bluuebunny/crossref_metadata_2025 huggingface dataset:
Developmental and evolutionary implications of labial, Deformed and engrailed expression in the Drosophila head
Authors: Diederich Robert J. , Pattatucci Angela M. , Kaufman Thomas C.
Published: April 2021

Prior developmental genetic analyses have shown that labial (lab) and Deformed (Dfd) are homeotic genes that function in the development of the embryonic (larval) and adult head. Using antibody probes to reveal the spatial distribution of the lab and Dfd proteins in embryonic and imaginai tissues, we have assessed the respective roles of these genes through an analysis of the correspondence of their expression patterns with their mutant phenotypes. With regard to imaginai develop-ment, lab and Dfd occupy adjacent non-overlapping expression domains in the peripodial cell layer of the eye-antennal disc, in patterns that are consistent with their adult mutant phenotypes and published fate maps. During embryogenesis, lab and Dfd exhibit limited overlapping expression in areas that are of no obvious significance to the development of larval head struc-tures, but also in areas that may have consequences for imaginai development. The head of Drosophila and other cyclorrhaphous Dipterans is characterized by an ex-treme morphological difference between the larval and adult stages. Given this unique ontogenetic and phylo-genetic history and the observation that homeotic transformations produced by the lab, Dfd, and proboscis pedia (pb) loci are manifested only in the adult, we suggest that distinct regulatory paradigms evolved for homeotic gene function in the development of the larval versus adult head. Finally, a detailed examination of the engrailed (en) expression pattern in the embryonic head strengthens the view of insect morphologists that the clypeolabrum evolved from the fusion of paired labral appendages.
Astrotactin provides a receptor system for CNS neuronal migration
Authors: Fishell Gord , Hatten Mary E.
Published: April 2021

CNS neuronal migration is a specialized form of cell motility that sets forth the laminar structure of cortical regions of brain. To define the neuronal receptor systems in glial-guided neuronal migration, an in vitro assay was developed for mouse cerebellar granule neurons, which provides simultaneous tracking of hundreds of migrating neurons. Three general classes of receptor systems were analyzed, the neuron-glial adhesion ligand astrotactin, the neural cell adhesion molecules of the IgG superfamily, N-CAM, LI and TAG-1, and the αsubunit of the integrin family. In the absence of immune activities, migrating cerebellar granule neurons had an average in vitro migration rate of 12 μm h-1, with individual neurons exhibiting migration rates over a range between 0 to 70 μm h-1. The addition of anti-astrotactin antibodies (or Fabs) significantly reduced the mean rate of neuronal migration by sixty-one percent, resulting in eighty percent of the neurons having migration rates below 8 μm h-1. By contrast, blocking antibodies (or Fabs) against LI, N-CAM, TAG-1 or β 1 integrin, individually or in combination, did not reduce the rate of neuronal migration.

By video-enhanced contrast differential interference contrast microscopy the effects of anti-astrotactin antibodies were seen to be rapid. Within fifteen minutes of antibody application, streaming of cytoplasmic organelles into the leading process arrested, the nucleus shifted from a caudal to a central position, and the extension of filopodia and lamellopodia along the leading process ceased. Correlated video and electron microscopy suggested that the mechanism of arrest by antiastrotactin antibodies involved the failure to form new adhesion sites along the leading process and the disorganization of cytoskeletal components. These results suggest astrotactin acts as a neuronal receptor for granule neuron migration along astroglial fibers.
Axon repulsion during peripheral nerve segmentation
Authors: Keynes Roger J. , Jaques Karen F. , Cook Geoffrey M. W.
Published: April 2021

The guidance of axons during embryonic development is likely to involve both adhesive and repulsive interactions between growth cones and their environment. We are characterising the role and mechanism of repulsion during the segmental outgrowth of motor and sensory axons in the somite mesoderm of chick embryos. Axons are confined to the anterior half of each somite by the expression in the posterior half of a glycoconjugate system (48×103Mr and 55×103Mr) that causes the collapse of dorsal root ganglion growth cones when applied in vitro. Enzymatic cleavage of this fraction with specific combinations of endo- and exoglycosidases removes collapse activity, suggesting that carbohydrate residues are involved in the execution of collapse. A similar activity is also detectable in normal adult grey matter, suggesting roles for repulsion beyond the development of spinal nerve segmentation.
Cell lineage in the cerebral cortex
Authors: Price Jack , Williams Brenda , Grove Elizabeth
Published: April 2021

We have studied cell lineage in the rat cerebral cortex using retroviral mediated gene transfer. By this method, a marker gene is inserted into dividing precursor cells such that their fate can be followed. We have applied this technique to two types of experiment. First, virus was used to label precursor cells of the cerebral cortex in situ during the period of neurogenesis. Second, cortical precursor cells were grown in dissociated cell culture, and virus was used to follow their development over the culture period. These experiments showed that the majority of precursor cells generate a single cell type - neurones, astrocytes, or oligodendrocytes. Moreover, this is true both in vivo and in dissociated cell culture. The only exception is a bipotential cell, which can generate both neurones and oligodendrocytes. These data suggest that the ventricular zone - the germinal layer of the embryonic cortex - is a mosaic of precursor cells of different restricted potentials.

