bluuebunny/crossref_metadata_2025 huggingface dataset:
Specification of anterior-posterior differences within the AB lineage in the C. elegans embryo: a polarising induction
Authors: Hutter Harald , Schnabel Ralf
Published: April 2021

In a C. elegans embryo the third cleavages of descendants of the anterior blastomere AB of the 2-cell stage create pairs of blastomeres that develop differently. By laser ablation experiments we show that the fates of all the posterior daughters of this division depend on an induction occurring three cleavages before these blastomeres are born. The time of induction precludes a direct effect on cell fate. Alternatively, we suggest that the induction creates a heritable cell polarity which is propagated through several divisions. We suggest a model to demonstrate how a signal could be propagated through several rounds of cell division. An important implication of our observations is that this early induction acts to specify blastomere identity, not tissue type. A detailed lineage analysis revealed that altering the inductive signal alters complex lineage patterns as a whole. The induction described here, together with two inductions described previously can be used to illustrate how the anterior portion of the C. elegans embryo can be successively subdivided into blastomeres with unique developmental potential.
Discrete Polycomb-binding sites in each parasegmental domain of the bithorax complex
Authors: Chiang Anne , O’Connor Michael B. , Paro Renato , Simon Jeff , Bender Welcome
Published: April 2021

The Polycomb protein of Drosophila melanogaster maintains the segmental expression limits of the homeotic genes in the bithorax complex. Polycomb-binding sites within the bithorax complex were mapped by immunostaining of salivary gland polytene chromosomes. Polycomb bound to four DNA fragments, one in each of four successive parasegmental regulatory regions. These fragments correspond exactly to the ones that can maintain segmentally limited expression of a lacZ reporter gene. Thus, Polycomb acts directly on discrete multiple sites in bithorax regulatory DNA. Constructs combining fragments from different regulatory regions demonstrate that Polycomb-dependent maintenance elements can act on multiple pattern initiation elements, and that maintenance elements can work together. The cooperative action of maintenance elements may motivate the linear order of the bithorax complex.
Regulation of HoxA expression in developing and regenerating axolotl limbs
Authors: Gardiner David M. , Blumberg Bruce , Komine Yuriko , Bryant Susan V.
Published: April 2021

Homeobox genes are important in the regulation of outgrowth and pattern formation during limb development. It is likely that homeobox genes play an equally important role during limb regeneration. We have isolated and identified 17 different homeobox-containing genes expressed by cells of regenerating axolotl limbs. Of these, nearly half of the clones represent genes belonging to the HoxA complex, which are thought to be involved in pattern formation along the proximal-distal limb axis. In this paper we report on the expression patterns of two 5′ members of this complex, HoxA13 and HoxA9. These genes are expressed in cells of developing limb buds and regenerating blastemas. The pattern of expression in developing axolotl limb buds is comparable to that in mouse and chick limb buds; the expression domain of HoxA13 is more distally restricted than that of HoxA9. As in developing mouse and chick limbs, HoxA13 likely functions in the specification of distal limb structures, and HoxA9 in the specification of more proximal structures. In contrast, during regeneration, HoxA13 and HoxA9 do not follow the rule of spatial colinearity observed in developing limbs. Instead, both genes are initially expressed in the same population of stump cells, giving them a distal Hox code regardless of the level of amputation. In addition, both are reexpressed within 24 hours after amputation, suggesting that reexpression may be synchronous rather than temporally colinear. Treatment with retinoic acid alters this Hox code to that of a more proximal region by the rapid and differential downregulation of HoxA13, at the same time that expression of HoxA9 is unaffected. HoxA reexpression occurs prior to blastema formation, 24-48 hours after amputation, and is an early molecular marker for dedifferentiation.
Expression of zebrafish nk2.2 is influenced by sonic hedgehog/vertebrate hedgehog-1 and demarcates a zone of neuronal differentiation in the embryonic forebrain
Authors: Barth Katrin Anukampa , Wilson Stephen W.
Published: April 2021

We have isolated zebrafish nk2.2, a member of the Nk-2 family of homeobox genes. nk2.2 is expressed in a continuous narrow band of cells along a boundary zone demarcating the location at which two of the earliest nuclei in the brain differentiate. This band of cells is located within a few cell diameters of cells expressing the signalling molecule sonic hedgehog/vertebrate hedgehog-1 (shh/vhh-1). Injection of shh/vhh-1 RNA results in ectopic expression of nk2.2 and concomitant abnormalities in the forebrain and eyes. Moreover, cyclops mutant embryos, which initially lack neurectodermal expression of shh/vhh-1, show a concomitant lack of nk2.2 expression. Together, these results suggest a requirement of shh/vhh-1 protein for the spatial regulation of nk2.2 expression.
Temporal regulation of the Xenopus FGF receptor in development: a translation inhibitory element in the 3′ untranslated region
Authors: Robbie Edward P. , Peterson Michael , Amaya Enrique , Musci Thomas J.
Published: April 2021

