Showing papers in "Development in 1977"

Limb-somite relationship: origin of the limb musculature

Abstract: Quail-to-chick grafting experiments performed during the third day of incubation demonstrate that somites can contribute to limb development. In orthotopic recombinations, migrating cells originating from the grafted unsegmented or segmented somitic mesoderm adjacent to the wing or leg field end up in the musculature respectively of the wing or the leg, where they express exclusively myogenic properties. Thus, in these heterospecific recombinations, the anatomical muscle has a double origin: muscle bulk of somitic origin; tendons and connective tissues of somatopleural origin. Similar features are observed in heterotopic recombinations with (segmented or unsegmented) somitic mesoderm located cranially or caudally to the limb levels. In the reverse chick-to-quail grafting experiments, the somitc participation to the limb mesoderm can also be observed. But it is less regular than that obtained in the quail-to-chick recombinations, and the muscle bulk is made up in various proportions of graft-originated somitic cells and of host somatopleural cells. The possible existence of juxtaposed and interdigitated myogenic and tendinogenic compartments is discussed in view of the dissimilarity between the results of the two kinds of heterospecific recombinations.

Gastrulation in the mouse: Growth and regionalization of the epiblast

Abstract: Histological determination of cell numbers in the mouse embryo between 4½ and 7½ days post coitum show that growth during this period, in which gastrulation occurs, is not uniform. Prior to primitive streak formation mean cell generation time is about 9 h. Co-incidental with the appearance of the primitive streak the embryo enters a period of rapid growth, lasting about 24 h, during which the mean cell generation time must be about 5 h in order to account for the increase in cell numbers. A more detailed study, in which variations in mitotic activity in different regions of the embryo have been analysed, has identified a small region, the so-called ‘proliferative zone’, constituting about 10% of the whole epiblast, in which cell generation time may average as little as 2–3 h over a 24 h period. The cell generation time for other epiblast regions is estimated at about 6·5 h. It is calculated that the proliferative zone, in the 24 h period commencing with primitive streak formation, could generate about half the cells in the 7½-day embryo. The topographical consequences of such a rapidly expanding region in the embryo are discussed in the light of other, circumstantial evidence, and it is postulated that the cells generated in the PZ may constitute the ectoderm of later stage embryos.

Analysis of endoderm formation in the avian blastoderm by the use of quail-chick chimaeras. The problem of the neurectodermal origin of the cells of the APUD series.

Abstract: The formation of the endoderm has been investigated in chimaeric embryos resulting from the combination of the lower and upper germ layers taken from chick and quail embryos at stages 2–6 of Vakaet (1962). The ability to recognize quail from chick cells made it possible to follow the fate of each germ layer during development. It appeared that the primitive hypoblast participates in the formation of the anterolateral extra-embryonic endoderm while the embryonic endoderm is formed later by migration of cells of the ectomesoblast through Hensen9s node and the primitive streak. Further interspecific combinations were carried out between ectoderm and endoderm + mesoderm from quail and chick embryos at stages 5–7 of Hamburger and Hamilton. The explants were grafted into chick embryos for several days and the intestinal structures which developed were observed. No contribution of cells from the neurectoderm to the endoderm was found. In contrast, cells coming from the neural crest colonized the intestinal structures and gave rise to the enteric ganglia. It was concluded from these observations that the enterochromaffin and endocrine cells of the gut epithelium do not originate from the neurectoderm.

Development of cytochalasin B-induced tetraploid and diploid/tetraploid mosaic mouse embryos

