bluuebunny/crossref_metadata_2025 huggingface dataset:
Neurotransmitter plasticity of cultured sympathetic neurones. Are the effects of muscle-conditioned medium reversible?
Authors: Vidal Simone , Raynaud Brigitte , Clarous Dominique , Weber Michel J.
Published: April 2021

Muscle-conditioned medium (CM) induces choline acetyltransferase (CAT) activity in primary cultures of new-born rat sympathetic neurones and depresses the development of tyrosine hydroxylase (TOH). By following these two enzymes, we have determined whether (1) the effects of CM are reversible and (2) the neurones progressively lose their sensitivity to CM with time in culture.

When neurones were cultured in the presence of 50 % CM (CM+ medium), TOH activity developed slowly but CAT activity developed at a high rate. When the cultures were then switched to unconditioned medium (CM− medium), CAT activity remained elevated and continued to develop at higher rate than in cultures that were never exposed to CM. On the other hand, the switch to CM− medium was accompanied by a transition from a low to a high rate of TOH development. CAT induction by CM was thus essentially irreversible, whereas the impairment of TOH development was fully reversible.

Conversely, we studied the effects of altering CM− to CM+ medium at progressively later culture days. CAT remained fully inducible for at least 2 to 3 weeks. On the other hand, TOH activity, which initially developed rapidly in CM− medium, first decreased to low levels after a switch to CM+ medium and then increased again, but at a slower rate.

Neuronal depolarization by elevated K+ and exposure to CM have mirror-image, and antagonistic, effects on both CAT and TOH developments (Raynaud et al. 1987a). Walicke, Campenot & Patterson (1977) showed that a previous depolarization reduced the induction of cholinergic traits by a subsequent exposure to CM. We found that (1) such a depolarization only delayed the induction of CAT by several days and did not prevent the transition to a state of low TOH expression caused by CM and (2) an exposure of the cultures to elevated K+ after exposure to CM did not cause a decline in CAT activity.

These data thus suggest that a state of high TOH expression can superimpose on a previously induced state of elevated CAT expression, but that the induction of CAT caused by a delayed exposure to CM is accompanied by a transition from a high to a lower state of TOH expression. In addition, neuronal depolarization does not stabilize the noradrenergic phenotype in a permanent manner and can not reverse cholinergic expression of sympathetic neurones to a purely noradrenergic phenotype.
A study in developing visual systems with a new method of staining neurones and their processes in fixed tissue
Authors: Godement Pierre , Vanselow Jens , Thanos Solon , Bonhoeffer Friedrich
Published: April 2021

Carbocyanine dyes, fluorescent lipophilic substances used for optical recordings of membrane voltage and for studies of membrane fluidity, have recently been shown to provide intense and long-lasting staining of neurones in vivo and in vitro (Schwartz & Agranoff, 1981; Honig & Hume, 1985,1986; Catsicas, Thanos & Clarke, 1986; Landmesser & Honig, 1986; Thanos & Bonhoeffer, 1987).

We report here that two of these dyes, dil (1,1′,dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate) and diO (3,3’-dioctadecyloxacarbocyanine perchlorate), can also label neurones in embryonic mouse and chicken brain tissue that has been previously fixed in aldehyde fixatives. Neuronal processes and perikarya can be labelled along considerable distances in both anterograde and retrograde directions. The staining of processes and cells, including their finest extensions is smooth and clear, rivalling intracellular injections of HRP or Lucifer Yellow. The appearance and time course of progression of the staining along axons suggest that the staining in fixed tissue occurs due to a process of diffusion of dyes along the plasma membranes of cells.

This technique has allowed us to study the first stages in the development of optic fibres in mouse embryos, especially at the optic chiasm. The early retinal projection (E13-E13I) is mainly crossed, but some optic fibres grow to the ipsilateral side of the brain at the outset. Retrogradely labelled ganglion cells from the dorsocentral area of the retina participate in the formation of both the ipsilateral and the contralateral projection. Thus, at early stages, crossed and uncrossed projections arise from identical subregions of the retina and the partition of the retina with respect to the laterality of its projection arises later.
Embryonic stem cell-mediated transfer and correct regulation of the chicken δ-crystallin gene in developing mouse embryos
Authors: Takahashi Yoshiko , Hanaoka Kazunori , Hayasaka Michiko , Katoh Kazuto , Kato Yoshihiro , Okada T. S. , Kondoh Hisato
Published: April 2021