Although precursor cells are restricted in terms of the cell types they generate, they seem not to be restricted in either the cortical laminae or cytoarchitectonie areas to which they can contribute. Both neuronal and grey matter astrocyte precursors contribute cells to multiple layers of both infra- and supragranular laminae. Moreover, in the hippocampal formation, neuronal precursors can contribute cells to more than one hippocampal field.
Genetic control of primary neuronal development in zebrafish
Authors: Kimmel Charles B. , Hatta Kohei , Eisen Judith S.
Published: April 2021

During the first day of embryogenesis in the zebrafish, a precise and relatively simple network of neurons develops, pioneering axonal pathways and apparently functioning to mediate reflexive motor responses to touch stimuli. We have begun to use zygotic lethal mutations to analyze the assembly of this ‘primary’ embryonic nervous system. Here we focus on spinal primary motoneurons, their inputs from hindbrain Mauthner neurons, and their outputs to segmental body wall muscle. The mutation nic-1 blocks synaptic transmission between nerve and muscle, yet embryonic primary motoneurons appear normal, suggesting that functional interactions with their targets are not involved in regulating their development. The mutation spt-1 directly disrupts development of this muscle, and the mutation cyc-1 appears to directly block specification of the floor plate. Both spt-1 and cyc-1 affect aspects of primary neuronal development, and they probably do so indirectly. The nonautonomous actions of these mutations are local and they produce variable neuronal phenotypes. The observations can be interpreted to mean that some cellular interactions that specify the neurons and their axonal paths occur at close range and involve multiple, possibly combinatorial, transmitterindependent pathways.
Dissection of the faint little ball (fib) phenotype: determination of the development of the Drosophila central nervous system by early interactions in the ectoderm
Authors: Raz Erez , Shilo Ben-Zion
Published: April 2021

The complex embryonic phenotype of mutations in the faint little ball (flb) locus, encoding the Drosophila EGF receptor homolog (DER), was dissected by temperature shifts of a temperature-sensitive allele. We show that the phenotype can be resolved into at least five components, which are temporally and spatially distinct. Most notably, the central nervous system (CNS) phenotype is determined at two separate phases. A severe collapse results from early defects in the DER-expressing ectodermal cells from which neuroblasts and midline glial cells delaminate. We thus suggest that DER activity is crucial for interactions that occur in the ectoderm at an early stage, and determine the fate of neuronal and glial cell lineages. This finding explains how a severe CNS phenotype is generated in fib embryos, in spite of the absence of expression of the protein in neuronal cells. In a second phase, during germ band retraction, the fib function is required specifically in the three pairs of midline glial cells (MG). In the absence of a functional DER protein, these cells die or fail to differentiate correctly, resulting in a fused commissure phenotype.
In vitro control of neuronal polarity by glycosaminoglycans
Authors: Lafont F. , Rouget M. , Triller A. , Prochiantz A. , Rousselet A.
Published: April 2021

We have studied the effects of proteoglycans (PGs) and glycosaminoglycans (GAGs) on the growth and morphology of neurons in culture. PGs from glial cells or Engelbreth-Hohn-Swarm tumor cells (EUS), pure bovine kidney heparan sulfate (HS), shark cartilage type C chondroïtin sulfate (CSc) and bovine mucosa dermatan sulfate (DS) added to embryonic rat neurons strongly enhanced total neurite growth after 48 h in vitro. No trophic effects were seen when PGs treated with a mixture of glycanases were used. PGs, CSc and HS not only enhanced neurite growth but induced the appearance of a majority of neurons with a single long axon whereas, in contrast, DS increased dendrite growth. GAGs bound to the cell surface and were rapidly internalized, a feature that correlated well with the absence of neurotrophicity of GAGs previously immobilized on the culture substratum. Although the mechanisms involved in GAGs neurotrophic effects and in the separate regulation of neuronal polarity by HS and DS were not elucidated, we found that, as opposed to HS, DS was able to enhance neuronal adhesion and spreading and to maintain a high level of expression of microtubule-associated protein 2 (MAP2), a specific dendritic marker. This finding confirms and extends our previous observations on the role of adhesion in the regulation of dendrite growth.
Commitment of mesoderm cells in Hensen’s node of the chick embryo to notochord and somite
Authors: Selleck Mark A. J. , Stern Claudio D.
Published: April 2021

Hensen’s node in the chick embryo contains prospective notochord cells in a ▿-shaped midline region of the mesoderm, and prospective medial half somite cells in the lateral portions of the node mesoderm, whilst lateral somite cells are derived from the rostral part of the primitive streak (Selleck and Stern, Development 112, 615-626, 1991). In the present study, we have investigated the commitment of these mesoderm cells to their fates by grafting between these regions of the node and primitive streak. By challenging the cells in this way, we have attempted to discover whether prospective notochord and somite cells can change their fates.