Abstract: By subjecting F1 (CBA × C57/BL) × A eggs at the time of 2nd cleavage to 10 μg/ml of cytochalasin B (CB), tetraploidy was produced in 52 % of 2-cell eggs and 35 % of 3-cell eggs. 2n/4n mosaic embryos were produced from 2-, 3- and 4-cell eggs and amounted to 20 % of all treated eggs. 80 % of tetraploid embryos developed in vitro into regular blastocysts with half the cell number of control diploids. The effectiveness of CB in producing tetraploid embryos is limited by the asynchrony of 2nd cleavage, both between eggs and between sister blastomeres. Two-cell presumed tetraploids were transplanted to recipients and examined between the 6th and 11th day of pregnancy. Up to 6½ days development is normal and most embryos form egg-cylinders. At 7½ days the embryonic part of the cylinders is underdeveloped and in later development fails to form an embryo. Development of foetal membranes is much less affected and in the most successfully developing egg-cylinders their formation can be fully accomplished. Failure of embryonic development appears to be due to subnormal activity of the primitive streak, resulting in shortage of mesoderm. Postimplantation development of 2n/4n mosaics was normal. While in embryos tetraploid cells were either absent or in very low proportion (below 4 %), their contribution to the foetal membranes amounted in some cases to up to 50 %. Elimination of tetraploid cells from mosaic embryos suggests that they have a lower proliferation rate than diploid cells.

Factors affecting the time of formation of the mouse blastocoele.

Abstract: In normal mouse embryos developing in vivo, the first appearance of the blastocyst cavity was found to be associated more closely with developmental age, judged by cell number, than with chronological age, i.e. elapsed time since ovulation. When development was slowed by in vitro culture, formation of the blastocoele was delayed. However, cell number itself was not a critical factor, since the number of cells per embryo could be doubled or tripled or halved by experimental manipulation without substantially affecting the timing of blastocoele formation. Experiments in which one cell division was suppressed with cytochalasin-B, leading to tetraploidy, showed that the number of cell divisions since fertilization was also not critical. A possible role is suggested either for nucleocytoplasmic ratio, or for the number of nuclear or chromosomal divisions or DNA replications since fertilization, all of which increase during cleavage.

An experimental investigation into the early development of the chick elbow joint

Abstract: The theory that differential growth of opposed chondrogenic centres is important in early joint formation has been tested experimentally by removing structures in relation to the chick elbow joint. The humerus and its cap of differentiating joint cells were found to develop independently of structures distal to them. Removal of the presumptive joint region at early stages resulted in fusion of the humerus with the radius and ulna. Results are discussed in terms of concepts concerning pattern formation of cell types in the early wing-bud.

Glycosaminoglycan localization and role in maintenance of tissue spaces in the early chick embryo

Abstract: Comparison of sections stained with Alcian blue at pH 1·0 or 2·5 demonstrates the distribution of sulfated and non-sulfated glycosaminoglycans in the extracellular matrix of the stage-8 (Hamburger & Hamilton, 1951) chick embryo. Both types of GAG are present in basement membranes throughout the embryo. Treatment of sections with Streptomyces hyaluronidase, reported to be specific for hyaluronic acid, prior to staining with Alcian blue at pH 2·5 reveals that hyaluronate is an important constituent of the extracellular matrix in basement membranes and in intercellular spaces within the mesoderm. Hyaluronate is shown to be the predominant glycosaminoglycan in the matrix of the head mesenchyme. In addition, examination by SEM and light microscopy of embryos after treatment in ovo with hyaluronidase shows that removal of hyaluronate from living embryos results in a dramatic decrease in cell-free spaces and a weakening of the association between mesoderm and ectoderm in the head.

Induction of melanogenesis in the epidermal melanoblasts of newborn mouse skin by MSH.

Abstract: The number of epidermal melanocytes positive to the dopa reaction increased when skin explants from newborn mice were cultured with MSH or dbc-AMP. These agents seem to induce melanogenesis in the pre-existing melanoblasts. This hormone-induced melanogenesis is suppressed by actinomycin D or cycloheximide, suggesting that the initiation of melanogenesis in the epidermal melanoblasts requires de novo transcription and translation.

Sex determination in germ line chimeras of Drosophila melanogaster.

