Search

One of the ways in which Fundulus deep cells move in vivo is by putting out long, fingerlike protrusions. This involves a change in the shape of the cell as a whole, with cytoplasmic flow, and is not just a local phenomenon. Moreover, particles on the cell surface move toward a protrusion as it is forming, suggesting surface flow. The role of surface flow is discussed both on a grown level and in respect to molecular fluidity. Long, stable protrusions can be pulled from cells by the application of negative pressure at a constant rate and these behave in a similar way to those formed during cell locomotion. Such long protrusions must be structured. The importance of contractile properties of the cytoplasm in the formation of protrusions was studied by treating cells with media that modify cellular contractility.

Antibodies to the myosin heavy chains of striated muscle were used to trace myogenic differentiation in the developing face and in cultures of cells from the facial primordia of chick embryos. In the intact face, myogenic cells differentiate first in the mandibular primordia and can be detected at stage 28. The early muscle blocks contain both fast and slow classes of myosin heavy chains. At stages 20 and 24, no myogenic cells are found in any of the facial primordia. However, when the cells are placed in micromass (high density) cultures, myogenic cells differentiate, revealing the presence of potentially myogenic cells in all the facial primordia. The number of myogenic cells bears no consistent relationship to the extent and pattern of chondrogenesis. Therefore the ability of the cell populations of the facial primordia to differentiate into cartilage when placed in culture is independent of the muscle cell lineage. The facial primordia represent a mixed cell population of neural crest and mesodermal cells from at least as early as stage 18.

Chondrogenesis of mesenchymal cells from the frontonasal mass, mandibles and maxillae of stage-24 chick embryos has been investigated in micromass (high-density) cultures. Distinct differences in the amount and pattern of cartilage differentiation are found. In cultures of frontonasal mass cells, a central sheet of cartilage develops; in cultures of mandible cells, less cartilage differentiates and nodules form; while in cultures of maxillae cells, virtually no chondrogenesis takes place. The same patterns of cartilage are found in cultures established from stage-20 embryos. At stage 28, frontonasal mass cultures form cartilage nodules and the number of nodules in mandible cultures is markedly decreased. There are striking parallels between the chondrogenic patterns of cells from the face and limb buds in micromass culture. The frontonasal mass cell cultures of stage-20 and -24 chick embryos resemble those established from the progress zone of limb buds. The progress zone is an undifferentiated region of the limb in which positional cues operate. Cultures established from the frontonasal mass of stage-28 chick embryos and from the mandibles of all stages resemble cultures of whole limb buds. These contain a mixture of committed and uncommitted cells. Ectoderm from facial primordia locally inhibits chondrogenesis in micromass cultures and this could provide a positional cue. The differences in chondrogenic potential of cells from facial primordia may underlie the specific retinoid effects on the frontonasal mass.

The formation of the nasal passages involves complex morphogenesis and their lining develops a spatially ordered pattern of differentiation, with distinct domains of olfactory and respiratory epithelium. Using antibodies to the neural cell adhesion molecule (N-CAM), keratan sulphate and heparan sulphate proteoglycan (HSPG) and a panel of lectins (agglutinins of Canavalia ensiformis (ConA), Dolichos biflorus (DBA), peanut (PNA), Ricinis communis (RCA1), soybean (SBA), Ulex europaeus (UEA1), and wheatgerm (WGA], we have documented cell surface characteristics of each epithelial domain. Binding of antibodies to N-CAM and to keratan sulphate, and the lectins ConA, PNA, RCA1, SBA and WGA marks the olfactory epithelial domain only. The restriction of N-CAM to the sensory region of the epithelium has also been reported in the developing ear. This striking similarity is consistent with the idea that N-CAM may be involved in the division of functionally and histologically distinct cell groups within an epithelium. We traced the olfactory-specific cell markers during development to gain insights into the origin of the epithelial lining of the nasal passages. All reagents bind at early stages to the thickened nasal placode and surrounding head ectoderm and then become progressively restricted to the olfactory domain. The expression of these characteristics appears to be modulated during development rather than being cell autonomous. The distribution of keratan sulphate was compared with collagen type II in relation to the specification of the chondrocranium. Keratan sulphate and collagen type II are only colocalized at the epithelial-mesenchymal interface during early nasal development. At later stages, only collagen type II is expressed at the interface throughout the nasal passages, whereas keratan sulphate is absent beneath the respiratory epithelium.