We find that the mesoderm cells In the midline ▿-shaped portion of Hensen’s node, when grafted into a different region, continue to integrate into the notochord, or form a separate notochord-like structure. By contrast, the prospective somitic cells do not appear to be committed to a somitic fate.
Basic fibroblast growth factor stimulates the sustained proliferation of mouse epidermal melanoblasts in a serum-free medium in the presence of dibutyryl cyclic AMP and keratinocytes
Authors: Hirobe Tomohisa
Published: April 2021

Basic fibroblast growth factor (bFGF) stimulated the sustained proliferation of mouse epidermal melanoblasts derived from epidermal cell suspensions in a serum-free medium supplemented with dibutyryl adenosine 3′,5′-cyclic monophosphate (DBcAMP). The melanoblasts could be subcultured in the serum-free medium supplemented with the two factors in the presence of keratinocytes, but not in the absence of keratinocytes. In these conditions, some melanoblasts proliferated without differentiating for more than 20 days including a subculture. This is the first report of a successful culture of melanoblasts from mammalian skin. This culture system is expected to clarify further markers for melanoblasts and requirements for their proliferation and differentiation.
LFB1 and LFB3 homeoproteins are sequentially expressed during kidney development
Authors: Lazzaro D. , De Simone V. , De Magistris L. , Lehtonen E. , Cortese R.
Published: April 2021

LFB1 (HNF-1/HNF-1 alpha/APF) and LFB3 (vHNF-1/HNF-1 beta) are two homeoproteins involved in the transcriptional regulation of several liver-specific genes. Both genes are expressed in the polarized epithelia of a wide range of tissues, including liver, the digestive tract and kidney. We have analyzed the expression pattern of LFB1 and LFB3 in the developing rat kidney by in situ hybridization. Our results show that LFB3 transcripts can be detected in mesoderm-derived cells as soon as they are induced to differentiate into a polarized epithelium, while LFB1 transcripts appear only at a later stage when the three different segments of the nephron become apparent. LFB1 transcripts are restricted to the proximal and distal tubules, whereas LFB3 is also detected in the collecting ducts. Neither LFB1 nor LFB3 are expressed in the glomeruli or in the transition epithelia of the ureters and of the urinary bladder, none of which are involved in active transport mechanisms. The sequential activation of these two genes is also observed in transfilter organ cultures of nephrogenic mesenchyme at different stages after induction. This expression pattern suggests that LFB3 and LFB1 play a role in two critical stages of the developmentally regulated conversion of the nephric mesenchyme into a polarized epithelium: the early inductory phase (LFB3) and the postinductory phase (LFB1+LFB3).
Identification and characterization of Drosophila female germ line transcriptional control elements
Authors: Frank Lawrence H. , Cheung Hung-Kam , Cohen Robert S.
Published: April 2021

The highly organized structure of the Drosophila ovary makes it an ideal system for studying mechanisms of differential gene expression. Here we report the identification of a 171 bp sequence from the 5′ end of the hsp26 gene that functions as a female germ-line-specific transcriptional regulator when linked in two copies to a basal promoter. The regulator is active only in nondividing cells of the germ line, i.e., only in nurse cells and oocytes. It is not active in any examined tissue or cell type outside of the female germ line. Copper nuclease footprinting studies show that the germ line regulator contains two binding sites for each of two different ovarian nuclear factors. Point mutations in the DNA target sites of either nuclear factor abolish in vitro binding and in vivo transcriptional activity, indicating that each factor is a positive activator of nurse cell/oocyte transcription. The two factors may represent different classes of activator proteins, since an increase in the copy number of one factor’s DNA target site cannot compensate for a decrease in the copy number of the other factor’s target site.
Ten different Polycomb group genes are required for spatial control of the abdA and AbdB homeotic products
Authors: Simon Jeffrey , Chiang Anne , Bender Welcome
Published: April 2021

Mutations in genes of the Polycomb (Pc) group cause abnormal segmental development due to ectopic expression of the homeotic products of the Antennapedia and bithorax complexes. Here the requirements for Pc group genes in controlling the abdA and AbdB products of the bithorax complex are described. Embryos containing mutations in the genes Polycomb (Pc), extra sex combs (esc), Enhancer of zeste [E(z)], polyhomeotic (ph), Sex comb on midleg (Scm), Polycomb-like (Pcl), Sex comb extra (See), Additional sex combs (Asx), Posterior sex combs (Psc) and pleiohomeotic (pho) were examined. In every case, both abdA and AbdB are expressed outside of their normal domains along the anterior-posterior (A–P) axis, consistent with these Pc group products acting in a single pathway or molecular complex. The earliest detectable ectopic expression is highest in the parasegments immediately adjacent to the normal expression boundary. Surprisingly, in the most severe Pc group mutants, the earliest ectopic AbdB is distributed in a pair-rule pattern. At all stages, ectopic abdA in the epidermis is highest along the anterior edges of the parasegments, in a pattern that mimics the normal abdA cell-specific pattern. These examples of highly patterned mis-expression show that Pc group mutations do not cause indiscriminate activation of homeotic products. We suggest that the ectopic expression patterns result from factors that normally activate abdA and AbdB only in certain parasegments, but that in Pc group mutants these factors gain access to regulatory DNA in all parasegments.
Spatial restriction of AChR gene expression to subsynaptic nuclei
Authors: Simon Alexander M. , Hoppe Peter , Burden Steven J.
Published: April 2021

