bluuebunny/crossref_metadata_2025 huggingface dataset:
Primitive streak mesoderm-like cell lines expressing Pax-3 and Hox gene autoinducing activities
Authors: Pruitt Steven C.
Published: April 2021

Differentiating P19 embryonal carcinoma (EC) cells transiently express an endogenous activity capable of inducing Pax-3 expression in adjacent P19 stem cells (Pruitt, Development 116, 573-583, 1992). In the present study, expression of this activity in mesodermal cell lineages is demonstrated. First, expression of the mesodermal marker Brachyury correlates with expression of Pax-3-inducing activity. Second, the ability of leukemia inhibitory factor (LIF) to block mesoderm differentiation at two different points is demonstrated and correlated with the inhibition of Pax-3-inducing activity. Finally, two mesodermal cell lines that express Pax-3-inducing activity were derived from P19 EC cells. Each of these lines expresses high levels of the mesodermal marker Brachyury and high levels of Oct-3/4 (which is down-regulated at early times during mesoderm differentiation) suggesting that these lines are early mesodermal derivatives.

Unlike EC or embryonic stem cell lines, each of the two mesodermal derivatives autoinduces Hox gene expression on aggregation even in the presence of LIF. Following aggregation, anterior-specific genes are expressed more rapidly than more posterior genes. These observations directly demonstrate the ability of murine mesodermal derivatives to autoinduce Hox gene expression in the absence of signals from other cell lineages. Similar to the Pax-3-inducing activity, signals from mesodermal cell lines were sufficient to induce HOX expression in adjacent P19 stem cells in cell mixing assays. These observations are consistent with the previous suggestion (Blum, M., Gaunt, S. J., Cho, K. W. Y., Steinbeisser, H., Blumberg, B., Bittner, D. and De Robertis, E. M. (1992)Cell 69, 1097-1106) that signals responsible for anterior-posterior organizer activity are localized to the anterior primitive streak mesoderm of the mouse embryo.
Dye-coupling in the root epidermis of Arabidopsis is progressively reduced during development
Authors: Duckett Catherine M. , Oparka Karl J. , Prior Denton A. M. , Dolan Liam , Roberts Keith
Published: April 2021

Epidermal cells of the Arabidopsis thaliana root are derived from a ring of 16 or so initials (stem cells) that are located just below the quiescent centre of four central cells. Derivatives of these initials divide transversely in the meristem-atic region of the root to produce files of cells that subsequently elongate and differentiate. There are two kinds of cell file in the epidermis, hair cells and non-hair cells, that are organised in an ordered pattern and become distinguishable within a few divisions of the initials. It is likely that local intercellular communication plays an important role in the early establishment of this pattern, enabling cells to register their position and fate. We have therefore examined the extent to which cells in the root apex are sym-plastically coupled through plasmodesmata. Using a variety of dye loading methods we show that the undifferentiated cells are dye-coupled i.e. cells in the meristem and the elongation zone are symplastically connected, but that cells gradually become symplastically isolated as the cells differentiate. By the time that visible hair outgrowth is observed the cells of the epidermis are symplastically isolated. This resembles the pattern of gap-junction connectivity observed in many animal systems where cells are coupled at the stage that cell patterns become established, but become progressively more isolated as they differentiate.
Relationship between retinoic acid and sonic hedgehog, two polarizing signals in the chick wing bud
Authors: Helms Jill , Thaller Christina , Eichele Gregor
Published: April 2021

Local application of all-trans-retinoic acid (RA) to the anterior margin of chick limb buds results in pattern duplications reminescent of those that develop after grafting cells from the zone of polarizing activity (ZPA). RA may act directly by conferring positional information to limb bud cells, or it may act indirectly by creating a polarizing region in the tissue distal to the RA source. Here we demonstrate that tissue distal to an RA-releasing bead acquires polarizing activity in a dose-dependent manner. Treatments with pharmacological (beads soaked in 330 μg/ml) and physiological (beads soaked in 10 μg/ml) doses of RA are equally capable of inducing digit pattern duplication. Additionally, both treatments induce sonic hedgehog (shh; also known as vertebrate hedgehog-1, vhh-1), a putative ZPA morphogen and Hoxd-11, a gene induced by the polarizing signal. However, tissue transplantation assays reveal that pharmacological, but not physiological, doses create a polarizing region. This differential response could be explained if physiological doses induced less shh than pharmacological doses. However, our in situ hybridization analyses demonstrate that both treatments result in similar amounts of mRNA encoding this candidate ZPA morphogen. We outline a model describing the apparently disparate effects of pharmcologic and physiological doses RA on limb bud tissue.
Products, genetic linkage and limb patterning activity of a murine hedgehog gene
Authors: Chang David T. , López Alric , Kessler Doris P. von , Chiang Chin , Simandl B. Kay , Zhao Renbin , Seldin Michael F. , Fallon John F. , Beachy Philip A.
Published: April 2021