To study regulation of δ-crystallin expression during ontogeny, we transferred the gene from chicken into developing mouse embryos by first transforming an embryonic stem (ES) cell line of mouse and then producing chimaeric embryos by combining them with normal mouse embryos. Using this technique, genes were transferred into a variety of developing mouse tissues with high efficiency. Two δ-crystallin gene constructs were used: the wild-type gene with 2200 bp of the 5’ flanking sequence, shown to be lensspecific in an assay using cultured mouse cells, and a mutant gene with 51 bp of the 5’ flanking sequence, lacking the sequence required for expression in lens cells. Five independent lines carrying the former and two lines carrying the latter were employed in producing chimaeras. In the chimaeric embryos having the wild-type gene, δ-crystallin was expressed in the lens and in specific regions of the primitive central nervous system (CNS) as is seen in embryonic expression in the chicken. In adult mouse chimaeras also, expression was restricted to the lens and the CNS, in the pyramidal neurones of the piriform cortex and the hippocampus, δ-crystallin expression in these tissues is due to proper transcriptional regulation, since no expression was observed when chimaeras were produced with the ES lines carrying the mutant gene. The experimental results reported here demonstrate the advantage of ES-cell-mediated gene transfer in the study of embryonic gene regulation, because a number of gene constructs and chromosomal sites can be analysed shortly after embryo manipulation without requiring gene transmission to the next generation.
De novo expression of a type IV collagen gene in Drosophila embryos is restricted to mesodermal derivatives and occurs at germ band shortening
Authors: Mirre Christian , Cecchini Jean-Pierre , Parco Yannick Le , Knibiehler Bernard
Published: April 2021

We have examined directly the expression of one collagen gene (DCgl) during Drosophila melanogaster embryogenesis by means of in situ hybridization. Transcripts of this gene, which were demonstrated to encode a basement membrane type IV collagen chain, began to accumulate specifically in mesodermal de-rivatives at stages 12-13 of embryogenesis, and not before. Cells expressing this gene overlap, or are closely intermingled with, somatic and visceral mesoderm in stages 12-14. In stages 15-17, in addition to the strongly positive fat bodies, highly labelled cell spots are found scattered around all the parts of the gut and symmetrically on each side of the ventral nerve cord. They correspond to circulating mesodermal cells which we consider to be haemocytes or mesoblasts.
A morphological and experimental study of the mesencephalic neural crest cells in the mouse embryo using wheat germ agglutinin–gold conjugate as the cell marker
Authors: Chan W. Y. , Tam P. P. L.
Published: April 2021

The distribution of the mesencephalic neural crest cells in the mouse embryo was studied by mapping the colonization pattern of WGA-gold labelled cells following specific labelling of the neuroectoderm and grafting of presumptive neural crest cells to orthotopic and heterotopic sites. The result showed that (1) there were concomitant changes in the morphology of the neural crest epithelium during the formation of neural crest cells in the 4- to 7-somite-stage embryos, (2) the neural crest cells were initially confined to the lateral subectodermal region of the cranial mesenchyme and there was minimal mixing with the paraxial mesoderm underneath the neural plate, (3) labelled cells from the presumptive crest region colonized the lateral craniofacial mesenchyme, the developing trigeminal ganglion and the pharyngeal arch, (4) the formation of neural crest cells was facilitated by the focal disruption of the basal lamina and the cell-cell interaction specific to the neural crest site and (5) the trigeminal ganglion was colonized not only by neural crest cells but also by cells from the ectodermal placode.
Growth factor superfamilies and mammalian embryogenesis
Authors: Mercola Mark , Stiles Charles D.
Published: April 2021

With the availability of amino acid and nucleotide sequence information has come the realization that growth factors can be clustered into superfamilies. Several of these superfamilies contain molecules that were not initially identified because of growth-promoting activities; rather they were discovered through their ability to regulate other processes. Certain members of these superfamilies are present during early mammalian embryogenesis. However, until recently, it has been difficult to manipulate the developing mammalian embryo to observe directly the effects of inappropriate, excessive, or reduced expression of these molecules. Despite this limitation, at least some of these molecules have been implicated in the control of differentiation and morphogenesis, two actions unpredicted from the cell biology of most of the growth factors. Moreover, these actions are reflected in nonmammalian species where homologues of the mammalian growth factors control crucial steps in the choice of developmental fate. This review describes five growth factor superfamilies and the role these molecules may have in controlling proliferation, differentiation, and morphogenesis during mammalian development.
Enhanced expression of TGF-β and c-fos mRNAs in the growth plates of developing human long bones
Authors: Sandberg M. , Vuorio T. , Hirvonen H. , Alitalo K. , Vuorio E.
Published: April 2021

The expression of mRNAs for type I and type II procollagens, transforming growth factor-β (TGF-β) and c-fos was studied in developing human long bones by Northern blotting and in situ hybridization. The cells producing bone and cartilage matrix were identified by hybridizations using cDNA probes for types I and II collagen, respectively. Northern blotting revealed that the highest levels of TGF-β mRNA were associated with the growth plates. By in situ hybridization, this mRNA was localized predominantly in the osteoblasts and osteoclasts of the developing bone, in periosteal fibroblasts and in individual bone marrow cells. These findings are consistent with the view that TGF-β may have a role in stimulation of type I collagen production and bone formation. Only a low level of TGF-β mRNA was detected in cartilage where type II collagen mRNA is abundant. In Northern hybridization, the highest levels of c-fos mRNA were detected in epiphyseal cartilage. In situ hybridization revealed two cell types with high levels of c-fos expression: the chondrocytes bordering the joint space . and the osteoclasts of developing bone. These differential expression patterns suggest specific roles for TGF-β and c-fos in osseochondral development.
Reinvestigation of the role of the optic vesicle in embryonic lens induction
Authors: Grainger Robert M. , Henry Jonathan J. , Henderson Robert A.
Published: April 2021