Acetylcholine receptors (AChRs) and the mRNAs encoding the four AChR subunits are highly concentrated in the synaptic region of skeletal myofibers. The initial localization of AChRs to synaptic sites is triggered by the nerve and is caused, in part, by post-translational mechanisms that involve a redistribution of AChR protein in the myotube membrane. We have used transgenic mice that harbor a gene fusion between the murine AChR delta subunit gene and the human growth hormone gene to show that innervation also activates two independent transcriptional pathways that are important for establishing and maintaining this non uniform distribution of AChR mRNA and protein. One pathway is triggered by signal(s) that are associated with myofiber depolarization, and these signals act to repress delta subunit gene expression in nuclei throughout the myofiber. Denervation of muscle removes this repression and causes activation of delta subunit gene expression in nuclei in non-synaptic regions of the myofiber. A second pathway is triggered by an unknown signal that is associated with the synaptic site, and this signal acts locally to activate delta subunit gene expression only in nuclei within the synaptic region. Synapse-specific expression, however, does not depend upon the continuous presence of the nerve, since transcriptional activation of the delta subunit gene in subsynaptic nuclei persists after denervation. Thus, the nuclei in the synaptic region of multinucleated skeletal myofibers are transcriptionally distinct from nuclei elsewhere in the myofiber, and this spatially restricted transcription pattern is presumably imposed initially by the nerve.
Wound closure in foetal rat skin
Authors: Ihara Setsunosuke , Motobayashi Yumiko
Published: April 2021

Foetal rat skin rapidly closes an open wound in organ culture and in vivo, this possibly being unique to organs still in the morphogenetic stage. In the present study, examination was made of morphological changes in foetal rat skin during closure of open wounds inflicted at day 16 of gestation. Phase-contrast microscopy of open-wounded skin cultured in vitro indicated inward spreading of the peripheral skin to be responsible for wound closure. Wound closure in vitro was inhibited by cytochalasin B (10 μg/ml), not by hydroxyurea (2 mM), indicating prenatal wound closure to be mediated by regulation of the microfilament system rather than cell proliferation. During wound closure in vitro and in vivo, light and scanning electron microscopy of the peripheral skin showed cells in the periderm, the outermost layer of the foetal epidermis, to elongate centripetally and en masse, whereas the shape of underlying epidermal cells not to change. Numerous spindle-shaped cells and fibrous matrices in the mesenchyme were redistributed, becoming oriented along the wound edge. Following isolation of the mesenchyme and epidermis by treatment with Dispase and separate culturing, the capacity for wound closure in vitro was found to be retained only by the mesenchyme. Cellular activity within the mesenchyme, rather than in the epidermis, would thus appear essential to wound closure in foetal rat.
Intracellular free Ca2+ changes during physiological polyspermy in amphibian eggs
Authors: Grandin Nathalie , Charbonneau Michel
Published: April 2021

We have made the first measurements of intracellular free calcium activity ([Ca2+]i) in urodele eggs during physiological polyspermic fertilization. Jellied eggs of the urodele amphibian Pleurodeles waltlii were impaled with intracellular Ca2+-selective microelectrodes and inseminated under various conditions of sperm:egg ratio to obtain various degrees of polyspermy. In 17 out of 45 cases the egg [Ca2+]i level (0.41 μM) showed no variation following fertilization. In 28 other cases, however, the egg displayed a slow increase in [Ca2+]i of 0.15 μM, starting around 15 minutes after fertilization and reaching a plateau level around 10 minutes later. The amplitude of the fertilization-associated increase in [Ca2+]i was found to be independent of the number of sperm interacting with the egg surface. Measurements with two Ca2+-microelectrodes impaled in single eggs showed that the increase in [Ca2+]i did not simultaneously occur at distinct places within the egg cortex and, in some cases, could be detected by only one of the two microelectrodes. This latter observation, as well as the absence of [Ca2+]i change at fertilization in some experiments, strongly suggested that each sperm interacting with the egg might, at various places, trigger a localized, non-propagating change in [Ca2+]i. Experiments in which eggs were locally inseminated, using a micropipette directed towards the site of impalement of one of the two Ca2+-microelectrodes, clearly established that [Ca2+]i changes, although incapable of propagating over the entire egg cortex, might nevertheless travel very slowly over short distances, their amplitude vanishing rapidly as they propagate from around the sites of sperm entry. The physiologically polyspermic egg of urodele amphibians appears to represent an exception to the universality of a fertilization-induced Ca2+ wave.
Compensatory synaptic growth in the rod terminals as a sequel to partial photoreceptor cell loss in the retina of chimaeric mice
Authors: Sanyal S. , Hawkins R. K. , Jansen H. G. , Zeilmaker G. H.
Published: April 2021

In the retina of chimaeric mice of rd and wild-type genotypic combination, selective loss of rd/rd photoreceptor cells, after initial development, leads to a mosaic retina with variable amounts of normal photoreceptor cells present over the retinal surface. In some of the rod terminals of these retinas the synaptic complexes with the second order retinal neurons are seen to contain multiple synaptic ribbons and an increased number of profiles of the postsynaptic elements. These changes are observed only in the rod terminals and not in the cone pedicles. Computer aided three-dimensional reconstruction of the altered synapses shows that these changes result from an increase in the number of synaptic sites, characterized by multiplication of the synaptic ribbons and enlargement of the second order neuronal processes. A quantitative analysis of such synapses, based on serial electron micrographs, shows that these are most frequently located in the retinal regions of the chimaeric individuals that have suffered maximum photoreceptor cell loss. Thus synaptic growth appears to take place as a reaction to the reduction of afferent input to the postsynaptic components. These findings demonstrate persistent synaptic plasticity in the rod terminals of mammalian retina during the maturational phase of late postnatal development. Compensatory synaptic growth in the rod terminals, as recorded here, can have important implications for the maintenance of visual sensitivity in the diseased or ageing retina.
Regulation of proneural gene expression and cell fate during neuroblast segregation in the Drosophila embryo
Authors: Skeath James B. , Carroll Sean B.
Published: April 2021