Abstract: Of 55 flies developing from blastoderms which had received male or female pole cell transplants, 15 (7 females and 8 males) wer shown by progeny testing to be germ line chimeras. Since donor and host pole cells were genetically marked with contrasting X- or Y-linked alleles, the progeny testing scheme enabled the genotypic sex of the donor component undergoing gametogenesis to be identified as either the same as ('homosexual' chimeras) or opposite ('heterosexual' chimeras) that of the host. All seven of the female chimeras were identified as 'homosexual' chimeras carrying only chromosomally female XYX donor and XX host germ cells. Similarly, all eight males were shown to be 'homosexual' chimeras with chromosomally male XY donor and XY host germ cells. The chromosomal sex of the donor component undergoing gametogenesis was in every case the same as the phenotypic sex of the host. Since there is an equal probability of constructing either a 'homosexual' or a 'heterosexual' chimera during pole cell transplantation, the ability of pole cells to differentiate functional gametes in hosts of the opposite sex was tested 50% of the time even if sex reversal of these donor pole cells could not be demonstrated. Thus the absence of 'heterosexual' chimerism strongly supports the interpretation that the phenotypic sex of a germ cell in Drosophila is determined entirely by its own chromosome constitution, not by that of the gonadal mesoderm.

Properties of extra-embryonic ectoderm isolated from postimplantation mouse embryos

Abstract: Extra-embryonic ectoderm isolated from the mouse embryo as late as 81/2 days post coitum can form cells with the morphological characteristics of trophoblast giant cells both in ectopic sites and in vitro. This similarity to the properties of ectoplacental cone tissue provides further support for the postulated common origin of both tissues from the trophectoderm of the blastocyst.

Mutations of Drosophila melanogaster that affect muscles

Abstract: Eight X-chromosome mutations (falling into five complementation groups) that affect the development and morphology of the indirect flight muscles of Drosophila melanogaster were investigated using histological, behavioural and genetic techniques All of these mutations result in flightlessness, in a marked reduction in the ability of the flies to jump, and in the wings being held in abnormal positions Mutations in each of the complementation groups have different effects on the morphology of the muscles Two (flapwing, vertical wing) result in absence of most of the indirect flight muscle fibres, a third (upheld) is required for the gross organization of muscle structure, another (heldup) is involved in the maintenance of muscle structure once formed, and the fifth seems to be necessary for the detailed architecture of the muscle fibre (indented thorax) The analysis of flies genetically mosaic with respect to each mutation by the technique of fate-mapping suggests that three (heldup, upheld and indented thorax) of the genes concerned have their primary site of action in the musculature itself, while the other two (flapwing and vertical wing) may function primarily in the fat-body and tracheae respectively

Embryonic malformations in rats, resulting from maternal diabetes: preliminary observations.

Abstract: Diabetes mellitus was induced in female Wistar rats by injections of either alloxan or streptozotocin, and their embryos were found to have significantly higher incidences (7.5%) of brain and heart abnormalities (non-closure of neural folds, and deformities of heart chambers) at mid-gestation than controls (2.2%). There were also increased numbers of resorptions (25% in diabetic animals: 7.2% in controls). Both drugs produced similar abnormalities. External and X-ray examination of 488 foetuses from streptozotocin-treated animals at 20 days showed eight cases of exomphalos, two cases of micrognathia with tongue protrusion, and 34 cases of incomplete sacral ossification. This last deformity occurred also in foetuses of mildly diabetic animals, and has been seen occasionally in infants of human diabetic mothers. Other evidence suggests that skeletal deformities may be due to hyperinsulinism in the foetuses of diabetic mothers. Even a mild or pre-diabetic condition may set the foetus at risk.

A method for enucleating oocytes of Xenopus laevis.

Abstract: A method is described for the enucleation and complete healing of Xenopus oocytes so that the enucleated oocytes withstand multiple injections and culture for several days. The oocytes are defolliculated and enucleated manually and allowed to heal in a potassium phosphate buffer. Oocytes enucleated in this way support RNA synthesis by injected HeLa-cell nuclei 3 days later. This method is valuable in providing a low-background system in which transcription of injected nuclei can be studied.