Early frog embryogenesis depends on a maternal pool of mRNA to execute critical intercellular signalling events. FGF receptor-1, which is required for normal development, is stored as a stable, untranslated maternal mRNA transcript in the fully grown immature oocyte, but is translationally activated at meiotic maturation. We have identified a short cis-acting element in the FGF receptor 3′ untranslated region that inhibits translation of synthetic mRNA. This inhibitory element is sufficient to inhibit translation of heterologous reporter mRNA in the immature oocyte without changing RNA stability. Deletion of the poly(A) tract or polyadenylation signal sequences does not affect translational inhibition by this element. At meiotic maturation, we observe the reversal of translational repression mediated by the inhibitory element, mimicking that seen with endogenous maternal FGF receptor mRNA at meiosis. In addition, the activation of synthetic transcripts at maturation does not appear to require poly(A) lengthening. We also show that an oocyte cytoplasmic protein specifically binds the 3′ inhibitory element, suggesting that translational repression of Xenopus FGF receptor-1 maternal mRNA in the oocyte is mediated by RNA-protein interactions. These data describe a mechanism of translational control that appears to be independent of poly(A) changes.
nanos is an evolutionarily conserved organizer of anterior-posterior polarity
Authors: Curtis, Daniel , Apfeld Javier , Lehmann Ruth
Published: April 2021

In Drosophila melanogaster, nanos functions as a localized determinant of posterior pattern. Nanos RNA is localized to the posterior pole of the maturing egg cell and encodes a protein that emanates from this localized source. Nanos acts as a translational repressor and thereby establishes a gradient of the morphogen Hunchback. Here we show that the mechanism by which nanos acts in Drosophila is a common developmental strategy in Dipteran insects. We used cytoplasmic transplantation assays to demonstrate that nanos activity is found in posterior poleplasm of five diverse Dipteran species. Genes homologous to nanos were identified from Drosophila virilis, the housefly Musca domestica, and the midge Chironomus samoensis. These genes encode RNAs that are each localized, like nanos, to the embryonic posterior pole. Most importantly, we demonstrate that these homologues can functionally substitute for nanos in D. melanogaster. These results suggest that nanos acts in a similar pathway for axis determination in other insects. Comparison of the Nanos sequences reveals only 19% overall protein sequence similarity; high conservation of a novel zinc finger near the carboxy terminus of the protein defines a region critical for nanos gene function.
Intracellular acidification of gastrula ectoderm is important for posterior axial development in Xenopus
Authors: Gutknecht D. R. , Koster C. H. , Tertoolen L. G. J. , Laat S. W. de , Durston A. J.
Published: April 2021

There is evidence suggesting that pHi elevation can induce differentiation to cement gland, an extremely anterior structure, during the early development of Xenopus laevis (Picard, J. J. (1975) J. Embryol. exp. Morphol. 33, 957-967; Sive, H. L., Hattori, K. and Weintraub, H. (1989) Cell 58, 171-180). We wanted to investigate whether axial development or neural induction are mediated in Xenopus via regulation of pHi. Our interest was stimulated further because certain signal transduction pathways, which are thought to mediate anterior neural induction (Otte, A. P., Van Run, P., Heideveld, M., Van Driel, R. and Durston, A. J. (1989) Cell 58, 641-648; Durston and Otte (1991), Cell-Cell Interactions in Early Development, pp. 109-127), are also known to modify the activity of proton extruders (Mitsuka and Berk (1991)Am. J. Physiol. 260, C562-C569; Wakabayashi, S., Sardet, C., Fafournoux, P., Counillon, L., Meloche, S., Pages, G. and Pouysségur, J. (1993) Rev. Physiol. Biochem. Pharmacol. Vol. 119, pp. 157-186). We therefore measured pHi in explants of gastrula ectoderm and neurectoderm and identified ion exchangers that regulate pHi in these tissues. The measurements showed that pHi decreases in explants of both neurectoderm and uninduced ectoderm during the time course of gastrulation, this pHi decrease thus fails to correlate with neural induction. One important regulator of this cytoplasmic acidification is the Na+/H+ exchanger. The pHi set point, at which the acid extrusion activity of this alkalizing exchanger is shut off, shifts to more acidic values during the time course of gastrulation, thus permitting cytoplasmic acidification. We found also that preventing cytoplasmic acidification and thereby elevating pHi in late gastrula cells led to the specific suppression of posterior development. Neural induction and anterior development were unaffected by treatments leading either to an elevation of or a decrease in pHi. These findings indicate that the cellular processes mediating anterior development and neural induction are pHi tolerant, while the signals mediating posterior development require a sustained pHi decrease for their action, suggesting that downregulation of pHi is nescessary for posterior axial development.
Cis-regulatory control of the SM50 gene, an early marker of skeletogenic lineage specification in the sea urchin embryo
Authors: Makabe Kazuhiro W. , Kirchhamer Carmen V. , Britten Roy J. , Davidson Eric H.
Published: April 2021