The induction of the lens by the optic vesicle in amphibians is often cited as support for the view that a single inductive event can lead to determination in a multipotent tissue. This conclusion is based on transplantation experiments whose results indicate that many regions of embryonic ectoderm which would normally form epidermis can form a lens if brought into contact with the optic vesicle. Although additional evidence argues that during normal development other tissues, acting before the optic vesicle, also contribute to lens induction, it is still widely held, on the basis of these transplantation experiments, that the optic vesicle alone can elicit lens formation in ectoderm. While testing this conclusion by transplanting optic vesicles beneath ventral ectoderm in Xenopus laevis embryos, it became apparent that contamination of optic vesicles by presumptive lens ectoderm cells can generate lenses in these experiments, illustrating the need for adequate host and donor marking procedures. Since previous studies rarely used host and donor marking, it was not clear whether they actually demonstrated that the optic vesicle can induce lenses. Using careful host and donor marking procedures with horseradish peroxidase as a lineage tracer, we show that the optic vesicle cannot stimulate lens formation in neurula- or gastrula-stage ectoderm of Xenopus laevis. Since the general conclusion that the optic vesicle is sufficient for lens induction rests on studies in many organisms, we felt it was important to begin to test this conclusion in other amphibians as well. Similar experiments were therefore performed with Rana palustris embryos, since it was in this organism that optic vesicle transplant studies had originally argued that this tissue alone can cause lens induction. Under conditions similar to those used in the original report, but with careful controls to assess the origin of lenses in transplants, we found that the optic vesicle alone cannot elicit lens formation. Our data lead us to propose that the optic vesicle in amphibians is not generally sufficient for lens induction. Instead, we argue that lens induction occurs by a multistep process in which an essential phase in lens determination occurs as a result of inductive interactions preceding contact of ectoderm with the optic vesicle.
Mesoderm-inducing factors: a small class of molecules
Authors: Godsave S. F. , Isaacs H. V. , Slack J. M. W.
Published: April 2021

Mesoderm-inducing factors (MIFs) from chick embryos, XTC cells and WEHI-3 cells were studied using various procedures. The object was to find whether they are similar to heparin-binding growth factors (HBGFs - the only known pure mesoderm-inducing substances) and, if not, whether they are similar to each other.

The major active components from all three MIF sources behave as somewhat hydrophobic, acid-stable molecules and do not bind to heparin. They all have relative molecular masses of about 13 000 measured by HPLC size exclusion chromatography. The isoelectric points measured by chromatofocusing were 6·7 (WEHI) and 7·7 (XTC). The chick MIF seemed somewhat heterogeneous by chromatofocusing and a portion of its activity bound to heparin sepharose.

All three MIFs have similar effects on explants of Xenopus blastula ectoderm to the heparin-binding growth factors, causing an elongation at the time of gastrulation followed by the development of mesenchyme, mesothelium and muscle cells, the proportion of muscle increasing with dose. Unlike the HBGFs they all also induce notochord if sufficiently high concentrations are used.

Our study shows that the MIFs examined here form a small group of potent agents distinct from the HBGFs and from other known growth and differentiation factors. Their occurrence in various tissues and cell lines suggests that they have functions in the adult organism as well as during early development.
The role of interstitial collagens in cleft formation of mouse embryonic submandibular gland during initial branching
Authors: Fukuda Yuh , Masuda Yukinari , Kishi Jun-Ichi , Hashimoto Yoko , Hayakawa Taro , Nogawa Hiroyuki , Nakanishi Yasuo
Published: April 2021

An interstitial collagenase was purified from the explant medium of bovine dental pulp and was shown to degrade collagens I and III but not IV and V. The enzyme halted cleft initiation in the epithelium of 12day mouse embryonic submandibular glands in vitro, indicating the active involvement of interstitial collagens in the branching morphogenesis. Transmission electron microscopic observation of the intact 12-day gland without any clefts showed the scattered localization of a few collagen fibrils at the epithelial-mesenchymal interface of the bulb and also revealed the presence of numerous microfibrils around the stalk. Collagen bundles were regularly seen close to the wavy basal lamina at the bottom of clefts of the intact 13-day gland and 12-day gland cultured for 17 h under normal conditions. Mesenchymal cells were found in the clefts together with the frequent localization of peripheral nerve fibres and capillary endothelial cells. The collagen bundles were more often observed in the 12-day gland cultured in the presence of bovine dental pulp collagenase inhibitor, which had been shown to enhance cleft formation. In contrast, collagen fibrils were rarely found at the epithelial-mesenchymal interface of the 12-day gland cultured in the presence of Clostridial or bovine dental pulp collagenase. The findings indicated that the formation of interstitial collagen bundles is essential to form clefts in the epithelium both in vivo and in vitro.
Spatial and temporal distribution of the adherens-junction-associated adhesion molecule A-CAM during avian embryogenesis
Authors: Duband Jean-Loup , Volberg Tova , Sabanay Ilana , Thiery Jean Paul , Geiger Benjamin
Published: April 2021

