bluuebunny/crossref_metadata_2025 huggingface dataset:
Serotonin and morphogenesis : I. Sites of serotonin uptake and -binding protein immunoreactivity in the midgestation mouse embryo
Authors: Lauder J. M. , Tamir H. , Sadler T. W.
Published: April 2021

The possible involvement of the neurotransmitter serotonin (5-HT) in morphogenesis of the craniofacial region in the mouse embryo has been investigated using the method of whole-embryo culture. Day-12 embryos were incubated for 3–4 h in the presence of 5-HT or its precursors L-tryptophan (L-TRP) or 5-hydroxytryptophan (5-HTP), followed by fixation, sectioning and staining with a specific antiserum to 5-HT. Sites of 5-HT immunoreactivity were found in a variety of locations in tissues of the head and neck, which are either epithelia derived from the non-neural ectoderm or are non-neuronal midline brain structures. These sites include the surface epithelia of the head, face, nasal prominences, branchial arches, oral cavity and associated parts of the nasal epithelium, the epithelium covering the eye, parts of the otic vesicle, the epiphysis and roof of the diencephalon. With the exception of the oral cavity, sites of immunoreactivity for serotonin-binding protein were identified in the mesenchyme adjacent to these sites. This mesenchyme consists of ectodermally derived neural crest cells, which are known to receive inductive influences from the epithelia with which they interact during their migration through the craniofacial region. The presence of 5-HT uptake sites in epithelia and adjacent sites of SBP in the underlying mesenchyme raises the possibility that 5-HT might be involved in those epithelial-mesenchymal interactions known to be important for the development of structures in the craniofacial region.
Interspecific micronuclear transplantation inParamecium:nucleogenesis and stomatogenesis in asexual and sexual reproduction
Authors: Chau M. F. , Ng Stephen F.
Published: April 2021

The micronucleus of Paramecium plays an essential role in the development of the oral apparatus in both asexual and sexual cycles. The present study analyses this somatic function of the micronucleus by interspecific transplantation of the micronucleus between two species, P. jenningsi and P. tetraurelia. The two species are similar in nucleogenesis in the sexual cycle, in the dependence of stomatogenesis on the micronucleus and in the pattern of the oral ciliature. P. jenningsi, however, has a longer oral apparatus. Renucleated cell lines were derived from heterospecific transplantation (P. jenningsi amicronucleates implanted with micronuclei of P. tetraurelia), and also from homospecific transplantation (P. jenningsi). Both homo- and heterospecific transplants exhibited abnormal micronuclear propagation during cell division. In the sexual cycle, the heterospecific transplants exhibited more severe micronuclear anomalies, suggesting interspecific incompatibility. On the other hand, the stomatogenic consequences of the two types of transplants in the asexual and sexual cycles were similar. It is concluded that micronuclear functions, in the assembly and normal patterning of the oral ciliature in the sexual cycle, are not species-specific. However, the oral apparatuses developed by the homo- and heterospecific transplants were similar in length, and approaching that of normal P. jenningsi. Hence, even though the micronucleus is necessary for developing normal oral length, the oral length characteristic of a species is determined by species-specific nonmicronuclear factors. The present findings resemble heterospecific dermal–epidermal inductive interactions in multicellular development, with the micronucleus exerting a nonspecies-specific ‘intracellular inductive stimulus’ on the oral anarchic field to promote oral development.
Honeybee morphogenesis: embryonic cell movements that shape the larval body
Authors: Fleig Richard , Sander Klaus
Published: April 2021

Crucial steps in honeybee morphogenesis are characterized by the migration of free epithelial edges while well-defined epithelial cell populations expand or translocate. For each step - dorsal strip invasion, gastrulation, formation of serosa, amnion and midgut - scanning electron microscopy reveals a highly specific syndrome of cell shapes and cell movements at the advancing epithelial edge. Epithelial translocation appears to result from various combinations of ameboid movement, cell flattening, cell rearrangement (intercalation) and cell contraction along the free epithelial edge. The direction of epithelial movements could result essentially from the initial shape and orientation of the edge combined with the cells’ tendency to close vacant spaces by extension and migration. These findings are discussed in relation to morphogenetic movements in other insects and to postembryonic wound healing in insects and amphibians.
The formation of pattern on the wing of the moth,Ephestia kühniella
Authors: Toussaint Neil , French Vernon
Published: April 2021

Transverse bands of coloured scales are a common feature of lepidopteran wing patterns and it has been suggested that their positions are specified by a propagated ‘Determination Wave’ (Kühn & von Englehardt), or a ‘Gradient’ of diffusible morphogen (Njjhout). We have assessed these models in an experimental study of the formation of bands in the moth, Ephestia kühniella.

The banding pattern can be altered by microcautery of the pupal wing between 1 h and 48 h postpupation (at 20°C) and effects are of two types:

Local modifications follow early (1–3 h postpupation) cautery located on or between the presumptive sites of the bands; operations more proximally or distally on the wing have no effect. Patterns consist of a loop deflecting the nearest band medially, around the site of cautery; or an isolated ring of band scales surrounding the operation site, with the two bands in their normal positions. The type of pattern formed depends only on the location of the cautery, with rings following medial cautery (midway between the bands).

