Cooke J et al. (1987), The organization of mesodermal pattern in Xenop...

XB-ART-27838

Development 1987 Dec 01;1014:893-908.

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The organization of mesodermal pattern in Xenopus laevis: experiments using a Xenopus mesoderm-inducing factor.

Cooke J , Smith JC , Smith EJ , Yaqoob M .

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In this paper, we study the mechanism by which a Xenopus cell line-derived mesoderm-inducing factor (MIF) might establish the spatial pattern of cellular differentiation in the mesoderm. The effects of the factor on competent animal pole tissue are consistent with it being identical to the natural mesoderm-inducing factor. The signal can only act on those membrane domains of the animal pole that face the blastocoel, but it can be stably recorded there, such that axial mesoderm is formed, after 15 min exposure or less. This exposure can end some hours, or several cell cycles, before the onset of RNA synthesis yet nevertheless be fully effective, although competence to respond also extends well after the onset of transcription. Exposure of the entire blastocoel lining of intact embryos to MIF causes a synchronous and sudden transformation of the behaviour and adhesive properties of all inner animal cap cells. This transformation mimics and is contemporaneous with the involution behaviour of normal mesoderm in the early gastrula marginal zone. Although high concentrations of MIF totally disorganize gastrulation, lower concentrations permit gastrulation to proceed. However, the pattern of mesoderm in these embryos is disrupted and ectopic mesoderm is formed around the blastocoel remnant. When MIF is injected directly into blastomeres, rather than into the blastocoel, it has no effect. This suggests that the molecule is secreted from source cells and affects target cells through an extracellular receptor. Finally, we show that small pieces of animal pole tissue recently exposed to MIF go on to produce morphogenetic signals perhaps distinct from MIF. We discuss the role of these signals in establishing and modifying the spatial pattern of cellular differentiation in the mesoderm of Xenopus.