The Drosophila embryonic central nervous system develops from sets of progenitor neuroblasts which segregate from the neuroectoderm during early embryogenesis. Cells within this region can follow either the neural or epidermal developmental pathway, a decision guided by two opposing classes of genes. The proneural genes, including the members of the achaete-scute complex (AS-C), promote neurogenesis, while the neurogenic genes prevent neurogenesis and facilitate epidermal development. To understand the role that proneural gene expression and regulation play in the choice between neurogenesis and epidermogenesis, we examined the temporal and spatial expression pattern of the achaete (ac) regulatory protein in normal and neurogenic mutant embryos. The ac protein is first expressed in a repeating pattern of four ectodermal cell clusters per hemisegment. Even though 5 –7 cells initially express ac in each cluster, only one, the neuroblast, continues to express ac. The repression of ac in the remaining cells of the cluster requires zygotic neurogenic gene function. In embryos lacking any one of five genes, the restriction of ac expression to single cells does not occur; instead, all cells of each cluster continue to express ac, enlarge, delaminate and become neuroblasts. It appears that one key function of the neurogenic genes is to silence proneural gene expression within the nonsegregating cells of the initial ectodermal clusters, thereby permitting epidermal development.
Expression of the two mannose 6-phosphate receptors is spatially and temporally different during mouse embryogenesis
Authors: Matzner U. , Figura K. Von , Pohlmann R.
Published: April 2021

Mammalian cells express two mannose 6-phosphate receptors, MPR46 and MPR300, both of which mediate the targeting of lysosomal enzymes to lysosomes. Additionally the receptors mediate the secretion (MPR46) and the endocytosis (MPR300) of lysosomal enzymes and the binding of IGFH (MPR300). We have analyzed the distribution of MPR46 and MPR300 transcripts during mouse embryogenesis by in situ hybridization. Up to day 15.5 of embryonic development we found a non-overlapping distribution of the transcripts for the two receptors. High expression of MPR46 was observed at sites of hemopoiesis and in the thymus while MPR300 was highly expressed in the cardiovascular system. Late in embryogenesis (day 17.5) a wide variety of tissues expressed the receptors, but still the expression pattern was almost non-overlapping. This unexpected complementary expression pattern points to specific functions of the two mannose 6-phosphate receptors during mouse embryogenesis.
Interactions between primordial germ cells play a role in their migration in mouse embryos
Authors: Gomperts Miranda , Garcia-Castro Martin , Wylie Chris , Heasman Janet
Published: April 2021

Primordial germ cells (PGCs) are the founder cell population of the gametes which form during the sexually mature stage of the life cycle. In the mouse, they arise early in embryogenesis, first becoming visible in the extraembryonic mesoderm, posterior to the primitive streak, at 7.5 days post coitum (d.p.c.). They subsequently become incorporated into the epithelium of the hind gut, from which they emigrate (9.5 d.p.c.) and move first into the dorsal mesentery (10.5 d.p.c.), and then into the genital ridges that lie on the dorsal body wall (11.5 d.p.c.). We have used confocal microscopy to study PGCs stained with an antibody that reacts with a carbohydrate antigen (Stage-Specific Embryonic Antigen-1, SSEA-1) carried on the PGC surface. This allows the study of the whole PGC surface, at different stages of their migration.

The appearance of PGCs in tissue sections has given rise to the conventional view that they migrate as individuals, each arriving in turn at the genital ridge. In this paper, we show that PGCs leave the hind gut independently, but then extend long (up to 40 μm) processes, with which they link up to each other to form extensive networks. During the 10.5 – 11.5 d.p.c. period, these networks of PGCs aggregate into groups of tightly apposed cells in the genital ridges. As this occurs, their processes are lost, and their appearance suggests they are now non-motile. Furthermore, we find that PGCs taken from the dorsal mesentery at 10.5 d.p.c. perform the same sequence of movements in culture. At first they are actively locomotory. They become linked to other PGCs via long processes and form clusters of non-motile cells. This aggregation together into closely apposed masses may be an important component of PGC migration from the gut into the genital ridges and would also allow signalling interactions between PGCs.