Global modifications follow a 36–48 h cautery anywhere on the wing surface and they consist of a medial displacement of both bands. The degree of displacement is very variable (with band separation reduced to 33–87 % of that on the contralateral control wing) but this does not depend on location of the cautery. Furthermore, the degree of modification does not depend on the precise age at cautery, as an equivalent range of effects is produced at 36 h, at 42 h and at 48 h.

The Determination Wave model is disproved since it explicitly assumes that the different degrees of global modification form a temporal sequence (from severe early to mild late) and they do not. Furthermore, this model cannot explain adequately the formation of loop or ring local modifications.

The Gradient model can account for many features of the results, but it does not readily explain the ring local modifications or the effect of (36–48 b) cautery in causing a global change in the banding pattern, independent of the site or precise time of operation.
Proliferation kinetics and cell lineages can be studied in whole mounts and macerates by means of BrdU/anti-BrdU technique
Authors: Plickert G. , Kroiher M.
Published: April 2021

S-phase cells in intact animals of the coelenterate species Eirene viridula, Hydractinia echinata, Hydra attenuate, and Hydra magnipapillata incorporate the thymidine analogue bromodeoxyuridine (BrdU) into newly synthesized DNA. BrdU-labelled nuclei divide and cells appear to undergo normal differentiation. Whole-mount preparations and macerated tissues were screened for S-phase cells by means of immunocytochemical detection of BrdU (Gratzner, 1982). It is demonstrated that spatial patterns of DNA replication can be evaluated easily. Cell lineages and pathways of cell migration could be traced.
The aetiology and pathogenesis of craniofacial deformity
Authors: Posw1llo David
Published: April 2021

Craniofacial malformations have been recorded since time immemorial. While observational studies have assisted in the recognition of syndromes, little light has been shed on the causal mechanisms which interfere with craniofacial development. Animal studies in which malformations occur spontaneously or have been induced by teratogenic agents have permitted step-by-step investigation of such common deformities as cleft lip and palate. The role of the ectomesenchymal cells of the neural crest and the possible phenomenon of disorganized spontaneous cell death are described in relation to lip clefts. The factors associated with isolated cleft palate, Pierre Robin syndrome and submucous clefts are described by reference to animal models.

The haemorrhagic accident preceding the onset of craniofacial microsomia is discussed as is the distinctly different phenomenon of disturbance to the migration or differentiation of neural crest cells in the pathogenesis of Treacher Collins syndrome. The more severe anomalies of the calvarium such as plagiocephaly, Crouzon and Apert syndrome still defy explanation, in the absence of an appropriate animal system to study; some thoughts on the likely mechanism of abnormal sutural fusions are discussed.
The role of glucose and pyruvate transport in regulating nutrient utilization by preimplantation mouse embryos
Authors: Gardner D. K. , Leese H. J.
Published: April 2021

Preimplantation mouse embryos utilize pyruvate preferentially during the early cleavage stages before switching to glucose at around the time of compaction. This switch in substrate preference has been studied using a non-invasive ultramicrofluorometric analytical technique on single mouse embryos.

On the basis of transport kinetic studies and inhibition by phloretin, cytochalasin B and sugar analogues, a component of glucose uptake by mouse blastocysts was found to be mediated by facilitated diffusion. The Jmax and Kt of this facilitated component were 3·53 pmol embryo −1 h −1 and 0·14 mm, respectively. At physiological concentrations of glucose, the facilitated component accounts for around 75 % of glucose uptake. Glucose uptake by blastocysts was found to be insensitive to insulin, added at a range of concentrations. There was no evidence for glucose active transport. The carrier-mediated component of glucose entry was detectable from the 2-cell stage onwards.

Pyruvate uptake was also mediated by a carrier throughout development. In the absence of glucose in the incubation medium, the characteristic decline in pyruvate uptake does not occur. The data are consistent with a role for embryonic cell transport in regulating glucose utilization prior to compaction, but do not exclude the involvement of metabolic factors, such as the allosteric regulation of the enzymes hexokinase and phosphofructokinase.
Studies in insect segmentation: from teratology to phenogenetics
Authors: Sander K.
Published: April 2021

Old and new data on the segmentation process in arthropods are considered mainly with reference to the genetical basis of segmentation in Drosophila. Problems discussed are the universality (or otherwise) of parasegmental metamery, homeotic genes in insects other than Drosophila, pair-rule traits in myriapods, various segmentation anomalies related to polar morphogenetic centres, and sequential segment determination in the horseshoe crab. The opening and closing paragraphs address the conceptual and terminological progress from the study of ‘developmental monsters’ to the study of ‘developmental genes’ as a means of analysing segmentation.
Homeotic genes and the control of segment diversity
Authors: Akam Michael , Dawson Iain , Tear Guy
Published: April 2021