A-CAM (adherens-junction-specific cell adhesion molecule) is a calcium-dependent adhesion molecule which is associated with intercellular adherens junctions in various tissues (Volk & Geiger, 1986, J. Cell Biol. 103. 1441–1450 and 1451–1464). In the present report, we have investigated the distribution of A-CAM during avian morphogenesis by immunofluorescence microscopy and immunoblotting. A-CAM appeared at the onset of gastrulation on developing mesodermal and endodermal cells and was then expressed on tissues derived from the three primary germ layers. During embryonic life, A-CAM was constitutively expressed in a number of tissues including the central and peripheral nervous system, myocardium, muscles, notochord, skin and lens whereas it was found transiently in many tissues ranging from the nephritic tubules and the endoderm of visceral arches to ectodermal placodes. In the adult, in addition to the nervous system, A-CAM was restricted to the skin, lens, heart and testis, and exhibited an apparent molecular weight higher than the one found in the embryo. The prevalence and cell-surface modulation of A-CAM could frequently be correlated with morphogenetic events such as mesenchyme condensation into epithelia or cell clusters (e.g formation of the somitic epithelium, kidney tubules and peripheral ganglia), dissociation of epithelia (e.g. dissociation of the somitic epithelium and segregation of neural crest from the neural tube), separation of cell populations (e.g. fibroblasts and myotubes in the heart) and reorganizations of epithelia (e.g. neurulation). In addition, using electron microscopy, the expression of A-CAM on the surface of aggregating and separating cells could be correlated with the formation and disappearance of adherens junctions. This precisèly scheduled control of A-CAM correlated with early morphogenetic events during embryogenesis suggests that this CAM could play a crucial role in these processes.
Tissue interactions and cell differentiation: neurone-sensory cell interaction during otic development
Authors: Water Thomas R. Van De
Published: April 2021

Statoacoustic ganglion neurones (SAG) are produced by the same group of cells (otic placode) that produce all of the receptor ceils that populate the sensory areas of the inner ear. The observation that ingrowth of SAG neurites to presumptive sensory areas of the inner ear preceded cytodifferentiation of those receptor cells suggested a causal relationship. Results from in vivo, in ovo and in vitro studies do not support a causal relationship. These studies support the hypothesis that the programme for labyrinthine sensory cell differentiation is intrinsic and does not require the extrinsic stimulus of neuronal interaction to trigger its expression. In contrast, developing statoacoustic ganglion neurones appear to require a trophic influence that is supplied by either their peripheral or central target tissues for their survival and maturation in vitro. A mechanism for the ingrowth of SAG dendrites to their appropriate target sites within the inner ear proposes that attractant fields produced by areas of differentiating sensory cells act to guide the nerve growth cones of ingrowing SAG neurites to the appropriate tissues. Preliminary results from a heterochronic series of SAG implants to common age otocysts suggest that these SAG neurones are capable of responding to the attractant fields which are produced by presumptive labyrinthine sensory epithelium over an extended period of otic development. Both in ovo and in vitro studies suggest that spatiotemporal patterns of extracellular matrix molecules may be important components of the attractant fields which are produced by the sensory areas of the developing inner ear and may ultimately result in the specificity of their neuronal connections.
Teratogens and craniofacial malformations: relationships to cell death
Authors: Sulik K. K. , Cook C. S. , Webster W. S.
Published: April 2021

Environmental agents including ethanol, 13-cis retinoic acid (RA, Accutane®), the antimetabolite methotrexate, periods of hypoxia, ionizing radiation or hyperthermic stress, when administered acutely to pregnant experimental animals, induce stagedependent craniofacial malformations comparable to those in corresponding human teratogen syndromes. Acute treatment regimens have allowed analysis of cell populations initially affected and subsequent dysmorphogenetic sequences as well as speculation relative to mechanisms of teratogenesis. In rodent models, ethanol and RA appear to affect similar cell populations and comparable malformations can be induced by both agents. When administered during gastrulation they cause a major insult to the anterior neural plate which results in characteristic ocular, brain and facial malformations comparable to those seen in the fetal alcohol syndrome. Exposure to these drugs at a time just prior to and during neural crest cell migration into the craniofacial and cervical regions results in malformations comparable to those seen in the DiGeorge sequence and/or retinoic acid embryopathy. Slightly later, at the time that the epibranchial placodes are active, insult results in mandibulofacial dysostosislike syndromes. We propose that the pattern of these malformations is related to the particular vulnerability of cells in the vicinity of normal programmed cell death. Cell death is also associated with ionizing radiation and hyperthermia-induced malformations. Both of these teratogens are particularly damaging to the early development of the eye and central nervous system. Teratogenic temperature elevations result in arrest of mitotic activity and death of cells in mitosis. Hypoxia is also associated with cell death in specific regions and subsequent malformation. For example, death of cells in the invaginating olfactory placode has recently been associated with cleft lip formation. The relationship of hypoxia-induced cell death to energy requirements is being explored. Acute treatment with methotrexate results in frontonasal dysplasia (median facial clefts). Combined effects of fluid imbalance, lack of proliferation or death of frontonasal mesenchyme appear to be involved. Although the mechanisms of craniofacial malformation are complex, a common feature for many is excessive cell death for which the embryo may be unable to compensate. Excessive cell death in regions of programmed cell death represents an important, yet little appreciated, mechanism of teratogenesis.
Abnormalities of craniofacial development: discussion report
Authors: Johnston M. C.
Published: April 2021

