin¢, ectopic fin; nt, neural tube; nt¢, ectopic neural tube; nc, notochord; s, somites. Bars, 250 mm (F), 100 mm (H).
	Fig. 3. Expression of tail bud genes in Notch ICD ectopic tails. (A) Xbra in tail bud, (B) Xhox3 in the distal tail bud, (C) Xwnt3a localised to the tail bud and dorsal side of the tail neural tube, (D) X-delta-1 is not restricted ventrally as in the host tail bud, but is expressed throughout the ectopic tail, (E) lfng expression is dorsal in the proximal tail, (F) X-shh expression in ectopic tails is much reduced compared to the host tail and may represent floor plate expression. (G) FGF-8 and (H) Xcad3 expression in ectopic tails resembles that of wild-type tail buds. Arrowheads indicate staining in ectopic tails formed from the grafts. All embryos are aged between stages 33 and 36. Posterior (host tail bud) is to the right and dorsal uppermost.
	Fig. 4. Xhox3 can induce ectopic tails and functions downstream of Notch ICD. (A-D) Animal cap grafts were made into the posterior neural plate of stage 13 hosts and assayed for formation of an ectopic tail bud showing expression of Xbra at stage 35. (A) Xhox3 grafts can generate an ectopic tail identical to those formed by Notch ICD. (B) Grafts expressing Xhox3EnR, a strong repressing version of Xhox3, also form ectopic tails. (C) Grafts expressing an activating (antimorphic) form of Xhox3, Xhox3VP16, do not generate ectopic tails. (D) Co-expression of the antimorphic Xhox3VP16 with Notch ICD prevents ectopic tail formation. (E-H) Expression of Xhox3VP16 in embryos causes tail, head and pigmentation defects and can be rescued by coinjection of wild-type Xhox3 mRNA. (E) Wild-type embryos at stage 40. Tails contain 42 somites on average. (F) Embryos injected with 100 pg of Xhox3VP16 have a reduced tail with 28-34 somites at stage 40, pigment cells are lacking and the head is slightly reduced. (G) Embryos injected with 50 pg Xhox3 mRNA have slightly reduced heads but normal tails, with 42 somites on average. (H) Co-injection of 50 pg of Xhox3 mRNA with 100 pg Xhox3VP16 completely rescues all the phenotypic effects of Xhox3VP16 and tails contain 42 somites. (I-L) Effect of inducing Xhox3VP16GR with dexamethasone at different developmental stages. (I) Injection of 400 pg of Xhox3VP16GR has no phenotypic effect if embryos are cultured in the absence of dexamethasone, or (J) treated with dexamethasone at stage 35. (K) Embryos injected with 400 pg Xhox3VP16GR and treated with dexamethasone at stage 26 have reduced tails and slight eye defects. (L) Embryos treated at stage 20 have vestigal tails and more severe eye defects.
	Fig. 5. Posterior restriction of the tail-forming ability of Notch ICD grafts can be overcome by coexpression of Xwnt3a. Animal cap pieces 100´600 mm are grafted into one of three positions along the anterior posterior axis of stage 13 hosts as shown. The final position of the graft is marked by Xgal staining (cyan, absent in H,I) and tail buds (host and ectopic) are marked by Xbra expression (dark blue). (A-C) Control animal cap pieces expressing b-galactosidase do not form tails when grafted into anterior (A), midtrunk (B) or posterior (C) positions. (D-F) Notch ICD animal caps do not form ectopic tails if grafted into the anterior (D) or trunk (E) positions, but ectopic tails are formed from 81% of posterior grafts (F). (G-I) Grafts expressing both Notch ICD and Xwnt3a can form ectopic tails in anterior (G), trunk (H) or posterior (I) positions.
	Fig. 6. Combined Notch and Wnt signalling induces formation of neural tube-like structures in animal caps. (A-D) Animal caps at stage 38. (A) Control caps expressing b-galactosidase; (B) caps expressing Notch ICD alone resemble controls. (C) Caps expressing Xwnt3a alone resemble controls. (D) Caps co-expressing Xwnt3a and Notch ICD elongate and swell. (E-I) Histology of injected caps at stage 38 compared to embryo of the same stage. (E) Transverse section through a Xenopus late embryo tail showing arrangement of tissues. (F) Section through uninjected animal cap showing atypical epidermis is formed. (G) Section through cap expressing Notch ICD showing poorly differentiated dense tissue. (H,I) Sections through animal caps co-expressing Notch ICD and Xwnt3a to show presence of fin-like mesenchyme (mc) and neural tube-like structures (arrows). (J-M) In situ hybridisation to NCAM, a marker of neural tissue (dark blue stain); (J) uninjected caps, (K) Notch ICD caps, (L) Xwnt3a caps, (M) caps injected with Notch ICD and Xwnt3a. (N-O) Sections of caps from M counterstained with methyl green (pale green) as in Beck and Slack (1998), to show that NCAM staining (dark blue) is restricted to the neural tube structures. ae, atypical epidermis; mc, mesenchyme; nc, notochord; nt, neural tube; s, somite. Bars, 100 mm (I), 50 mm (N,O).
	Fig. 7. Combined Notch and wnt signalling induces neural tissue in animal caps. RNAse protections showing Xhox3 and NCAM are strongly induced in the presence of both Notch ICD and Xwnt3a but absent from uninjected caps, or caps injected with either RNA alone. Whole stage-38 control embryonic RNA is included for comparison; tRNA lane is a negative control.
	Fig. 8. A model for tail outgrowth in Xenopus. (A) The NMC model for tail bud initiation. At stage 13 three regions, N, M and C, shown here in a section through the dorsal midline, come into alignment so that the junction of N and M overlies C. This interaction is essential for tail formation and is also required for onset of expression of lfng and Xwnt3a at stage 26. (B) Rearrangement of N, M and C occurs by the time of outgrowth. By stage 26, closure and extension of the neural tube results in formation of the posterior wall of the neural tube from the lateral blastopore lips, and movement of the N-M junction to the tip of the tail. (C) A mechanism for tail bud outgrowth mediated by Notch signalling. At around stage 26, lfng expression begins in the dorsal roof of the neural tube. Expression terminates at the posterior of the embryo in a sharp boundary, which slightly overlaps expression of the early genes X-Notch-1 and X-delta-1 in the posterior wall. This arrangement results in localised activation of Notch signalling at the leading edge of the tail bud. In the presence of Xwnt3a, also turned on at stage 26 in the extreme posterior dorsal roof and distal tip of the tail bud, this results in Xhox3 expression in the distal tip of the tail bud. Xhox3 is required at least for outgrowth of the tail, and formation of the tail bud-derived neural tube. In addition, Xwnt3a may be required for dorsal-ventral patterning of the tail bud-derived neural tube. (D) Summary of the expression of key genes at the time of tail outgrowth. Tailbud expression of lfng, Xwnt3a, Notch, Delta and Xhox3 is shown in blue for comparison. Drawings are based on sections of stage-30 embryos shown in Beck and Slack (1998). cnh, chordoneural hinge; end, endoderm; nt, neural tube; pw, posterior wall.