We show also that the first PGCs to emigrate from the hind gut, between 9 and 9.5 d.p.c., do so directly into the area where the genital ridges will form. This suggests that an adhesive interaction between these ‘pioneer germ cells’ and the target tissue may play a role in the localisation of PGCs into the genital ridges as they aggregate.
A functional test for maternally inherited cadherin in Xenopus shows its importance in cell adhesion at the blastula stage
Authors: Heasman Janet , Ginsberg Dorit , Geiger Benjamin , Goldstone Kim , Pratt Travis , Yoshida-Noro Chikako , Wylie Chris
Published: April 2021

We report here on the consequences of reducing the expression of EP-cadherin at the earliest stages of Xenopus development. Injection of oligodeoxynucleotides antisense to maternal EP-cadherin mRNA into full-grown oocytes reduced the mRNA level in oocytes, and the protein level in blastulae. Adhesion between blastomeres was significantly reduced, as seen in whole embryos, and in assays of the ability of blastomeres to reaggregate in culture. This effect was especially conspicuous in the inner cells of the blastula and included the disruption of the blastocoel. The severity of the EP-cadherin mRNA depletion and of the disaggregation phenotype was dose dependent. This phenotype was rescued by the injection into EP-cadherin mRNA-depleted oocytes of the mRNA coding for a related cadherin, E-cadherin, that is normally expressed at the gastrula stage in the embryonic ectoderm.
A central role for epidermal segment border cells in the induction of muscle patterning in the Drosophila embryo
Authors: Volk Talila , VijayRaghavan K.
Published: April 2021

The correct patterning of muscles in the Drosophila embryo depends on the migration of developing muscles over the ectoderm and on the attachment of these muscles to specific attachment sites. We investigate the mechanisms that are involved in this process and describe experiments that allow a genetic dissection of the role of the ectoderm in muscle migration and attachment.

We show that cells along the segmental border in the ectoderm are used by the developing muscles to reach their attachment sites. These segment border cells are recognized by dissociated myotubes in single suspensions in culture. Thus, developing muscles have properties that allow the specific recognition of the segment border cells and migrate to attach to these cells. The segment border cells are absent in the mutant wingless and naked. In these mutants, the muscles are severely disorganized. We show that this is not a mere consequence of disruption of the epidermis, since, in the mutant patched, where segmental patterning is affected, the segment border cells are present near their normal position; the muscles in this mutant are relatively organized. Similarly, in the mutant lines where ectopic segment border cells are present, the observed muscle derangement correlates well with the ectopic attachment sites that are present.

Finally, we have analyzed mutants at the stripe locus and have shown that lethal alleles disrupt muscle organization during embryogenesis. Enhancer-trap alleles of stripe that we have analyzed show reporter gene expression in the segment border cells.

Our results indicate a role for the segment border cells in guidance of migrating muscle fibers to their attachment sites.
Primordial germ cell migration in Drosophila melanogaster is controlled by somatic tissue
Authors: Jaglarz Mariusz K. , Howard Kenneth R.
Published: April 2021

In Drosophila, as in many other organisms, primordial germ cells show invasive and migratory behavior moving from their site of origin to the somatic component of the gonad. At a characteristic time in development, the primordial germ cells pass across the primordium of the gut and migrate on its outer surface toward the mesoderm, where they eventually associate with the somatic tissues of the gonad. Here we demonstrate that the exit and migration are specific behaviors of the primordial germ cells and that they are controlled by the somatic tissue of the embryo rather than by a germ cell autonomous clock. Using mutations, we show that these controlling somatic events probably occur in the tissue of the gut primordium itself.
The mouse tissue plasminogen activator gene 5′ flanking region directs appropriate expression in development and a seizure-enhanced response in the CNS
Authors: Carroll Pamela M. , Tsirka Stella E. , Richards William G. , Frohman Michael A. , Strickland Sidney
Published: April 2021

Tissue plasminogen activator (t-PA) is a secreted serine protease implicated in multiple aspects of development. In the adult rat brain, transcription of t-PA is an immediate-early response in the hippocampus following treatments that induce neuronal plasticity. To study the sequence elements that govern transcription of this gene, in situ analysis was used to define t-PA’s temporal and spatial expression pattern in midgestation embryos. Transgenic mice were then generated carrying t-PA 5′ flanking sequences linked to the E. coli lacZ gene. Constructs containing 4 kb of the flanking sequences (4.0TAMGAL) confer β-galactosidase activity mostly to the same tissues that exhibit high levels of t-PA mRNA by in situ analysis. In 4.0TAMGAL embryos from embryonic day 8.5 (E8.5) to 13.5 (E13.5), the majority of expression observed is localized to neural ectoderm-derived tissues. β-galactosidase activity is first detected in restricted neuromeres in the midbrain and diencephalon, at E8.5 and E9.5 respectively. At E10.5, transgene expression is observed in neural crest-derived cranial nerves and dorsal root ganglia, but not placode-derived cranial nerves. From E10.5 to E13.5, β-galactosidase activity is observed in postmitotic neurons of the midbrain, spinal cord, neural retina and the developing olfactory system. β-galactosidase activity is also detected in areas undergoing tissue remodeling such as the pinna of the ear, whisker follicles and the limbs. In adult mice, lacZ is expressed in the hippocampus and this expression was found to be enhanced upon seizure in the giant pyramidal neurons of CA3. These results reinforce the concept that t-PA plays a role in neurogenesis and morphogenesis, and identifies the promoter region that directs its transcriptional regulation both in development and in the CNS.
Distribution of, and a putative role for, the cell-surface neutral metallo-endopeptidases during mammalian craniofacial development
Authors: Spencer-Dene Bradley , Thorogood Peter , Nair Sean , Kenny A. John , Harris Malcolm , Henderson Brian
Published: April 2021