The following topics arose as major areas of discussion after individual papers and in the general discussion at the end of the session. Discussion from both sources has been merged here to facilitate easier reading.
Craniofacial development: a summing up
Authors: Wolpert L.
Published: April 2021

It is convenient to distinguish between three related problems in development: cell differentiation; pattern formation, which is about spatial organization; and morphogenesis in the strict sense, which is about change in form, particularly of cell sheets, but includes cell migration (Wolpert, 1981; Wolpert & Stein, 1984). All these need to be linked to gene action. If one looks forward over the next five to ten years then the future of craniofacial biology lies in molecular cell biology. This is not to say that all the problems at the tissue level have been solved, quite the contrary, but rather that the emphasis must now be at the cell and molecular level.
Craniofacial growth, evolutionary questions
Authors: Gans Carl
Published: April 2021

Understanding the growth of craniofacial systems in mammals, particularly in man, has always posed problems. Such craniofacial systems are formed ontogenetically of multiple tissue types, and the contributions of these tissues do not obviously match the divisions of adult skeletal elements (see Thorogood, this volume). Even the kind and number of segments in the head region continue to attract attention (Maderson, 1987). Furthermore, craniofacial systems appear to show trends toward an unusual number of developmental abnormalities or teratologies. Many of these teratologies suggest that we are not looking at a simple coordinated whole (Salinas, 1982; Shprintzen, 1982); rather, it seems as if multiple cranial and facial components incur differential growth either symmetrically or asymmetrically.

It seems instructive to treat the basis of this curious array of complications from an evolutionary viewpoint, considering two aspects, adaptation and history.
Morphogenesis of the head and face: discussion report
Authors: Wedden S. E.
Published: April 2021

The discussion following the session on evolution and morphogenesis of the head and face concentrated upon two major issues: How can one test models of development, particularly at biochemical and molecular levels?Are the cell populations of the early facial primordia heterogeneous and when might this heterogeneity arise?
Cellular and molecular aspects of cephalic neural crest development: workshop report
Authors: Bee Jim , Newgreen Don
Published: April 2021

The neural crest (NC) is derived from the ectoderm at the lateral borders of the neural plate and is first distinct during the later stages of neurulation when prospective NC cells segregate from the surrounding epidermal ectoderm and neurectoderm along the entire dorsolateral aspect of the forming and newly formed neural tube. From this origin, NC cells migrate extensive distances throughout the embryo to give rise to a large number of differentiated cell types and contribute to a diverse series of organ systems. This unique cell population therefore poses fundamental questions of the mechanisms underlying morphogenesis and differentiation during embryonic development.

The developmental fate of NC cells reflects their point of origin along the anterior–posterior axis of the embryo. Of particular significance to the present volume is the major contribution of cells derived from the cephalic NC to the facial skeleton.
Mesoderm induction in Xenopus laevis. responding cells must be in contact for mesoderm formation but suppression of epidermal differentiation can occur in single cells
Authors: Symes K. , Yaqoob M. , Smith J. C.
Published: April 2021

When Xenopus embryos are cultured in calcium- and magnesium-free medium (CMFM), the blastomeres lose adhesion but continue dividing to form a loose heap of cells. If divalent cations are restored at the early gastrula stage the cells re-adhere and eventually form muscle (a mesodermal cell type) as well as epidermis. If, however, the cells are dispersed during culture in CMFM, muscle does not form following reaggregation although epidermis does. This suggests that culturing blastomeres in a heap allows the transmission of mesoderm-induction signals from cell to cell while dispersion effectively dilutes the signal.

In this paper, we have attempted to substitute for cell proximity by culturing dispersed blastomeres in XTC mesoderm-inducing factor (MIF). We find that dispersed cells do not respond to XTC-MIF by forming mesodermal cell types after reaggregation, but the factor does inhibit epidermal differentiation. One interpretation of this observation is that an early stage in mesoderm induction is the suppression of epidermal differentiation and that formation of mesoderm may require contact-mediated signals that are produced in response to XTC-MIF.

We have gone on to study the suppression of epidermal differentiation in more detail. We find that this is a dose-dependent phenomenon that can occur in single cells in the absence of cell division. Animal pole blastomeres become more difficult to divert from epidermal differentiation at later stages of development and by stage 12 they are ‘determined’ to this fate. Fibroblast growth factor (FGF) also suppresses epidermal differentiation in isolated animal pole blastomeres and transforming growth factor-β l acts synergistically with FGF in doing so.
Segmentation in leech development
Authors: Weisblat David A. , Price David J. , Wedeen Cathy J.
Published: April 2021

