164
Pattern formation
The dependence of DNA and protein biosynthesis on cytoplasmic pH
during the cell cycle in Dictyostelium discoideum
Rob Aerts, Tony Durston and Wouter Moolenaar, Hubrecht Laboratory, Uppsalalaan 8, 3584 CT Utrecht,
The Netherlands
We measured cytoplasmic pH in Dictyostelium discoideum under various conditions, using the digitonin
null-point method (Rink, T. J., Tsien, R. Y., Pozzan, T. (1982) /. Cell Bioi, 95,189. In synchronised cell
populations the cytoplasmic pH clearly fluctuates during the cell cycle. These fluctuations coincide with
fluctuations in the rates of biosynthetic processes; the rates of DNA synthesis and protein synthesis show a
sharp optimum in the cytoplasmic pH range 7-40 to 7-45.
The cytoplasmic pH of Dictyostelium discoideum can be changed by certain weak acids and bases. The
effect of ammonia is concentration dependent; a low concentration is able to increase cytoplasmic pH, high
concentrations have the opposite effect. Artificial manipulation of cytoplasmic pH influences the rate of
DNA and protein synthesis and shows the same correlation between these processes and cytoplasmic pH as
mentioned above. On the other hand, if DNA synthesis is inhibited in synchronous populations of cells then
the cell cycle fluctuations in cytoplasmic pH continue.
The interrelation between cytoplasmic pH and DNA synthesis in Dictyostelium discoideum as revealed by
the experiments summed up above corresponds strikingly to the relation between intracellular pH and DNA
synthesis established in a taxonomically quite different system, namely lymphocytes of the mouse (Gerson,
D. F., Kiefer, H., Eufe, W. (1982) Science 216,1009). That in Dictyostelium discoideum, on the one hand,
artificial manipulation of cytoplasmic pH influences the rates of biosynthetic processes, and on the other
hand, fluctuations in cytoplasmic pH continue after DNA synthesis inhibition suggests that the cytoplasmic
pH may be a general regulator of the cell cycle.
The differentiation direction of a Dictyostelium discoideum cell depends on the cell cycle phase in which it
enters development (McDonald, S., Durston, A. J., /. Cell ScL, in press) and as we have shown, the
cytoplasmic pH (and the concomitant rates of biosynthetic processes) changes with the cell cycle phase.
Furthermore a recent model proposes that a low intracellular pH favours stalk cell differentiation and a high
intracellular pH favours spore differentiation (Gross, J. D. etal. (1983).
Nature, 303, 244). With regard to
this it is particularly interesting to note that we find higher rates of 3H-uridine incorporation in the prespore
region of the slug than in the prestalk region.
Morphogenesis in the chick limb
Karen Bell and J. C. McLachlan, Oxford University, Dept. Zoology, South Parks Road, Oxford
Two regions of the chick wing bud have been shown to influence pattern and growth during development.
The apical ectodermal ridge (AER) is essential for the outgrowth of the limb and the formation of the
proximo-distal axis (Summerbell, 1974). The AER appears to influence the positional value of cells in the
distal mesoderm by controlling their density and division rates (Summerbell & Lewis, 1975). The zone of
polarizing activity (ZPA) is an area of cells at the base of the limb bud which, when transplanted anteriorly,
induces the formation of supernumerary limb structures along the antero-posterior axis (Saunders &
Gasseling, 1968). Considerable growth is observed in the limb bud following ZPA grafting, which results in
the host tissue doubling within 36 hours (Cooke & Summerbell, 1980).
To investigate cell division and pattern formation in vitro we cultured ZPA and AER cells with an
embryonic cell line (mouse 3T3 cells) and measured changes in the initiation of DNA synthesis by the
uptake of labelled thymidine. At the same time distal and posterior tissue were grafted anteriorly into host
wing buds. These were either labelled with tritiated thymidine 16-19 hours after operating, and sectioned,
or allowed to develop for 7 days to assess gross cartilage patterns. Anterior wing tissues was used as a
control in all the experiments. So far we have shown that chick tissue can induce 3T3 cells to initiate DNA
synthesis, while conditioned media and non-contact experiments suggest that a diffusible substance is
involved.
COOKE, J. & SUMMERBELL, D. (1980). Cell cycle and experimental pattern duplication in the chick wing
during embryonic development. Nature, 287, 697-701.
SAUNDERS, J. W. & GASSELING, M. T. (1968). Ectodermal-mesenchymal interactions in the origin of limb
symmetry. In Epithelial-Mesenchymal Interactions (ed. R. Fleischmajer & R. E. Billingham), pp. 78-97.
Baltimore, Williams & Wilkins.
SUMMERBELL, D. (1974). A quantitative analysis of the effect of excision of the AER from the chick limb
bud. /. EmbryoL exp. Morph. 32, 651-660.
SUMMERBELL, D. & LEWIS, J. H. (1975). Time place and positional value in the chick limb bud. /. Embryol.
exp. Morph. 33, 621-643.
Pattern formation
Metamorphosis and pattern formation in Hydractinia echinata
165
S. Berking, Zoologisches Institut, INF'230, D-6900 Heidelberg, West Germany
Hydractinia echinata is a marine, colony forming Coelenterate. Fertilized eggs develop into freely
swimming planula larvae, which undergo metamorphosis to+ a sessile (primary) polyp. Metamorphosis can
be triggered by means of certain marine bacteria and by Cs ions. Half a day after such a treatment a larva
will have developed into a polyp.
The induction of metamorphosis can be prevented by addition of inhibitor I, a substance partially purified
from tissue of Hydra. The larvae of Hydractinia echinata also appear to contain this substance. Inhibitor I
applied
after the onset of metamorphosis blocks its continuation as long as it remains in the culture medium.
Cs + ions applied within the same time period also block the continuation of metamorphosis. However, these
two agents have opposite
effects on the body pattern of the resultant polyps. The experiments indicate that
the application of Cs + ions trigger the generation of the prepattern. Inhibitor I appears to be an element of
this prepattern.
Anterior-like cells may play a role in cell type proportioning in
Dictyostelium
Angela Blaschke, Cornells Weijer, Harry MacWilliams*, Zoologisches Institut, Luisenstrasse 14,
8000Munchen2, West Germany
Most discussions of cell type proportioning in Dictyostelium consider only the prestalk and prespore cells;
the 'anterior-like' cells (resembling prestalk cells but dispersed in the prespore zone) are ignored. We have
investigated the role of anterior-like cells by studying these cells in chimeric slugs; the chimeras were
mixtures of prestalk-prespore proportioning mutants with their parental type or of axenic cells grown with
(G+) and without (G—) glucose. The cells were mixed in the vegetative phase; in each case one of the cell
types had been labelled with tetramethylrhodamine isothiocyanate. Chimeric slugs were dissected into
prestalk and prespore zones and disaggregated into single cells. MUD-1 (a monoclonal antibody against
prespore cells, donated by M. Krefft) was used to distinguish prespore from anterior-like cells in the
disaggregated prespore tissue. We could thus determine the ratio of mutant to wild type (or G+ to G - )
among prestalk, prespore, and anterior-like cells.
The short-prestalk mutant HS2 shows a reduced fraction of prestalk cells but a normal complement of
anterior-like cells. In mixtures of HS2; with its parent HS1 the prespore cells are enriched in HS2; the ratio
of mutant to parent among anterior-like cells is essentially the same as that among prespore cells.
The long-prestalk mutant HS3 shows an increased fraction of both prestalk and anterior-like cells. In
mixtures of HS3 and HS1 the prestalk zone is enriched in HS3; the HS3: HS1 ratio in the anterior-like cells
is essentially the same as in the prestalk cells. Similar behaviour is seen in mixtures of G - and G+ cells.
These findings cannot be explained with a 'two-threshold model' in which different levels of the same
parameter are necessary for prestalk and anterior-like differentiation. The findings support a model in which
two. independent feedback loops regulate cell type proportions in slime molds. One loop controls the
proportion of anterior-like cells to prespore cells; the other controls the ratio of anterior-like cells to
prestalk cells. HS3 and G— cells are modified in sensitivity to the feedback signal in the first loop (but not in
the second); HS2 is modified in sensitivity to the feedback signal of the second loop (but not in the first). It is
conceivable that there is no direct interaction between prestalk and prespore cells.
166
Pattern formation
Proportion regulation in hydra is complex
Hans R. Bode* and Patricia M. Bode, Developmental Biology Center, University of California, Irvine,
California, CA 92717, U.S.A.
Excision of a piece of the body column of hydra results in regeneration of a diminutive version of the
animal in a morphallactic manner. As part of the patterning that occurs, tissue is allocated to each of the
structures: hypostome, tentacle zone, tentacles, body column, and basal disk. An approach to the processes
underlying this allocation is to examine the extent to which proportion regulation occurs. If a high degree
proportion regulation exists, a constant fraction of the tissue will be allotted to each structure independent
of the size of piece and the regenerate would be a quite precise miniature of a normal adult. Excised pieces
of the body column varying 40-fold in size were analyzed to determine quantitatively how the pattern
changes as it is constrained to form in less and less tissue. Alterations were found in tissue allocation, body
shape and tentacle number. Only the total number of cells in the basal disk and in the tentacles remained
proportional to the whole animal. With decreasing size, the hypostome and tentacle zone increased
allometrically at the expense of body tissue. The body column circumference followed the circumference of
the head, which was proportionately larger in smaller animals, resulting in increasingly wider and squatter
columns. The size of the individual tentacles decreased gradually with decreasing size, as did the number of
tentacles, but the distance between them remained the same. The complexity of these proportioning results
indicate that patterning in hydra is more complicated than can be accounted for by models proposed to
explain these processes.
Topospecific antigens in insect appendages
1
D. Bulliere andF. Bulliere*2. ^Universiti des Antilles etde la Guyane, Lab. Biologie et Physiologie
Animates, BP592, F 97167 Pointe a Pitre Cedex Guadeloupe, French West Indies. 2Universiti Scientifique et
Mtdicale de Grenoble, Lab. Zoologie et Biologie Animale, ERA 621, BP 68 38402 Saint Martin d'Heres
Cedex, France
Grafting of tegument pieces have shown that the position of epidermal cells in cockroach appendages is
specified in such a manner that each cell shares characteristics with many others, but that the position of
each of them is unique.
Thus, the various cells of the tibia and the femur can belong to the same generatrix (appendage
assimilated to an articulated cylinder), to the same level in the segment, but differ according to the segment
character.
Experimental results have led us to search the molecular basis for these character types on the plasma
membranes.
