Cooke J and Smith EJ (1988), The restrictive effect of early exposure to lit...

XB-ART-27709

Development 1988 Jan 01;1021:85-99.

Show Gene links Show Anatomy links

The restrictive effect of early exposure to lithium upon body pattern in Xenopus development, studied by quantitative anatomy and immunofluorescence.

Cooke J , Smith EJ .

???displayArticle.abstract???
We have carried out an anatomical study of Xenopus larval and gastrula stages resulting from treatment of synchronous early blastulae for brief periods with Li+. We confirm the proposal that such treatment causes a particular transformation, and partial elimination, of the normal body pattern. Coordinated restriction of pattern, without appreciable loss of cell number, is seen in all three germ layers. The distortion has been investigated by quantitative study of mesoderms at a standard stage, in relation to the normal fate map for mesoderm, and with the help of immunofluorescence on sections for somitic muscle and for blood. In the extreme syndrome, mesoderm arises from all around the blastula as usual, but is symmetrical and corresponds to that arising near the dorsal/anterior meridian of the normally specified egg or embryo with a large posterior subset of the normal pattern values thus missing. The effect is independent of any inhibition of archenteron formation or mesoderm migration (i.e. the cell mechanics of gastrulation) incurred by the treatment. It is also quite separate from a syndrome caused by more prolonged exposure to Li+ during gastrulation. A small, but distinctive, anterior pattern region is also not expressed and, anomalously in relation to their general nature, these forms differentiate considerable blood tissue. We consider the implications of some details of the pattern restriction for our understanding of interaction in the normal development and propose that the Li+ embryo is likely to be useful as a specific 'differential screen', in relation to the normal, during the search for those gene products that mediate initial regionalization of the body.

???displayArticle.pubmedLink??? 3416774

Species referenced: Xenopus laevis
Genes referenced: tbx2 uqcc6

???attribute.lit??? ???displayArticles.show???