Segments in glossiphoniid leeches, such as Helobdella triserialis, are the products of stereotyped cell lineages that yield identifiable cells from first cleavage. Cell lines generating segmental tissues are separated from those generating prostomial tissues early in development. Segments arise from five bilateral pairs of longitudinal columns of primary blast cells that are generated by five bilateral pairs of embryonic stem cells called teloblasts. There are four ectodermal cell lines (N, O, P and Q) and one mesodermal cell line (M) on each side of the embryo. In normal development, each cell line generates a segmentally iterated set of identified definitive progeny comprising a mixture of cell types. In the M, O and P cell lines, each blast cell generates one segment’s worth of definitive progeny (segmental complement). But the clones of blast cells in each of these three cell lines interdigitate longitudinally with cells of the adjacent clones from the same line, so that the clone of an individual m, o and p blast cell is distributed across more than one segment. Thus, there is no simple clonal basis for morphologically defined segments. In the N and Q cell lines, two blast cells are required to produce one segmental complement of definitive progeny; in each of these two cell lines, two classes of blast cells (nf and ns, qf and qs) are produced in exact alternation. Primary n and q blast cells are about the same size and are produced at the same rate as blast cells for the o and p bandlets, but the longitudinal extent of their clones is roughly half that of the o and p blast cells’ clones. During division of the blast cells, the n and q bandlets become compressed relative to the o and p bandlets, so that the segmental complements of the different cell lines can come into register. This compression movement is manifest as a movement of n and q bandlets relative to o and p bandlets in the posterior portion of the germinal band. The number of true segments in leech is fixed at 32; the counting mechanism is not known, but several hypotheses have been disproved. Segmentation in annelids and arthropods differs extensively at the cellular level, yet these phyla are presumed to share a common segmented ancestor. One strategy to identify homologous processes in annelid and arthropod segmentation is to compare the patterns of expression of evolutionarily conserved, developmentally important genes. Preliminary observations using a cross-reacting antibody that is thought to recognize a highly conserved region of a Drosophila segmentation gene, engrailed, labels nuclei of some blast cells early in development and, later, some neurones in the differentiating suboesophageal ganglion.
Spatially restricted domains of homeo-gene transcripts in mouse embryos: relation to a segmented body plan
Authors: Gaunt Stephen J. , Sharpe Paul T. , Duboule Denis
Published: April 2021

By use of in situ hybridization experiments, the transcripts of several different mouse homeo-genes (Hox-1.2, -1.3, -1.4, -1.5, -3.1 and -6.1) have been localized in 121-day mouse embryos. In a comparison of these genes on adjacent or nearby embryo sections, it is found that their transcripts occupy domains which are usually different, although overlapping, along the anteroposterior axis of the body. The domains are not limited to single segments (assumed to be represented by single prevertebrae) but they encompass regions of adjacent segments. In addition to the prevertebral column, the transcript domains extend into the central nervous system and at least some of the organs (pharynx, thyroid, trachea, lung, stomach and kidney).

Within the prevertebral column, a striking feature of most of the domains is that the abundance of transcripts rises (anteriorly) and falls (posteriorly) over a distance of several adjacent prevertebrae. For Hox-1.4 and Hox-1.3 the rise is over prevertebrae within the cervical region. For Hox-6.1, Hox-1-2 and Hox-3.1, the rise is over prevertebrae within the thoracic region. For each of the genes examined, transcripts in the central nervous system extend to a more anterior position in the body than transcripts in the prevertebral column. The myelencephalon of the hindbrain contains at least three different anterior boundaries for homeo-gene transcript domains. The positions of these are defined by Hox-1.5 (most anterior), Hox-1.4 and Hox-1.3. Anterior boundaries for Hox-6.1 and Hox-1.2 are apparently located at the Hox-1.3 position. Homeo-gene transcript domains extend into several structures known to be derived, at least in part, from the neural crest. These include the ventral pharynx, thyroid, aortic trunk and, probably, the sympathetic nerve chain and thymus.

For several genes of the Hox-1 cluster, we note a correspondence between the serial arrangement of genes on the chromosome and the arrangement of their transcript domains in the developing embryo. We also note some striking similarities between the transcript domains of different homeo-genes that share the same subfamily (Duboule et al. 1988). These observations, and others, offer possible clues about the molecular mechanisms that might underlie the formation and maintenance of homeo-gene transcript domains.
Murine genes with homology to Drosophila segmentation genes
Authors: Dressler Gregory R. , Deutsch Urban , Balling Rudi , Simon Dominique , Guenet Jean-Louis , Gruss Peter
Published: April 2021

As a potential method to define genes that control vertebrate segmentation processes during embryogenesis, murine genes containing a protein domain homologous to the Drosophila paired box sequence were isolated. The mouse genome contains at least three genes with highly conserved paired box like sequences, termed Pax genes, that are also conserved in other vertebrates. During embryogenesis, the Paxl gene is expressed in ventral sclerotome cells and in the intervertebral disks of the vertebral column. Thus, Paxl clearly shows a segmented pattern of expression. A second gene, Pax2, also is expressed in segmented structures of the intermediate mesoderm and in certain regions of ectoderm derived tissues. The Paxl and Pax2 genes map to chromosomes 2 and 7, respectively. Interestingly, several known mutations that affect vertebral column development map near the Paxl locus.
Gradients and insect segmentation
Authors: French Vernon
Published: April 2021

‘Morphogen’ gradients have long been invoked as a means of specifying spatial patterns of developmental fate, and it has now been demonstrated that they are indeed involved in the early steps of insect segmentation.

In many insects, including Drosophila, ligature and transplantation experiments have shown that the segment pattern develops through interactions between the ends of the egg. These results, plus those from irradiation and centrifugation of chironomid eggs, suggest that specific maternally synthesized RNAs are localized at the ends of the oocyte, and act as sources of opposing anterior and posterior gradients in the early egg.