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Species referenced: Xenopus laevis
Genes referenced: amh gnao1 mif
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	Fig. 1. Differentiation in explanted animal caps after various conditions of early exposure to MIF. (A.B) Sections transverse and longitudinal to axial structure in explants exposed to MIF for 20min immediately after excision from the 128-cell-stage embryo. All explants were extensively washed after exposure and cultured in 67% NAM until controls reached stage 35. (C) Differentiation indistinguishable from that of controls (67% NAM only) and (D) differentiation corresponding to the 'weakest' grade of mesoderm induction, as observed after culture in limiting dilutions of MIF. These appearances follow continuous culture in the same MIF preparation that gives rise to A and B but beginning 30min after dissection from the donor, when little or no original 'inner' blastomere membrane was exposed to the medium, nc, notochord; s, somite; ab, abnormal, wrinkled epidermis; ep, properly stretched, bilayered epidermis over a fluid-inflated cavity, not seen in complete absence of induction. Scale bar, 300 um.
	Fig. 2. Thin-sectional appearances at gastrula stages and the organization of somite muscle seen by immunofluorescence, after blastocoelic injection of MIF or control (XL-derived) dialysate. A-C show the marginal regions of stage-10 (early) gastrulae. A is a control embryo. B and C are, respectively, at very early and slightly more advanced stages in the minutes-long sequence of behaviour change in the inner blastocoel roof and wall, following injection with MIF at stage 7. Inner cells first become more loosely adhesive and surfaceactive, and then form a recognizably separate layer without localized, organized involution. This is followed by loss of the normal layered structure at the junction of endoderm, involuting mesoderm and blastocoel wall (arrows). D and E are higher power views of, respectively, the normal and abnormal blastocoel roof at the slightly later stage 10+. The inner, looser layers of cells in (E), consisting of ectopic mesoderm, how have the appearance of newly involuted mesoderm, except that there is no uninduced ectoderm available for them to migrate over. The large-celled yolky blastocoel floor appears at bottom. (F) Immunofluorescence for somite muscle in the tail of a normal larva, in grazing parasagittal section. Note the regular positioning of the fusiform cells in chevron-shaped blocks. (G) Image as in F but from an embryo that received a low dose (0-004 units) of MIF into the blastocoel (see Fig. 4A, second embryo). Note inferior extension and regularity of cell positioning, and the crazing and disorganization of the pattern. (H) Muscle immunofluorescence in the ventral site of the abnormally occluded blastocoel of an embryo that only just completed gastrulation and tailbud formation after MIF injection. An ectopic layer of striated muscle is visible. This forms from what would have normally produced anteroventral ectoderm. Scale bar, 300/im for A-C, 100^m for D,E, 125 pm for F,G and 80 um for H.
	Fig. 3. The altered structure during the first half of normal gastrulation, and the morphology at larval stages, after intrablastocoelic injection of MIF. (A) Sagittal sections at control stage 10—, 101 and 111 in normal (sham-injected) and MIF-injected embryos. Presumptive dorsal side, where normal mesoderm involution occurs first, is to the right. The upper control drawing represents the onset of stage 10, up to which point control and MIFinjected development are morphologically identical. Second and third rows represent typical stage-10\| and -111 embryos, with normal on the left and MIF-injected on the right, but with a split representation of the latter at stage 111 to show events typical of higher (left half) and lower (right half) doses. Tissue that is specified as mesoderm and has involuted or undergone the analogous change in cell behaviour is shown stippled throughout. Ectoderm and uninvoluted mesoderm is unshaded, and endoderm is hatched. The stage of development of external blastoporal lips and archenteron is shown in the outlines. At the time of MIF-induced abnormality, the normal mesoderm has just started to create an interface by migrating up the outer shell of preinvoluted mesoderm and presumptive ectoderm. The endodermal mass has a sharply limited border just ahead of the emerging mesoderm, because it has preferential affinity for that tissue. The two principal sequences of abnormal behaviour concern mesoderm, and then a consequent one by endoderm. There is a massive, synchronous 'recruitment' of mesodermal cells out of both the deep presumptive ectoderm and, we believe, the presumptive mesoderm as yet unrecruited by the normal process. Normal mesoderm already in a new layer then ceases its involution and movement, for lack of appropriate space and substrate. The normal development of a sharp preand post-involution interface (arrowheads), the deepening of the archenteron, and the advance of the blastopore over the yolk plug, all driven by mesoderm involution (Keller, 1986), are halted. Meanwhile the anterior edge of the endoderm mass alters its normal sharp outline, and its cells rapidly and abnormally achieve contact with the entire lining of the former blastocoel. The volume of the embryo is preserved by the appearance of an abnormal cavity within the endoderm in the vegetal region. After lower MIF doses (about 0-006 units), dorsal lip activity reappears and a pre- and post-involution interface forms by stage 111-12 (arrowheads), whereas with doses above 0-02 units this never occurs, and the ectopic mesoderm may reveal its 'dorsal' status by its semiorganized convergent behaviour. (B) Camera