The hedgehog (hh) segmentation gene of Drosophila melanogaster encodes a secreted signaling protein that functions in the patterning of larval and adult structures. Using low stringency hybridization and degenerate PCR primers, we have isolated complete or partial hh-like sequences from a range of invertebrate species including other insects, leech and sea urchin. We have also isolated three mouse and two human DNA fragments encoding distinct hh-like sequences. Our studies have focused upon Hhg-1, a mouse gene encoding a protein with 46% amino acid identity to hh. The Hhg-1 gene, which corresponds to the previously described vhh-1 or sonic class, is expressed in the notochord, ventral neural tube, lung bud, hindgut and posterior margin of the limb bud in developing mouse embryos. By segregation analysis the Hhg-1 gene has been localized to a region in proximal chromosome 5, where two mutations affecting mouse limb development previously have been mapped. In Drosophila embryos, ubiquitous expression of the Hhg-1 gene yields effects upon gene expression and cuticle pattern similar to those observed for the Drosophila hh gene. We also find that cultured quail cells transfected with a Hhg-1 expression construct can induce digit duplications when grafted to anterior or middistal but not posterior borders within the developing chick limb; more proximal limb element duplications are induced exclusively by mid-distal grafts. Both in transgenic Drosophila embryos and in transfected quail cells, the Hhg-1 protein product is cleaved to yield two stable fragments from a single larger precursor. The significance of Hhg-1 genetic linkage, patterning activity and proteolytic processing in Drosophila and chick embryos is discussed.
Signal transduction during shrimp oocyte activation by extracellular Mg2+: roles of inositol 1,4,5-trisphosphate, tyrosine kinases and G-proteins
Authors: Lindsay LeAnn L. , Clark Wallis H.
Published: April 2021

Oocytes of the shrimp Sicyoniaingentis are naturally activated upon contact with seawater Mg2+. We investigated the mechanism of Mg2+-induced intracellular Ca2+ release and cortical contraction through treatment of oocytes with various activators and inhibitors of signal transduction pathways. Injection of oocytes with the second messenger inositol 1,4,5-trisphosphate resulted in an immediate rise in intracellular Ca2+ and normal cortical contraction. By contrast, injection of the GTP analog guanosine 5′-O-(3′-thiotriphosphate) to activate G-proteins did not affect intracellular Ca2+ levels but did induce cortical contraction. Tyrosine kinase inhibitors (tyrphostin and staurosporine) suppressed the Mg2+-induced Ca2+ rise and contraction, and the inhibition could be overcome by injection with inositol 1,4,5-trisphosphate. Immunoprecipitation of phosphotyrosine-containing proteins from oocyte lysates showed that a 110×103Mr protein was phosphorylated within seconds of oocyte exposure to Mg2+, and tyrphostin inhibited the phosphorylation of this protein. Pre-treatment of oocytes with the protease trypsin abolished their ability to release Ca2+ in response to extracellular Mg2+, indicating a role for a cell surface protein during normal oocyte activation; treated oocytes could be rescued by inositol 1,4,5-trisphosphate injection. These results suggest that S. ingentis oocytes are activated through a Mg2+ ‘receptor’ which activates a tyrosine kinase and results in the production of inositol 1,4,5-trisphosphate to release intracellular Ca2+ stores and induce cortical con-traction. A G-protein/GTPase may also be involved in the pathway leading to cortical contraction.
FGF2 regulates proliferation of neural crest cells, with subsequent neuronal differentiation regulated by LIF or related factors
Authors: Murphy Mark , Reid Kate , Ford Miriam , Furness John B. , Bartlett Perry F.
Published: April 2021

Two of the key early events in the development of the peripheral nervous system are the proliferation of neural crest precursor cells and their subsequent differentiation into different neural cell types. We present evidence that members of the fibroblast growth factor family, (FGF1 or FGF2) act directly on the neural crest cells in vitro to stimulate proliferation in the presence of serum. These findings correlate with in situ hybridisation analysis, which shows FGF2 mRNA is expressed in cells both in the neural tube and within newly formed sensory ganglia (dorsal root ganglia, DRG) at embryonic day 10 in the mouse, when neural crest precursors are proliferating within the DRG. This data infers an autocrine/paracrine loop for FGF regulation of proliferation. Evidence supporting this notion is provided by the finding that part of the endogenous proliferative activity in the NC cultures is related to FGF. It was also found, in early neural crest cultures, that exogenous FGF completely inhibited neuronal differentiation, probably as a direct consequence of its mitogenic activity. In order to stimulate neuronal differentiation significantly, it was necessary to remove the FGF and replace it with leukemia inhibitory factor (LIF) or related factors. Under these conditions, 50% of the cells differentiated into neurons, which developed a sensory neuron morphology and were immunoreactive for the sensory markers CGRP and substance P. These data support a model of neural crest development, whereby multipotential neural crest precursor cells are stimulated to divide by FGF and subsequent development into sensory neurons is regulated by LIF or other cytokines with a similar signalling mechanism.
Regulation of scute function by extramacrochaete in vitro and in vivo
Authors: Cabrera Carlos V. , Alonso María C. , Huikeshoven Hella
Published: April 2021