Retinoic acid is a good candidate for a morphogen in chick limb bud development. The challenge now is to determine how retinoic acid interacts with limb bud cells and how the retinoic acid signal is integrated with other signals to mould and pattern the developing limb.

The invasiveness of mouse lymphocytes and thymocytes, rabbit peritoneal neutrophil granulocytes (PMNs), mouse peritoneal macrophages (both activated and non-activated) and pig endothelial cells was assayed by implanting these cells to the chick wing bud. Cells of each type moved into the wing mesenchyme, although activated macrophages invaded poorly. PMNs were the most invasive cells and had moved well into the limb after only a few hours. PMNs, lymphocytes and thymocytes were ingested by wing mesenchyme cells. Endothelial cells, however, ingested chick blood cells. The implanted cells showed differences in ability to survive in the limb: PMNs disappeared rapidly, lymphocytes and thymocytes sometimes persisted for 24 h, while grafts of macrophages and endothelial cells were present at 24 h. Mechanisms which might be involved in the invasiveness of these cells, and also in their different abilities to survive in the chick wing, are discussed with particular reference to the production of plasminogen activator.

A method of controlled release that allows the continuous local application of retinoids (vitamin A derivatives) in living tissues has been developed. Several biocompatible 200-microns-diameter polymeric beads have been tested as possible carriers. Each type of bead was loaded by soaking in an isotopically labeled retinoid solution, washed, and then transferred into tissue culture medium for quantitative release measurements. Positively-charged ion-exchange resins of the Dowex 1 type were found to be the most suitable for the controlled release of retinoic acid, a negatively charged compound. For the controlled release of uncharged retinoids such as retinyl acetate, uncharged acrylic ester polymer beads are preferred; these beads can also be used to release the negatively charged compounds retinoic acid and prostaglandin E1. In all cases, a prolonged release is obtained that persists for more than a day. During this interval, the release is diffusion-controlled, and the total amount of compound released is directly proportional to the amount of the compound that the bead is exposed to during the initial loading step. High-performance liquid chromatography has been used to analyze the nature of the released retinoid. When the positively charged beads are loaded with all-trans-retinoic acid, there is a time-dependent decrease in the proportion of the all-trans isomer released which is due to an increased release of two cis isomers. This isomerization reaction occurs at a considerably slower rate when the uncharged beads are used as carriers. To mimic the conditions under which the local release of retinoic acid causes striking pattern duplications in developing chick wings, beads loaded with isotopically labeled retinoids were manually implanted into a slit cut into wing buds of stage-20 chick embryos. The release rate obtained was comparable to that found in vitro, and a time-dependent accumulation of the released radioactive compound was measured that was confined to the tissue near the site of implantation. All of the beads tested were readily accommodated by the tissue and could be easily removed at any time to terminate the treatment. It is believed that the controlled release of chemicals from such tiny biocompatible implants has a wide potential range of applications in biology.

Implanting inert carriers soaked in retinoic acid into the anterior margin of the developing limb of chicken embryos leads to orofacial malformations as well as affecting pattern formation in the limb. Using anion-exchange beads as carriers, and soaking solutions of 1-10 mg/ml retinoic acid, almost 100% of the embryos have malformations of the face. The effects on the treated limbs range from symmetrical patterns of duplicated digits (maximum number of digits being four) to truncations in which no digits were formed at all. Typically, in the malformed faces the upper beak is completely absent, no nostrils are present and the front of the face forms a scalloped rim of tissue above the mouth. By reference to normal beak development, the seven bulges of tissue that make up the rim can be identified as derivatives of the masses of tissue that normally would fuse to form the upper beak. The roof of the mouth consists of three bulges of tissue flanked by widely separated palatal shelves. The defect can thus be classified as severe bilateral clefting of the primary palate. By examining the morphology of the faces of treated embryos, the origin of the defect can be traced to failure of the frontonasal mass to enlarge. Thus, the oronasal fissures are very wide and fusion across them to form the primary palate cannot occur. The way in which retinoic acid brings about the defect is discussed in relation to possible mechanisms involved in the production of cleft palate. The parallel is noted between the associated effects of retinoic acid on beak and limb morphogenesis and the chick mutation cpp, that also affects both face and limbs.