Homeotic genes control the diversity of segment development, but the domains of action of homeotic genes do not obviously correspond with the major morphological subdivisions of the insect body. We suggest that this lack of correspondence is misleading, because the spatial domains defined by genetics mask fundamental differences in the roles played by individual genes in different regions. In one or more parasegments, each homeotic gene is expressed ‘metamerically’; that is, it is expressed from blastoderm stages onwards in all or virtually all cells of the parasegment primordium. Elsewhere, the same homeotic gene may be deployed adventitiously, only in subsets of cells and at later stages of development. We argue that the early ‘metameric’ domains of gene expression do correlate with the major morphological subdivisions of the fly. This suggests a relatively direct relationship between the expression of particular homeotic genes and the establishment of the ‘ground plan’ that characterizes segments within each major tagma of the body. This relationship allows us to suggest a scenario for the evolution of homeotic genes in relation to the evolving morphological organization of the arthropod body plan in the insect–myriapod lineage.
Expression of homeobox gene Hox 1.1 during mouse embryogenesis
Authors: Mahon Kathleen A. , Westphal Heiner , Gruss Peter
Published: April 2021

Many of the genes controlling segmentation and pattern formation in Drosophila contain a conserved 183 bp sequence known as the homeobox. Homeobox sequences have been found in a range of metazoan species, including the vertebrates mouse and man. This striking conservation suggests that homeobox genes may play a fundamental role in developmental processes. If this is the case then it might be expected that vertebrate homeobox genes will be differentially expressed during embryogenesis and that the timing of their expression will coincide with major morphogenetic events. Here the spatial and temporal patterns of expression of murine homeobox genes will be explored, concentrating on the Hox 1.1 gene as an example. Using in situ hybridization to localize RNA transcripts, it has been found that Hox 1.1 is expressed in a region-specific manner during the formation and differentiation of the embryonic anteroposterior axis. Although striking patterns of expression of Hox 1.1 and other homeobox genes are seen in overtly segmented structures of the embryo (i.e. somites, prevertebral elements, neural tube and dorsal spinal ganglia) expression is also seen in tissues with no obvious segmental origin. The results suggest that homeobox genes probably do not play an exclusive role in segmentation in vertebrates, but are consistent with a role in the assignment of positional identity along the axis of the embryo.
Models for maternally supplied positional information and the activation of segmentation genes in Drosophila embryogenesis
Authors: Meinhardt Hans
Published: April 2021

Recent experiments concerned with early Drosophila development require some modification of models proposed earlier. The addition of a positional information system spreading out from the anterior pole (as indicated by the bicoid mutation) allows very good size regulation. Additional steep gradients at both poles (as suggested by the mutations of the torso group) allow simpler mechanisms for the activation of the gap (or cardinal) genes. Simulations of pattern alterations due to mutations in one of these systems agree well with the observed phenotypes. Mechanisms that can lead to bicaudal phenotypes as well as mechanisms that keep the anteroposterior and the dorsoventral axes perpendicular to each other are discussed.

According to the model, the sharpness of the region specific activation of the gap genes results from local competition of the gene products. For the correct activation of the pair-rule genes, cooperation between gap genes is required. Basic features of the pair-rule pattern can be described by two binary sequences whose phase shift contains the polarity information. Segmentation results from the reiteration of at least three, but probably four, cell states. Each of these cell states is induced at two different positions in the pairrule pattern, such that 14 segments are formed from 7 double segments. A computer simulation is provided.
Peptidergic properties expressed in vitro by embryonic neuroblasts after neural induction
Authors: Pituello Fabienne , Deruntz Philippe , Pradayrol Lucien , Duprat Anne-Marie
Published: April 2021

As an immediate consequence of neural induction, some neurectodermal cells acquire the ability to develop a number of characteristic neuronal features, without requiring any subsequent embryonic cues (Duprat et al. 1987). Thus, adrenergic, cholinergic and gabaergic traits are expressed in cultures of neural fold and neural plate isolated from amphibian embryos immediately after induction and grown in a defined medium.

The aim of the present study was to determine, using the same in vitro model, their abilities to develop peptidergic phenotypes.

Using immunocytochemical techniques, we show that substance P-, enkephalin- (leu-enkephalin, met-enkephalin), and somatostatin-like immunoreactivities are expressed in subpopulations of neurones grown in vitro, whereas VIP (vasoactive intestinal polypeptide) is not detected under the same conditions. The appearance and development of the somatostatinergic phenotype has been quantified by RIA both in cell extracts and in the culture medium. Somatostatin-like immunoreactivity (SLI) undetectable at the late gastrula stage, can be measured in cells after 4 days of culture and continues to increase over the next 10 days. In culture medium, SLI is present at a constant level from day 4 up to day 14.

These data reveal that some neuronal precursor cells acquire, during neural induction, the potentiality to biosynthesize, store and release neuropeptides. Furthermore, the expression of these peptidergic phenotypes in distinct subpopulations of neurones suggests that certain neuronal precursors become committed to different metabolic pathways at the earliest steps of neurogenesis.
5-azacytidine affects the programming of expression of the somatic nucleus of Paramecium
Authors: Kwok Fanii W. K. , Ng Stephen F.
Published: April 2021