The SM50 gene encodes a minor matrix protein of the sea urchin embryo spicule. We carried out a detailed functional analysis of a cis-regulatory region of this gene, extending 440 bp upstream and 120 bp downstream of the transcription start site, that had been shown earlier to confer accurate skeletogenic expression of an injected expression vector. The distal portion of this fragment contains elements controlling amplitude of expression, while the region from −200 to +105 contains spatial control elements that position expression accurately in the skeletogenic lineages of the embryo. A systematic mutagenesis analysis of this region revealed four adjacent regulatory elements, viz two copies of a positively acting sequence (element D) that are positioned just upstream of the transcription start site; an indispensable spatial control element (element C) that is positioned downstream of the start site; and further downstream, a second positively acting sequence (element A). We then constructed a series of synthetic expression constructs. These contained oligonucleotides representing normal and mutated versions of elements D, C, and A, in various combinations. We also changed the promoter of the SM50 gene from a TATA-less to a canonical TATA box form, without any effect on function. Perfect spatial regulation was also produced by a final series of constructs that consisted entirely of heterologous enhancers from the CyIIIa gene, the SV40 early promoter, and synthetic D, C, and A elements. We demonstrate that element C exercises the primary spatial control function of the region we analyzed. We term this a ‘locator’ element. This differs from conventional ‘tissue-specific enhancers’ in that while it is essential for expression, it has no transcriptional activity on its own, and it requires other, separable, positive regulatory elements for activity. In the normal configuration these ancillary positive functions are mediated by elements A and D. Only positively acting control elements were observed in the SM50 regulatory domain throughout this analysis.
Identification of a neurogenic sublineage required for CNS segmentation in an Annelid
Authors: Ramírez Felipe-Andrés , Wedeen Cathy J. , Stuart Duncan K. , Lans Deborah , Weisblat David A.
Published: April 2021

In embryos of leeches (phylum Annelida), metameric structures arise sequentially from a germinal plate comprising the descendants of five pairs of embryonic stem cells called teloblasts. It has been shown that transverse stripes of cells expressing ht-en (a homolog of engrailed, a Drosophila segment polarity gene), arise in the germinal plate prior to the appearance of segmental ganglia and that, in the main neurogenic lineage (derived from the N teloblasts), the stripe of cells expressing ht-en demarcates the boundary between prospective segmental ganglia. Previous lineagetracing experiments had suggested that the clones of nf and ns primary blast cells in the N lineage are confined to within segmental borders. This conclusion was called into question by the observation that the cells expressing ht-en do not appear to be at the very posterior edge of the nf clone, from which they arise. To resolve this issue, we have injected individual primary blast cells with fluorescent lineage tracers; we find that cells in the nf clone actually straddle two adjacent ganglia. Moreover, using photoablation techniques, we find that the nf clone is required for proper morphogenesis of the segmentally iterated central nervous system (CNS).
phantastica: a gene required for dorsoventrality of leaves in Antirrhinum majus
Authors: Waites Richard , Hudson Andrew
Published: April 2021