Two retinoids, all-trans-retinoic acid and a synthetic analog, TTNPB, were locally applied to different positions along the proximo-distal axis of embryonic chick wing buds using controlled release carriers. Truncations or limbs with duplicated structures across the antero-posterior axis develop after retinoid application to distal positions in buds from stage 20-24 embryos. Phocomelic limbs develop when the retinoids are applied more proximally to buds of stage 23-24 embryos. Duplications of the pattern of structures along the proximo-distal axis never occur.Using TTNPB that is relatively stable, the amount of retinoid in the wing tissue when phocomelia is induced was measured. There is twice as much retinoid per cell in the proximal half of the bud as in the distal half of the bud. The concentration of TTNPB in proximal tissue is estimated to be three times higher than in distal tissue in which pattern formation and cartilage morphogenesis are relatively normal.At early stages in the development of phocomelia, the shape of the bud changes and the indentation that marks the elbow does not arise. Neither retinoid-induced cell killing nor effects on the pattern of programmed cell death were detected.The induction of phocomelia by retinoids appears to be based on effects on proximal cells, whereas retinoids produce pattern changes by acting on distal cells. Furthermore, compared with pattern changes, higher concentrations of retinoid in the bud tissue are required to produce phocomelia.

The interpretation of positional information can provide the basis for pattern formation in limb morphogenesis. The gradient in positional information along the antero-posterior axis, which is specified with respect to a localised boundary region, can be modified by grafting this region to successive positions along the axis. The pattern of digits obtained is consistent with a model based on diffusion of a labile morphogen and is thus similar to models proposed for the development of pattern in invertebrates.

A survey of the behaviour of a variety of normal and malignant tumours and cells has been carried out to gain insights into the mechanisms of tumour invasiveness. The tumours and cells were implanted into the developing chick wing bud, which is a loose mesenchyme bounded by ectoderm. The distribution of the grafted cells was examined histologically after one or two days. The special feature of this assay is that the behaviour of cells is tested in a 3-dimensional tissue. Cells from 3 different carcinomas, mouse lung tumour, rat bladder tumour and human breast tumour did not invade the mesenchyme, whereas trophoblast, sarcoma 180, cultured hamster fibroblasts (BHK, PyBHK, Nil 8, HSV Nil 8) and neuroblastoma cells did. Cells from embryonic pigmented retina and heart ventricle were non-invasive. These results suggest that cell movement may not be a common feature of all invasive tumours. The cells that did move into the mesenchyme appeared to do so by various mechanisms. Lack of contact inhibition of movement, although probably involved in the invasiveness of sarcoma 180 cells, does not appear to be necessary for invasion: cells that have been shown to exhibit contact inhibition of movement (BHK and PyBHK) also invade. Both normal and transformed cells (BHK and PyBHK; Nil 8 and HSV Nil 8) moved into the mesenchyme. Other invading cells, such as trophoblast, neuroblastoma and to a small extent, HSV Nil 8 cells, destroy the adjacent host tissue and this may be important in the invasiveness of these cells. The patterns of invasion and interactions with the host tissue were varied. Trophoblast and the fibroblasts were often elongated along the basement membrane at the ectoderm/mesenchyme border and also closely apposed to the endothelial linings of blood vessels. Sarcoma 180 and neuroblastoma cells clustered around nerves. The embryonic tissues and neuroblastoma cells were often associated with blood vessels. These results are discussed in relation to tumour invasion. A striking finding was that the carcinoma cells were frequently found positioned within the wing ectoderm on the basement membrane. This affinity of carcinoma cells for the epithelium rather than the mesenchyme leads to a reappraisal of the mechanisms involved in the invasiveness of carcinomas.

The interaction of cells from embryonic liver, neural retina and mesonephros with cells from limb-bud mesenchyme has been investigated in vivo by grafting these tissues into the developing chick wing-bud. The implanted cells were in all cases from quail tissue which can be recognized histologically. As embryonic liver and neural tube are tissues that sort externally to limb-bud mesenchyme in mixed aggregates, it would be expected, from a differential adhesiveness hypothesis, that heterotypic adhesions along the borders of graft and host would be favoured over cell-cell adhesions in the graft. No morphological signs of this were evident: rather the grafted cells maximized like-like contacts. The cells of the grafts, including those from control mesenchyme, did not invade into the wing. The results were the same irrespective of whether the graft was a fragment of tissue or a pellet of reaggregated cells. This supports the idea that cells within tissues are not actively moving around and also provides controls for assaying the invasiveness of other cell types, such as malignant cells into the wing.