In Drosophila, different groups of maternal ‘seg mentation’ genes are required for depositing within the oocyte terminal, anterior and posterior spatial cues. Injection of wild-type cytoplasm into mutant eggs which lack the anterior (bicoid) or posterior (oskar) cue suggests that these are normally distributed as gradients from strictly localized sources. It has now been shown directly that bicoid RNA passes into the oocyte from the nurse cells, remains localized in the anterior tip, and is later translated into protein which forms an exponential concentration gradient down the early egg. Genes required for posterior spatial information have not yet been cloned, so a posterior gradient (most likely to consist of nanos product) has yet to be directly demonstrated.

Analysis of zygotic ‘segmentation’ genes has shown that the different segment primordia are not directly specified by small changes in the anterior or (postulated) posterior gradient. It seems likely that the maternal cues specify a few bands of expression of zygotic gap genes such as hunchback, Krüppel and knirps, and that the pattern is then elaborated through interactions between these.

The anterior gradient seems to form by diffusion of bicoid protein, but the posterior signal seems to be capable of reorganization in some injection experiments. This could imply a diffusion/reaction mechan ism, or could result simply from the way in which the terminal, anterior and posterior cues act via gap gene activity. Hence the segment pattern formed after injection (and after irradiation of chironomid eggs) will not always correspond to the gradient profile.

Other types of insect egg develop with no nurse cells or external anterior source of RNA and, in these, there is some evidence of a posterior gradient but not of a similar signal from the anterior end.

It is now clear from the analysis of segmentation in Drosophila that the determinants and gradients in ferred from earlier studies do provide a positional framework within which the segment pattern is gradu ally elaborated. Investigation of segmentation in other eggs will be greatly assisted if the molecular techniques can be transferred from Drosophila.
Modifying expression of the engrailed gene of Drosophila melanogaster
Authors: Poole Stephen J. , Kornberg Thomas B.
Published: April 2021

The engrailed gene is required for segmentation of the Drosophila embryo and is expressed in cells constituting the posterior developmental compartments. In mutant embryos lacking engrailed function, portions of the cuticular pattern in each segment are deleted, resulting in fusion of adjacent denticle bands. Using P-element-mediated transposition, we generated flies that express the engrailed gene under the control of an hsp70 promoter, and found that ectopic, heat-shock-induced, engrailed expression caused pattern defects similar to those in embryos lacking engrailed function. Sensitivity to heat shock was only during the cellular blastoderm and early gastrulation periods. This window of sensitivity corresponds to the time when wildtype engrailed protein localizes into segmentally reiterated stripes and represents only a small portion of the normal period of engrailed gene expression.
Expression and role of E- and P-cadherin adhesion molecules in embryonic histogenesis: I. Lung epithelial morphogenesis
Authors: Hirai Yohei , Nose Akinao , Kobayashi Shigeyasu , Takeichi Masatoshi
Published: April 2021

The role of Ca2+ -dependent cell-cell adhesion mol ecules, E- and P-cadherins, in the histogenesis of mouse embryonic lung was studied. All epithelial cells of the lung express both E- and P-cadherin at the early developmental stage. P-cadherin, however, gradually disappears during development, initially from the main bronchi and eventually from all epithelial cells. When a monoclonal antibody to E-cadherin (ECCD-1) was added to monolayer cultures of lung epithelial cells, it induced a partial disruption of their cell-cell adhesion, while a monoclonal antibody to P-cadherin (PCD-1) showed a subtle effect. A mixture of the two antibodies, however, displayed a synergistic effect. We then tested the effect of the antibodies on the morphogenesis of lung primordia using an organ culture system. In control media, the explants formed typical bronchial trees. In the presence of ECCD-1, the explants grew up at the same rate as in the control, but their morphogenesis was affected. The control explants formed round epithelial lobules with an open luminal space at the tips of the bronchial trees, whereas the lobules of explants incu bated with ECCD-1 tended to be flat and devoid of the luminal space. PCD-1 showed a similar but very small effect. A mixture of the two antibodies, however, showed a stronger effect: the branching of epithelia was partially suppressed and the arrangement of epithelial cells was distorted in many places. These results suggest that E and P-cadherin have a synergistic role in the organiz ation of epithelial cells in lung morphogenesis.
Initiation of mesodermal cell migration and spreading relative to gastrulation in the urodele amphibian Pleurodeles waltl
Authors: Shi De-Li , Darribère Thierry , Johnson Kurt E. , Boucaut Jean-Claude
Published: April 2021