The pattern of adult sensilla in Drosophila is established by the dosage-sensitive interaction of two antagonistic groups of genes. Sensilla development is promoted by members of the achaete-scute complex and the daughterless gene whereas it is suppressed by whereas extramacrochaete (emc) and hairy. All these genes encode helix-loop-helix proteins. The products of the achaete-scute complex and daughterless interact to form heterodimers able to activate transcription. In this report, we show that (1) extra-macrochaete forms heterodimers with the achaete, scute, lethal of scute and daughterless products; (2) extra-macrochaete inhibits DNA-binding of Achaete, Scute and Lethal of Scute/Daughterless heterodimers and Daughter-less homodimers and (3) extramacrochaete inhibits tran-scription activation by heterodimers in a yeast assay system. In addition, we have studied the expression patterns of scute in wild-type and extramacrochaete mutant imaginal discs. Expression of scute RNA during imaginal development occurs in groups of cells, but high levels of protein accumulate in the nuclei of only a subset of the RNA-expressing cells. The pattern is dynamic and results in a small number of protein-containing cells that correspond to sensillum precursors. extramacrochaete loss-of-function alleles develop extra sensilla and correspondingly display a larger number of cells with scute protein. These cells appear to arise from those that in the wild type already express scute RNA; hence, extramacrochaete is a repressor of scute function whose action may take place post-tran-scriptionally.
Regulation of a decapentaplegic midgut enhancer by homeotic proteins
Authors: Manak J. Robert , Mathies Laura D. , Scott Matthew P.
Published: April 2021

The clustered homeotic genes encode transcription factors that regulate pattern formation in all animals, conferring cell fates by coordinating the activities of downstream ‘target’ genes. In the Drosophila midgut, the Ultrabithorax (Ubx) protein activates and the abdominalA (abd-A) protein represses transcription of the decapentaplegic (dpp) gene, which encodes a secreted signalling protein of the TGFβ class. We have identified an 813 bp dpp enhancer which is capable of driving expression of a lacZ gene in a correct pattern in the embryonic midgut. The enhancer is activated ectopically in the visceral mesoderm by ubiquitous expression of Ubx or Antennapedia but not by Sex combs reduced protein. Ectopic expression of abd-A represses the enhancer. Deletion analysis reveals regions required for repression and activation. A 419 bp subfragment of the 813 bp fragment also drives reporter gene expression in an appropriate pattern, albeit more weakly. Evolutionary sequence conservation suggests other factors work with homeotic proteins to regulate dpp. A candidate cofactor, the extradenticle protein, binds to the dpp enhancer in close proximity to homeotic protein binding sites. Mutation of either this site or another conserved motif compromises enhancer function. A 45 bp fragment of DNA from within the enhancer correctly responds to both UBX and ABD-A in a largely tissue-specific manner, thus representing the smallest in vivo homeotic response element (HOMRE) identified to date.
Isoform-specific induction of a retinoid-responsive antigen after biolistic transfection of chimaeric retinoic acid/thyroid hormone receptors into a regenerating limb
Authors: Pecorino Lauren T. , Lo Donald C. , Brockes Jeremy P.
Published: April 2021

Retinoic acid (RA) induces secretory differentiation in the wound epidermis of a regenerating amphibian limb. We investigated the role of individual RA receptor (RAR) types in the newt wound epidermis by introducing chimaeric RA/thyroid hormone (T3) receptors (χ α1 and χ δ1) that can be activated by T3. A biolistic particle delivery system was employed to transfect cells in the wound epidermis of a regenerating limb and approximately 10% of the cells in targeted surface areas expressed marker genes. Both χ α1 and χ δ1 were comparable in their ability to stimulate transcription of a synthetic reporter construct through a RA response element after activation with T3 in situ. This activation was also comparable to that obtained by the endogenous complement of RARs in the RA-treated, transfected wound epidermis. The RA-inducible WE3 antigen, a marker for secretory differentiation, which distinguishes the wound epidermis from normal skin (Tassava, R. A., Johnson-Wint, B. and Gross, J. 1986, J. Exp. Zool. 239, 229-240), was used to assess the functional role of χ α1 and χ δ1. Chimaeric receptors were transfected with an alkaline phosphatase marker gene, activated with T3, and the expression of both the marker and WE3 was analyzed by double-label immunofluorescence. Newt limbs transfected with χδ1 showed many double-labelled cells dependent on the presence of T3, whereas contralateral limbs transfected with an alkaline phosphatase marker lacking chimaeric receptor sequences did not. Limbs transfected with χ α1 did not show double-labelled cells in the presence or absence of T3, whereas in an earlier study χ α1, and not χ δ1, inhibited growth. These results indicate that specific effects of RA can be mediated by particular types of RARs and demonstrate a novel approach for studying the action of RA on its target tissues.
Overlapping patterns of IGF2 and H19 expression during human development: biallelic IGF2 expression correlates with a lack of H19 expression
Authors: Ohlsson Rolf , Hedborg Fredrik , Holmgren Lars , Walsh Colum , Ekström Tomas J.
Published: April 2021

The spatial patterns of IGF2 and H19 gene expression are strikingly similar during parts of human embryonic/fetal and early postnatal development. Notable exceptions were found with the ciliary anlage of the embryonic retina and the choroid plexus/leptomeninges, where transcripts from the IGF2 but not the H19 locus could be detected. Moreover, in contrast to the other tissue samples examined, the choroid plexus/leptomeninges expressed both parental IGF2 alleles. Whilst RNase protection analysis revealed a weak activity of the P1 promoter in the choroid plexus/leptomeninges, the P2, P3 and P4 promoters were all active wherever IGF2 was expressed. We discuss these observations with respect to a hypothesized coordinated control of the reciprocally imprinted and closely linked IGF2 and H19 loci.
Expression of maize KNOTTED1 related homeobox genes in the shoot apical meristem predicts patterns of morphogenesis in the vegetative shoot
Authors: Jackson David , Veit Bruce , Hake Sarah
Published: April 2021

