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A central topic of embryology is the establishment of the body plan during embryogenesis. Starting with maternal factors distributed in the early cleavage stages in distinct patterns and gradients cell-to-cell interactions including early embryonic induction result in the formation of mesoderm and the organizer area. While many facts are known about the role of growth factors like activin (closely related to the vegetalizing factor), processed Vg1, BMPs and FGF for mesoderm formation, the establishment of the central nervous system is not yet well understood. However, there is growing evidence that neural induction is a multistep process at the level of the dorsal mesoderm (organizer) and the reacting neuroectoderm. Therefore the existence of only one neuralizing factor is unlikely. We report about data that follistatin protein is not a direct neural inducer. Furthermore our comparative studies of Xenopus and Triturus exogastrulae indicate that planar signals are unlikely in the Triturus embryo (urodeles) during the early steps of neural induction. Vertical signals emanating from the chordamesoderm are essential for the terminal neuralization and regionalization of the central nervous system during gastrulation for both Xenopus and Triturus. The putative role of neuralizing factors and BMP/activin-like molecules for the stabilization or shift of neuroectoderm into different pathways of differentiation (epidermis or neural default state) is discussed.
Fig_ 1. Test methods. (AI Imp/antation-method (Einsteck-Experiment
after Spemann and Mangold, 1924): 1)blastopore lip; 2) pef/et (insoluble
factors or so-called heterogeneous inducers). !B) Sandwich-Technique
(Holtfreter, 1933a) (insoluble factors or heterogeneous inducers). an, animal
cap (ectoderm). IC) Nuclepore@-chamber (Grunz and Tacke, 1986;
modified after Saxen, 1961) for tests of soluble factors, which can only
act on the former blastocoelicside of the explant. The Nuclepore@-filter
prevents the curling up of the ectoderm. (DJ Test plate (1) with flat -bottom
wefls (Terasaki-plate) Test of soluble factors in smaff amounts of
medium (10 IJI)like FGFor activin (anima/cap assay); (2) one weff at higher
magnification. N. Nuclepore-membrane; Pa. outer Plexiglas-ring; Pi,
inner Plexiglas-ring; CM. culture medium; M, O2 and COTpermeable
membrane (Petriperm@, Fa. Heraeus). Further test methods are
described by Hildegard Tiedemann (1986).
Fig. 2. Differentiation of ectoderm
after dissociation and reaggregation
(details of the treatment see Table
1). (AI Disaggregated cells treated
with 0.02 ng/ml activin (EDF) (Table 1,
series 19). The reaggregated cells differentiated
mainly into neural (archencephalic
brain, neu) structures. A tiny
notochord (no) was found in addition.
ce, cement gland. !B) Disaggregated
cells treated with 0.2 ng/mf activin
(EDF) (series 18). Both archencephalic
brain structures (neu) and a substantial
amount of notochord (no) were found
in this series 18. ce, small cement
gland; ep, epidermis. (CI Disaggregated
cells treated with 2 ng/ml
activin (EDF) (series 17). The reaggregated
cells differentiated totally into
notochord. No other differentiation
could be found in addition. tD)
Disaggregated cells treated with 150
tJM Suramin (series 22). The reaggregated
cells differentiated into neural
derivatives as untreated controls. IE)
Disaggregated cells treated with 10
ng/ml basic fibroblast growth factor
(bFGF) (series 20). The reaggregated
cells formed mesenchyme (me) in
addition to the brain structures (neu).
The presence of mesenchyme and the
histological features suggest that
these differentiations are deuterenchepllc
brain structures. FGF is able
to shift the pattern from anterior to
posterior brain structures. ep. epidermis.
(F) Disaggregated cells treated
with 10 -7 M retinoic acid (series 2)
The reaggregated cells formed neural
derivatives, which differ distinctly from
the archencephalic structures shown
in (A and B). We suggest that the
structures are similar to deuterencephalic
(deu) brain structures
Fig. 3. Animal caps (competent ectoderm) of Xenopus- or Triturusmiddle
or late gastrulae were treated with 1, 10, 100 and 1000 ng/ml follistatin (recombinant human follistatin) for 6-8 h. Several explants were cultured for 3 days for histological analysis. The cases shown above (Xenopus-early gastrulaectoderm treated with 1000 ng/ml follistatin) were fixed in HEMFA after 24 h for whole-mount in situ preparation
with a neural specific {3-tubulin (Richter et al., 1988). In contrast to
the larva (control) the explants (an) show no neural specific signals. Also
by histological analysis (not shown) we could confirm that Xenopus- and
Triturus- ectoderm treated with different concentrations of folfistatin (1-
10.000 ng/mf) differentiated in atypical (ciliated) epidermis only. neu.
neural tube; br. brain with eye vesicle; ot, otic vesicle
Fig. 4. Comparison of the gastrulation processes of Urodela
(Tr;turus alpestris) (A-D) and Anura (Xenopus laevis) IE-H). bp,
blastoporus, m, marginal zone; neu, neuro-ectoderm; pres. Ep., presumptive
epidermis. (A,E) Very early gastrula (stage 10, Nieuwkoop and
Faber, 1956) In Xenopus the ectoderm and the marginal zone consists of
two cell/ayers. Mesoderm is already located inside of the embryo. (S,F)
Early phase of neuralinduction. Rolling-in of the marginal zone at stage
10-10.5. In Xenopus (F) the internal marginal zone only is movingto the
animal pole (compare with plate 18, Hausen and Riebesefl, 1991) #,
zone of vertical signals, *' , zone of postulated planar signals. fC,G)
Advanced stage of neural induction by vertical signals. The primary steps
of neural induction are nearly finished. (D,H) Small yolk plug stage, The
primary steps of neural induction are now finished. The 3 germ layers
have reached their final position
Fig. 5. Exogastrulae of Xenopus Jaevis have formed neural structures in addition to mesoderm derivative tissue. (AI Whole-mount in situ
preparation with the neural-specific marker np 187 (control embryo). (Dissertation C. Schiiren, Essen). IB) Exogastrula, which shows the marker not
only in the ectodermal zone (a) but also in the intermediate (b) and the meso-endodermal part (c). IC) Histological section of an exogastrula of the
same series after 3 days' culture at 20�C. The transversal section (see arrow b in 58) shows that neural structures are accompanied by notochord
(D) Transversal section of an exogastrula in the area (c) (see 58). In this part of the endo-mesoderm neural structures were not observed. Transversal
sections {not shown] of the distal part of the ectoderm (zone (a) in 58) contain neural structures only- However, notochord together with neural structures
is found in the neighbouring sections in the zone between (b) and (c). The differentiation of neural structures accompanied by notochord in both
the ectodermal and meso-endodermal part explains the expression of neural markers and indicates that the neural structures were induced by vertical
signals emanating from the chordamesoderm (notochord) no, notochord; ne, neural structures; so, somites; en, yolk-rich material (endodermderivative)
Fig. 6. The ectoderm of an early gastrula of Triturus alpestris was
separated from the meso-endoderm by micro dissection. A bridge
remained between the ectoderm and meso-endoderm in the area of the
blastopore (Grunz et aI., 1995). (AI Pseudoexogastrula after 11 days' culture
at 20"C The ectoderm separated from the endo-mesoderm has differentiated
into ciliated (atYPlcaf)epidermis. (8) Neural structures(brain
structures and an eye with lens) have differentiated in the intermediate
zone between endo- mesoderm and ectoderm only. The neural structures
are found close to notochord (see inset) en, endoderm or derivatives;
ee, ectoderm (atYPical epidermis); no, notochord; ne, brain structures;
ey, eye; Ie. lens