This report introduces a new system in the study of programming of genomic function during development of the somatic nucleus of Paramecium tetraurelia. Previous works have established a definite, but replaceable, role of the germ nuclei (micronuclei) in oral development in the asexual cycle; their removal from the cell generates viable amicronucleate cell lines, which characteristically suffer a transient period of growth depression marked by abnormal oral development. Such cell lines gradually recover, showing that a compensatory mechanism is activated in the absence of the germ nuclei to bring the cell back to near-normal. To test the notion that the somatic nucleus (macronucleus) is involved in this compensation, cells possessing micronuclei were treated with 5-azacytidine during sexual reproduction when new somatic nuclei develop. These cells were then propagated asexually for a number of fissions in the absence of the drug, and thereafter micronuclei were removed from them. The amicronucleate cell lines generated in this manner clearly did not suffer a depression as severe as the untreated controls did in terms of growth rate and oral development, and they recovered much sooner. This supports the notion that the somatic nucleus is the physical basis of the compensatory mechanism. This study suggests that the stomato-genic sequences in question normally become repressed in the somatic nucleus developing in sexual reproduction, and that 5-azacytidine administered to the cells at this time could alter this programme which then persists during subsequent asexual propagation. The possibility that the somatic nucleus is programmed by methylation of cytosine at the 5’ position is discussed.
Developmental expression of tissue inhibitor of metalloproteinase (TIMP) RNA
Authors: Nomura Shintaro , Hogan Brigid L. M. , Wills Alan J. , Heath John K. , Edwards Dylan R.
Published: April 2021

Single-stranded antisense RNA probes have been used to study the expression of the metalloproteinase inhibitor TIMP (tissue inhibitor of metalloproteinases), during mouse embryogenesis and in adult tissues. Using a sensitive RNase protection assay, low levels of transcript can be detected in a variety of tissues, including maternal deciduum, embryonic kidney, lung and amnion. Higher levels are seen in osteogenic tissues such as calvaria, while the highest level in any tissue is found in the ovary, though even here expression is an order of magnitude below that observed in growth factor-treated fibroblasts in vitro. Using the technique of in situ hybridization, TIMP transcripts can first be detected in osteogenic tissues in the head and limb at about 15-5 days post coitum, and increase in amount until birth. The high levels of TIMP RNA in the ovary are localized to cells of the corpora lutea.
Targeted ablation of α-crystalIin-synthesizing cells produces lens-deficient eyes in transgenic mice
Authors: Kaur Satbir , Key Brian , Stock Jeffrey , McNeish John D. , Akeson Richard , Potter S. Steven
Published: April 2021

Genetic ablation techniques were used to study the role of the lens in mammalian eye development. Ablation was accomplished by microinjecting murine eggs with chimeric DNA constructs in which the aA-crystallin gene regulatory sequence (−366 to +46) was fused to the highly cytotoxic diphtheria toxin gene coding sequence. For genetic ablation to be successful the promoter regulating expression should be specific and completely silent in cells necessary for normal mouse development. In this report, we describe the generation and analysis of transgenic mice with this readily discernible phenotype: aphakia or eyes without lens. Of the 109 live-born pups, eight carried the transgene and could be grouped according to the apparent severity of eye malformations. Lines 4, 5 and 6 founder (F0) mice had the most severe phenotype. Histological analysis revealed: marked reduction in eye size, total absence of lens, increased retinal cell density and extensive whorling of the retinal fibre layers. The line 1 F0 mouse displayed a distinct lens opacity and lines 2, 3 and 8 F0 mice were mosaics with a relatively mild, but most unusual phenotype. Their eyes contained a small, highly vacuolated lens. The progeny of these mosaics that inherited the transgene, however, again exhibited the severe phenotype. The aberrant structures of the eyes in which complete genetic ablation of the lens has been achieved suggest that the lens plays a pivotal role in the development of multiple components of the murine eye.
Computer-aided 3-D reconstruction of interphase microtubules in epidermal cells of Datura stramonium reveals principles of array assembly
Authors: Flander David J. , Rawlins David J. , Shaw Peter J. , Lloyd Clive W.
Published: April 2021

From immunofluorescence microscopy it has been suggested that cortical microtubules form whole-cell arrays. This has been most clearly seen in cylindrical hairs where the existence of helical arrays testifies to the continuity of the array around the side walls of the cell. It is not, however, clear how microtubules pack in ‘typical’ polyhedral cells with multiple, angled facets. In addressing this problem, elongated and isodiametric cells in the epidermis of Datura stramonium L. were subjected to anti-tubulin immunofluorescence avoiding distortion by cellulase treatment and air-drying. Serial focal sections were then deblurred by computer, the information being digitized, reconstructed and then rotated in order to observe the arrangement of microtubules along the anticlinal walls (in the z-axis). This established several things. Microtubules tend to be parallel upon any one cell face; they form transverse, oblique or longitudinal arrays except that some walls bear a crisscross arrangement. In subepidermal cells, microtubules clearly form helices. In the elongated epidermal cells, transversely wound microtubules are confirmed by rotation to be continuous from one face to another and probably, therefore, also constitute helices. Microtubules on oblique end walls can be seen to continue onto the side walls and do not form a separate set. Although microtubules can be ordered upon two adjacent facets, the orientation with respect to the stem’s axis need not necessarily be identical on both facets, i.e. overall alignment can change at the cell edge.

In isodiametric epidermal cells, microtubules can similarly be traced from one cell facet to another. However, where microtubules from two anticlinal walls spill over onto a periclinal wall at divergent angles, a crisscross arrangement is set up. This is attributed to the geometrical problem of fitting parallel lines around irregular polyhedra. Despite crossing over one another, the microtubules on these walls are nevertheless continuous with MTs on the side walls.