By incubating membrane fractions of epidermal cells adsorbed on slides with antisera or monoclonal
antibodies, it has been shown that tibia membranes possess an antigenicity different from that of femur
ones. These differences have been shown again in situ on tibia and femur sections. Moreover, it has been
observed that some antibodies with tibia or femur specificity, link not only to epidermal cells but also to
internal tissues such as muscles and nerves. These observations lead to the idea that a specific adhesivity
associated with membrane molecular structures regulate recognition processes. These would allow
epidermal cells to form harmonious regenerates, muscle cells to find their place of insertion and nerve cells
to arrive at their zones of innervation, resulting in the appendage forming a functional unit.
Pattern formation
167
Dynamics of the pattern specifications system in the egg and embryo of
Xenopus
J. Cooke* andJ. Webber, Developmental Biology Division, National Institute for Medical Research, The
Ridgeway, Mill Hill, London NW71AA
The useful distinction has recently been made between states of 'specification' in embryonic tissue and the
later, cellular phenomenon of commitment or determination for particular pathways of development (Slack,
1983). Specified tissue has been given information which in the absence of subsequent stimuli will ensure
that its development corresponds with a particular part of the body-pattern, even though its cells, having as
yet no commitment, can attain new specifications in response to new (normal or experimentally imposed)
positional cues. The nature of commitment and the determined state is most relevant to those interested
primarily in the molecular biology of cell differentiation, but since normal development occurs via
unchallenged processes of specification, the nature of the specifying system and of the specified state must
be understood if we are to comprehend the origin and regulation of pattern.
Material may, in normal development, pass through states of specification which do not correspond with
its presumptive fate in the pattern, and thus its final committed state. This will be so if the specification
system develops gradually, either as a sequence of inductions or as a 'morphogen gradient' that is 'read'
locally by cells to give them positional values (Wolpert, 1971), but whose profile evolves only slowly towards
its final condition. Several lines of recent experimentation on early Xenopus (amphibian) development
suggest that specification of both the latter gradual kinds is in operation from the time of the early,
post-fertilisation events onwards. The understanding emerging from the experiments differs from the
classical or 'textbook' understanding of vertebrate embryogenesis, including that assumed in much of the
first author's previous work. According to this, only the 'organiser' region was specified (and determined)
from early stages, and this then acted as a reference position for the slow subsequent build-up of positional
specification elsewhere. In fact, at least in Xenopus, considerable though variable specification for general
body patterning is achieved right across the egg by the post-fertilisation events of the single cell stage, while
detailed specification is reached within presumptive mesoderm by the onset of gastrulation.
Feather pattern formation
Duncan Davidson* and Allyson Ross, M.R.C. Clinical and Population Cytogenetics Research Unit, West
General Hospital, Edinburgh EH42XU
The pattern of feathers in avian skin is intermediate in complexity between simple, irregular
arrangements of organs (e.g. primary hair follicles in mouse dorsal skin) and complex geometrical patterns
which display size-invariant regulation of the number of elements (e.g. cartilages in the veretebrate limb).
The triangular array of feathers in the skin is highly regular, but geometrically simple and the number of
feathers formed depends on the area of tissue available. The system provides an opportunity to study the
generation of a regular pattern without the burden of having to account for size-invariant regulation.
In chick dorsal skin, feather primordia first appear medially on the sixth day of development and over the
next two days a morphogenetic wave sweeps across either side of the back forming successive
anterior-posterior rows of primordia. Evidence from in vitro experiments suggests that there is (1) a local
spacing mechanism which operates between incipient feather sites, and (2) an overall age gradient in the
integument which predates the pattern of feather sites. According to one class of model these two levels of
organisation are sufficient to generate the pattern: by restricting local spacing interactions to a moving
frontier the temporal organisation provides the global order necessary for the formation of a regular spatial
pattern. Evidence for this class of model, its implications for understanding the relation between patterns of
different complexity, and the prospects for exploring it further in the feather system are discussed.
168
Pattern formation
Reticulate scales of the foot pads of the birds could be neotenic feathers
Danielle Dhouailly 1 and Roger H. Sawyer 2 . 1 ERA CNRS 621, Laboratoire de Biologie animale, University
de Grenoble 1, 38402 St. Martin d'Heres Cedex, France. ^Department of Biology, University of South
Carolina, U.S.A.
In birds, the integument can give rise to two types of cutaneous appendages, scales and feathers. Scales
occur only on the foot, while feathers cover the rest of the body, and can be found within scale tracts, under
genetic or experimental conditions. Two major types of scales are usually distinguished: large rectangular
scutellate scales, which cover the metatarsus and the anterior face of the toes, and small rounded reticulate
scales, which cover the plantar face. Scutellate scales and feathers are made up of beta-keratins, while
reticula contain alpha-keratins only, as does the inter-appendage integument. As scutellate scale and feather
epidermis acquire the ability to synthesize beta-keratins in a succession of developmental stages, the
question arises whether reticula are accomplished appendages. In order to answer this question, heterotopic
dermoepidermal recombinations involving plantar tissues from 10-day chick embryos have been performed
and the results analysed with specific alpha- and beta-keratin antibodies. Reticula made up of alpha-keratins
formed in recombinants of plantar dermis and foot epidermis. When plantar dermis was associated with
midventral apteric epidermis, short feathers made up of beta-keratins differentiated. As the midventral
epidermis is able to form either scutellate scales or feathers, depending on the type of dermis it is associated
with, 10-day metatarsal or 6-day dorsal (Dhouailly & Sengel, 1983), the present results allow to advance the
hypothesis that reticula are not scales, but feathers arrested at an early stage of their development.
D. & SENGEL, P. (1983). Feather-forming properties of the foot integument in avian embryos.
In Epithelial-mesenchymal Interactions in Development (eds. R. H. Sawyer & J. F. Fallon), pp. 147-161.
New York: Praeger Press.
DHOUAILLY,
Differentiation and the cell cycle in Dictyostelium discoideum
A. Durston 1 , A. Timmermans \ 5. McDonald 2 , R. Aerts l and C. Weijer 3.1Hubrecht Laboratory,
Uppsalalaan8, 3584 CT Utrecht, The Netherlands.2Department of Biology, Princeton University, Princeton,
NJ08544, U.S.A. 3Zoologisches Institiit der Universitat Munchen, Luisenstrasse 14, 8 Munchen 2, West
Germany
Development of the cellular slime mould Dictyostelium discoideum is triggered by starving
undifferentiated (vegetative) cells. These then aggregate, undergo morphogenesis and differentiate to two
cell types: stalk cells and spores.
We have shown that the differentiation direction of a D. discoideum cell depends on the cell cycle phase in
which it enters development. Synchronised D. discoideum cells were starved in various phases, labelled with
TRITC and allowed to co-develop with unlabelled asynchronous cells. They showed a maximum tendency
for (pre)stalk differentiation if starved about 2 h before mitosis (in a 7-8 h cycle). More than 90 % of
labelled cells then differentiated in this direction (McDonald, S. and Durston, A. J. Cell Sci., in press).
We also investigated the D. discoideum cell cycle using flow cytofluorimetry (Durston et al. Roux Arch.,
accepted). We found, contrary to previous belief, that nearly all the vegetative cycle is G2 . S is > 6 % and
G! is less. The cycle of developing cells does not differ detectably from the vegetative cycle in the stages
(aggregation and slug) examined. Both (pre) differentiated cell types in the slug are mainly G2 , but they
differ in cytoplasmic (mitochondrial) DNA: prespore cells having less. It is possible that these cell types are
concentrated in different phases (i.e., at different points in G2): a possibility also consistent with our finding
that the few S phase cells in the slug are exclusively prespore localized (Durston, A. and Vork, F., Exp. Cell
Res., 115, 454 (1978)). This is being investigated further.
We examined the rdle of the cell cycle for differentiation by applying EUDR at concentrations up to
5 raM. This can block at least about 90 % of DNA synthesis and all cell division in growing and developing
D. discoideum cells, but does not prevent differentiation, at least as far asprestalk and prespore, even if
applied 24 h before cells start development. We suspect that, in D. discoideum as in other cells, there is a
cell cycle oscillator that regulates, but does not depend on, DNA synthesis and cytokinesis and that in D.
discoideum, this also regulates differentiation. We have shown that cytoplasmic pH(pHj)i which possibly
regulates differentiation direction in D. discoideum (Gross, J. etal. Nature, 303, 244 (1983)), also oscillates
during the D. discoideum cell cycle, and regulates but is not dependent on, DNA synthesis.
Pattern formation
Initiation of the pigment pattern in the axolotl larva
169
Hans Henning Epperlein 1 and Jan Lofberg 2.1Department of Anatomy, Freiburg University, D-7800
Freiburg, West Germany.2Department of Zoology, Uppsala University, Uppsala, Sweden
Developing larval pigment patterns of certain amphibians provide suitable systems for the study of
migration, differentiation and arrangement of neural crest cells. We analyzed the initial steps in the
formation of the barred pigment pattern of the axolotl embryo to find out how the bars, i.e. alternating
transverse bands of yellow xanthophores and black melanophores become arranged along the dorsal half of
larvae of advanced age (stage 41 onward). By DOPA incubation and pteridine fluorescence at high pH
(ammonia treatment) melanophores and xanthophores, respectively, can be visualized before they become
externally visible. We found that melanophores occurred already at stage 30/31 along the trunk neural crest
in a premigratory position. With proceeding development the melanophores had migrated laterally, and at
stage 35-36 they were scattered uniformly in the dorsolateral flank and dorsal head. At this stage several
DOPA positive, black groups of melanophores emerged along the premigratory trunk neural crest.
Xanthophores appeared first at stage 35-36 in a pattern of several distinct cell groups along the trunk neural
crest. Their occurrence was restricted to these groups. Combined DOPA incubation and pteridine
fluorescence in the same embryos showed that DOPA positive melanophore groups and fluorescing
xanthophore groups were identical, but that melanophores and xanthophores existed separately in these
groups. The temporally restricted occurrence and elevated appearance of the mixed chromatophore groups
could be morphologically correlated in the SEM with humps along the premigratory neural crest. We can
now relate the barred pigment pattern of the older larvae to the 'prepattern' of the premigratory
chromatophore groups. Xanthophores initiate the yellow bar component of the pigment pattern by causing
melanophores in and around the groups to fade and become invisible while melanophores are retained in
spaces without xanthophores where they form the black bars.