In this paper we describe the expression patterns of a family of homeobox genes in maize and their relationship to organogenic domains in the vegetative shoot apical meristem. These genes are related by sequence to KNOTTED1, a gene characterized by dominant neomorphic mutations which perturb specific aspects of maize leaf development. Four members of this gene family are expressed in shoot meristems and the developing stem, but not in determinate lateral organs such as leaves or floral organs. The genes show distinct expression patterns in the vegetative shoot apical meristem that together predict the site of leaf initiation and the basal limit of the vegetative ‘phytomer’ or segmentation unit of the shoot. These genes are also expressed in the inflorescence and floral meristems, where their patterns of expression are more similar, and they are not expressed in root apical meristems. These findings are discussed in relation to other studies of shoot apical meristem organization as well as possible commonality of homeobox gene function in the animal and plant kingdoms.
Induction of chondrogenesis: requirement for synergistic interaction of basic fibroblast growth factor and transforming growth factor-beta
Authors: Frenz Dorothy A. , Liu Wei , Williams James D. , Hatcher Victor , Galinovic-Schwartz Vera , Flanders Kathleen C. , Water Thomas R. Van De
Published: April 2021

Interactions between the epithelial anlage of the developing mouse inner ear and its associated periotic mesenchyme control the differentiation of the cartilaginous otic capsule. Transforming growth factor-beta1 (TGF-β1) is a naturally occurring signal peptide that is present in these tissues at times of active differentiation and morphogenesis. Previous studies have shown that TGF-β1 alone is not a sufficient stimulus to initiate chondrogenesis in cultured periotic mesenchyme. In this study, we provide evidence that basic fibroblast growth factor (bFGF) can elicit a specific but limited chondrogenic response in cultured periotic mesenchymal cells. We also demonstrate that simultaneous addition of bFGF and TGF-β1 to cultured periotic mesenchyme results in a full chondrogenic response comparable to that which occurs when periotic mesenchyme is grown in the presence of its natural inductor tissue (i.e. otic epithelium). Utilizing antibodies directed against bFGF, we show localization of endogenous bFGF in the otic epithelium in vivo and in mixed epithelial-mesenchymal cultures. Additionally, we demonstrate the presence of FGF-like activity in medium conditioned by otic epithelium. Blocking of epithelial elicited chondrogenesis by a combination of both αbFGF and αTGF-β1 antibodies provides further evidence of the necessity for these growth factors in the chondrogenic differentiation of periotic mesenchyme in vitro. Our results suggest a role for both bFGF and TGF-β1 in the regulation of chondrogenesis during otic capsule formation in situ.
Microtubule and chromatin behavior follow MAP kinase activity but not MPF activity during meiosis in mouse oocytes
Authors: Verlhac Marie-Hélène , Kubiak Jacek Z. , Clarke Hugh J. , Maro Bernard
Published: April 2021

Oocyte meiotic maturation is triggered by different stimuli (hormones, unknown signals through cell interactions) in different species. These stimuli indirectly lead to the activation of a major cell cycle regulating activity, the maturation promoting factor (MPF). Other factors, such as the product of the proto-oncogene c-mos or enzymes of the MAP kinase family, are also involved in the process of maturation. MAP kinase activation occurs during meiotic maturation in oocytes from different species with different kinetics. The relationships between MPF activation and MAP kinase activation have been well studied in species such as clam and Xenopus. In this paper, we study the precise timing of MAP kinase activation (as measured by phosphorylation of exogenous myelin basic protein and shifts in mobility of ERK 1 and ERK 2) versus MPF activation (as measured by phosphorylation of exogenous histone H1) during mouse oocyte maturation and, in parallel, morphological events such as changes in microtubule organization and chromatin condensation. We observed that MAP kinase activation was delayed after MPF activation and that this activity persisted throughout maturation whereas MPF activity dropped between the two meiotic metaphases. After parthenogenetic activation of ovulated eggs, MAP kinase inactivation was very slow compared to MPF inactivation. During the first mitotic cell cycle, a rise in myelin basic protein kinase activity at M-phase was observed but it was not related to MAP kinase activation. Furthermore, microtubules and chromatin remained in a metaphase-like state during the complete period of maturation (including the period between the two meiotic metaphases) and a few hours after activation. Thus, during meiosis but not during mitosis, the changes in microtubule organization and chromatin condensation correlate with MAP kinase activity rather than with MPF activity. We discuss the possible role of MAP kinase in the maintenance of a metaphasic state during meiosis when MPF is inactive.
A maternal factor affecting mouse blastocyst formation
Authors: Renard J.-P. , Baldacci P. , Richoux-Duranthon V. , Pournin S. , Babinet C.
Published: April 2021

Normal development of the mouse embryo requires the presence of both paternal and maternal genomes. This is due to functional differences having their origin in a differential imprinting of parental genomes. Furthermore, several lines of evidence show that the very early interactions between egg cytoplasm and pronuclei may influence the programming of the embryonic genome and modulate the functional inequality of the parental contribution even during preimplantation stages. In this paper, we show that a factor present in ovulated oocytes of the mouse mutant strain DDK and therefore of maternal origin prevents the formation of the blastocyst. This factor, which acts via an interaction with the paternal genome, is present in oocytes as an RNA and is still active in preimplantation embryos. This is the first direct evidence of such a maternal control in the mouse.
Neuroectodermal transcription of the Drosophila neurogenic genes E(spl) and HLH-m5 is regulated by proneural genes
Authors: Kramatschek Bernd , Campos-Ortega José A.
Published: April 2021