To understand better the mechanisms that lead to dorsoventrality in the lateral organs of plants, mutants at the phantastica (phan) locus of Antirrhinum majus have been identified and characterised. The leaves, bracts and petal lobes of phan mutants show varying degrees of reduction in dorsal tissues, indicating that phan is required for establishing dorsal cell identity. Each phan mutant produces a variety of different leaf morphologies, but has a characteristic and relatively constant floral phenotype. In several different forms of phan mutant leaves and petal lobes, novel boundaries between dorsal and ventral cell types form ectopic axes of growth, suggesting that phan-dependent dorsal cell identity is required for lateral growth of the wild-type leaf and petal lobe. Comparisons between the development of wild-type and mutant petals or leaves reveal that phan acts early in development of these lateral organs. The possible role of the phan gene in evolution of different leaf forms is discussed.
Xwnt-8b: a maternally expressed Xenopus Wnt gene with a potential role in establishing the dorsoventral axis
Authors: Cui Yanzhen , Brown Jeffrey D. , Moon Randall T. , Christian Jan L.
Published: April 2021

In amphibian embryos, establishment of dorsal-ventral asymmetry is believed to involve dorsal-ventral differences in vegetally derived mesoderm-inducing signals and/or differences in the competence of animal hemisphere (ectodermal) cells to respond to these signals. Previous studies have shown that certain Wnt proteins can generate an ectopic dorsal axis when misexpressed, and that they do so by modifying the response of ectodermal cells to inducers. None of these Wnt proteins are expressed at an appropriate time to do so in vivo. In this study, we describe the isolation and characterization of a full length cDNA for the Xenopus Wnt gene, Xwnt-8b, whose biological activity and expression pattern suggest that it may be involved in establishment of the dorsoventral axis. Both maternal and zygotic Xwnt-8b transcripts undergo alternative splicing to generate mRNAs which encode two different forms of Xwnt-8b protein. During early cleavage stages Xwnt-8b transcripts are confined primarily to animal hemisphere blastomeres, while zygotically derived Xwnt-8b transcripts are restricted almost exclusively to a band of cells in the prospective forebrain of neurula and tailbud stage embryos. Ectopically expressed Xwnt-8b can completely rescue dorsal development of embryos ventralized by exposure to ultraviolet light, and can induce a complete secondary axis in wild-type embryos. Axis induction is observed only if Xwnt-8b is supplied prior to the onset of zygotic gene transcription. This biological activity, together with the presence of maternal Xwnt-8b transcripts in cells that will be induced to form the dorsal mesoderm, is consistent with the possibility that Xwnt-8b may be the endogenous agent that establishes asymmetry in the response of ectodermal cells to mesoderm-inducing signals, thereby initiating dorsal development.
Induction of prostatic morphology and secretion in urothelium by seminal vesicle mesenchyme
Authors: Donjacour Annemarie A. , Cunha Gerald R.
Published: April 2021

Mesenchymal-epithelial interactions are essential for the development of the male reproductive tract. Tissue recombination experiments have been used to define the characteristics of these interactions. When mesenchyme, embryonic connective tissue, is recombined with epithelium from another organ an instructive induction may occur in which the developmental fate of the epithelium is altered. Instructive inductions are most common when the epithelium that is removed from the mesenchyme and the epithelium that is recombined with the mesenchyme are from the same germ layer. All of the mesenchyme of the male reproductive tract is of mesodermal origin. The epithelia of these organs are derived from either the mesodermal Wolffian duct epithelium or the endodermal urogenital sinus epithelium. Urogenital sinus mesenchyme can instructively induce bladder and urethral epithelium to form prostate (Donjacour, A. A. and Cunha, G. R. (1993) Endocrinol. 132, 2342-2350) and seminal vesicle mesenchyme can instructively induce epithelium from the ductus deferens and ureter (Cunha, G. R., Young, P., Higgins, S. J. and Cooke, P. S. (1991) Development 111, 145-158) to form seminal vesicle. To see whether inductive interactions could occur across germ layers in this system, seminal vesicle mesenchyme, normally associated with a mesodermal epithelium, was recombined with epithelium from neonatal or adult bladder or urethra, which are of endodermal origin. The resulting tissue recombinants were analyzed histologically and by immunocytochemistry and western blotting with antibodies to prostatic and seminal vesicle secretory proteins. Full prostatic differentiation was observed in tissue recombinants made with seminal vesicle mesenchyme plus either adult or neonatal bladder or urethral epithelium. These tissue recombinants made dorsolateral but not ventral prostatic secretory proteins. None of them developed into seminal vesicle. To examine whether epithelial androgen receptors were required for this process, seminal vesicle mesenchyme was recombined with urothelium from mice with the testicular feminization mutation. These mice lack functional androgen receptors. While these tissue recombinants grew, no prostatic proteins were detected. Seminal vesicle mesenchyme acted as a potent prostatic inducer, indicating that similar mesenchymal signals can induce prostatic and seminal vesicle development. Urogenital epithelia of both endodermal and mesodermal origin appeared to be able to respond to these inductive signals produced by the seminal vesicle mesenchyme; however, their responses differed depending on their germ layer of origin: mesodermal epithelium yielding seminal vesicle (Cunha, G. R., Young, P., Higgins, S. J. and Cooke, P. S. (1991) Development 111, 145-158) and endodermal epithelium yielding prostate.
Regulation of mouse egg activation: presence of ryanodine receptors and effects of microinjected ryanodine and cyclic ADP ribose on uninseminated and inseminated eggs
Authors: Ayabe Takuya , Kopf Gregory S. , Schultz Richard M.
Published: April 2021