We have investigated the autonomous migration of marginal cells and their interactions with extracellular matrix (ECM) located on the inner surface of the blastocoel roof in the urodele amphibian, Pleurodeles waltl, using a novel in vitro migration assay. Animal hemispheres containing equatorial cells removed at different cleavage stages and dorsal marginal zone (DMZ) expiants of early gastrula stage were cultured either on fibronectin (FN)-coated or ECM-conditioned substrata. In explanted animal hemispheres, dorsal marginal cells showed autonomous migration on FN-coated substratum at the same time as the onset of gastrulation in control embryos. They acquired this capacity at least at the 32-cell stage, whereas lateral and ventral marginal cells acquired it after the 64-cell stage. DMZ outgrowths of early gastrula stage exhibited auton-omous spreading on both substrata. In addition, we showed that they spread preferentially toward the ani-mal pole when deposited on substratum conditioned by the dorsal roof of the blastocoel. By culturing dissociated marginal cells on ECM-conditioned substratum, we also found that increased spreading capacity of marginal cells was related to the initiation of their migration. A comparative study of the migration of marginal cells in ultraviolet (u.v.)-irradiated and normal embryos was also made. The results indicate that dorsal marginal cell migration was absent or dramatically reduced by u.v.-irradiation. These results suggest that the differential acquisition in the spreading capacity both in timing and in intensity around the marginal zone was correlated with the sequential involution of mesodermal cells in the course of gastrulation.
Cloned mouse melanocyte lines carrying the germline mutations albino and brown: complementation in culture
Authors: Bennett Dorothy C. , Cooper Philip J. , Dexter Tim J. , Devlin Linda M. , Heasman Janet , Nester Barry
Published: April 2021

We have established two new immortal lines of mouse melanocytes, melan-b and melan-c, from mice homozygous for the brown (b) and albino (c) mutations respectively. Both lines were derived through differentiation in vitro of embryonic epidermal melanoblasts. The brown melanocytes are visibly brown by light microscopy, and centrifuged cell suspensions form brown pellets. The albino melanocytes form white pellets and contain abundant unpigmented premelanosomes as shown by transmission electron microscopy. Like normal, non-immortal melanocytes and like the immortal black melanocyte line melan-a, both lines show little or no growth in a standard, serum-supplemented medium, but proliferate well in the presence of 12-o-tetradecanoyl phorbol-13-acetate (TPA). Sustained growth of the albino cells also requires either keratinocyte feeder cells or 2-mercaptoethanol (2-ME). The modal chromosome numbers are 39 for melan-b and 40 (diploid) for melan-c. Neither line is tumorigenic in nude mice. Hetero-karyons between the two lines can be constructed and form wild-type, black pigment. Melanocyte lines can now be reproducibly generated from mice of different strains, and provide tools for molecular studies of germline coat-colour mutations. These two lines provide elegant means to study the developmentally controlled expression of the two complementary genes, B and C, with black melanin pigment as a readily detectable natural marker.
Cyclic-AMP-induced elevation of intracellular pH precedes, but does not mediate, the induction of prespore differentiation in Dictyostelium discoideum
Authors: Campagne Michiel M. Van Lookeren , Aerts Rob J. , Spek Wouter , Firtel Richard A. , Schaap Pauline
Published: April 2021

Prespore gene expression in Dictyostelium is induced by the interaction of cAMP with cell surface cAMP receptors. We investigated whether intracellular pH (pHi) changes mediate the induction of prespore gene expression by cAMP. It was found that cAMP induces a 0 ·15 unit increase in pHi within 45 min after stimulation. The cAMP-induced pHi increase precedes the induction of prespore gene expression, measured by in vitro transcription, by about 15 ·30 min. Cyclic-AMP-induced pHi changes can be bypassed or clamped by addition of, respectively, the weak base methylamine, which increases pHi, or the weak acid 5,5-dimethyl-2,4-oxazolidinedione (DM0), which decreases pHi. Bypass of the cAMP-induced increase of pHi with methylamine does not induce the expression of prespore genes, while

inhibition of the pHi increase with DMO does not inhibit the induction of prespore gene expression. Cyclic-AMP-induced prespore protein synthesis and the proportion of prespore cells in multicellular aggregates are also not affected by bypassing or inhibiting the cAMP-induced pHi increase. These results show that although a morphogen-induced pHi increase precedes the induction of prespore gene expression, this increase does not mediate the effects of the extracellular cAMP signal on the transcription or translation of prespore genes in Dictyostelium discoideum.
Neural crest development in Xiphophorus fishes: scanning electron and light microscopic studies
Authors: Sadaghiani Bahram , Vielkind Juergen R.
Published: April 2021

We have studied neural crest development in two teleost fish species, Xiphophorus maculatus (platyfish) and X. heUeri (swordtail), and found similarities to that in other vertebrates but also some important differences. Unlike in other vertebrates, segregation of neural crest cells occurs in masses or groups from the dorsal-lateral part of the neural keel (tube) except in the mesencephalon region, where neural crest cells segregate from the dorsal-midline and in the most anterior trunk region, where they segregate individually. However, the cells were found in the usual neural tube-somite and somite-ectoderm migration pathways. Notably numerous cells, presumed in part to be neural crest cells, were found in a third location, dorsally on the neural tube. These cells exhibit a series of morphological stages referred to as ‘covering’, ‘condensation’, and ‘differentiation’. A great amount of ECM was observed in these fish and can be temporally and regionally correlated with the appearance of the neural crest cells. No major differences could be detected between the two fish species with the exception that segregation and appearance of neural crest cells in various locations occur earlier in the platyfish. This time difference could lead to perturbations in neural crest cell development in certain platyfish-swordtail hybrids and may contribute to the formation of neural-crest-derived pigment cell tumours, melanomas, in these hybrids.