The Enhancer of split gene complex (E(SPL)-C) of Drosophila comprises seven genes encoding bHLH proteins, which are required by neuroectodermal cells for epidermal development. Using promoter and gene fusions with the lacZ gene, we determined the location of cis-acting sequences necessary for normal expression of two of the genes of the E(SPL)-C, E(spl) and HLH-m5. About 0.46 kb of E(spl) and 1.9 kb of HLH-m5 upstream sequences are necessary to reproduce the normal transcription pattern of these genes. The gene products of achaete, scute and lethal of scute, together with that of ventral nervous system condensation defective, act synergistically to specify the neuroectodermal E(spl) and HLH-m5 expression domains. Negative cross- and autoregulatory interactions of the E(SPL)-C on E(spl) contribute, directly or indirectly, to this regulation. Interactions involve DNA binding, since mutagenesis of binding sites for bHLH proteins in the E(spl) promoter abolishes neuroectodermal expression and activates ectopic expression in neuroblasts. A model for activation and repression of E(spl) in the neuroectoderm and neuroblasts, respectively, is proposed.
Cell intercalation during Drosophila germband extension and its regulation by pair-rule segmentation genes
Authors: Irvine Kenneth D. , Wieschaus Eric
Published: April 2021

After the onset of gastrulation, the Drosophila germband undergoes a morphological change in which its length along the anterior-posterior axis increases over two-and-a-half fold while its width along the dorsal-ventral axis simultaneously narrows. The behavior of individual cells during germband extension was investigated by epi-illumination and time-lapse video microscopy of living embryos. Cells intercalate between their dorsal and ventral neighbors during extension, increasing the number of cells along the anterior-posterior axis while decreasing the number of cells along the dorsal-ventral axis. Mutations that reduce segmental subdivision of the embryo along the anteriorposterior axis decrease both germband extension and its associated cell intercalation. In contrast, cell intercalation and germband extension are still detected in embryos that lack dorsal-ventral polarity. Characterization of germband extension and cell intercalation in mutant embryos with altered segmentation gene expression indicates that these processes are regionally autonomous and are dependent upon the establishment of striped expression patterns for certain pair-rule genes. Based on these observations, we propose a model for germband extension in which cell intercalation results from the establishment of adhesive differences between stripes of cells by pair-rule genes.
The Drosophila 18 wheeler is required for morphogenesis and has striking similarities to Toll
Authors: Eldon Elizabeth , Kooyer Sandra , D’Evelyn Diana , Duman Molly , Lawinger Patrick , Botas Juan , Bellen Hugo
Published: April 2021

We have isolated and characterized a novel gene, named 18 wheeler (18w) for its unique segmental expression pattern in Drosophila embryos and expression in cells that migrate extensively. 18 wheeler transcripts accumulate in embryos in a pattern reminiscent of segment polarity genes. Mutations in 18w cause death during larval development and early adulthood. Escaping mutant adults often display leg, antenna, and wing deformities, presumably resulting from improper eversion of imaginal discs. Sequence analysis indicates that 18w encodes a transmembrane protein with an extracellular moiety containing many leucine rich repeats and cysteine motifs, and an intracellular domain bearing homology to the cytoplasmic portion of the interleukin-1-receptor. Expression of 18W protein in non-adhesive Schneider 2 cells promotes rapid and robust aggregation of cells. Analysis of the expression of 18w in different mutant backgrounds shows that it is under control of segment polarity and homeotic genes. The data suggest that the 18W protein participates in the develop-mental program specified by segmentation and homeotic genes as a cell ahesion or receptor molecule that facilitates cell movements.
Selective disruption of E-cadherin function in early Xenopus embryos by a dominant negative mutant
Authors: Levine Elena , Lee Chung Hyun , Kintner Chris , Gumbiner Barry M.
Published: April 2021

E-cadherin function was disrupted in vivo in developing Xenopus laevis embryos through the expression of a mutant E-cadherin protein lacking its cytoplasmic tail. This truncated form of E-cadherin was designed to act as a dominant negative mutant by competing with the extracellular interactions of wild-type endogenous E-cadherin. Expression of truncated E-cadherin in the early embryo causes lesions to develop in the ectoderm during gastrulation. In contrast, expression of a similarly truncated N-cadherin protein failed to cause the lesions. The ectodermal defect caused by the truncated E-cadherin is rescued by overexpression of wild-type E-cadherin, by co-injection of full-length E-cadherin RNA along with the RNA for the truncated form. Overexpression of full-length C-cadherin, however, is unable to compensate for the disruption of E-cadherin function and can actually cause similar ectodermal lesions when injected alone, suggesting that there is a specific requirement for E-cadherin. Therefore, E-cadherin seems to be specifically required for maintaining the integrity of the ectoderm during epiboly in the gastrulating Xenopus embryo. Differential cadherin expression reflects, therefore, the requirement for distinct adhesive properties during different morphogenetic cell behaviors.
Hoxa-2 expression in normal and transposed rhombomeres: independent regulation in the neural tube and neural crest
Authors: Prince Victoria , Lumsden Andrew
Published: April 2021

In this study we have cloned the chick Hoxa-2 gene and analysed its expression during early development. We find that Hoxa-2 has a rostral limit of expression in the rhombencephalic neural tube corresponding precisely to the boundary between rhombomeres (r)1 and 2; a limit further rostral than any other Hox gene reported to date. Neural crest migrates from r2 to populate the first branchial arch, yet although Hoxa-2 is expressed down the full dorsoventral extent of r2 during the phase of neural crest emigration, there is no Hoxa-2 expression in either the emergent neural crest or in the first branchial arch. Conversely, at the level of r4, both the neural tube and the neural crest cells, which migrate out of this rhombomere to populate the second branchial arch, express Hoxa-2.