Experimental investigations on the role of Hensen's node in
somitogenesis
Ileana Fazakas-Todea and S. Sandor, Laboratory of Embryology, Center of Hygiene and Public Health,
1900 Timisoara, Romania
1. The UV irradiation of the node area in chick embryos of early somite stages (1-7 somites) prevents, by
necrotizing the cell population of the irradiated zone, the further regression of the node. This result attests
the existence of a real, distinct node cell population and the real character of regression movement.
2. The subnodal transsection of similar embryos at about 0-1-0-2 mm caudal to the node leads (as
observed also by several other authors) to the development of a 'tail', projecting into the hole formed after
the intervention. The 'tail' contains axial organs and represents the results of an 'autonomous' regression of
the node area. The previous irradiation of the node area prevents the shaping of the tail.
3. In both experimental models segmentation and somite differentiation is possible caudal to the arrested
node area (with the development of median somite blocks) and on the edges of the hole respectively. Thus
the node seems not to be an absolute contributor - by its regression - to the determination of somitogenesis.
4. The arrest of the node area regression does not influence (during the developmental stages studied) the
rate of somitogenesis in the anterior part of the segmental plate.
5. The lengthening of the prenodal segmental plate, during the period of development investigated,
questions the existence of a constant number of 'somitomeres' within the segmental plate (Packard &
Jacobson, 1976; Meier, 1979) as the size increase of more caudal somites cannot be confirmed.
170
Pattern formation
Epithelial-mesenchymal interactions regulate the differentiation of
vertebrate palatal medial edge epithelial cells
M. W. Ferguson 1 and L. S. Honig 2.1Anatomy Department, Queen's University Belfast, Medical Biology
Centre, 97 Lisburn Road, Belfast BT9 7BL and from 1st June 1984 - Department of Basic Dental Science,
Turner Dental Hospital, University of Manchester, Higher Cambridge Street, Manchester Ml5 6FH.
2
Laboratory for Developmental Biology, University of Southern California, Los Angeles, California, U.S.A.
During closure of the embryonic secondary palate the medial edge epithelial cells (MEE) fuse and die in
mammals, become cobblestoned, villous and migrate in alligators but keratinise in birds (resulting in
physiological cleft palate in the aves). We performed experimental epithelial-mesenchymal recombinations,
both within and between mouse, chicken and alligator embryos, followed by in vitro culture of the
recombinants. Recombination of palatal epithelia with a neutral (e.g. mandibular) mesenchyme resulted in
no MEE differentiation in alligators and chicks, but normal MEE differentiation in mice, indicating that
murine palatal epithelium is determined from the onset of palatogenesis. Recombination of mandibular
epithelium and palatal mesenchyme resulted in the appropriate MEE differentiation in all three species
indicating that palatal mesenchyme specifies MEE differentiation. Further recombinations revealed that
mesenchymal specificity was present (1) after the time of normal palatal closure, (2) in all parts of the
mesenchyme (not just in an isolated cell population on the medial edge), and could influence the
differentiation of epithelia recombined upside down. If palatal epithelia were recombined at right angles to
the medial edges of the palatal mesenchymes (crossed recombinations), MEE differentiation occurred in the
area overlying the mesenchymal edge and not along the 'original' medial edge of the epithelium. In
interspecies palatal recombinations with the medial edges coincident, differentiation of the MEE was
characteristic of the vertebrate from which the mesenchyme originated and independent of epithelia origin.
Similar results were obtained for interspecies crossed recombinations except for mouse palatal epithelia on
either alligator or chick mesenchyme. The latter showed two medial edges: one a line of cell death
corresponding to the original medial edge of the mouse epithelium and at right angles to it a new medial
edge corresponding to the medial edge of the underlying mesenchyme and with an MEE phenotype
characteristic of the vertebrate from which the mesenchyme originated. The nature of the mesenchymal
signal is unknown but defective tissue interactions may be responsible for natural cleft palate in birds and
pathological cleft palate in man. This work is supported by M.R.C. (U.K.) grant 8113610CB, EHSSB grant
EP109. 74-75, and NIH grants DE-02848 and DE-03569.
Formation of the pigment cell pattern in internal organs of the White
Silkie fowl embryo
Raymond Ferrand and Alain L'Hermite, Laboratoire de Biologie du Diveloppement, University de Nantes,
2 rue de la Houssinie're, 44072 Nantes, France
Contrary to lower vertebrates in which pigment cells are widely distributed, in upper vertebrates these
cells are found in limited areas, especially in both epidermal and dermal layers. However, in some species,
melanocytes populate muscular clefts, perineural and perivascular sheets, mesenteries and, very scarcely,
some organs. Among these species the Silkie fowl exhibits a particular internal pigment cell pattern,
resulting into a heavy pigmentation of numerous organs. This feature offers a good opportunity to test the
two following hypotheses. According to the first one this extensive pigmentation depends on high invading
capacities of Silkie fowl melanoblasts and, according to the second, the pigment cell pattern is determined
by an environmental process.
The experiments were performed in Japanese quail and White Wilkie fowl embryos. The White Silkie
fowl only differs from other Silkie fowl strains by its unpigmented plumage. Fragments of anlages of
pigmented organs (proventriculus, gizzard, intestine, rectum, caeca, lungs, ovary, testis, mesonephros) and
non-pigmented organs (ventricular muscle, spleen, liver) were isolated from 5 to 6-day-old White Silkie fowl
embryo, i.e. just prior to, or at the onset of melanoblast homing. The anlages normally invaded by
melanoblasts in Silkie fowl embryo, were populated by melanoblasts that differentiated into melanocytes
exhibiting the quail nuclear marker after processing by the Feulgen-Rossenbeck reaction. The anlages of
normally unpigmented organs were not colonized by quail pigment cells. Quail melanoblasts exhibit the
same behaviour (invading or not-invading) as Silkie fowl melanoblasts when they are submitted to identical
environmental cues. In some cases, the grafted organ developed in the vicinity of the same organ of the quail
embryo host. The fact that the grafted organ became heavily pigmented contrary to the host one, strongly
suggests that environmental cues are not only permissive but also attractive for prepigment cells.
The formation of internal pigment cell pattern in Silkie fowl embryo is not due to particular invading
capacities of melanoblasts. The homing and definite localization of pigment cells seems to be regulated by an
environmental process.
Pattern formation
Calcium is a candidate for the morphogen in Acetabularia
171
B. C. Goodwin*, Biology Department, The Open University, Walton Hall, Milton Keynes, MK76AA
Morphogens have proved to be unexpectedly elusive substances despite the widespread expectation that
they are involved in the generation of form in developing and regenerating organisms. Our studies
(Goodwin & Pateromichelakis, 1979; Goodwin etal. 1983) on the morphogenesis of whorls in regenerating
Acetabularia plants provide evidence that the substance primarily involved in whorl initiation is cytosolic
free calcium. Experimental and theoretical (Goodwin et a). 1984; Goodwin & Trainor, 1984) investigations
suggest that this pattern-forming process is to be understood as a combination of electrophysiological,
diffusional, and visco-elastic aspects of cellular organisation which results in a periodic pattern of free
calcium, solation of the cytoplasm, and cell-wall buckling which results in a periodic ring of hair primodia.
Calcium is also involved in tip and hair elongation by a related but spatially simpler process. These results
and their implications for morphogenesis in other systems (Odell et al. 1981; Oster et al, 1983) will be
discussed, particularly the possibility that calcium may be a universal primary morphogen.
GOODWIN, B. C. & PATEROMICHELAKIS, S. (1979). The role of electrical fields, ions and the cortex in the
morphogenesis of Acetabularia. Planta 145, 425-435.
GOODWIN, B. C , SKELTON, J. L. & KIRK-BELL, S. M. (1983). Control of regeneration and morphogenesis by
divalent cations in Acetabularia.Planta 157, 1-7.
GOODWIN, B. C , MURRAY, J. D. & BALDWIN, D. (1984). Calcium: the elusive morphogen in Acetabularia?
Nature (submitted).
GOODWIN, B. C. & TRAINOR, L. E. H. (1984). Tip and whorl morphogenesis in Acetabularia by
calcium-induced strain fields. In preparation.
ODELL, G., OSTER, G. E., BURNSIDE, B. & ALBERCH, P. (1981). The mechanical basis of morphogenesis
Devi Biol. 85, 446-462.
OSTER, G. F., MURRAY, J. D. & HARRIS, A. (1983). Mechanical aspects of mesenchymal morphogenesis. /.
Embryol. exp. Morph. 78, 83-125.
Interactions between melanoblasts and tissue factors in the formation of
internal pigment cell pattern in two breeds of fowl and in their hybrids. A
genetic analysis of the results
Marie-Martine Hallet and Raymond Ferrand, Laboratoire de Biologie du Diveloppement, University de
Nantes, 2, rue de la Houssinidre, 44072 Nantes, France
Factors involved in the formation of the internal pigment cell pattern were analysed associating neural
crest graft technique with a genetic approach. Quail neural tube grafts were performed into White Silkie,
White Leghorn and hybrids chick embryos. Silkie melanoblast migration was studied by grafting White
Silkie neural tubes into White Leghorn embryos.
White Silkie chickens exhibit an extensive internal pigmentation; White Leghorn chickens are
apigmented. Hybrids were obtained by crossing White Silkie males with White Leghorn females; in such Fx
birds, females are nearly as deeply pigmented as the Silkie parent, while the male progeny contains no or
very little internal pigment.
Neural tube segments (5 somites long) were excised at the level of the last formed somites from 20 to 27
somite embryos and grafted isotopically and isochronically. Host embryos were sacrificed from the 14th to
the 16th day of incubation and the donor pigment cells were located in the internal migratory region
corresponding to the transverse strip of donor-like pigmented feathers. The Feulgen-Rossenbeck technique
made it possible to distinguish quail from chick cells.
In White Silkie and in male and female hybrid embryos, the quail melanocytes invaded all the connective
tissues and gave an extensive internal pigmentation in the migratory region, while White Leghorn host
tissues showed a limited internal pigmentation. By grafting Silkie neural tube into White Leghorn embryos,
we never detected any internal pigmentation in the migratory zone.
The analysis of these results permitted us to dissociate and to distinguish melanoblast and tissue factors
that interact in the formation of the pigment cell pattern:
- The pattern differences exhibited by quail pigment cells, when grated into Silkie, Leghorn and ¥x
embryos, proved the tissue regulation of the internal pigmentation trait in the Silkie strain. This genetic
trait, given by the Silkie parent to its hybrid offsprings, is a dominant character.
- Differences between Silkie and quail melanoblast patterns in the Leghorn tissue environment exhibit
genetical differences for migrating and/or differentiating capacities.