In vitro development of haploid mouse embryos produced by bisection of one-cell fertilized eggs

Abstract: F1(CBA × C57BL'10) mouse eggs originating from spontaneous or induced ovulation and fertilized by CBA-T6T6 or PO spermatozoa were bisected with a glass needle into halves each containing a pronucleus. This technique offers a unique opportunity of producing both androgenetic and gynogenetic haploid embryos from one egg. Out of 600 operated eggs, in 406 (67·7%) both halves survived. During 96 h of culture in vitro the fragments were inspected once daily and finally examined in air-dried preparations. Eighty-seven per cent of halves underwent first cleavage but their further development was to a large extent affected by extrinsic factors connected with experimental procedure (mainly by suboptimal and variable culture conditions) and by the origin of eggs (those from spontaneous ovulation being superior). For this reason developmental capabilities of egg halves were assessed in a selected group of pairs in which at least one partner reached the stage of four or more blastomeres. The observed ratio between pairs with both or only one sister embryo developing successfully suggests that androgenetic embryos carrying Y rather than X chromosome can cleave twice but do not survive beyond 4-cell stage. None of the metaphase plates from older embryos contained a Y chromosome. These observations imply that the X chromosome is genetically active during early cleavage and that a full haploid set is required for preimplantation development to be completed. Formation of blastocysts varied from batch to batch, with an average of 12·8% and maximal incidence of 29·5% . In 34 pairs both fragments developed beyond the 4-cell stage but in only one case did both form blastocysts. Haploid blastocysts were composed of 27 cells on average which was about a half of the number of cells in control diploid zona-free whole eggs. Ten out of 51 embryos with metaphase plates proved to be haploid/diploid mosaics.

Development of interspecific hybrids of Mus

Abstract: Artificial insemination has been used to produce interspecific mouse hybrids. Mus musculus x Mus cervicolor cervicolor hybrids failed to complete more than a few cleavage divisions but both M. musculus x M. dunni and M. musculus x M. caroli hybrids completed preimplantation development. These hybrid embryos are heterozygous for various X-linked enzymes and may provide a useful genetic system for studying X-chromosome inactivation during early development. Further development of M. muscuius x M. caroli hybrids was studied: several completed foetal development; a few survived to maturity but none has yet reproduced.

Fertilization of immature frog eggs: cleavage and development following subsequent activation.

Abstract: Frog eggs are normally fertilized after reaching metaphase II. When eggs are inseminated prior to that, several sperm enter, but entry does not activate the egg. When such inseminated, immature eggs were maintained until they became mature and then were artificially activated, the eggs began to cleave. The cleavage furrows were irregular and often multiple, but the eggs developed to blastulae or partial blastulae. About 2 leads to 5% of the eggs developed to tadpoles. Typical asters were not associated with the entering sperm; rather, asters appeared only after activation. The sperm nucleus often formed chromosomes which were attached to small spindles. It is clear that sperm which remain for a time in unactivated egg cytoplasm, retain their ability to promote cleavage and development. Aster formation required not only sperm centrioles but also activated egg cytoplasm. Sperm which entered either near the equator or in the animal half of mature eggs usually produced normal cleavage furrows. Sperm which entered the animal half of immature eggs produced multiple animal half furrows when the egg was subsequently activated. In contrast, sperm which entered near the equator of immature eggs often failed to induce furrowing on subsequent activation or produced unusual equatorial furrows. The difference in the type of furrow between eggs inseminated in the animal half or at the equator is interpreted as a consequence of dissociating sperm entry from the cortical contraction which occurs in activation.

Interdigital cell death during limb development of the turtle and lizard with an interpretation of evolutionary significance

Abstract: Cell death accompanies the formation of free digits in birds and mammals. However, in species with webbing between the adult digits, little or no cell death occurs in the prospectively webbed region of the developing interdigit. Cell death does not occur during the formation of free digits in amphibians. In this paper we report that cell death accompanies the formation of the digits in snapping and painted turtles and in the skink (a lizard). We conclude that cell death accompanying the formation of free digits had its origin at the point of amniote emergence during evolution.