In conclusion, in elongated cells the arrays still approximate helices of various pitch: in isodiametric cells (where the walls subtend variable non-orthogonal angles to one another) the integrity of the array appears to be preserved by microtubules crossing over each other upon what is termed a ‘sacrificial’ face. The overriding tendency is for microtubules to form an integral array regardless of cell shape.
Altered expression of spatially regulated embryonic genes in the progeny of separated sea urchin blastomeres
Authors: Hurley David L. , Angerer Lynne M. , Angererf Robert C.
Published: April 2021

We have examined the importance of the extracellular environment on the ability of separated cells of sea urchin embryos (Strongylocentrotus purpuratus) to carry out patterns of mRNA accumulation and decay characteristic of intact embryos. Embryos were dissociated into individual blastomeres at 16-cell stage and maintained in calcium-free sea water so that daughter cells continuously separated. Levels of eleven different mRNAs in these cells were compared to those in control embryos when the latter reached mesenchyme blastula stage, by which time cells in major regions of the intact embryo have assumed distinctive patterns of message accumulation. Abrogation of interactions among cells resulted in marked differences in accumulation and/or turnover of the individual mRNAs, which are expressed with diverse temporal and spatial patterns of prevalence in intact embryos. In general, separated cells are competent to execute initial events of mRNA accumulation and decay that occur uniformly in most or all blastomeres of the intact embryo and are likely to be regulated by maternal molecules. The ability of separated cells to accumulate mRNAs that appear slightly later in development depends upon the presumptive tissue in which a given mRNA is found in the normal embryo. Messages that normally accumulate in cells at the vegetal pole also accumulate in dissociated cells either at nearly normal levels or at increased levels. In one such case, that of actin Cylla, which is normally restricted to mesenchyme cells, in situ hybridization demonstrates that the fraction of dissociated cells expressing this message is 4- to 5-fold higher than in the normal embryo. In contrast, separated cells accumulate significant levels of a message expressed uniformly in the early ectoderm but are unable to execute accumulation and decay of different messages that distinguish oral and aboral ectodermal regions. These data are consistent with the idea that interactions among cells in the intact embryo are important for both positive and negative control of expression of different genes that are early indicators of the specification of cell fate.
Detection of age-related changes in the distributions of keratan sulfates and chondroitin sulfates in developing chick limbs: an immunocytochemical study
Authors: Sorrell J. Michael , Caterson Bruce
Published: April 2021

A panel of four separate monoclonal antibodies, all known to specifically recognize epitopes on keratan sulfate glycosaminoglycans, were employed in an immunocytochemical study of developing chick hind limbs. In addition, two monoclonal antibodies specific for epitopes on chondroitin/dermatan sulfate glycosaminoglycans were employed on equivalent sections to determine the degree of colocalization of keratan and chondroitin/ dermatan sulfates. The spatial distributions of keratan sulfate and chondroitin/dermatan sulfate differed to some extent. In younger embryos, high extracellular concentrations of keratan sulfate occurred in joints and articular cartilages, with diminishing amounts being present in epiphyseal and diaphyseal regions. The high concentration of keratan sulfate in joints and articular cartilage corresponded to equally high concentration of chondroitin-6 sulfate. With advancing age, the above mentioned distribution was modified, most notably by increased amounts of keratan sulfate within diaphyseal regions. Finally, the use of four different anti-keratan sulfate monoclonal antibodies made it possible to compare keratan sulfate epitope expression. Differences in keratan sulfate epitopes were noted in some regions of bones, mostly in diaphyseal regions of younger bones and epiphyseal regions of older bones. This pattern of keratan sulfate expression suggests that different types of keratan sulfate may be present and their expression may be developmentally regulated.
Localized synthesis of the Vg1 protein during early Xenopus development
Authors: Tannahill D. , Melton D. A.
Published: April 2021

The Xenopus Vgl gene encodes a maternal mRNA that is localized to the vegetal hemisphere of both oocytes and embryos and encodes a protein related to the TGF-β family of small secreted growth factors. We have raised antibodies to recombinant Vgl protein and used them to show that Vgl protein is first detected in stage IV oocytes and reaches maximal levels in stage VI oocytes and eggs. During embryogenesis, Vgl protein is synthesized until the gastrula stage. The embryonically synthesized Vgl protein is present only in vegetal cells of an early blastula. We find that Vgl protein is glycosylated and associated with membranes in the early embryo. Our results also suggest that a small proportion of the full-length Vgl protein is cleaved to give a small peptide of Mr= —17×103. These results support the proposal that the Vgl protein is an endogenous growth-factor-like molecule involved in mesoderm induction within the amphibian embryo.
Immunohistochemical localisation of chondroitin sulphate proteoglycans and the effects of chondroitinase ABC in 9- to 11-day rat embryos
Authors: Morriss-Kay Gillian , Tuckett Fiona
Published: April 2021