Endopeptidase-24.11 (neutral endopeptidase, neprilysin, ‘enkephalinase’, EC 3.4.24.11) and endopeptidase-24.18 (endopeptidase-2, meprin, EC 3.4.24.18) are cell-surface zinc-dependent metallo-endopeptidases able to cleave a variety of bioactive peptides including growth factors. We report the first study of the cellular and tissue distribution of both enzymes and of the mRNA for NEP during embryonic development in the rat. Endopeptidase-24.11 protein was first detected at E10 in the lining of the gut and, at E12, the enzyme was present on the notochord, medial and lateral nasal processes, otocyst, mesonephros, heart and neuroepithelium. In contrast, at this time endopeptidase-24.18 was present only on the apical surface of the neuroepithelial cells. By E14 and E16, NEP was also detected in a wide range of craniofacial structures, notably the palatal mesenchyme, the choroid plexus, tongue and perichondrium. The distribution of endopeptidase-24.18 at these stages was restricted to the inner ear, the nasal conchae, and ependymal layer of the brain ventricles and the choroid plexus. Although endopeptidase-24.11 had been detectable in the craniofacial vasculature at E12 and E14, this was no longer apparent at E16. Significantly, the distribution of endopeptidase-24.11 mRNA closely matched the immunolocalization of the protein at all stages investigated.

In order to explore the functional role of these enzymes, inhibition studies were carried out using two selective inhibitors of endopeptidase-24.11, phosphoramidon and thiorphan. E9.5 and E10.5 embryos exposed to either inhibitor displayed a characteristic, asymmetric abnormality consisting of a spherical swelling, possibly associated with a haematoma, predominantly on the left side of the prosencephalon, and the severity of this defect appeared to be a dose-dependent phenomenon. This study suggests that these enzymes play previously unrecognized roles during mammalian embryonic development.
YIGSR domain of laminin binds surface receptors of mesenchyme and stimulates migration during gastrulation in sea urchins
Authors: Crawford Bryan D. , Burke Robert D.
Published: April 2021

During gastrulation in sea urchins, cells at the tip of the archenteron extend filopodia that attach to the blastocoel wall and are thought to assist in the elongation of the archenteron. Upon completion of gastrulation, these cells migrate into the blastocoel. Time-lapse video records were made of preparations from which ectodermal cells have been removed, leaving the archenteron, mesenchyme cells and blastocoelar extracellular matrix (ECM) bounded by the basal lamina. In preparations of late gastrulae, cells at the tip of the archenteron extend filopodia that attach to the basal lamina and pull it inward, collapsing the preparation. This collapse does not occur in preparations made prior to the elongation phase and can be inhibited with cytochalasin B and azide, but not with colchicine. Migratory behavior increased in preparations treated with the laminin-derived peptide Tyr-Ile-Gly-Ser-Arg (YIGSR). Cells extend and retract filopodia, collapse the ECM and migrate out of the preparation. This behavior was not observed in preparations treated with whole laminin, fibronectin or Arg-Gly-Asp-Ser (RGDS) peptides. Cells in preparations treated with YIGSR extend significantly more processes than those incubated in RGDS, laminin, fibronectin or BSA. This effect is titratable between 10−3 and 10−6 M. Whole laminin has a significant inhibitory effect on the number of cell processes observed. Double labelling experiments with biotinylated laminin or biotinylated CDPGYIGSR and a mesenchyme-specific monoclonal antibody (Sp12) reveal that laminin and CDPGYIGSR label mesenchymal and non-mesenchymal cells. A CDPGYIGSR affinity column binds a 125I-labelled cell surface component, which elutes with YIGSR and has an Mr of about 80×103 on SDS-PAGE. We propose that cells at the tip of the archenteron attach to the basal lamina during archenteron elongation, and that domains of laminin containing YIGSR in the basal lamina of the target region stimulate migratory behavior in these cells.
Disruption of morphogenesis and transformation of the suspensor in abnormal suspensor mutants of Arabidopsis
Authors: Schwartz Brian W. , Yeung Edward C. , Meinke David W.
Published: April 2021

The suspensor is the first differentiated structure produced during plant embryogenesis. In most angiosperms, the suspensor functions early in development to provide nutrients and growth regulators to the embryo proper. In Arabidopsis, the suspensor undergoes programmed cell death at the torpedo stage and is not present in mature seeds. We have identified at least 16 embryo-defective mutants of Arabidopsis that exhibit an enlarged suspensor phenotype at maturity. In this report, we focus on seven abnormal suspensor mutants, which define three genetic loci (sus1, sus2 and sus3). Recessive mutations at each of these loci disrupt morphogenesis in the embryo proper and consistently result in the formation of a large suspensor. Defects in the embryo proper appear by the globular stage of development; abnormalities in the suspensor are detected soon after at the heart stage. Storage protein and lipid bodies, which normally accumulate only in the embryo proper late in embryogenesis, are present in both the arrested embryo proper and enlarged suspensor. Therefore, cell differentiation in the embryo proper can proceed in the absence of normal morphogenesis, and the suspensor can be transformed into a structure with features normally restricted to the embryo proper. These observations are consistent with a model in which normal development of the embryo proper limits growth and differentiation of the suspensor. Altered development of the embryo proper in mutant seeds leads indirectly to proliferation of suspensor cells and expression of properties characteristic of the embryo proper. Ultimately, growth of the transformed suspensor is limited by the same genetic defect that disrupts development of the embryo proper. The availability of multiple alleles of sus1 and sus2, including T-DNA tagged alleles of each, will facilitate the cloning of these essential genes and molecular analysis of interactions between the embryo proper and suspensor early in development.
Discrete endogenous signals mediate neural competence and induction in P19 embryonal carcinoma stem cells
Authors: Pruitt Steven C.
Published: April 2021