172
Pattern formation
Control of leg bud development by the zone of polarising activity (ZPA)
/. R. Hinchliffe, P. J. Griffiths and A. Sansom, Zoology Department, University College of Wales,
Aberystwyth SY23 3DA
It has recently been argued that the chick leg bud develops according to different principles from the wing
bud, and that it is not under the overall control of the zone of polarising activity (ZPA). Barriers inserted at
stages 20-1 at the level of somites 30/31 or mid-31 resulted in normal development of the skeleton anterior
to the barrier (Rowe & Fallon, 1982).
We carried out impermeable barrier insertion at stages 20-1 at intersomite level 29/30 (somite levels as
defined in Rowe & Fallon, 1982, Fig. 1), or alternatively, we amputated the posterior half of the leg bud at
the same level. Within 24 h, the anterior half showed apical ectodermal ridge (AER) regression and death
of many of the underlying mesodermal cells. Such anterior halves developed poorly (forming only a
shortened tibia: digits 1 and 2 were absent), while in controls the half posterior to the barrier developed
normally.
We mapped the leg bud ZPA by transplanting small blocks of posterior marginal mesenchyme anteriorly
to chick wing buds and assessing the degree of duplication of the wing skeleton produced in the host. Blocks
taken from the flank posterior to the leg bud and from the angle between posterior flank and proximal base
of the leg bud, and from the distal position under the AER opposite somite 30, all had very low activity.
Blocks taken from the posterior margin of the leg bud, plus those taken under the AER opposite somite 31,
had high activity.
We consider that barrier insertion at the level of intersomite 29/30 thus excludes the ZPA from the
anterior half, which in consequence regresses. Insertion at the level of mid-31 might well include ZPA
material anterior to the barrier, resulting in normal development anteriorly. As in the wing bud, ZPA
amputation/exclusion experiments support the view that the normal development of the leg bud is
dependent on the continuing presence of the ZPA.
ROWE, D. A. & KALLON, J. F. (1982). Normal anterior pattern formation after barrier placement in the
chick leg: further evidence on the action of polarizing zone. /. Embryol. exp. Morph. 69, 1-6.
Cleavage in growing normal and experimentally deformed naked eggs of
a paedogenetic dipteran insect
Pablo Junquera, Department of Entomology, Swiss Federal Institute of Technology Zurich, 8092 Zurich,
Switzerland
The eggs of the paedogenetic gall midge Heteropeza pygmaea develop parthenogenetically inside the
mother larva. They are not provided with a chonon, and the cleaving eggs remain enveloped by the
follicular epithelium. Egg growth and yolk accumulation continue during the whole of embryonic
development. After experimental elimination of the follicular epithelium, 'naked' eggs are formed, which
also grow (though less than normal eggs) but remain spherical insteaad of assuming normal elongated shape.
Naked eggs are nevertheless able to attain the blastoderm stage. The ultrastructure of both normal and
naked eggs has been studied during cleavage and blastoderm formation.
The number of elements of Golgi apparatus and endoplasmic reticulum strongly increases during early
cleavage, both in normal and naked eggs. Their association with cleavage furrows and nuclei suggests a
preponderant role of these organelles in membrane production. Egg yolk consists of lipids and glycogen,
whereas proteins are absent. Cleaving normal and naked eggs contain numerous lysosomal vesicles
indicating intense autophagic processes which may be related with the recycling of structures during
cleavage. Cleavage furrow formation is independent from the positioning of the cleavage nuclei. In the
course of cleavage the number of microtobules in the ooplasm increases considerably. They become
especially numerous in association with the cleavage furrows, surrounding the cleavage and blastoderm
nuclei, in the intercellular bridges connecting the blastoderm cells with the central part of the egg, and in the
cytoplasmic sheet immediately underlying the blastoderm layer. It is suggested that the microtobules as
representatives of the cytoskeleton are involved in the progressive formation of a kind of scaffold during
cleavage. This scaffold may be responsible for the varying egg architecture and for the final accurate
distribution of the egg components in the blastoderm. Since these processes are also accomplished in naked
spherical eggs, they can be considered as independent (i) from the follicular epithelium as a limiting
envelope and as involved in egg metabolism, and (ii) from the normal egg shape.
Pattern formation
Dictyostelium morphogens and how they might act
173
Robert Kay*, Julian Gross, Mike Peacey, Keith Jermyn, Babu Dhokia, Will Kopachik, Jenny Brookman and
Ralph Pogge, Imperial Cancer Research Fund, Burtonhole Lane, London NW71AD
During Dictyostelium development an aggregate of similar cells becomes partitioned into a zone of
prestalk cells and a zone of prespore cells which respectively give rise to the stalk and spore mass of the
fruiting body. Like other morphogenetic fields, the Dictyostelium aggregate can form a normal pattern from
a wide range of input cell numbers and restore the pattern after parts are removed and it possesses a
dominant, axis-organising region, the tip.
Our aim is to discover the nature of the Dictyostelium morphogens and how they act. Using in vitro
assays, in which isolated amoebae are submerged in a simple salts medium, we have found that cyclic AMP
(the chemotactic agent during aggregation) is essential for both stalk and spore cell differentiation and is
indeed a sufficient inducer for spore differentiation in certain sporogenous mutant strains. However stalk
cell differentiation requires in addition DIF, a low MW, non-polar morphogen which has been purified and
whose unusual chemical nature will be described. DIF appears to be the natural regulator of the stalk/spore
choice because it is capable of redirecting amoebae from spore to stalk differentiation in vitro, is made at the
appropriate time in development and because its absence from certain mutants blocks prestalk but not
prespore cell differentiation. Perhaps paradoxically, dissected prespore regions have higher DIF levels than
prestalk regions. This distribution could reflect intracellular DIF stores, but if true for extracellular DIF, it is
suggestive of DIF being a depleted-substrate in one class of reaction-diffusion mechanism. Ammonia is
produced during development as a result of catabolism by the starving cells and we find that in vitro it
opposes the action of DIF and favours spore formation. Thus, like DIF, it could somehow be involved in
proportion regulation and patterning in the aggregates.
We have proposed that intracellular pH (pHj) is the parameter which governs whether a cell differentiates
into a stalk or spore cell, with low pHj favouring stalk. Ammonia should elevate intracellular pHj by
permeation of the uncharged from (thus favouring spore) whilst we hypothesise that DIF somehow reduces
pHj. Recently we have discovered a plasma-membrane, outwardly-directed proton pump in Dictyostelium,
which could be involved in pH; regulation. Various inhibitors of this pump, such as diethylstilbestrol, mimic
DIF and induce stalk cell differentiation but DIF itself does not directly inhibit the pump in vitro. However
it remains possible that DIF could inhibit the pump indirectly.
Studies on vitamin A-induced pattern duplication in the regenerating
axolotl limb
5. KeebleandM. Maden, National Institute for Medical Research, Mill Hill, London NW71AA
The effect of retinoic acid (RA^ on the regenerating axolotl limb is to cause serial reduplications in the
proximodistal axis (Maden, 1983). Several histological changes are apparent both in the epidermis and
mesoderm of RA treated limbs and we have shown by tissue grafts that the epidermal effects play no part in
pattern formation alterations. The most obvious histological effect on the mesoderm is to cause clumping of
the blastemal cells. We show here that this is caused by a local increase in fibronectin production.
Studies on the local application of RA in silastin blocks have revealed concentration and time effects
similar to those obtained by immersion of animals in vitamin A solutions. By using radioactive retinoic acid
we have determined its rate of release from silastin blocks and thus we have been able to estimate the
absolute amount of RA needed to cause specific pattern changes. Further studies with labelled RA to
discover its site of action within blastemal cells will be described.
MADEN, M. (1983). The effect of vitamin A on the regenerating axolotl limb. /. Embryol. exp. Morph. 77,
273-295.
174
Pattern formation
The embryo forming potency of the postero-marginal area of the area
pellucida of stages X through XII E. C&K of the chick
O. Kahner and H. Eyal-Giladi, Zoology Department Hebrew University of Jerusalem, Jerusalem 91904,
Israel
The bilateral symmetry of a stage X E. G&K chick blastoderm is expressed during the first hours of
incubation by the sequential posterio-anterior appearance of the hypoblast, primitive-streak and embryonic
axis.
Experiments on stage X through XII blastoderms, were carried out to assess the involvement of the
posterio-marginal (p.m.) region of the area pellucida (a.p.) in the formation of an embryonic axis:
1. A p.m. square 0-3x0-2 mm was removed without replacement. In all stages studied axes developed from
the original healed posterior side.
2. Equally sized p.m. and latero-marginal squares of the same blastoderm were mutually exchanged. At
stage X only one axis developed from the p.m. fragment at its lateral location. At stage XI two axes
developed at 90 ° to each other from both lateral and posterior. At stage XII again one axis developed but
from the original posterior side.
3. Blastoderms were cut into fa) a circular peripheral ring including the area opaca and adjacent margin of
the area pellucida (a.p.) and (b) a central disc of the a.p. (a) was rotated by 90 ° in relation to (b) prior to
incubation. At stage X only one axis developed, from the posterior side of (a). At stage XI two axes
developed from the posterior sides of (a) and (b). At stage XII one axis developed from the posterior side of
(b).
Conclusion: a small p.m. fragment of a stage X blastoderm can initiate the development of an ectopic axis
and inhibit the regeneration of an axis at the original posterior side. At stage XI both the axis-forming and
inhibitory capacities of the p.m. fragment become weaker as an axis-forming capacity starts to build up
anterior to the p.m. At stage XII this tendency is more pronounced.
Developmental phase specific response of the avian limb pattern to
cytostatic treatment
W. Kocher, D. Braasch, B. Preusser, S. Storm and U. Kocher-Becker, Inst. fur Embryonal-Pharmakologie,
Freie Universitdt Berlin, Garystr. 5, D-1000 Berlin 33, Germany
Chick embryos of various stages were treated with the alkylating substances triethylene-melamine (TEM)
by a single injection into the air chamber of the egg. Treatment was performed in half day steps from the
unincubated egg until day 5 of incubation, then in whole day steps (days 6 and 7). The doses were about the
respective embryonal LD 50. The effects of TEM treatment on the formation of the cartilaginous skeleton
were investigated in cleared methylene blue preparations of day-10 embryos. Though typical alterations
were found in all parts of the body and of the skeleton, the scope of this presentation is confined to the
limbs, since treatment phase specificity is demonstrated most clearly in the alterations of their pattern.