Other Hox genes expressed in the rhombencephalic neural tube demonstrate a transfer of expression from neural tube to neural crest at all axial levels of expression. Hoxa-2 is thus unusual in demonstrating separate anterior expression limits in neural tube and neural crest; this allowed us to test whether Hox gene expression patterns in neural crest are determined by migratory pathways or are prespecified by the site of origin in the neuroepithelium. Grafting experiments in which pairs of rhombomeres were transplanted to ectopic sites at the time of rhombomere boundary formation reveal a prepatterning of the neural crest with respect to Hoxa-2 expression. The decision to down-regulate Hoxa-2 expression in r2-derived neural crest, but to maintain Hoxa-2 expression in r4-derived neural crest is intrinsic to the premigratory crest cell population. Thus, following grafting of r4 to the r2 site and vice-versa, Hoxa-2 expression is maintained in r4-derived neural crest, but lost in r2-derived neural crest.
Differential response of embryonic and fetal myoblasts to TGFβ: a possible regulatory mechanism of skeletal muscle histogenesis
Authors: Angelis M. G. Cusella-De , Molinari S. , Donne A. Le , Coletta M. , Vivarelli E. , Bouche M. , Molinaro M. , Ferrari S. , Cossu G.
Published: April 2021

Embryonic and fetal skeletal myoblasts were grown in culture in the presence of TGFβ. Under the conditions employed, TGFβ inhibited differentiation of fetal but not of embryonic myoblasts. To investigate the possible relevance of these data to skeletal muscle histogenesis in vivo, we studied the proliferation/differentiation state of mesodermal cells in the proximal region of the limb bud at the time of primary fiber formation. BrdU labeling and immunostaining for myosin heavy chains revealed that very few mesodermal cells enter the S phase of the cycle when differentiated primary fibers fist appear. However, a few hours later, many cells in S phase surround newly formed muscle fibers, suggesting that the latter may be a source of mitogens for undifferentiated myoblasts. Co-culture experiments supported this hypothesis, showing that medium conditioned by fiber-containing explants can stimulate myoblast proliferation.

Taken together these data suggested a possible mechanism for the regulation of muscle fiber formation. The model assumes that fibers form in the proximal region of the limb bud, where TGFβ is known to be present, and BrdU labeling experiments did not reveal cells in S phase.

It is conceivable that non-dividing embryonic myoblasts (which do not respond to TGFβ) can undergo differentiation, while fetal myoblasts are inhibited by TGFβ. Once formed, primary fibers may stimulate a new wave of proliferation in fetal myoblasts, in order to expand the pool of cells needed to form secondary fibers.

To test this model we developed an organ culture for limb buds which resulted in the production of myotubes with a phenotype similar to embryonic (primary) and fetal (secondary) fibers, roughly at the time when primary and secondary fibers form in vivo. When these cultures were treated with TGFβ embryonic myotubes did form (as expected), but fetal myotubes never appeared. Conversely, when these cultures were treated with anti-TGFβ neutralizing antibodies, fetal myotubes developed earlier than in control cultures, suggesting that endogenously produced TGFβ may repress differentiation of fetal cells in vitro and, possibly, in vivo.
Subcellular localization of the segment polarity protein patched suggests an interaction with the wingless reception complex in Drosophila embryos
Authors: Capdevila Javier , Pariente Félix , Sampedro Javier , Alonso José Luis , Guerrero Isabel
Published: April 2021

The product of the segment polarity gene patched is a trans-membrane protein involved in the cell communication processes that establish polarity within the embryonic segments of Drosophila. Monoclonal antibodies have been raised against the patched protein, and by immunoelectron microscopy part of the patched staining is found associated with discrete regions of the lateral plasma membrane of the embryonic epidermal cells. Using a mutation affecting endocytosis (shibire) we find that patched is a membrane-bound protein, which is internalized by endocytosis, and that the preferential sites of accumulation resemble the described localizations of the cell-cell adhesive junctions of the epidermal cells. patched partially co-localizes with the wingless protein in the wingless-expressing and nearby cells, in structures that seem to be endocytic vesicles. These data suggest the interaction of patched protein with elements of the reception complex of wingless, as a way to control the wingless expression.
Violation of cell lineage restriction compartments in the chick hindbrain
Authors: Birgbauer Eric , Fraser Scott E.
Published: April 2021