Endogenous signals capable of inducing neuroectodermal differentiation are expressed by differentiating P19 EC cells in vitro. The present study demonstrates that at least two discrete signals are required. One is expressed by isolated primitive streak mesoderm-like cell lines and has the capacity to induce the expression of Pax-3 but, alone, induces neural differentiation inefficiently. The second signal is not expressed by the primitive streak mesoderm-like cell line but is present in conditioned media from differentiating P19 EC cells following DMSO treatment. This signal does not induce either Pax-3 expression or morphological differentiation and does not commit stem cells to a neuroectodermal fate. Rather, it acts synergistically with the signal derived from the primitive streak mesoderm-like cells to increase the efficiency with which stem cells respond initially by Pax-3 expression and subsequently by differentiation towards neural lineages. The activity of this second signal can be replaced by forskolin and 3-isobutyl-1-methyl-xanthine suggesting that its effects are transduced by a cyclic nucleotide-dependent pathway.
Pollen-pistil interactions and developmental regulation of pollen tube growth in Arabidopsis
Authors: Kandasamy Muthugapatti K. , Nasrallah June B. , Nasrallah Mikhail E.
Published: April 2021

A developmental analysis of pollination responses in Arabidopsis implicates pollen as well as stigma maturation factors in the acquisition of reproductive function. In the anther, competence of pollen to germinate and to produce pollen tubes in situ occurred late in development. In the pistil, competence to support pollen germination and tube growth extended over a broad developmental window, and abundant as well as efficient pollen tube development was observed on pistils at anthesis and for a period of 1-2 days prior to flower opening. In contrast, pollen tube growth on immature pistils was found to proceed at low efficiency, at reduced growth rates, and with lack of directionality. Based on the pattern of pollen tube growth at different stages of pistil maturation, temporally regulated signals emanating from specialized cells of the pistil are inferred to be operative in each of the four identified phases of pollen tube growth. In the stigma and the stylar transmitting tissue, these signals directed the path of intra-specific pollen tubes as well as pollen tubes from another cruciferous genera, Brassica. By contrast, in the ovary, signaling by the ovule was effective only on intra-specific pollen tubes and was thus identified as the basis of inter-specific incompatibility. Furthermore, the acquisition of reproductive function was found to involve, in addition to the induction of a variety of stimulatory signals, a heretofore unrecognized developmental restriction in the capacity of epidermal surfaces of the flower to support pollen tube growth.
dachshund encodes a nuclear protein required for normal eye and leg development in Drosophila
Authors: Mardon Graeme , Solomon Noah M. , Rubin Gerald M.
Published: April 2021

Neural specification and differentiation in the Drosophila eye sweep across the unpatterned epithelial monolayer of the eye imaginal disc following a developmental wave termed the morphogenetic furrow. The furrow begins at the posterior margin of the eye imaginal disc and moves anteriorly as a linear front. Progression of the furrow requires the function of hedgehog, which encodes a secreted signaling protein. We characterize mutations in dachshund, a gene that encodes a novel nuclear protein required for normal cell-fate determination of imaginal disc cells. In the absence of dachshund function, cells at the posterior margin of the eye disc fail to follow a retinal differentiation pathway and appear to adopt a cuticle fate instead. These cells are therefore unable to respond to pattern propagation signals such as hedgehog and furrow initiation does not occur. In contrast, cells in more anterior portions of the eye disc are able to differentiate as retinal cells in the absence of dachshund activity and respond normally to patterning signals. These results suggest that posterior margin cells are distinct from other cells of the eye imaginal disc by early stages of development. dachshund is also necessary for proper differentiation of a subset of segments in the developing leg. Null mutations in dachshund result in flies with no eyes and shortened legs.
The Notch signalling pathway is required for Enhancer of split bHLH protein expression during neurogenesis in the Drosophila embryo
Authors: Jennings Barbara , Preiss Anette , Delidakis Christos , Bray Sarah
Published: April 2021

The Enhancer of split locus is required during many cell-fate decisions in Drosophila, including the segregation of neural precursors in the embryo. We have generated monoclonal antibodies that recognise some of the basic helix-loop-helix proteins encoded by the Enhancer of split locus and have used them to examine expression of Enhancer of split proteins during neurogenesis. The proteins are expressed in a dynamic pattern in the ventral neurogenic region and are confined to those ectodermal cells that surround a neuroblast in the process of delaminating. There is no staining in the neuroblasts themselves. We have also examined the relationship between Enhancer of split protein accumulation and the Notch signalling pathway. Protein expression is abolished in a number of neurogenic mutant backgrounds, including Notch, but is increased as a result of expressing a constitutively active Notch product. We conclude that Notch signalling activity is directly responsible for the accumulation of basic helix-loop-helix proteins encoded by the Enhancer of split locus.