Studies on cell behaviour in vitro have indicated that the chondroitin sulphate proteoglycan (CSPG) family of molecules can participate in the control of cell proliferation, differentiation and adhesion, but its morphogenetic functions had not been investigated in intact embryos. Chondroitin/chondroitin sulphates have been identified in rat embryos at low levels at the start of neurulation (day 9) and at much higher levels on day 10. In this study we have sought evidence for the morphogenetic functions of CSPGs in rat embryos during the period of neurulation and neural crest cell migration by a combination of two approaches: immunocytochemical localization of CSPG by means of an antibody, CS-56, to the chondroitin sulphate component of CSPG, and exposure of embryos to the enzyme chondroitinase ABC. Staining of the CS-56 epitope was poor at the beginning of cranial neurulation; bright staining was at first confined to the primary mesenchyme under the convex neural folds late on day 9. In day 10 embryos, all mesenchyme cells were stained, but at different levels of intensity, so that primary mesenchyme, neural crest and sclerotomal cells could be distinguished from each other. Basement membranes were also stained, particularly bright staining being present where two epithelia were basally apposed, e.g. neural/surface ectoderms, dorsal aorta/neural tube, prior to migration of a population of cells between them. Staining within the neural epithelium was first confined to the dorsolateral edge region, and associated with the onset of neural crest cell emigration; after neural tube closure, neuroepithelial staining was more general. Neural crest cells were stained during migration, but the reaction was absent in areas associated with migration end-points (trigeminal ganglion anlagen, frontonasal mesenchyme). Embryos exposed to chondroitinase ABC in culture showed no abnormalities until early day 10, when cranial neural crest cell emigration from the neural epithelium was inhibited and neural tube closure was retarded. Sclerotomal cells failed to take their normal pathway between the dorsal aorta and neural tube. Correlation of the results of these two methods suggests: (1) that by decreasing adhesiveness within the neural epithelium at specific stages, CSPG facilitates the emigration of neural crest cells and the migratory movement of neuroblasts, and may also provide increased flexibility during the generation of epithelial curvatures; (2) that by decreasing the adhesiveness of fibronectin-containing extra-cellular matrices, CSPG facilitates the migration of neural crest and sclerotomal cells. This second function is particularly important when migrating cells take pathways between previously apposed tissues.
Molecular characteristics of cytostatic factors in amphibian egg cytosols
Authors: Shibuya Ellen K. , Masui Yoshio
Published: April 2021

In amphibians, zygotes microinjected with cytosol of unactivated eggs are arrested at metaphase of mitosis. The factor responsible for this effect has been designated ‘cytostatic factor, (CSF)’. CSF is inactivated by Ca2+ addition to cytosols. During storage of the Ca2+-containing cytosols, a stable CSF activity develops. Therefore, the first Ca2+-sensitive CSF and the second Ca2+-insensitive CSF have been referred to as primary CSF (CSF-1) and secondary CSF (CSF-2), respectively. We have partially purified CSF-1, which had been stabilized with NaF and ATP, and CSF-2 from cytosols of Rana pipiens eggs by ammonium sulphate (AmS) precipitation and sucrose density gradient centrifugation or gel filtration, and investigated their molecular characteristics. CSF-1 was sensitive to protease, but resistant to RNAse, and inactivated within 2h at 25°C. CSF-1 could be sedimented in a sucrose density gradient from a fresh cytosol or its crude fraction precipitated at 20-30% saturation of AmS, showing the sedimentation coefficient 3S. When analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), all the proteins in partially purified CSF-1 samples entered the gel and were separated into numerous peptide bands. In contrast, CSF-2 was an extremely large molecule, being eluted from Sepharose columns as molecules larger than 2×106, and failed to enter the gel when analyzed by SDS-PAGE. It could be purified 40 times from cytosols. CSF-2 was a highly stable molecule, being neither inactivated nor dissociated at pH 11’5 or by 4m-NaCl and L1C1 and 8 M-urea. It was also resistant to RNAse treatment. However, CSF-2 could be broken down into small peptides of variable sizes by trypsin, a-chymotrypsin, and papain, but not by S. aureus V8 protease, although it was less sensitive to proteases than CSF-1. The dose-dependency test showed that the activity of CSF-2 is independent of its concentration and that an amount of CSF-2 could cause cleavage arrest earlier when injected into a blastomere in a larger volume.
Pair-rule expression of a cell surface molecule during gastrulation of the moth embryo
Authors: Carr Jeffrey N. , Taghert Paul H.
Published: April 2021

The TNI monoclonal antibody recognizes a cell surface epitope that is present on subsets of growing axons in the developing nervous system of moth embryos. This antigen is also found in a variety of other developing tissues: in all cases its expression is cell-specific and transient. Here we show that the first expression of the TNI epitope in moth embryos occurs specifically on the surfaces of mesodermal cells during gastrulation, and that it is limited to alternate segments. Creation of this pair-rule pattern of expression includes indications of an initial 4-segment periodicity, and transient immunoreactivity in ‘off’ segments. The alternating pattern is most dramatic at the end of gastrulation. It changes rapidly such that, during organogenesis, the TNI antigen(s) is expressed in many developing tissues of all segments, with little segment-specific variation. Immunolabelling of living embryos under culture conditions demonstrated that the TN1 epitope(s) is associated with cell surfaces, both during neurogenesis and during the earlier period of gastrulation. These observations indicate that pair-rule gene functions operate in insects other than Diptera and suggest that cell surface molecules may be utilized early in insect embryogenesis in the initial establishment of large body regions.
Ontogeny of immunoglobulin expression in the Mexican axolotl
Authors: Fellah Julien S. , Vaulot Daniel , Tournefier Annick , Charlemagne Jacques
Published: April 2021