After treatment in early stages (day 0-li), malformations of the limbs appeared only sporadically and
were developed mostly unilaterally. Preaxial duplications in the hind feet were found after treatment on
days 0-1, pero- and amelia after treatment on day li. After treatment during organogenesis, limb defects
occurred in the majority of the fetuses and, on the whole, showed a bilateral manifestation. Treatment on
day 2 led to a loss of proximal elements (tibia, fibula, radius, ulna), resulting in pre- or postaxial hemimelia,
phocomelia, and other cases of meromelia. After treatment on days 2\ and 3, aplasia of fibula and radius
and fusions of elements in the knee and elbow were frequent, slight reductions in feet and hands being found
only rarely. After trreatment between days 3£ and 4i, the zone of deficiency shifted distally (Kocher, W.
(1970). Wilhelm Roux' Archiv Entwmech. Org. 165,53-86): after treatment on day 3i, it was situated in the
radius and in the proximal part of the metatarsus, after treatment on day 4£, in the terminal metapodium of
the hind limb and of the wing. At the same time, the phalangeal region was increasingly included in the zone
of deficiency. This shift of the teratological effect into more terminal structures continued after treatment on
days 5 to 7. After treatment on days 5 and 6, segregation of the phalanges did not occur, 1st toe and 2nd
finger (= preaxial marginal ray of the wing) were strongly reduced or missing. Treatment on day 6 led to the
persistence of the interdigital webs. After treatment on day 7, fusions and reductions were limited to the
terminal phalanges. The proximo-distal shift of the main effects with increasing age at treatment obviously
corresponds to the proximo-distal sequence in development of the segments of the limbs. (Supported by
DFG, Sfb 29).
Pattern formation
175
Supernumerary outgrowth after grafting anterior leg bud tissues into
wing bud of the chick embryo
J. Laborda-val and N. L. Murillo-Ferrol, Departamento de Anatomia y Embryiologia, Facultad de
Veterinaria, Miguel Servet, 177, Zaragoza-13, Spain
A mesoblastic zone located in the posterior region of the limb bud (the Zone of Polarizing Activity, ZPA)
seems to control limb polarity across the antero-posterior axis, possibly in the form of a difusion gradient of
morphogen (Wolpert et al, 1975). It has been shown that grafting a ZPA to the anterior margin of a host
limb bud alters the pattern so that it forms a mirror image reduplication of the host limb field causing the
formation of a supernumerary limb of contralateral polarity. It is well known that apical ectodermal ridge in
avian leg bud presents constant anterior and posterior thickness. Otherwise, in wing bud it is asymmetric,
thicker posteriorly than anteriorly. To test if anterior leg mesoderm (subjacent to a thick apical ridge)
displays some kind of inductive properties as might be found in polarizing mesoderm, a wedge containing
mesoderm and overlying ectoderm from preaxial localization near the body wall of a stage 19 to 20
(Hamburger & Hamilton, 1951) chick leg bud was grafted to the anterior margin of a host chick wing bud at
the same stages. It resulted in duplication of stylopodial skeletal elements, with distal ends fused between
them. Likewise, radius showed increase in width and, in some cases, ulnalike morphological appearance on
proximal end. In no case were duplicated autopodial structures obtained. The leg bud, without an anterior
wedge allowed to grow, showed lack of preaxial elements like toes I and II, and tibia in one case. On the
other hand, when leg bud anterior wedge was grafted on a flank host chick embryo or on an 8 day CAM, an
unidentified short piece of cartilage developed. Could the presence of over-developed or reduplicated
proximal host wing elements be attributed to special morphogenetic characters of anterior leg mesoderm?
V. & H. L (1951). A series of normal stages in the development of the chick embryo. /.
MorphoL, 88, 49-92.
WOLPERT, L., LEWIS, J. H. & SUMMERBELL, D. (1975). Morphogenesis of the vertebrate limb. In Cell
Patterning, Ciba Foundation Symp. vol. 29 (ed. R. Porter & J. Rivers), pp. 95-130. Amsterdam:
Elsevier-North Holland.
HAMBURGER,
Pattern modification in hydroidpolyps caused by retinoids
Werner A. Muller, Zoolog. Institut, University, INF230, D-6900 Heidelberg, West Germany
Retinoids alter pattern specification not only in vertebrates. In the hydroid Hydractinia at least three
pattern forming systems are affected by pulsetype treatment with e.g. retinoic acid 10~10 to 10~5M:
1) The radial pattern: metamorphosing primary polyps form more tentacles but fewer stolons per unit
circumference.
2) The longitudinal body pattern: the position of the hydranth/stolon junction is shifted for the benefit of
stolon elongation. The final expression of this shift is a total transformation of the hydranth into a giant
stolon.
3) The colonial pattern; new hydranth buds arise in higher frequency and at positions closer to the founder
hydranth.
It is argued that the effect on the longitudinal and colonial pattern is due to a reduction of the range of
spacing signals emitted by the primary hydranth. Dose response10curves display optima and some effects are
reverted when the
optimum is surpassed. Very low doses (10~ to 1 0 " ^ ) e.g. promote head formation,
high doses (10~6 to 10~5M) cause reduction of existing head structures.
Several effects are counteracted by a putative morphogen, the Hydra-derived inhibitor (Berking, 1977,
1983). The 'head activator' (Schaller & Bodenmuller 1981), on the other hand, displayed no stimulating
effect on the number of tentacles and buds being formed.
176
Pattern formation
Cellular contribution to supernumerary limbs of developing and
regenerating limbs in the axolotl
Ken Muneoka, Developmental Biology Center, University of California, Irvine, CA 92717, USA
The cellular contribution to supernumerary limbs resulting from anterior-posterior opposed contralateral
grafts of limb buds and blastemas, and contralateral grafts between limb buds and blastemas has been
analyzed using the triploid cellular marker in the axolotl. Cellular contribution was analyzed using a new
technique of isolating the limb dermis for whole mount analysis and in some cases in paraffin sections of the
skinned limbs. The results were the same for all three types of grafting operations. Overall, the cellular
contribution from the graft and stump was approximately equal in all supernumerary limbs analyzed,
although the position of the boundary was variable from limb to limb. The pattern of cellular contribution in
these supernumerary limbs was found to be asymmetrical in the dorsal-ventral axis with posterior tissue
contributing predominately to the posterior and dorsal part of the supernumerary limbs and anterior tissue
contributing predominately to the anterior and ventral part of the supernumerary limbs. These results are
consistent with pattern formation models which suggest that intercalary regeneration is the driving force for
pattern regulation of the vertebrate limb. Furthermore, it is suggested that intercalary regeneration in the
anterior-posterior axis displays a directional bias similar to that observed in the proximal-distal limb axis.
Finally, the triploid cell marker was used in conjunction with the black/white pigmentation marker. It was
shown that these two markers behave independently, suggesting that the position of pigment cells following
grafting is not a good indicator of the origin of surrounding non-pigmented cells.
Spacing control in pattern formation of hydroids
G. Plickert, Zoologisches Institut der Universitat, Heidelberg, Im Neuenheimer Feld 230, D-6900 Heidelberg,
West Germany
The spatial arrangement of polyps in a hydroid colony is regular - new polyps are formed according to
certain rules: polyp buds occur periodically in the terminal region of elongating stolons at typical distances
to preceding buds, and intercalary between existing polyps.
Spacing is controlled by an inhibitory field emanating from polyps and polyp buds. The field extends into
the stolon tissue and prevents polyp budding within a critical range. Experimental results reveal additional
field characteristics and indicate an endogenous inhibitor as the molecular basis of spacing control:
the field range depends on age and size of the controlling polyp,
the field breaks down after removal of the polyp or only of the polyp head,
volume and convection of the ambient medium strongly influence interpolyp distances,
from tissue homogenates a non-toxic low molecular weight substance which increases the field range can
Pattern formation
111
Mapping of the oxygen consumption in the intrauterine chick
blastoderms
E. Raddatz \ H. Eyal-Giladi 2 and P. Kucera 1.x Institute of Physiology, 1011 Lausanne CHIN,
Switzerland, institute of Zoology, The Hebrew University of Jerusalem, Israel
The spatio-temporal pattern of the oxygen uptake during the formation of the area pellucida of the very
early chick embryo has been studied. The developmental stages investigated were stage VI and stage X
according to Eyal-Giladi and Kochav (1976), i.e., 10 and 20 hours uterine-age respectively. The blastoderms
were carefully dissected out from aborted or just laid eggs, cleaned from adherent yolk and then mounted in
a special culture chamber thermostabilized at 42 °C for stage VI (oviduct temperature) or 38 °C for stage X
(incubation temperature). The oxygen consumption in minute regions of the intact blastoderms has been
measured in vitro by scanning microspectrophotometry using haemoglobin as oxygen donor as well as
indicator of respiration (Kucera & Raddatz, (1980). Resp. Physiol. 39, 199-215).
The regional oxygen fluxes through the embryonic tissue range from 14-8 ± 6-3 to
25-4 ± 5nl.h-l.mm-2 at stage VI and from 9-8 ± 4-4 to 15-7 ± 5 nl.h-l.mm-2 at stage X. This
temporal decrease probably reflects a change in cellular compactness (shedding process) and/or spreading
out of the blastoderm itself. Nevertheless, the oxygen consumption of the whole blastoderm
(130 ± 70 nl.h-1) remains constant throughout the studied period of development, although the number of
cells doubles. Moreover, at stage VI the oxygen flux in the center of the blastoderm (25 nl.h-l.mm-2) is
significantly higher than in the posterior and anterior regions (17 nl.h—l.mm—2). At stage X the oxygen
flux of the posterior part is significantly higher than it is in the anterior region (16 and 10 nl.h-l.mm-2,
respectively). This remains true also when the values are corrected for cell number (Qc) or for cytoplasmic
volume (Qv) estimated from histological sections. For the anterior, central and posterior regions, the Qc
values are 3-6, 7 and 4-4 at stage VI and 1-2, 1-6 and 1-5 pl.h—l.cell—1 at stage X, respectively; Qv values
are 126, 334 and 148 at stage VI and 287, 380 and 365 nl.h-l.mm-3 at stage X, respectively.
These antero-posterior variations of metabolic activity may be related to the early morphogenetic
processes leading to the embryonic axis formation.
PS formation and maturation following the removal of the primary
hypoblast in chick blastoderms with and without marginal zone
Zehava Rangini and Hefzibah Eyal-Giladi, Department of Zoology, Hebrew University, 91904, Jerusalem,
Israel
Earlier studies indicated that in case of removal of a stage XIII E.G&K primary hypoblast (hy), the
presence of the marginal zone (mz) seems to be essential for further normal regeneration and development.