	Fig. 1. Graded external morphologies following early blastular lithium treatment. (A) A group of Li+-treated embryos at late tailbud stages, surrounding a control of normal morphology. The upper group of four have severe versions of pattern restriction (grades 4, 3) while the lower pair represent grades 2 and then 1, the attenuated versions. (B) Sketches of the external forms seen at stage 32. Reading from left to right and top to bottom, these are the normal; gTade 4; the versions of grade 3 with incomplete (iii) and complete (iv) blastopore closure (see text); and a grade 2 (v) and grade 1 (vi) pair as in A. (C) Lateral views of a normal early gastrula stage 10 (upper) and of a synchronous embryo due to develop into a severe (gTade 4) pattern-restricted form. Note the high, near-equatorial position and synchronous, ring-shaped development of the zone of blastoporal activity. Scale bar for the drawings, approx. 1 mm. eg, cement gland; ec, eyecup; rn, radial neuralized area; ev, ear vesicle; g, gill architecture; fl, prominent flange-shaped zone of gut morphology (see text); bl, original blastocoel cavity; pn, pronephros; 5, somites; bp, blastopore or open blastoporal rim.
	Fig. 2. Amounts of somite and notochord, and occurrence of blood, in fully pattern-restricted bodies. (A,B) Phase-contrast and immunofluorescent images for Xenopus somite muscle in a stage-40 grade-4 Li+ body. Note massiveness of notochord (nc) and very small allocation to somite, whose position is indicated by an arrow on the phase picture. (C,D) Immunofluorescence for muscle in transverse sections of stage-40 normal development, at ear vesicle/hindbrain and trunk axial levels respectively. Note the small allocation of somite, in relation to the more constant-sized notochord allocation, at the former more anterior level. (E) Part of a 'horizontal' section (stage 40) through that portion of the fully pattern-restricted body that hangs downmost in gravity, showing cells resembling the normal immature blood of the mid 30s stage larva in Feulgen, light green and orange G staining. Note intense nuclear staining and free, nonadhesive appearance of the cells concerned. (F,G) Immunofluorescence for Xenopus larval and adult globins at control stage 40, in part of the grade-4 Li+ body near that seen in E and in a grazing section near the heart of the normal larva, respectively. Note relatively massive development but immaturity of the blood tissue in F as opposed to the brightly fluorescing, circulating blood cells of G. Magnifications, (A,B,F,G) xl60, (C,D) X100, (E) x250.
	Fig. 3. Sections through severely pattern-restricted lithium bodies. (A) Horizontal section at notochord level through the normal stage-32 body; br, brain. (B) Section through the turret-shaped neural area and grazing the ring of cement gland formations, in a grade-4 Li+ embryo. The evaginations from a central neural cavity and the peripheral formations with the appearance of eyecup indicate the diencephalic level of development. (C) A subsequent level of section and from, a body showing somewhat more anteroposterior directionality. Note the massive notochord allocation, the more clearly retinal structures (more than two are typically seen), the narrow strips of unorganized somite material and the transversely sectioned tunnel of archenteron traversing the posterior 'side' of the notochord. The notochord shows no gradation of differentiation stage; e, eye; /, lens. (D) Similar section level to C but showing an ear vesicle in addition to eyecup and midbrain profiles, and a wider archenteron tunnel which is not passing 'posterior' to the notochord. (E) Horizontal section through the pharynx or gill architecture of the normal body. Note shape of the thin endodermal wall and character of the relatively massive investing mesenchymes. (F,G) Sections through the pharynx or gill region in two almost radial patternrestricted bodies. Note the shape and thinness of the endodermal wall of the main archenteric cavity and the mesenchymal areas. The thick downwardly projecting 'peg' of notochord and its accompanying minor archenteron, passing down through the larger endodermlined region, is also seen. (H) Sectional level beneath that of F and G, and in a body with more evidence of directionality of pattern. The now thick-walled endoderm is invested with broad, squamous endothelial-lined cavities (ei) reminiscent of the portal system between liver and heart regions of the normal body. A grazing section of endoderm-like the normal posterior pharynx floor (not illustrated) is also seen. Other labels as in Fig. 1. Magnification, x63 throughout. Scale bar, 1mm approx.
	Fig. 4. Sections through bodies with attenuated versions of the pattern restriction. (A) Grade-2 body at notochord level. Four somite segments are apparent opposite the fully differentiated, anterior notochord sector. The single focused cement gland is seen. The placode of singlelayered, cuboidal epithelium at top left, though open, almost certainly represents ear vesicle. (B) Higher power view of the rear end of the body of A at a more ventral level near the blastopore. This shows an obliquely sectioned portion of the impacted, immature notochord, with small allocations of disorganized somite tissue, characteristic of the grade 2 or 1 body behind the zone of properly formed pattern. (C) Grade-1 body at notochord level. About ten proper somite segments are apparent. The mid- and hindbrain and one ear vesicle are sectioned horizontally. A zone of impacted notochord as in A is encountered between the most posterior somite seen and the blastopore. Disorganized pronephros is seen on one side. (D) Horizontal section through the middle of the optic chiasm, normally opposite the anterior boundary of the notochord in a normal stage-40 larva. (E) Section 50 fim 'ventral' to D in same larva. This passes through the significant forebrain cavity, i.e. a more 'anterior' brain region where no notochord underlies the brain. An appearance like G below is not seen until 40 fim beyond this level. (F,G) Sections through midoptic chiasm (c/i) level, and only 40 jim ventral or 'anterior' to this, in a grade-1 pattern-restricted stage-40 larva. Note that in contrast to the normal, notochord is coextensive with brain, and the anterior section grazes the anterior end wall of the nervous system in which no forebrain formation exists in front of the optic chiasm. Other labels as in previous figures. Magnifications, (A,C) x63, (B,D-G) X160.
	Fig. 5. The nature of the pattern restriction. (A) Fate map from the presumptive right-hand side showing the general way in which material from around the meridians of the pregastrular embryo is used in normal construction of the axial mesodermal pattern (see Cooke & Webber, 1985). The two heavy curved lines mark off restricted sectors of this map and represent subsets of mesodermal pattern that occur in the limit form and in more attenuated versions of the Li+ syndrome. The restricted pattern is then produced, however, from mesoderm all around the embryo. (B,C) Longitudinal sections of normal neurula (sagittal plane) and of synchronous form fully radialized after blastula lithium treatment (compare with Cooke & Smith, 1987, fig. 7 for u.v.-induced nonaxial development). Axial (notochordal) mesoderm is shown hatched, other mesoderm stippled, and endoderm indicated by crosses. Differences in thickness between axial and other mesoderm, and between neuralized and epidermal ectoderm, are somewhat exaggerated in relation to the early neurula stage 14 actually dissected. In C, the cavity of the original blastocoel remnant, pushed ventrally and free of mesoderm in the normal case, is obliterated by contact from the endoderm onto its epidermal wall whose inner face appears to have been ectopically mesodermalized. A new intraendodermal cavity forms to conserve the volume of the embryo (see also embryos treated with soluble mesoderm-inducing factor - Cooke et al. 1987). The embryos are drawn as they orientate in gravity, as the annular, shallow archenteron of form C causes its blastopore to face upwards as well as being surrounded by the neuralized and notochordal areas. There is later a transformation to give an archenteric passage by-passing a notochordal mass, because of the latter's demand for convergence - minimization of surface area in relation to cell number. (D,E) Exploded diagrams of notochord, induced neural and endodermal (gut) structure in the normal and the limit pattern-restricted larval body. Since gastrulation effectively turns the presumptive gut and mesoderm inside out, reversing its polarity, the normal body is drawn mirror-imaged with respect to A. Labelling of structures is as in previous figures, with addition of pc, prechordal region, h, heart-forming region, b, bloodforming region and/, m, h, fore- mid- and hindbrain regions of neuralized ectoderm and CNS. Scale bar, 1 mm approx. for drawings of A-C.