The ontogeny of immunoglobulin (Ig) synthesis was followed at both cellular and serological levels in the Mexican axolotl (Ambystoma mexicanum) using polyclonal antibodies recognizing all Ig molecules and a set of monoclonal antibodies (Mabs) specific for the Cμ and Cv heavy Ig chain isotypes and for the light chain constituents shared by IgM and IgY molecules. Clusters of IgM- and of IgY-synthesizing lymphocytes, often located in separate sites, are first present in spleen sections of 7-week-old 25 mm larvae, about one month after differentiation of the spleen anlage (stage 39–40). In 12-week-old 30–35 mm larvae, the relative proportion of IgM- and IgY-synthesizing cells in the spleen is the same as that in adult animals. However, a marked enhancement of the spleen B cell compartment occurs from 5 to 9 months when Ig-positive cells represent about 88 % of the lymphocyte population compared to 60 % in adults. No structures equivalent to B cell germinal centers were observed at any stage of the spleen differentiation and cells, although often clustered in small groups, remain dispersed in the entire organ. The relative proportions of IgM and IgY B cells throughout the spleen remain constant during development (about 1 IgY+ cell for 5–6 IgM+ cells) and IgM molecules are first detected in the serum of 2·5-month-old larvae. The enhancement of the serum IgM level correlates well with the absolute number of IgM+ cells in the growing spleen. IgY molecules cannot be detected in the serum before the 7th month but their level quickly increases to reach about 60 % of the adult value at 10 months. Thyroxine-induced metamorphosis or hyperimmunization of 4-to 6-month-old larvae had no effect upon the temporal expression of the Ig classes in serum.
Pigment pattern expression in the plumage of the quail embryo and the quail-chick chimaera
Authors: Richardson Michael K. , Hornbruch Amata , Wolpert Lewis
Published: April 2021

The pattern of pigmentation in birds is dependent on the migration and differentiation of a population of neural crest cells that develop into melanoblasts. On the basis of previous grafting experiments Rawles (1948) concluded that the pigment pattern of the chimaera is determined by the genotype of the donor melanocyte. This led Wolpert (1981) to suggest that melanoblasts from one bird can read the positional value of the ectoderm in the feather papillae of another bird. An alternative view is that an isomorphic prepattern in the feathers determines the pigment pattern. We have examined these ideas in relation to the local pigment patterns of the embryonic quail wing, distal to the elbow, where several rows of feather papillae are consistently unpigmented.

Melanin pigment is first seen at stage 35. By stage 39 a characteristic pigment pattern has been established. Most of the dorsal feather papillae are heavily pigmented, whereas many ventral papillae are unpigmented. Of the ventral papillae three rows (E2, E3 and H2) are always unpigmented, and it is these three rows that form the basis of the quail local pattern. The DOPA reaction indicates that no melanoblasts are present in these white feathers, although they are present in all the feathers of the White Leghorn wing.

When quail neural crest cells are grafted to the chick, either isotopically or to the wing bud, all or nearly all rows of ventral papillae become pigmented by stage 39. The only evidence of donor influences in the pattern is that, in some grafts, rows E2–3 have a high proportion of unpigmented papillae, and wings from earlier stages resemble the quail. When unpigmented papillae are present, histology shows that they contain undifferentiated crest cells. When introduced into a quail wing bud, chick crest cells enter all the feather papillae of the wing, including those in rows E2–3 and H2.

We suggest that neither the positional information nor the prepattern theory alone can account for all of our findings. Contrary to previous claims, local cues may be important in determining crest-cell differentiation. We have established that crest cells migrate into all feather papillae of the quail-chick chimaera, including those that will remain unpigmented. We show that neither differential migration nor differential proliferation is involved in pattern formation in the quail-chick chimaera.
G-protein-linked signal transduction systems control development in Dictyostelium
Authors: Johnson R. L. , Gundersen R. , Lilly P. , Pitt G. S. , Pupillo M. , Sun T. J. , Vaughan R. A. , Devreotes P. N.
Published: April 2021

G-protein-linked cAMP receptors play an essential role in Dictyostelium development. The cAMP receptors are proposed to have seven transmembrane domains and a cytoplasmic C-terminal region. Overexpression of the receptor in cells, when the endogenous receptor is not present, results in a 10- to 50-fold increase in cAMPbinding sites. Antisense cell lines, which lack cAMP receptors, do not enter the developmental program. Ligand-induced phosphorylation is proposed to occur on serine and threonine residues in the receptor C-terminus. The kinetics of receptor phosphorylation and dephosphorylation correlate closely with the shift of receptor mobility and the adaptation of several cAMP-induced responses. Two α-subunits, G-α-1 and G-α-2, have been cloned and specific antisera developed against each. Both subunits are expressed as multiple RNAs with different developmental time courses. The mutant Frigid A has a functional defect in G-α-2 which prevents it from entering development. We propose that G-protein-linked receptor systems will be a major component in the development of many organisms.
Determinative mechanisms in secondary muscle lineages of ascidian embryos: development of muscle-specific features in isolated muscle progenitor cells
Authors: Nishida Hiroki
Published: April 2021