The purpose of the present study was to follow the regeneration of a secondary lower layer macroscopically
and microscopically and relate it to further development. In two experimental series the lower surface of
blastoderms operated on at stages XIII E.G&K to 3+H&H was studied.
In all operated blastoderms a massive extrusion of yolk platelets from the ventral side of the epiblast was
followed by the rapid formation of a continuous yolky sheet. Polyinvaginating epiblastic cells populated the
space between the epiblast and the yolky layer.
1) In series (a) only the hy was removed. In all stages primitive streak (PS) formation continued
uninterruptedly, but its basal lamina remained intact until it reached its full length. Normal differentiation
of axial mesoderm and neural plate followed. PS elongation and lower layer regeneration occurred
simultaneously. In stages XIII-2 a narrow posterior strip of lower layer regenerated rapidly. The rest of
the lower layer was completed later by endodermal cells invaginated via the PS, thus leaving the mesoderm
ventrally exposed except for the yolky layer.
2) In series (b) the hy+mz were removed. Blastoderms operated at stage 3~H&H and later, continued
development as in series (a). In earlier operated blastoderms the PS initially continued to elongate, then
development was arrested followed by PS regression, being more pronounced the younger the operated
blastoderm was. Despite the regeneration of a lower layer from the cut margins no mesodermal or neural
axes formed.
Conclusions: A stage 3 H&H blastoderm can continue its development even after the removal of the
hy+mz. In younger blastoderms the initial induction of PS by the hy is not sufficient for the maturation of a
PS. In case of an early removal of the hy, the rapid growth of a posterior lower strip associated with the mz
can replace the function of the primary hypoblast, by supporting PS maturation, normal gastrulation and
embryonic differentiation.
178
Pattern formation
Determinative events and size-independence of pattern formation
during planarian regeneration
E. Said and J. Bagund, Department Genetica, Univ. de Barcelona, Diagonal 645, Barcelona-28, Spain
It has been known for a long time that grafted heads inhibit the formation of a new head when the host is
cut near the graft. This inhibition is a temperature-dependent process appearing to be related to distance
between graft and cut, and to the interval of time between grafting and cutting. Using this inhibitory test we
have studied the time of determination of head and pharynx during regeneration.
We found that determinative processes are extremely rapid and temperature dependent, 3-12 h being
needed for head determination and 6-24 h for pharynx determination. Since at 3-24 h of regeneration the
blastema is barely distinguishable, we suggest that determination takes place in a very narrow piece of distal
stump tissue behind the wound. Moreover, X-ray irradiated organisms which do not have mitotic activity
make blastemas of different sizes (down to a value of 13 % of controls) that differentiate head and pharynx
as an unirradiated blastema and stump. This suggests that determination and pattern formation in
planarians are size-independent processes.
The rapid determination and the size-independence of pattern formation suggest that pattern first appears
by morphallaxis within a small region of distal tissue, being amplified later on by an epimorphic process that
includes cell division and cell migration.
Duplicitas cruciata and other riddles of embryonic patterning in insects
explained by intercalation of pattern elements
K. Sander and H. O. Gutzeit, Institutfur Biologie I (Zoologie), Albertstr. 21a, D 7800 Freiburg,
West Germany
Intercalation of missing positional values or pattern elements has been well established as a formal
principle for postembronic patterning in insects but hardly so for embryonic stages except in leg
development (French, 1983). We will show that several vexing monstrosities of the insect trunk obtained by
perturbation of the earliest stages of development can be explained using the polar co-ordinate model.
Duplicitas cruciata, a cross-shaped axial duplication with opponent heads and tails, has been analyzed by
Krause (1962) in the stone cricket. It invariantly results from anterior median incision through both germ
anlage and amnion, followed by healing of the cut margins of each half-germ anlage and its half-amnion.
The subsequent 'assimilatory induction' of the annealed amnion parts to form the complementary body half
(Krause, 1962) can be viewed as transverse (= latitudinal) intercalation between discrepant positional
values of median body plane and amnion. At the level of the base of incision, the posterior rim of the
induced parts is bordering the non-induced cells of the uncut posterior amnion, and this would trigger
longitudinal intercalation between the positional values of the amnion (= most posterior meridional value)
and the induced anterior parts, leading to intercalation of an additional end of body. The latitudinal values
might be assigned either bilaterally in the germ anlage, in keeping with bilateral body symmetry, or in clock
face fashion through germ anlage and amnion (which together represent a circular epithelium), with the
amnion representing a distinct but uniform positional value. The latter assumption would explain two other
patterning riddles, namely the unilateral insertion of a supernumerary body axis after oblique cuts through
germ anlage and amnion followed by annealing in the stone cricket (Krause & Krause, 1957), and the
formation of two 'anterior' outgrowths at either side of the dorsal midline in the mirroring plane of dipteran
double abdomens (Bischoff & Gutzeit, unpublished observation).
V. (1983). A model of insect limb regeneration. In Primers in Developmental Biology (eds.
Malacinsky & Bryant) (in press).
KRAUSE, G. (1962). Die Entwicklungsphysiologie kreuzweise verdoppelter Embryonen. Embryologia 6,
355-386.
KRAUSE, G. & KRAUSE, J. (1957). Die Regulation der Embryonalanlage von Tachycines (Saltatoria) im
Schnittversuch. Zool. Jb. (Anat.) 75, 481-550.
FRENCH,
Pattern formation
The organization of morphogenesis in Dictyostelium
179
Pauline Schaap, Zoological Laboratory, Kaiserstraat 63, 2300 RA Leiden, The Netherlands
Starved amoebae of the cellular slime moulds aggregate to form multicellular structures, which perform a
series of shape changes that lead to formation of slug-shaped structures and fruiting bodies. The cells
differentiate into two types; stalk cells and spores, which become arranged into a simple pattern. Pattern
formation is closely interlinked with changes in shape. The upper extremity of the structures (tip) seems to
organize cell movement that leads to shape changes (Raper, 1940). The aggregation process of some species
such as D. discoideum is mediated by oscillatory secretion of cAMP by the aggregation centre and cAMP
relay by surrounding cells (review Van Haastert & Konijn, 1982). It was suggested that the tip might
organize morphogenesis by continuing to emit cAMP signals (Rubin & Robertson, 1975). We recently
observed oscillatory cell movement in completed aggregates of D. minutum. Cells inside the aggregate were
attracted to the signalling centre, which resulted in the formation of tips and slug-shaped structures. Shortly
before tip formation, cAMP receptors and cAMP phosphodiesterase appeared, the cells became
chemotactically sensitive towards cAMP (Schaap et al, 1984) and acquired the capacity to relay cAMP
signals. It thus seems very likely that cell movement in the multicellular stage of D. minutum is coordinated
by oscillatory cAMP signalling and cAMP relay. A variety of other Dictyostelium and Polysphondy Hum
species also seems to make use of cAMP signalling during multicellular development. When comparing
different species, a positive correlation was evident between the appearance of oscillatory signalling at an
early stage of development, the capacity of the tip to organize a relatively large number of cells into a
fruiting body and the early appearance of a prespore/prestalk pattern. The implications of these findings will
be discussed.
K. B. (1949). Pseudoplasmodium formation and organization in Dictyostelium discoideum. J. Elisha
Mitchell Sci. Soc. 56, 241-282.
RUBIN, J. & ROBERTSON, A. (1975). The tip of the Dictyostelium discoideum pseudoplasmodium as an
organizer. /. Embryol. exp. Morphol. 33, 227-241.
VAN HAASTERT, P. J. M. & KONIJN, T. M. (1982). Signal transduction in the cellular slime molds. Mol. and
Cell. Endocr. 26, 1-17.
SCHAAP, P., KONIJN, T. M. & VAN HAASTERT, P. J. M. (1984). cAMP pulses coordinate morphogenetic
movement during fruiting body formation of Dictyostelium minutum. Proc. Natl. Acad. Sci. USA (in
press).
RAPER,
Comparative study of patterns of cell differentiation in
several slime mould species
Pauline Schaap and Johan Pinas, Zoological Laboratory, Kaiserstraat 63, 2300 RA Leiden, The Netherlands
Different cellular slime mould species can show considerable morphological variety (Olive, 1975). The
species Dictyostelium discoideum has been widely used as a model system for the study of cell differentiation
and pattern formation. A general disadvantage of the study of only one species is the difficulty to separate
main features of development from specific processes which are the characteristics of that single species. We
therefore performed a detailed study of the arising of patterns of cell differentiation in a variety of cellular
slime mould species.
The investigated species can be subdivided into two groups. 1) Species as D. minutum, D. lacteum and D.
vinaceo-fuscum, in which all cells develop prespore properties. Once started, prespore differentiation
proceeds rapidly until evident in more than 90 % of the cell population. In D. minutum and D. lactecum, the
transformation of prespore cells into stalk cells proceeds gradually after cells have reached the tip. In D.
vinaceo-fuscum ^differentiation of prespore cells starts somewhat below the tip. 2) In species as D.
discoideum, D. mucoroides and D. purpureum, only a proportion of the cells develop prespore properties; a
prepattern is formed consisting of a region behind the tip of cells which retained the properties of
aggregative cells ('prestalk' region) and a more distal region of prespore cells. More complex processses
seem to operate in these species. The proportion of 'prestalk' cells/prespore cells is subjected to regulation.
The boundary between 'prestalk' cells and prespore cells is usually clearly defined and prespore cells that
cross this boundary immediately lose their prespore properties (Gregg & Davis, 1982) Prespore
differentiation, once started does not proceed rapidly and levels off during the slug stage to 60 % of the
attainable levels, only to increase again during culmination. Stalk cell differentiation occurs after cells have
reached the tip, except in D. discoideum where it also takes place at the base of the structure.
Another characteristic which separates the first group from the second group is that the latter group
makes use of oscillatory cAMP signalling during aggregation, while the first group uses other
chemoattractants than cAMP. The possible significance of this difference for differences in pattern
formation is currently being investigated.
OLIVE, L. S. (1975). The mycetozoans. New York: Academic Press.
GREGG, J. H. & DAVIS, R. W. (1982). Dynamics of cell redifferentiation in Dictyostelium mucoroides.
Differentiation 21, 200-205.