Origine des ceintures scapulaires et pelviennes chez l'embryon d'oiseau

Abstract: The development and the origin of the pectoral and pelvic girdles have been studied in bird embryos by homo- or heterotopic transplantations of somitic and somatopleural mesoderm. Experiments consisted in implanting a piece of somitic or of somitic and somatopleural mesoderm obtained from a 2- to 2·5-day quail or chick embryo into a chick host of equal age. Results showed that the scapula derives from the somitic mesoderm, while clavicle, coracoid, sternum and pelvic girdle originate from the somatopleural mesoderm. The longitudinal span of the territories of the various skeletal elements, expressed as the number of the corresponding somites, was found to be as follows: scapula 15–24, clavicle 10–15, coracoid 15–17, sternum 12–26, and pelvic girdle 26–32. It was also demonstrated that both somitic and somatopleural mesoderm are regionalized as early as 2 days of incubation, prior to somitic segmentation, with respect to their ability to give rise to the skeletal elements of the girdles. These results were compared to those acquired previously concerning the other morphogenetic potentialities (vertebrae, ribs, limb musculature, dorsal plumage) of the para-axial and lateral mesoderm.

Inhibition of cranial neural crest cell development by vitamin A in the cultured chick embryo.

Abstract: Chick embryos at stage 8, prior to neural crest cell migration, were explanted on whole egg medium with or without vitamin A and cultured for 3 days. Sections through the head regions showed that the cranial neural crest cells had migrated into the first visceral arch in the controls but were absent from this structure in the treated embryos. These observations suggest that vitamin A inhibits neural crest cell development or migration, an effect which may in part account for the facial malformations produced by excess vitamin A.

The establishment of the oral-aboral axis in the ctenophore embryo.

Abstract: In a small percentage of normal embryos and in a higher percentage of embryos centrifuged prior to the first cleavage the positions of the polar bodies and the site of the first cleavage furrow do not coincide These cases have been used to establish whether polar body formation sites or first cleavage initiation sites correlate best with the oral-aboral axis of the embryo In all cases when the first cleavage is initiated at a site different from the site where the polar bodies were given off, the pattern of the first four cleavages is normal, the segregation of comb plate potential at these stages is normal, and the larvae that form are normal The extent to which comb plate potential is localized along the oral-aboral axis of the embryo prior to the first cleavage, during the first cleavage and at the 2-cell stage was also examined These experiments demonstrate that the oral-aboral axis is established at the time of the first cleavage, that cleavage plays a causal role in setting up the axis, and that comb plate-forming potential begins to be localized in the aboral region of the embryo at this time

In vitro development of palatal tissues from embryonic mice. II. Tissue isolation and recombination studies.

Abstract: The epithelial and mesenchymal tissues of the secondary palate from 12-, 13-, and 14-day embryonic mice were enzymatically separated and cultured in isolation and in homochronic and heterochronic recombinations. In both homochronic and heterochronic recombinations, epithelial differentiation was similar to that in vivo . In heterochronic recombinations, epithelium differentiated according to a schedule appropiiate for the age of the epithelium rather than for the age of the mesenchyme, suggesting that differentiation of palatal epithelium is temporally ‘determined’ as early as 12 days of gestation. Palatal epithelium cultured in isolation was capable of limited differentiation. Potential for differentiation therefore exists within the isolated palatal epithelium at an early stage of palatal development, and epithelial-mesenchymal interactions are required during palatal development to support full epithelial differentiation.

Evaluation of the technique of immunosurgery for the isolation of inner cell masses from mouse blastocysts.

Abstract: Inner cell masses (ICMs) immunosurgically-isolated from 31/2-day mouse blastocysts were examined for trophoblast cell contamination and developmental capacity. Blastocysts were preincubated in rabbit anti-mouse antiserum, washed thoroughly and then incubated in complement. The ICMs were then easily dissected by drawing through a fine pipette. Various experiments confirmed that the trophectoderm had been completely removed by this treatment. Firstly, the ICMs did not bind a fluorescein-conjugated antibody directed against rabbit IgG, indicating the absence of cells exposed to the rabbit antiserum during the immunosurgical procedure. Secondly, ICMs dissected from blastocysts preincubated in a suspension of melanin granules did not include any of the trophoblast cells that had phagocytosed the granules. And, thirdly, the protein synthetic profile of these ICMs was similar to microsurgically dissected ICMs, and in particular, trophoblast specific spots were absent. The developmental capacity of immunosurgically-isolated ICMs was tested by injecting them into blastocysts and transferring to the uterus of 2 1/2-day pseudopregnant recipients. Extensive chimaerism was detected in the majority of implants, 5-6 days after transfer, but only in ICM-derived tissues. This demonstrates both the lack of trophoblast cell contamination and functional viability of these ICMs.