Muscle cells of the ascidian larva originate from three different lines of progenitor cells, the B-line, A-line and b-line. Experiments with 8-cell embryos have indicated that isolated blastomeres of the B-line (primary) muscle lineage show autonomous development of a musclespecific enzyme, whereas blastomeres of the A-line and b-line (secondary) muscle lineage rarely develop the enzyme in isolation. In order to study the mechanisms by which different lines of progenitors are determined to give rise to muscle, blastomeres were isolated from embryos of Halocynthia roretzi at the later cleavage stages when conspicuous restriction of the developmental fate of blastomeres had already occurred. Partial embryos derived from B-line muscle-lineage cells of the 64-cell embryo (B7.4, B7.5 and B7.8) showed autonomous expression of specific features of muscle cells (acetylcholinesterase, filamentous actin and musclespecific antigen). In contrast, b-line muscle-lineage cells, even those isolated from the 110-cell embryo (b8.17 and b8.19), did not express any muscle-specific features, even though their developmental fate was mainly restricted to generation of muscle. Isolated A-line cells from the 64-cell embryos (A7.8) did not show any features of muscle differentiation, whereas some isolated A-line cells from the 110-cell embryos (A8.16) developed all three above-mentioned features of muscle cells. This transition was shown to occur during the eighth cell cycle. These results suggest that the mechanism involved in the process of determination of the secondary-lineage muscle cells differs from that of the primary-lineage muscle cells. Interaction with cells of other lineages may be required for the determination of secondary precursors to muscle cells.

The presumptive b-line and A-line muscle cells that failed to express muscle-specific features in isolation did not develop into epidermial cells. Thus, although interactions between cells may be required for muscle determination in secondary lineages, the process may represent a permissive type of induction and may differ from the processes of induction of mesoderm in amphibian embryos.
An extracellular matrix molecule of newt and axolotl regenerating limb blastemas and embryonic limb buds: immunological relationship of MT1 antigen with tenascin
Authors: Onda Hiroaki , Goldhamer David J. , Tassavat Roy A.
Published: April 2021

Several well-characterized extracellular matrix (ECM) components have been localized to the amphibian limb regenerate, but the identification and characterization of novel ECM molecules have received little attention. Here we describe, using mAb MT1 and immunocytochemistry, an ECM molecule expressed during limb regeneration and limb development. In limb stumps, mAb MT1 reactivity was restricted to tendons, myotendinous junctions, granules in the basal layers of epidermis, periosteum (newts) and perichondrium (axolotls). In regenerating limbs, reactivity in the distal limb stump was first detected 5 days and 1 day after amputation of newt and axolotl limbs, respectively. In both species, mAb MT1 recognized what appeared to be an abundant blastema matrix antigen, localized in both thin and thick cords between and sometimes closely associated with blastema cells. Reactivity was generally uniform throughout the blastema except for a particularly thick layer that was present immediately beneath the wound epithelium. During redifferentiation stages, mAb MT1 reactivity persisted among blastema cells and redifferentiating cartilage but was lost proximally in areas of muscle and connective tissue differentiation. During the entire period of embryonic limb development, mAb MT1 reactivity was seen in the ECM of the mesenchyme and in a layer beneath the limb bud ectoderm, similar to its distribution during regeneration. Considerable mAb MT1 reactivity was also associated with the developing somites. The reactivity of mAb MT1 in blastema and limb bud was similar if not identical to that of a polyclonal Ab against tenascin (pAbTN), a large, extracellular matrix glycoprotein implicated in growth control, inductive interactions, and other developmental events. This pAbTN effectively competed against mAb MT1 binding on blastema sections. In immunoblots, both mAb MT1 and pAbTN recognized a very high molecular weight (approximately Mr 1000 × 103) protein in blastema extracts of both newts and axolotls. mAb MT1 immunoprecipitated a protein of Mr 1000K size which reacted to both mAb MT1 and pAbTN in immunoblots. These data show that tenascin is in the matrix of the urodele blastema and limb bud, and suggest that mAb MT1 identifies urodele tenascin.
In vivo regulation of MPF in Xenopus oocytes
Authors: Johnson Andrew D. , Smith L. Dennis
Published: April 2021

Entry into M phase in the eukaryotic cell cycle is controlled by the oscillating activity of MPF. The active component of MPF is now known to be the p34cdc2 protein kinase originally found in yeast. The p34cdc2 protein kinase displays a characteristic M-phase-specific histone Hl kinase activity when it interacts with cyclins, which are proteins that oscillate through the cell cycle and are thought to regulate p34cdc2 activity. Cyclins can induce M phase when introduced into fully grown Xenopus oocytes and cyclin may play a role in normal oocyte maturation. Small Xenopus oocytes do not mature in response to the hormonal triggers which act on stage 6 oocytes. We introduced cyclin into stage 4 (small) Xenopus oocytes and showed that it activates MPF in these cells, probably by interacting with endogenous p34cdc2 kinase. yye made labelled extracts from cyclin-mRNA-injected stage 4 oocytes and used them to show differential stability of clam cyclins A and B at oocyte maturation. The relative stability of the two forms of cyclin related directly to their ability to stabilize crude MPF preparations from injected stage 6 oocytes.