180
Pattern formation
The formation of patterns in Drosophila
Gerold Schubiger*, Richard G. Fehon, Ann Gauger and Caroline P. Kiehle, Department Zoology,
University of Washington, Seattle WA 98195, USA
A pattern is an exact display of different structures, which is repeated during development in each
individual of a particular species. Pattern formation is not only genetically controlled, but also depends on
the cytoplasm and the cell's environment. As development progresses, changes occur in all three
components in an orchestrated way, thereby guaranteeing the formation of proper patterns. Drosophila,
like all insects, develops a specific number of segments which are morphologically well characterized. The
cells which produce these patterns become determined to form certain segments at a specific time during
development. The initial determination depends on proper coordination between epigenetic events and
changes in genomic activity. Once established, this determination is quite faithfully propagated by a cell to
all its daughters.
Cell determination not only expresses itself in the cell's restricted fate but can also be expressed on the cell
surface. For example, dissociated single cells determined to form a wing sort out from cells differently
determined for leg. Furthermore, thoracic imaginal disc cells bind preferentially to thoracic embryonic
segments, while genital disc cells bind preferentially to abdominal embryonic segments. Thus cells with
similar states of determination can recognize each other even if they are of different developmental stages.
Another property of determined cells is the ability to regenerate structures of a pattern. Regeneration
allows us to study aspects of normal development; because processes such as cell lineage restrictions are
established both in normal development and in pattern regulation. We found that the ability to regenerate
missing patterns is compartment dependent. Only cells of the anterior compartment can regenerate
posterior cells, indicating that posterior cells are more rigidly determined, as was predicted from studies on
normal development.
Morphological improvement and proximalization of regeneration in
frog tadpoles treated with vitamin A
K. K. Sharma, Zoologisches Institut der Universitdt zu Koln, 1. Lehrstuhl: Experimentelle Morphologie,
Weyertalll9, 5000 Koln, 41, West Germany
Vitamin A palmitate has been found to promote regenerative processes in the limbs of even
post-metamorphic frogs. The present study was made on tadpoles of the frog Rana breviceps at stage
VIII/IX, when thigh, shank and ankle are recognizable in the hind limbs and rudiments of toes 3,4 and 5 are
separated by indentation. Surprisingly, at this stage in this species most of the ankle level regenerates are
oligodactylous whereas thigh and shank level regenerates are all pentadactylous. The objective was to find
out whether treatment of tadpoles with vitamin A for different periods prior and post-amputation would
improve the morphology of the ankle level regenerates. The hind limbs were amputated through ankle.
Following amputation controls were reared in tap water throughout the experimental period (15 days).
Treated groups were reared in 15 i.u./ml solution of vitamin A palmitate (i) for 24 h, 48 h or 72 h prior to
amputation or (ii) for 1 h, 6 h, 12 h, 24 h, 48 h or 72 h post- amputation after which they were transferred
to water for the remaining period. In controls, only 3 (11.5 %) out of 26 regenerates were pentadactylous.
Vitamin A treatment prior to amputation or post-amputation up to 24 h, significantly improved the
morphology of the defective oligodactylous regenerates. The maximum number (85 %) of normal
pentadactylous regenerates were obtained in the tadpoles treated for 6 h after amputation. In these cases
the regenerated portion consisted only of the part distal to the level of amputation. However, regeneration
of whole limbs consisting of all elements from girdle to toes in continuation with the ankle stump
(proximalization of regeneration) was found in 72-7 % and 32-5 % cases in the tadpoles treated with
vitamin A after amputation for 48 h and 72 h, respectively. In the remaining cases post-blastemic
development was inhioited. To conclude, vitamin A treatment prior to amputation or post-amputation up to
24 h improves only the morphology of the regenerates, further treatment causes proximalization and
simultaneous inhibition of regeneration.
Pattern formation
181
Pattern formation in feathers of peacock and wings of peacock butterfly
A. Sibatani, CSIRO Division of Molecular Biology, North Ryde, Sydney, Australia
Similar patterns on lepidopteran wings or bird feathers may vary in size in related taxa. The
pattern-forming fields may be small and of variable size in different individuals and taxa to produce similar
patterns. Analysis of wing homoeosis in butterflies indicated that a unified ocellar pattern ranging over more
than one cellule materialized through cooperation among pattern-forming entities set in individual cellules
bordered by nervures which may act as a diffusion barrier. These entities can either form an independent
ocellus or become part of a larger ocellus.
The ocellus-like pattern in peacock feather is laid on parallel-running barbes which are physically discrete
elements and formed individually in the feather follicle, indicating that a unified pattern must be produced
by integration of activities of at least partially independent pattern-forming entities with attending cell
proliferation. The colour pattern of some birds is such that more than one pattern may appear at different
postures of the bird in rest and flight on the same set of contour feathers, which may in turn vary serially but
systematically in their own local patterning so as to produce the composite pattern. The same applies to
lepidopteran patterns from which such a unified, meaningful shape merges only at the species-specific
resting position of the insect, and which is composed of the patterns laid on different, and hence, distant,
parts of the body including fore and hind wings. Homologies of wing pattern elements in Lepidoptera
indicate that in some taxa, separate pattern elements on a wing may also generate a new pattern unit by
partial recombination involving a shift in relative positions among these elements.
Thus, an ostensibly unified pattern does not necessarily point to a unitary pattern-forming process.
Coordination of parts, almost like a 'designed' process, should be operative in both ontogeny and phylogeny
and thus be crucial in biological pattern formation, suggesting that its basic mechanism could be much more
complicated and organized than a simple physical process in a pattern-forming field occurring at a single
level of organizational hierarchy.
Positional signaling in the chick limb bud by retinoic acid and by the
Zone of Polarising Activity
Dennis Summerbell, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW71AA
When tissue is grafted from the posterior margin of the amniote limb bud to the anterior margin of a chick
embryo host is produces an anterior supernumerary limb with mirror image symmetry to the host limb. A
local source of retinoic acid implanted at the anterior margin gives a similar result. Various experiments
suggest a quantitative relationship between the strength of activity of the graft or implant and the degree of
mirror image reduplication obtained. This has been formalised in a bio-assay called the Strength of Activity
Index. Various experiments have shown that it is possible to reduce the strength of activity of a graft to
obtain incomplete supernumerary limbs with digits of an originally more posterior identity missing.
Similarly reducing the molarity of retinoic acid in the implant reduces the expressed strength of activity. In
the latter case it is also possible to elevate the molarity of the retinoic acid above that needed for full mirror
image reduplication. This causes the loss or reduction of supernumerary and even host limb. Removing the
source of retinoic acid after increasing exposure time in the host causes an increase in the expressed strength
of activity. Implanting the second hand source in a secondary host demonstrates that the retinoic acid has
been utilised, as the first value increases the second value declines. This can also be shown directly by
measuring the movement of radiolabelled retinoic acid from implant to host. Using the Index one can also
map activity along the posterior margin and in the flank of early embryos. The wing Zone of Polarising
Activity seems to arise as a diffuse low activity source in the flank. It gradually condenses into a full strength
source just posterior to the limb bud when it first appears. It then moves out along the limb margin gradually
declining in activity and again growing more diffuse. It is not yet clear whether the source acts as an
analogue of a zone of polarising activity, whether it creates a zone of polarising activity or whether the
similarity of action is unrelated and coincidental.
182
Pattern formation
Retinoic acid and pattern formation in the developing chick wing
C. Tickle 1, J. Lee l and G. Eichele 2}Department of Anatomy and Biology as applied to Medicine, The
Middlesex Hospital Medical School, London W1P6B. 2Department of Biochemistry, University of
California, San Francisco, USA
Retinoic acid has been locally applied to wing buds on controlled-release carriers. Local application to the
anterior margin of the bud leads to a series of dose-dependent changes in pattern formation that are highly
reproducible. First, at low concentrations of retinoic acid, additional digits are sequentially formed,
followed, at higher concentrations, by the progressive thinning of the symmetrical wing. The concentration
of 8retinoic 7acid in the tissue required for the formation of additional digits has been found to be
10~ - 10~ M. This is a steady-state level and exposure for 14-20 h is necessary. The formation of
additional digits appears to be a two-step process, involving a reversible priming phase of about 12 h
followed by a second phase in which digits are added and/or promoted.
Local application of retinoic acid to the apex of the bud also brings about changes in pattern formation.
When low concentrations are applied additional digits may form but truncations are obtained with higher
concentrations. Application of a range of retinoic acid concentrations to the posterior margin leads to the
development of wings with normal digit patterns but these are smaller.
To gain insights into how retinoic acid brings about these effects, wing buds to which retinoic acid has
been locally applied have been examined at short times after treatment. Bud outgrowth is affected and the
shape of the outgrowth depends on the concentration of retinoic acid and the position to which it is applied.
Underlying these differences in bud outgrowth is an effect of retinoic acid on the apical ectodermal ridge,
which leads to changes in its thickness and the distance it extends around the limb margin.
The effect of retinoic acid on pattern formation in the vertebral column
of the chick embryo
S. WealeandS. Wedden, Department of Anatomy and Biology as Applied to Medicine, The Middlesex
Hospital Medical School, Cleveland Street, London W1P6DB
Retinoic acid was locally applied to early stage chick embryos using controlled release carriers placed
either in unsegmented or segmented somitic mesoderm. Carriers placed in unsegmented mesoderm had a
variety of effects on development inducing abnormal vertebral segmentation and the formation of extra ribs
and truncated trails. When placed in somites these effects were not seen. These results will be discussed in
terms of current ideas on the formation of vertebrae.
Pattern formation
Morphogenesis of the developing face of the chick embryo and
malformations following treatment with retinoic acid
183
5. E. Wedden, Department of Anatomy and Biology as applied to Medicine, The Middlesex Hospital Medical
School, London W1P6DP
The development of the upper beak involves fusion of a central mass of tissue, the frontonasal mass, with
the maxillary processes on either side, thereby establishing a continuous upper lip and two nostril slits.
Subsequent beak development can be viewed as an exaggerated version of nose morphogenesis. Arising
from the centre of the frontonasal mass, the upper beak is bilaterally symmetrical with such well defined
midline structures as the egg tooth and a central rod of cartilage, the prenasal cartilage.
To study how the midpoint in the face is established, small pieces were taken from the frontonasal mass,
either from the centre or from lateral positions abutting the nasal placodes. These were then grafted to
another site in the embryo to continue development. At early stages, all pieces give rise to midline
structures, whereas later this ability becomes restricted to the central region of the frontonasal mass. This
suggests that at these later stages, the position of the midline has now been specified.