Weight differences in rat embryos prior to sexual differentiation

Abstract: Sex of day-12 rat embryos was determined using Barr body counts made on spreads of amniotic membranes examined histologically. Embryonic weight, protein content and rate of thymidine incorporation were compared in male and female embryos. Male embryos were found to be heavier and accordingly to contain more protein on absolute but not on per unit weight basis. The rate of thymidine incorporation did not differ in the two sexes. Since gonadogenesis in day-12 rat embryos is rudimentary, with gonadal differentiation of sex not yet apparent, the increased weight suggests that sex-linked genes exist which influence body growth prior to gonadal endocrine activity.

Pattern formation in Dictyostelium discoideum: I. Development of prespore cells and its relationship to the pattern of the fruiting body

Abstract: Immunofluorescent staining of the prespore cells of the cellular slime mould Dictyostelium discoideum was carried out using a heterologous spore antibody. The highly specific staining of the prespore vesicles (PSVs) within the prespore cells enabled quantitative determinations to be made of the rate and extent of development of these cells throughout the life cycle. The results showed that PSVs first appeared in a large proportion of the cells shortly after the cells had chemotactically aggregated into multicellular masses. During the later phases of the life cycle, the proportion of cells containing PSV increased, as did the fluorescent intensity of their PSVs, until the early culmination stage of development when 85–90 % of the total cell population contained PSVs. Lowering the temperature of development delayed the onset of vesicle formation and decreased the proportion of prespore cells in the total cell population. Changing the growth conditions of the cells prior to multicellular development also had a significant effect on the proportions of prespore cells, as did the use of a mutant known to give rise to fruiting bodies with a reduced number of spores. The comparability between these estimates of prespore cell proportions at culmination and previously reported spore:stalk ratios within fruiting bodies confirms the view that PSVs are reliable indicators of prespore cells. The finding that temperature and growth conditions and the use of mutants all of which are known to affect spore:stalk ratios, also all affected prespore proportions in the expected direction, adds further weight to this argument. The fact that prespore cells are beginning to differentiate early in the multicellular phase of the life cycle and the related finding that such differentiation always precedes formation of the grex tip are results of considerable importance to the development of a model for pattern formation in D. discoideum .

On the possibility of sperm aster involvement in dorso-ventral polarization and pronuclear migration in the amphibian egg.

Abstract: Sperm aster involvement in the determination of the plane of bilateral symmetry of the future embryo as well as pronuclear movement and orientation has been investigated in the amphibian egg. The procedure involved injecting very small amounts of disrupted sperm cells into the subcortical region of the egg of the toad Bufo arenarum . Using this procedure we have previously shown that structural integrity of the sperm cell is not required for the determination of the symmetry plane. The present report deals with the cytological effects induced by the injection of sperm homogenate into the egg. As expected, an aster was always present in these eggs. This aster closely resembled that produced in the fertilized egg as the sperm nucleus migrates through the egg mass. Sections of injected eggs have also shown the presence of two pigment trails similar in appearance to those produced by pronuclear movement after normal fertilization. The streak analogous to the sperm entrance track emerged from the point where the pipette was introduced. None of these effects were observed in Ringer's injected eggs. At the time of the rotationof symmetrization, the female pronucleus was seen within a clear area of cytoplasm located at a short distance below the egg cortex. An aster field organized around this clear area was probably formed as a result of the egg centriole activation. In no case was the egg nucleus seen to migrate towards the copulation site as was seen to occur in sperm homogenate-treated eggs. The active agent was found to be a particulate component which sediments with the head but not the tail fraction of Bufo arenarum sperm. Injection of blood cells caused a similar response to that elicited by the sperm homogenate.