lucida drawings of normal and a range of MIF-injected embryos at stage 35. The least abnormal case has all major head parts and all germ layers present, but heart and blood do not form. Increasing doses reduce the anterior axial pattern, the pronephros disappears and somite segmentation is disrupted. A significant accessory trunk-tail formation is often present, centred on the ectopic mesoderm region but running into what remains of the embryo's 'own' axis anteriorly where there is no brain. At intermediate doses where gastrulation has only just recovered, the tailbud may be the only externally recognizable structure, although massive, normally positioned notochord and somite tissue are found internally. The ectoderm and nervous system have few cells and often disintegrate. Above this dose, gastrulation never occurs and a thick pad of disorganized mesoderm lies between the thin shell of abnormal ciliated epidermis (stippled) and the unenclosed endodermal mass. e, eyecup; ev, ear vesicle; g, gills and pharynx; pn, pronephros; s, somite segments; fb, forebrain above cement gland; bp, blastopore. Scale bars, 1mm.
	Fig. 4. The effect of blastocoelic MIF injection on morphogenesis of embryos, and on properties of grafted blastocoel roof tissue. (A) A group of one normal and five abnormal siblings at stage 37. The normal individual is second from bottom. The upper group show external appearances after low (top) and moderate (2nd and 3rd) doses of MIF. Even individuals such as that at top have no heart or blood and little pronephric development, while somite disorganization (see Fig. 2G) and reduced epidermal and CNS cell populations are evident. As the syndrome becomes more severe, only posterior axial structure and notochord is recognizable, while unorganized muscle or significant accessory tail and CNS formation may occur anteroventrally. Higher MIF doses abort gastrulation. Above and below the control embryo are two individuals with highly organized but anteriorly incomplete second axial patterns, following the grafting operation described in B. (B) An operation in which a piece of blastocoel roof tissue from a donor blastula, previously injected with MIF or a control solution, is grafted into the ventral marginal zone of a late blastula host. Grafts (100-300 cells) from control injected donors or from those receiving less than about 006 units MIF, integrate into host posteroventral regions with no disturbance to morphogenesis (see lower larval sketch). Grafts treated with higher doses of MIF, even after careful washing before implantation, act like dorsal blastoporal lip tissue and organize the development of second axial patterns (see upper larval sketch and A,C). The host blastula is represented in sagittal section with its own prospective dorsal lip site to left. (C) Anterior tissue structure in horizontal sections from the two secondary axes following grafting shown in (A). The level of anterior structure reached in these depends upon graft size and original donor MIF dosage. Both those shown are incomplete, one reaching pronephric levels but showing a small notochord piece in its otherwise fused somite axis, the other reaching ear vesicle and hind brain induction levels, without notochord. Note highly organized somite segmentation, which does not occur anywhere within donor embryos after the doses of MIF used in these experiments (see Fig. 2G). eg, cement gland; nc, notochord tissue; ev, ear vesicle; pn, pronephros; s, somites; tb, tailbud. Scale bar: 1 mm for drawings of B.
	Fig. 5. Injection of poly(A)+ RNA from XTC, but not XL, cells into animal pole blastomeres causes mesoderm induction. Poly(A)+ RNA from XL cells (A,B) or XTC cells (C,D) was injected into the animal hemisphere of Xenopus embryos at the 2- to 4-cell stage. At the mid-blastula stage (stage 8) the animal pole regions of these embryos were dissected out and allowed to develop in 67 % NAM. (A) DAPI staining. (B) The same section stained with 12/101; no muscle is formed. (C) DAPI staining. (D) The same section stained with 12/101; large amounts of muscle are formed in response to XTC poly(A)+ RNA. Scale bar in (D) is 200um and applies to all frames.
	Fig. 6. Explants combining MIF-treated and lineagelabelled animal cap tissue. (A) Preparation of explant combinations. The left-hand side shows how one donor embryo is lineage labelled with RLDx and allowed to develop to stage 8 (about 7h). The centre column shows how competent animal cap tissue at stage 7 is exposed to MIF and excised with careful washing after about 1 h at stage 8+. The right-hand side shows how the same tissue, but past its competent period at stage 11 + , is similarly exposed to MIF. Experimental combinations are prepared from left and centre components, control combinations from left and right. (B) Upper (muscle immunofluorescence) and lower (lineage label) images from a sectioned combination at control larval stages. The bulk of muscle develops from the directly MIFexposed component, but morphogenesis frequently results in such muscle being overlain by lineage-labelled epidermis. In C-H, muscle immunofluorescent images from sections are on the left, and their paired lineage label images on the right. (C,D) Even within the mesodermal tissue that fills the explant combinations, nonmuscle regions including lineage-labelled cells frequently abutt onto muscle. Freely diffusing transfer of the original MIF does not seem to cause systematic muscle induction adjacent to treated tissue. (E,F and G,H) Two examples of the incorporation of lineagelabelled cells into the periphery of a region of muscle development. Arrowheads indicate cells labelled with RLDx that are also stained with anti-muscle antibody. Scale bar: 1 mm in drawings of A, and 65um on photomicrographs B-H.