Application of retinoic acid on controlled release carriers routinely leads to upper beak defects. The
normal enlargement of the frontonasal mass required for fusion with the maxillary processes is inhibited and
outgrowth of the beak does not occur. The resulting defect is severe bilateral clefting of the primary palate.
One possibility is that retinoic acid is cytotoxic and reduces cell numbers in the frontonasal mass. This has
been investigated by vital staining. An understanding of how retinoic acid causes this malformation may
lead to greater insights into oronasal development.
Dependence of cell type proportioning and sorting on cell cycle phase in
Dictyostelium discoideum
Cornells J. Weijer, Gertrud Duschl and Charles N. David, Zoologisches institut Universitat Munchen,
Luisenstrasse 14, 8000 Munchen 2, West Germany
The relationship between cell cycle phase of vegetative amoebe and prestalk and prespore sorting and
differentiation in the slug stage were investigated in the slime mold Dictyostelium discoideum. Due to
discrepancies between our results and published data about the organisation of the Dictyostelium cell cycle,
we found it necessary to reinvestigate the cell cycle using fluorometric determinations of cellular and nuclear
DNA contents in exponentially growing cultures and in synchronized cultures. Almost all cells are in G2
during both growth and development. There is no Gl period,. S-phase is less than 0-5 h, and G2 has an
average length of 6-5 h in axenically grown cells. Mitocnondrial DNA, which constitutes about half of the
total DNA, is replicated throughout the cell cycle. There is no difference in the nuclear DNA content of
axenically grown and bacterially grown cells.
To investigate the relationship between cell cycle phase and sorting cells were synchronized by release
from stationary phase. Samples were taken at various times during the course of a synchronous cell
doubling, fluorescently labelled and mixed with cells of random cell cycle phase from exponentially growing
cultures. The fate of the fluorescently labelled cells was recorded in the slug stage. Cells early in the cycle
exhibit strong prestalk sorting; cells taken later in the cycle exhibit strong prespore sorting. The period of
prestalk sorting occurs immediately following mitosis and lasts about 1 h in a cell cycle of about 7 h
duration. Accompanying the altered sorting behavior is a marked change in the prestalk-prespore
proportions in slugs formed from populations of synchronized cells. Cells synchronized early in the cycle
form slugs with 55 % prespore cells; cells synchronized late in the cycle form slugs with 90 % prespore. The
results are discussed in terms of models for the formation of the prestalk-prespore pattern in slugs.
184
Pattern formation
The pattern of embryonic dentition development in two reptilian species
B. Westergaard 1 andM. W.J. Ferguson 2.1Institute ofCell Biology and Anatomy, University of
Copenhagen, Universitetsparken 15, DK-2100, Copenhagen 0. 2Department of Basic Dental Science,
University of Manchester
Development of the dentition from initiation to hatching was investigated in Cnemaspis kandiana
(Gekkonidae) and Alligator mississippiensis (Crocodylia) by macroscopy, radiography, SEM, light
microscopy and detailed reconstructions, utilising a close series of staged embryos. The pattern of dentition
development was similar in the two species, but markedly different from that proposed by previous studies,
e.g. Zahnreihen, perfect alternation or clones. In each jaw quadrant adult alligators have 20 functional
teeth, each of which are continuously replaced (20 tooth families). In the lower jaw, the first formed tooth
germ appears at embryonic stage 14 in tooth family 3; the second at stage 15 in family 6, the 3rd, 4th, 5th,
6th and 7th at stage 17 in families 12, 2, 4, 9 and 16 respectively. At stage 21 all 20 families have been
established, 33 tooth germs initiated, but two of these embryonic teeth have been resorbed. Indeed, the first
18 teeth to form are ultimately resorbed or shed without becoming functional. The next 8 teeth to form
function only for a short period. The first formed embryonic tooth germs are discernible by macroscopy and
SEM as surface elevations (denticles). These consist of the usual dental tissues except enamel which first
develops at stage 21 on the second generation tooth of family 3. The enamel organs of earlier teeth
degenerate after dentine formation so heralding the onset of resorption. Medial to the invaginating dental
epithelium lies a distinct line of cobblestoned epithelial cells, which is visible by SEM on the jaw surfaces.
Where new teeth are initiated between two already existing ones, the distances between them were
measured and correlated with the sizes and developmental stages of the latter. This analysis suggests that
each tooth has a size and stage dependent zone of inhibition around it. It also indicated that the pattern of
establishing tooth families is related to differential jaw growth. Comparison between Alligator
mississippiensis and Cnemaspis kandiana revealed that differential jaw growth facilitates positional changes
in tooth initiation and morphology, thus endowing the dentition with considerable evolutionary plasticity.
The detailed fate maps generated by this study highlight the value of the developing reptilian dentition for
studies of pattern formation and positional information. This work was supported by the Danish Natural
Science Research Council.
Computer assisted analysis of restorative growth in the chick embryo
limb after antimitotic insult
Susan M. Wilde, John H. Satchell, John Sowden and Dennis Summerbell. National Institute for Medical
Research, TheRidgeway, Mill Hill, London NW71AA
Restorative growth has been demonstrated in the early mouse embryo (Snow & Tam, 1979). Primitive
streak embryos treated with mitomycin C (MMC) exhibit asynchronous increased growth of the various
organs until at 13-5 days they achieve a normal weight and size. There is good evidence for regulation of
growth in the chick limb bud (Summerbell, 1981) and we have now used antimitotic drugs to create a
situation in which restorative growth might be observed during fetal stages.
1-B-D-arabinofuranosylcytosine (ara-c) is droped onto limb bud stages. Whole mounts stained to show
the skeleton are prepared at regular intervals up to 14 days. The lengths of 7 skeletal elements for wings and
legs are measured using a computer assisted method. The skeleton is displayed on a monochrome VDU. A
computer generated cursor is superimposed on the image using a BBC microcomputer. The position of the
cursor is adjusted using a digitising tablet, and the ends of each skeletal element digitised. This provides data
from which the lengths of skeletal elements (and other parameters such as angle of articulation of joints) can
be obtained.
The treated limbs are shorter than the control limbs and there is no evidence for an increase in the growth
rate at any stage. There is therefore no restorative growth.
We are now using [5-3H] ara-c to trace how long the drug stays in the embryo. In the case of the mouse the
antimitotic agent may be removed across the placenta. The difference between the mouse and chick results
does not seem to be drug dependent, so far MMC has produced similar results to ara-c.
SNOW, M. H. L. & TAM, P. P. L. (1979). Is compensatory growth a complicating factor in mouse teratology?
Nature (Lond.) 279, 555-557.
SUMMERBELL, D. (1981). Evidence for regulation of growth size and pattern in the developing chick limb
bud. /. Embryol. exp. Morph. 65 (suppl.) 129-150.
Pattern formation
Pattern formation during lateral line development in Xenopus
185
Rudolf Winklbauer, Max-Planck-InstitutfiirEntwicklungsbiologie, Abt. VSpemannstrasse35, D-7400
Tubingen, West Germany
The lateral line system of Xenopus is an example of a periodic pattern, a kind of pattern widely distributed
among living organisms. The development of the supraorbital lateral line system has recently been studied
quantitatively at the cellular level (Winklbauer & Hausen, 1983a, b). The organ system has been shown to
develop from an epidermal primordium which elongates to form a streak of cells. This streak is subdivided
into a linear series of cell groups containing only about eight cells each, thus forming a row of primary
organs. These studies have been extended here by asking which rules govern pattern formation in this
system. In triploids, cell size is 1-5 x normal. When the primordium is fragmented into primary organs in
triploids, these organs do not contain eight cells, as normally, but only about five cells, i.e. about
§ x normal. Thus, the increase in cell size is compensated by a corresponding reduction in cell number,
keeping constant the organ size in terms of total cell mass or volume. This result excludes a cell counting
mechanism for determining organ size. If (in diploid embryos) the size of the primordium is reduced
experimentally, the size of the primary organs formed is not adjusted in such a way as to allow the formation
of a normal number of organs. Instead, the size of the forming organs is kept normal, and the number of
organs is correspondingly reduced, i.e. the primordium is not capable of 'size regulation'. This result is
consistent with a 'prepattern' type of mechanism underlying the formation of the periodic lateral line
pattern.
R. & HAUSEN, P. (1983a). Development of the lateral line system in Xenopus laevis. I.
Normal development and cell movement in the supraorbital system. /. Embryol. exp. Morph. 76,
265-281.
WINKLBAUER, R. & HAUSEN, P. (19836). Development of the lateral line system in Xenopus laevis. II. Cell
multiplication and organ formation in the supraorbital system. /. Embryol. exp. Morph. 76, 283-296.
WINKLBAUER,
Cell death in regulating chick wings buds
Bridget Yallup, Department of Biology, Medical and Biological Sciences Building, The University,
Southampton SO93TU
Cell death is a characteristic feature of chick wing development. Examination of the cell death pattern in
wing buds with an experimentally created deficiency or excess of cells along the antero-posterior (a-p) axis is
one way to test the possibility that cell death plays an important developmental role by controlling the
amount of mesenchyme available to form skeletal elements. Deficiencies and excesses were created at stage
21 (this stage regulates well for both skeletal pattern and size) by respectively either removing or duplicating
a central 1-5 somite width slice of tissue (Yallup & Hinchliffe, 1983). Deficient, excess and normal wing buds
stained in ovo with Neutral Red were examined at successive 3 to 4-h intervals during the first 48 or 72 h
after operation. A modification of the normal cell death pattern is seen from 6 h after operation in
regulating wing buds. The main effect is on the Anterior Necrotic Zone (ANZ), the Opatjue Patch being
slightly affected, whilst the Posterior Necrotic Zone appears relatively normal. In deficiencies, the ANX is
in half the cases completely inhibited until 48 h after operation, an additional small area of anterior cell
death being present between 6 to 12 h in the remaining cases. The ANZ in an excess enlarges from 8 h, to 2
to 3 times the linear dimensions of the normal ANZ and remains persistently enlarged. These results suggest
that the ANZ may well play an important role in the regulation process, possibly by controlling the amount
of mesenchyme available to form skeletal elements. However, cell death is not proposed as either the sole or
the primary mechanism by which regulation may occur, since post-operative changes in other
developmental events are seen concurrently with changes in the normal cell death pattern in regulating wing
buds.
B. L. & HINCHLIFFE, J. R. (1983). Regulation along the antero-posterior axis of the chick wing
bud. In Limb Development and Regeneration, A (eds. J. F. Fallon & A. I. Caplan), pp. 131-140. New
York: Alan R. Liss Inc.
YALLUP,