Cooke J and Smith JC (1987), The midblastula cell cycle transition and the c...

XB-ART-28286

Development 1987 Feb 01;992:197-210.

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The midblastula cell cycle transition and the character of mesoderm in u.v.-induced nonaxial Xenopus development.

Cooke J , Smith JC .

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Xenopus embryos (UV embryos) resulting from u.v. (254 nm) irradiation to the vegetal egg hemisphere and thus developing little or no axial pattern (UV5-Scharf & Gerhart, 1983), have been compared histologically with synchronous normal siblings at each of three stages. In addition, the relative amounts of blood-forming tissue produced in normal and in UV embryos have been studied by Western blotting total protein from larval stages and by immunofluorescence on sections. The observations on midblastulae (around 5000 cells) were aimed at detecting any systematic retardation, due to u.v., of the slowing of the cell cycle that normally commences at the 2-4000 cell stage and makes possible zygotic transcription and the preparation for gastrulation. No such retardation was apparent. Observations on postgastrular stages gave an assessment of the size and character of the population of mesoderm founder cells, in relation to the control, for embryos visibly undergoing entirely nonaxial development. Little deficit in total mesodermal cell number was found, though the entire mesoderm adopted the histological character proper to only some 40% of that in the normal pattern i.e. trunk lateral plate. Blood-forming capacity appears to be enhanced out of all proportion to the size of the mesoderm as a whole. The results are discussed in terms of the probable nature of the primary positional system for axial pattern and the later mechanisms of mesodermal patterning.

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Species referenced: Xenopus
Genes referenced: alb mhc2-dab uqcc6