When retinoic acid is locally applied to the anterior margin of developing chick wing buds on ion-exchange beads, dose-dependent changes in the skeletal pattern result. At low doses, additional digits develop. At high doses, there is thinning of the symmetrical wing. Local application of retinoic acid to the apex of the bud also leads to pattern changes, but in contrast normal wing patterns are almost always obtained following application posteriorly. These effects are manifest at 6-7 days after the operation although only a brief exposure (14-20 h) to retinoic acid is required. Therefore the morphology of wing buds was studied at shorter times after the start of treatment. The local application of retinoic acid to the wing bud margin leads to changes in extent of the apical ridge that can be detected at 24 h after application. The behaviour of the apical ridge with varying doses and positions of retinoic acid application has been analysed quantitatively and dose response curves obtained. At low doses of retinoic acid, the length of the apical ridge increases or remains constant, but then progressively decreases with higher doses. The progressive obliteration of the ridge starts first near the bead and then involves more distant parts of the bud. Thus the region of the ridge affected depends on the position at which the retinoic acid is applied. We propose that these effects on the apical ridge reflect dose-dependent responses to the local concentration of retinoic acid that varies with distance from the source. At high doses, the apical ridge disappears but at low doses it is maintained. Since grafts of polarizing region tissue also have a graded effect on ridge morphology, a possible interpretation of the retinoic acid effects is that tissue adjacent to the source is converted into polarizing region tissue. Alternatively, retinoic acid may act directly on the ridge cells. The changes in the extent of the apical ridge produced by retinoic acid lead to different forms of bud outgrowth. The form of the outgrowth depends on the dose of retinoic acid, the position of application and the interaction between the effects of the local source of retinoic acid and those of the polarizing region of the host bud. These considerations give some insights into why anterior application of retinoic acid leads to the development of additional digits whereas posterior application generally gives normal wings.

Cells from normal rat brain tissue did not survive and few cells could be found 1 d after grafting. In contrast, cells from a glioma and a carcinogen-treated rat brain survived well and many mitoses were observed. These malignant cells also invaded the limb. The behaviour of normal and malignant cells was followed at shorter times after grafting and some invasion by the normal cells was detected. The first signs of degeneration of normal cells were apparent around 7 h after grafting, and after this the grafts progressively deteriorated. These results support the ideal that the ability of cells to survive and grow in embryonic tissues is a characteristic of malignant cells. The findings are discussed in relation to mechanisms of tumour formation.

All-trans-retinoic acid causes striking digit pattern changes when it is continuously released from a bead implanted in the anterior margin of an early chick wing bud. In addition to the normal set of digits (234), extra digits form in a mirror-symmetrical arrangement, creating digit patterns such as a 432234. These retinoic acid-induced pattern duplications closely mimic those found after grafts of polarizing region cells to the same positions with regard to dose-response, timing, and positional effects. To elucidate the mechanism by which retinoic acid induces these pattern duplications, we have studied the temporal and spatial distribution of all-trans-retinoic acid and its potent analogue TTNPB in these limb buds. We find that the induction process is biphasic: there is an 8-h lag phase followed by a 6-h duplication phase, during which additional digits are irreversibly specified in the sequence digit 2, digit 3, digit 4. On average, formation of each digit seems to require between 1 and 2 h. The tissue concentrations, metabolic pattern, and spatial distribution of all-trans-retinoic acid and TTNPB in the limb rapidly reach a steady state, in which the continuous release of the retinoid is balanced by loss from metabolism and blood circulation. Pulse-chase experiments reveal that the half-time of clearance from the bud is 20 min for all-trans-retinoic acid and 80 min for TTNPB. Manipulations that change the experimentally induced steep concentration gradient of TTNPB suggest that a graded distribution of retinoid concentrations across the limb is required during the duplication phase to induce changes in the digit pattern. The extensive similarities between results obtained with retinoids and with polarizing region grafts raise the possibility that retinoic acid serves as a natural "morphogen" in the limb.