Sperm-induced activation of mammalian eggs is associated with a transient increase in Ca2+ concentrations thought to be derived from inositol 1,4,5-trisphosphate-sensitive and -insensitive intracellular stores. Whereas the importance of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores has been evaluated, the identity and role of inositol 1,4,5-trisphos- phate-insensitive stores are poorly understood. To explore the role of the ryanodine-sensitive Ca2+ store, we first used reverse transcription-polymerase chain reaction to identify transcripts of the ryanodine receptor in eggs and deter- mined that transcripts for the type 2 and 3 receptor were present. Immunoprecipitation of radioiodinated egg extracts with an antibody that recognizes both type 2 and 3 receptors detected specifically a band of Mr=520,000. Immunolocalization of the receptor(s) using laser-scanning confocal microscopy revealed that the receptor(s) was uniformly distributed in the cortex of the germinal vesicle- intact oocyte, but became asymmetrically localized to the cortex in a region apposed to the meiotic spindle in the metaphase II-arrested egg; this asymmetrical localization developed by metaphase I. The role of the ryanodine receptor in mouse egg activation was examined by deter- mining the effects of microinjected ryanodine or cyclic ADP ribose on endpoints of egg activation in either unin- seminated or inseminated eggs. Ryanodine induced the conversion of the zona pellucida glycoprotein ZP2 to its postfertilization form ZP2f in a biphasic concentration- dependent manner; nanomolar concentrations stimulated this conversion, whereas micromolar concentrations had no stimulatory effect. Cyclic ADP ribose also promoted the ZP2 conversion, but with a hyperbolic concentration dependence. Neither of these compounds induced cell cycle resumption. Inhibiting the inositol 1,4,5-trisphosphate- sensitive Ca2+ store did not inhibit the ryanodine-induced ZP2 conversion and, reciprocally, inhibiting the ryanodine- sensitive Ca2+ store did not inhibit the inositol 1,4,5- trisphosphate-induced ZP2 conversion. Last, treatment of eggs under conditions that would block the release of Ca2+ from the ryanodine-sensitive store had no effect on any event of egg activation following fertilization. Results of these experiments suggest that although ryanodine receptors are present and functional, release of Ca2+ from this store is not essential for sperm-induced egg activation.
Distinct expression and shared activities of members of the hedgehog gene family of Xenopus laevis
Authors: Ekker Stephen C. , Lynn McGrew L. , Lai Cheng-Jung , Lee John J. , von Kessler Doris P. , Moon Randall T. , Beachy Philip A.
Published: April 2021

The hedgehog family of signaling proteins is associated with a variety of spatial patterning activities in insects and vertebrates. Here we show that new members of this family isolated from Xenopus laevis are expressed embryonically in patterns suggestive of roles in patterning in the ectoderm, nervous system and somites. Banded hedgehog is expressed throughout the neural plate and subsequently in both the nervous system and in the dermatome of somites. Cephalic hedgehog is expressed in anterior ectoderm and endodermal structures, and sonic hedgehog is expressed in patterns which parallel those in other species. Injection of RNAs encoding Xenopus hedgehogs induces ectopic cement gland formation in embryos. Similar to reported activities of noggin and follistatin, Xenopus hedgehogs share a common ability to induce cement glands in animal cap explants. However, hedgehog activities in naive ectoderm appear capable of acting independently of noggin and fol-listatin since, although all three are induced by activin in animal cap explants, X-hh expression does not induce noggin or follistatin.
Isolation of novel tissue-specific genes from cDNA libraries representing the individual tissue constituents of the gastrulating mouse embryo
Authors: Harrison S. M. , Dunwoodie S. L. , Arkell R. M. , Lehrach H. , Beddington R. S. P.
Published: April 2021