Previous cell lineage studies indicate that the repeated neuromeres of the chick hindbrain, the rhombomeres, are cell lineage restriction compartments. We have extended these results and tested if the restrictions are absolute. Two different cell marking techniques were used to label cells shortly after rhombomeres form (stage 9+ to 13) so that the resultant clones could be followed up to stage 25. Either small groups of cells were labelled with the lipophilic dye DiI or single cells were injected intracellularly with fluorescent dextran. The majority of the descendants labelled by either technique were restricted to within a single rhombomere. However, in a small but reproducible proportion of the cases (greater than 5%), the clones expanded across a rhombomere boundary. Neither the stage of injection, the stage of analysis, the dorsoventral position, nor the rhombomere identity correlated with the boundary crossing. Judging from the morphology of the cells, both neurons and non-neuronal cells were able to expand over a boundary. These results demonstrate that the rhombomere boundaries represent cell lineage restriction barriers which are not impenetrable in normal development.
Rat olfactory neurons can utilize the endogenous lectin, L-14, in a novel adhesion mechanism
Authors: Mahanthappa Nagesh K. , Cooper Douglas N. W. , Barondes Samuel H. , Schwarting Gerald A.
Published: April 2021

L-14 is a divalent, lactosamine-binding lectin expressed in many vertebrate tissues. In the rat nervous system, L-14 expression has been observed previously in restricted neuronal subsets within the dorsal root ganglia and spinal cord. In this study we report that L-14 is expressed by non-neuronal cells in the rat olfactory nerve. We demonstrate that L-14 binds and co-localizes with two ligands in the rat olfactory system: a β-lactosamine-containing glycolipid, and a putative member of the laminin family. The former is expressed on the surfaces of nascent olfactory axons originating from neuron cell bodies in the olfactory epithelium. The latter is present in the extracellular matrix of the axonal path leading to synaptic targets in the olfactory bulb.

In vitro, we find that recombinant L-14 promotes primary olfactory neuron adhesion to two laminin family members, and promotes intercellular adhesion. Both activities are dose-dependent, and are independent of integrinmediated mechanisms. We have thus found that L-14 can serve two distinct adhesive functions in vitro, and propose that L-14 in vivo can promote olfactory axon fasciculation by crosslinking adjacent axons and promote axonal adhesion to the extracellular matrix.
lola encodes a putative transcription factor required for axon growth and guidance in Drosophila
Authors: Giniger Edward , Tietje Kathleen , Jan Lily Yeh , Jan Yuh Nung
Published: April 2021

Mutations in the gene longitudinals lacking (lola) lead to defects in the development of axon tracts in the Drosophila embryonic central nervous system. We now show that lola mutations also cause defects of axon growth and guidance in the peripheral nervous system, and causes a particular cluster of embryonic sense organs (lch5) to be oriented improperly. Axonal aberrations caused by lola are similar to those caused by mutations of three other genes, logo, Notch and Delta, raising the possibility that lola works in the same genetic pathway as do these other molecules. The lola gene encodes at least two nuclear protein products, apparently by differential RNA splicing. The predicted proteins contain an amino-terminal motif similar to that recently described for a family of transcription factors, including the products of the Drosophila genes tramtrack and the Broad Complex. Like Ttk and BR-C, one of the two characterized products of the lola locus bears sequences similar to the zinc-finger motif, but the other (neuronal) form of the protein has no recognizable DNA-binding motif.
Axons arrest the migration of Schwann cell precursors
Authors: Bhattacharyya Anita , Brackenbury Robert , Ratner Nancy
Published: April 2021

The neural crest gives rise to a variety of cell types including Schwann cells of the peripheral nervous system. Schwann cell precursors begin to differentiate early and migrate along specific pathways in the embryo before associating with nerve trunks. To determine whether motor axons direct the migration of Schwann cell precursors along specific pathways, we tested the effect of ablating the ventral half of the neural tube, which contains motor neuron cell bodies. The ventral neural tube was removed unilaterally from lumbar regions of chicken embryos at stage 17, when neural crest cells are just beginning to migrate and before motor axons have extended out of the neural tube. At several stages after ventral tube ablation, sections of the lumbar region of these embryos were stained with anti-acetylated tubulin to label developing axons, HNK-1 to label migrating neural crest cells and 1E8 to label Schwann cell precursors. In many embryos the ablation of motor neurons was incomplete. The staining patterns in these embryos support the idea that some Schwann cells are derived from the neural tube. In embryos with complete motor neuron ablation, at stage 18, HNK-1-positive neural crest cells had migrated to normal locations in both control and ablated sides of the embryo, suggesting that motor axons or the ventral neural tube are not required for proper migration of neural crest cells. However, by stage 19, cells that were positive for HNK-1 or 1E8 were no longer seen in the region of the ventral root, nor ventral to the ventral root region. Because Schwann cell precursors require neural-derived factors for their survival in vitro, we tested whether neural crest cells that migrate to the region of the ventral root in ventral neural tube-ablated embryos then die. Nile Blue staining for dead and dying cells in ventral neural tube-ablated embryos provided no evidence for cell death at stage 18. These results suggest that motor axons arrest the migration of Schwann cell precursors during neural crest migration.
Developmental control of a G1-S transcriptional program in Drosophila
Authors: Duronio Robert J. , O’Farrell Patrick H.
Published: April 2021