It has been suggested that positional information along the proximo-distal axis of the limb-bud is specified by time spent in the progress zone. Mesenchyme cells have been killed by X-irradiation, reducing the rate cells leave the zone. The time spent there by some cells is thus increased. When limbs, stage 18/19, stage 21, or tips of stage 24, are treated with increasing doses of X-irradiation, from 1000 rads to 2500 rads proximal structures are progressively lost, whereas distal ones--the digits--are relatively unaffected. There was no evidence for intercalation of missing parts. These effects are due to killing or damage of mesenchyme cells: the ectoderm is not affected at these doses. The results are consistent with a quantitative analysis based on the progress zone model, in which viable cells repopulate the progress zone and gradually restore it to normal as non-dividing cells are diluted out. It is suggested that any treatment causing damage to the mesenchyme at early stages will give similar results. The mesenchyme cells appear to be surprisingly resistant to radiation damage. The form of the limb-bud is not altered by damaging the mesenchyme. Differences in the development of structures at similar proximo-distal levels, following irradiation, is considered in terms of the requirement of a threshold number of cells.

Retinoids have been applied to stage 20 chick embryos by using beads that act as controlled release carriers. With beads soaked in high concentrations of all-trans-retinoic acid, the face, in addition to the wing, is affected. Severe bilateral clefting of the primary palate results; the upper beak is completely missing, whereas the lower beak is unaffected. By using a scoring system that quantitates the severity of the upper beak defect, dose-response curves have been obtained. With beads soaked in progressively higher concentrations of retinoic acid, the upper beaks are increasingly truncated. The synthetic retinoid TTNPB also causes beak defects and is 30 times more potent than all-trans-retinoic acid. By removing beads soaked in retinoids at different times after implantation, the effect of varying the length of exposure of the developing face to retinoids has been examined. The production of beak defects is a two-step process and only a short exposure to retinoid is required to produce clefting. There are striking similarities in the dose-time relationships of retinoid treatments that are required to bring about beak defects and pattern changes in the limb. The outgrowth and development of spatially defined patterns of connective tissue within the upper beak appear analogous to processes involved in limb morphogenesis. We propose that retinoids may act by the same mechanisms in both systems. An unsolved puzzle is why retinoids specifically affect the development of the upper beak.

Small, positively charged beads that slowly release known amounts of all-trans-retinoic acid have been implanted below the apical ectodermal ridge at the anterior margin (opposite somite 16) of wing buds of 3 1/2 day-old chick embryos. The continuous release of retinoic acid is shown to create an anteroposterior concentration gradient of retinoic acid in the limb field that is stable with time, despite the fact that this compound is metabolized by the limb tissue. With beads that release increasing amounts of retinoic acid, the normal 234 digit pattern is progressively altered to a 2234, to a 32234, and then to a 432234 pattern. The tissue concentrations of all-trans-retinoic acid required to change the digit pattern in this way range between 1 and 25 nM. When the same amounts of retinoic acid are released from posteriorly implanted beads (placed below the apical ectodermal ridge opposite somite border 19/20 or somite 20), the normal digit pattern is unaffected. Implantations of beads that release all-trans-retinoic acid are thus identical in their effect to grafts of cells from the limb polarizing region, which cause similar dose-dependent changes in the digit pattern when grafted to the anterior margin of the bud (but not when grafted opposite somites 19 or 20). Because of the low concentrations of retinoic acid required for its biological effect, the graded response observed, and the fact that a concentration gradient is established across the limb field, all-trans-retinoic acid closely mimics the putative morphogen that has been postulated to be emitted by polarizing region cells during normal development.

Three different kinds of carcinoma cell, human breast tumours, mouse lung tumour and rat bladder tumours have been implanted into the developing chick wing to assay their invasive behaviour. We found that the majority of the carcinoma cells did not invade the mesenchyme but were positioned in the ectoderm. We examined the ultrasturcture of the interaction of the carcinoma cells with both the ectoderm and the mesenchyme to see if there are any differences. We found no specialized adhesive junctions between carcinoma cells and mesenchyme cells. In contrast, the carcinoma cells seem to be adhesive to the ectoderm cells; desmosomes between the two have been found and also frequent parallel alignment of membranes over long distances. This difference between the interaction of the carcinoma cells with the mesenchyme and the ectoderm may explain why the carcinoma cells can penetrate into the ectoderm but rarely do so into the mesenchyme. The carcinoma cells formed a smooth border with the mesenchyme even in the absence of a basal lamina. These results are discussed in relation to the mechanisms of invasion of epithelial tumours in vivo. In particular, the relative importance of the basal lamina, and cell adhesion within the tumour, in limiting invasiveness are assessed.