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	Fig. 1. Cell number and mitotic incidence in synchronous normal and UV siblings during midblastula transition. Nuclear number, on the ordinate, refers to the sum of nuclei actually transected in every fifth 7/an section, in that portion of the blastula shown in the inset diagram (ap, animal pole). Estimates of total cells in the whole blastulae, derived by including the animal cap and using the appropriate Abercrombie correction (1946), lie in the expected range at 5000-8400. Each time point is represented by three control (•) and three u.v.-irradiated (O) blastulae, and the first time point is at the projected onset of the 13th round of cleavage extrapolated from the pre-MBT cleavage intervals. Thus it is seen that in the population as a whole, somewhat over two thirds of the blastomeres initially present divide over the hour spanned by the observations, whereas pre-MBT cycles were 27 min long at 20°C in the egg batch concerned. The overall lag in cell number in the UV samples is barely statistically significant (0-05 <P<O-1), though a similar marginal deficit was seen in the other MBT experiment. Control sample mitotic incidences are given above each set of cell number points, UV, below, but no difference at any time point was significant. A photograph of the cellular texture of the marginal region of midblastular material is included, but the delicate Feulgen staining of nuclear figures at such early stages is not well revealed. Bar, 100 Um.
	Fig. 2. Histological structure of UV5 embryos and controls at control stage 13. (A-C) Anterior, middle and posterior levels of transverse section through a normal stage 13, early neurula. A includes only prechordal mesoderm, B includes notochord and dorsally converging somitogenic tissue and C is in the thick circumblastoporal zone of mesodenn recruitment. (D-F) Three equivalent levels of section through a UV5 sibling at an equivalent stage of blastopore closure, 20min older than the control. Little evidence of deviation from radial symmetry of architecture is seen. Bar, 100 um.
	Fig. 3. The mesoderm at stage 13. In A-F, tracings of the sections of Fig. 2 are laid out as in that figure, with the mesodenn profile shaded to demarcate it from the inner endoderm mass (which may exhibit an archenteric cavity), and from the enclosing ectoderm. Below this are diagrammatic midsagittal sections of the normal (G) and UV5 (H) development. These show, respectively, the dorsal-ventral and axial character of mesoderm and endoderm, and its absence in the experimental development, pc, prechordal, nc, notochord, r, recruitment zone, bl, blastocoel remnant. Bars: A-F, 100um; G,H, 300um.
	Fig. 4. Histological structure of UV5 embryos and controls at control stage 32. (A-C) Head, middle (pronephric) and rx)sterior levels of transverse section through a normal axial larva. (D-F) Three positions of transverse section through a UV5 of the same age, containing early-, middle-, and late-recruited mesoderm. nc, notochord, 5, somite, pn, pronephros, Ipl, lateral plate character of mesoderm, br, brain parts including eye. Bar, 100 um.
	Fig. 5. Muscle myosin and larval globin synthesis in normal and UV5 development at control larval stage 38. (A) Western blot of total larval protein, probed with anti-muscle myosin antiserum. Two sets of four tracks bearing serial twofold dilutions are shown. On the left, the sample was prepared from a set of ten normal larvae. On the right, preparation was from ten synchronous UV5 examples. The arrow marks the band revealing myosin, and the first (right-hand) track of each series, representing one embryo's worth of protein, is indicated above. (B) Lanes from a Western blot probed with anti- Xenopus larval globins. The left-hand lane represents one embryo's worth of protein from a normal larval sample as in (A). The right-hand lane represents one embryo's worth of protein from a UV5 sample. The arrow marks the band revealing globin concentration. Larvae were solubilized in Laemmli sample buffer and loaded onto linear 5-15 % polyacrylamide gradient gels 1-5 mm thick. The separated proteins were transferred electrophoretically to nitrocellulose. The membrane was 'blocked' overnight at room temperature in 10 % normal goat serum and 4% bovine serum albumin in PBS-A. It was incubated for 2 h in a 1/200 dilution of anti-globin serum in 1 % BSA in PBS-A. After five washes in PBS-A over 1 h, it was incubated in a 1/500 dilution of HRPconjugated affinity-purified goat-anti-rabbit IgG (Miles) for 2h, then re washed. The nitrocellulose was probed for peroxidase activity using DAB.
	Fig. 6. Distribution of larval globin production in normal and UV5 development at stage 34. 10/an wax sections transverse to the long axis, treated by indirect immunofluorescence with rabbit anti-Xenopus globin and FITC-conjugated goat anti-rabbit IgG and counterstained with aqueous eriochrome black. (A) The normal heart level, showing absence of red blood cells in the circulatory spaces at this stage. (B) Level midway between heart and blastopore in normal larva, showing the restricted and compact midventral sector from which blood originates. The sector is never wider than shown and diminishes towards the blastopore. The tissue is not structurally distinguishable from adjacent lateral plate at these stages. (C-F) Successive sectional levels through typical UV5 body, from anterior grazing section of mesoderm and covering ectoderm to a level halfway back to the terminal blastopore. Distribution of globin is ubiquitous and apparently solid in mesoderm of first two levels, but the appearance at levels around E shows that there is a rather sharp though irregular posterior boundary to the globin-producing region, again unrevealed by any structural distinction in ordinary light microscopy although the tissue tends more towards a multilayered and open texture anteriorly. Globin is never seen from level F back. The fluorescence associated with globin production, while perfectly distinct, tends to be slightly less in the UV5 case at an identical time after fertilization, as might be expected in view of the nonspecific delay of 3-4 h in developmental stage shown by partial axial UV forms at these stages, h, heart; end, yolky endoderm; Ipl, mesoderm of lateral plate character; ect, ectoderm; Hb, fluorescent appearance marking haemoglobin production. Bar, 100um.
	Fig. 7. Suggested events delimiting the area of larval blood production in normal and in radialized UV development. Longitudinal sections of normal (A) and UV5 (B) embryos are shown at the close of gastrulation. The mesoderm in section is shown stippled and, in (A), this corresponds to the sagittal plane of left-right symmetry. The anterior mesoderm border, corresponding to the earliest involution, is shown at the left by a heavy dashed line in each case, while arrows more posteriorly indicate the direction and vigour of migration (and dorsal convergence in the normal case) of the later-involuting tissue. The final area of production of haemoglobincontaining cells, extending back from the anterior mesodermaJ border, is delimited by a thin black line. In the normal case, the dorsal and posterior zone devoted to somite tissue is delimited by a single dashed line, and this incorporates the later-involuting mesoderm from almost all around the blastopore. Normally, 'dorsal' pattern elements probably act to limit by suppression the area in which 'ventral' developments such as blood occur, whereas such signals are absent in the UV5 embryo. But the mechanism whereby blood is specified only in earlyrecruited or 'anterior' mesoderm remains. In the normal neurula, mesoderm initially behind this is swept upwards past the blastopore to join the somite axis in the dorsalization process, thus giving the definitive blood domain a posterior border that abuts on the proctodaeum or blastopore remnant in the later larva.