We have defined a coordinate program of transcription of S-phase genes (DNA polymerase α, PCNA and the two ribonucleotide reductase subunits) that can be induced by the G1 cyclin, cyclin E. In Drosophila embryos, this program drives an intricate spatial and temporal pattern of gene expression that perfectly parallels the embryonic program of S-phase control. This dynamic pattern of expression is not disrupted by a mutation, string, that blocks the cell cycle. Thus, the transcriptional program is not a secondary consequence of cell cycle progression. We suggest that developmental signals control this transcriptional program and that its activation either directly or indirectly drives transition from G1 to S phase in the stereo-typed embryonic pattern.
Characterization of a novel Dictyostelium discoideum prespore-specific gene, PspB, reveals conserved regulatory sequences
Authors: Powell-Coffman Jo Anne , Firtel Richard A.
Published: April 2021

While Dictyostelium discoideum has been studied as a developmental system for decades, and many regulatory proteins have been cloned, the molecular mechanisms of cell-type-specific gene expression are poorly understood. In this paper we characterize a novel prespore gene, PspB, and undertake a comparative analysis of the regulatory regions in prespore-specific D. discoideum promoters. Sequence alignment of the PSPB gene product with other prespore-specific proteins identifies a conserved, repeated 12 amino acid cysteine-containing motif that may be involved in spore coat function or assembly. Analysis of the PspB promoter identifies two domains essential for developmentally induced promoter activity. The first region includes two CA-rich elements (CAEs) that we show to be functionally homologous to the cAMP-inducible elements previously identified in the SP60 (cotC) promoter. The PspB CAEs compete with the SP60 (cotC) CAEs for binding in vitro to a developmentally regulated nuclear activity. We identify this activity as G-box Binding Factor, a developmentally induced transcription factor. The PspB CAEs and adjacent nucleotides direct a very low level of prespore-enriched expression, but high levels of cell-type-specific expression requires a second promoter region: a 46-bp AT-rich sequence that does not resemble the CAEs or any other previously described late gene promoter elements. Com-parison of the PspB AT element with regulatory regions of the SP60 (cotC), SP70 (cotB), and D19 (pspA) promoters reveals an extensive consensus sequence. We suggest that these AT-rich sequences may represent a common regulatory element (or elements) required for prespore gene activation.
Role of transforming growth factor-beta isoforms in regulating the expression of nerve growth factor and neurotrophin-3 mRNA levels in embryonic cutaneous cells at different stages of development
Authors: Buchman Vladimir L. , Sporn Michael , Davies Alun M.
Published: April 2021

We have investigated if transforming growth factor-beta (TGF-β) isoforms influence the level of expression of nerve growth factor (NGF) mRNA and neurotrophin-3 (NT-3) mRNA in embryonic tissues innervated by neurons that depend on NGF and NT-3 for survival. Presumptive dermal and epidermal cells from the maxillary territory of the embryonic mouse trigeminal ganglion were cultured in defined medium during the early stages of innervation when trigeminal neurons switch their survival dependence from NT-3 to NGF. In E11 and E12 cultures, when the in vivo levels of NGF mRNA and NT-3 mRNA are increasing, TGF-β1, TGF-β2 and TGF-β3 each increased the level of NGF mRNA but had no effect on NT-3 mRNA. In E13 cultures, when the in vivo levels of NGF mRNA and NT-3 mRNA reach a peak (relative to actin mRNA) prior to a marked fall in the level of NT-3 mRNA and a gradual decrease in the level of NGF mRNA, TGF-βs promoted further increases in the level of NGF mRNA but caused a decrease in the level of NT-3 mRNA. All three TGF-β mRNAs were detected in the maxillary territory in vivo before the arrival of the earliest axons and their levels rose throughout the period in which sensory axons reach this territory. Our findings demonstrate age-related changes in the influence of TGF-βs on the expression of neurotrophins in developing cutaneous cells and raise the possibility that TGF-βs play a role in regulating the changing patterns of neurotrophin gene expression in sensory neuron target fields.
Characterisation of the second messenger pathway underlying neurite outgrowth stimulated by FGF
Authors: Williams Emma J. , Furness Josie , Walsh Frank S. , Doherty Patrick
Published: April 2021

Cerebellar neurons, cultured on monolayers of 3T3 fibroblasts or on a polylysine/laminin-coated substratum, responded to recombinant basic FGF by extending longer neurites. The response was biphasic reaching a maximum at 5 ng/ml FGF, but desensitising at 100-200 ng/ml FGF. The response to FGF could be inhibited by a tyrosine kinase inhibitor (the erbstatin analogue), by a diacylglycerol lipase inhibitor (RHC-80267) and by a combination of N- and L-type calcium channel antagonists or other agents that negate the effects of calcium influx into neurons. The response to FGF could be fully mimicked by arachidonic acid added directly to the cultures, or generated via activation of phospholipase A2 with melittin. The response to melittin, but not to FGF or arachidonic acid, was inhibited by 4-bromophenacyl bromide, a phospholipase A2 inhibitor. The response to arachidonic acid was also biphasic and high concentrations of this agent could cross-desensitise the FGF response and vice versa. The response to arachidonic acid could be fully inhibited by the agents that block or negate the effects of calcium influx into neurons, but was not inhibited by the tyrosine kinase or diacylglycerol lipase inhibitors. These data suggest that FGF stimulates neurite outgrowth by activating a cascade that involves activation of phospholipase Cγ to produce diacylglycerol, conversion of diacylglycerol to arachidonic acid by diacylglycerol lipase and the activation of voltage-gated calcium channels by arachidonic acid.