Application of a morphological time scale to hereditary differences in prenatal mouse development

Abstract: A standardized morphological time scale for prenatal mice is presented, which is useful from 13·0 to 17·0 days chronological age with an accuracy of 0·1 day. Morphological age for an embryo or fetus is shown to correlate highly with ages estimated from body weight and crown-rump length. The time scale is used to study the comparative development from 14 to 17 days prenatal age of an F2 and four inbred mouse strains. The F2 mice average 0·5 day ahead of C57BL/6 mice, and C57BL/6 mice average 0·5 day ahead of A, BALB/c, and, at some ages, DBA/2 mice. Using reciprocal F1 crosses and reciprocal backcrosses, it is also shown that both the fetal heredity and the maternal environment contribute significantly to the more advanced development of hybrid mice.

The eversion and differentiation of Drosophila melanogaster leg and wing imaginal discs cultured in vitro with an optimal concentration of β-ecdysone

Abstract: The stages of the eversion and differentiation of prothoracic leg and wing discs of Drosophila melanogaster , in Shields and Sang9s medium 3, are described. The range of specific imaginal structures produced, including patterns of sensilla trichodea and sensilla campaniformia, are noted, and the relationship of these structures to those differentiated in situ is discussed.

Delayed appearance of ectodermal cell death as a mechanism of polydactyly induction

Abstract: The temporal program of cell death in the apical ectodermal ridge and mesoderm of rat embryo hindlimbs was documented using supravital staining with Nile blue sulfate. Dye uptake indicative of cell death began postaxially at about 290 h of development and was followed in a few hours by preaxial staining, which became more extensive and intense up to 313 h. Two agents which cause preaxial polydactyly, cytosine arabinoside and 5-fluorodeoxyuridine, postponed the onset of preaxial ectodermal cell death while at the same time having the expected cytotoxic effect on limb-bud mesoderm. In addition, a zone of deep preaxial mesodermal necrosis, thought to control the size of digit 1 in normal embryos, was usually absent in cytosine-arabinoside-treated embryos. The results suggest that the prolonged survival of ectodermal cells effected an increased inductive activity on the underlying mesoderm, leading to the formation of excess digital tissue. The data further suggest that the rate at which mesodermal cells were killed affected the subsequent delay of ectodermal cell death.

Genetic analysis of developmental mechanisms in hydra: III. Characterization of a regeneration deficient strain

Abstract: Mutant hydra strains showing abnormal development can be isolated through sexual inbreeding of wild hydra. One such mutant strain, called reg-16, regenerates tentacles very poorly following amputation of the head and foot. Tentacle regeneration, however, is significantly enhanced by subdividing the regenerating fragment longitudinally. Lateral tissue implants that induce head formation in wild-type hydra either regress or induce foot formation in reg-16 polyps. These results suggest that regeneration deficiency in reg-16 is due to a defective polarity gradient. A chimaeric strain of hydra was produced by combining interstitial cells (and thus their differentiation products, nerve cells and nematocytes) of reg-16 hydra with epithelial cells of another strain which is capable of normal regeneration. The chimaeras regenerate normally, suggesting that the defect of reg-16 is not located in the interstitial or nerve cells.

Ultrastructural analysis of the apical ectodermal ridge during vertebrate limb morphogenesis: II. Gap junctions as distinctive ridge structures common to birds and mammals

Abstract: The fine structure of the apical ectodermal ridge of five phylogenetically divergent orders of mammals and two orders of birds was examined using transmission and freeze fracture electron microscopy. Numerous large gap junctions were found in all apical ectodermal ridges studied. This was in contrast to the dorsal and ventral limb ectoderms where gap junctions were always very small and sparsely distributed. Thus, gap junctions distinguish the inductively active apical epithelium from the adjacent dorsal and ventral ectoderms. The distribution of gap junctions in the ridge was different between birds and mammals but characteristic within the two classes. Birds, with a pseudostratified columnar apical ridge, had the heaviest concentration of gap junctions at the base of each ridge cell close to the point where contact was made with the basal lamina. Whereas mammals, with a stratified cuboidal to squamous apical ridge, had a more uniform distribution of gap junctions throughout the apical epithelium. The difference in distribution for each class may reflect structural requirements for coupling of cells in the entire ridge. We propose that all cells of the apical ridges of birds and mammals are electrotonically and/or metabolically coupled and that this may be a requirement for the integrated function of the ridge during limb morphogenesis.