A total of 5 conventional, directionally cloned plasmid cDNA libraries have been constructed from the entire embryonic region of the mid-gastrulation mouse embryo and from its four principal tissue constituents (ectoderm, mesoderm, endoderm and primitive streak). These libraries have been validated with respect to the number of independent clones, insert-size and appropriate representation of diagnostic marker genes. Subtractive hybridisation has been used to remove clones common to the Endoderm and Mesoderm cDNA libraries resulting in an Endoderm minus Mesoderm subtracted library. Probe prepared from this subtracted library has been hybridised to a grid containing approximately 18,500 Embryonic Region library clones. Three novel clones have been recovered as well as expected genes already known to be highly expressed in the primitive endoderm lineage at this stage of development. In situ hybridisation to early postim-plantation embryos has revealed the expression patterns of these novel genes. One is highly expressed exclusively in visceral endoderm, one is expressed in ectodermal and endodermal tissues, and the third proves to be an early marker of prospective and differentiated surface ectoderm as well as being expressed in endoderm and its derivatives.
Epithelial-mesenchymal conversion of dermatome progenitors requires neural tube-derived signals: characterization of the role of Neurotrophin-3
Authors: Brill Gilat , Kahane Nitza , Carmeli Chana , von Schack David , Barde Yves-Alain , Kalcheim Chaya
Published: April 2021

Development of the somite-derived dermatome involves conversion of the epithelial dermatome progenitors into mesenchymal cells of the dermis. In chick embryos, neural tube-derived signals are required for this conversion, as the interposition of a membrane between neural tube and somites results in a failure of the dermatome to lose its epithelial arrangement. However, dermis formation can be completely rescued by coating the membranes with Neurotrophin-3, but not with the related molecule Nerve growth factor. Neurotrophin-3 was also found to be necessary for dermatome dissociation using in vitro explants or partially dissociated dermomyotomes. The functional relevance of these observations was investigated by neutralizing endogenous Neurotrophin-3 using a specific blocking antibody. Antibody-treated embryos revealed the presence of tightly aggregated cells between myotome and ectoderm instead of the loose dermal mesenchyme observed in embryos treated with control antibodies. As previous studies have demonstrated the presence of Neurotrophin-3 in the neural tube, these results suggest that it may be a necessary neural tube-derived signal required for early stages of dermis formation.
Fusion from myoblasts to myotubes is dependent on the rolling stone gene (rost) of Drosophila
Authors: Paululat Achim , Burchard Susanne , Renkawitz-Pohl Renate
Published: April 2021

The development and differentiation of the body wall musculature in Drosophila are accompanied by changes in gene expression and cellular architecture. We isolated a Drosophila gene, termed rolling stone (rost), which, when mutated, specifically blocks the fusion of mononucleated cells to myotubes in the body wall musculature. β3 tubulin, which is an early marker for the onset of mesoderm differentiation, is still expressed in these cells. Gastrulation and mesoderm formation, as well as the development of the epidermis and of the central and peripheral nervous systems, appear quite normal in homozygous rolling stone embryos. Embryonic development stops shortly before hatching in a P-element-induced mutant, as well as in 16 EMS-induced alleles. In mutant embryos, other mesodermal derivatives such as the visceral mesoderm and the dorsal vessel, develop fairly normally and defects are restricted to the body wall musculature. Myoblasts remain as single mononucleated cells, which express muscle myosin, showing that the developmental program of gene expression proceeds. These myoblasts occur at positions corresponding to the locations of dorsal, ventral and pleural muscles, showing that the gene rolling stone is involved in cell fusion, a process that is independent of cell migration in these mutants. This genetic analysis has set the stage for a molecular analysis to clarify where the rolling stone action is manifested in the fusion process and thus gives insight into the complex regulating network controlling the differentiation of the body wall musculature.
Mosaic analysis of the let-23 gene function in vulval induction of Caenorhabditis elegans
Authors: Koga Makoto , Ohshima Yasumi
Published: April 2021

The let-23 receptor tyrosine kinase gene is required for vulval induction and larval survival in the nematode Caenorhabditis elegans. We carried out genetic mosaic analyses of the let-23 gene function by using the cloned let-23 and ncl-1 genes. The wild-type let-23 gene was required in a vulval precursor cell to adopt the 1° vulval fate in animals carrying a let-23 vulvaless or lethal chromosomal mutation. In almost all the animals, vulval precursor cells adjacent to a 1° fate cell were induced to the 2° vulval fate regardless of the let-23 genotypes. These findings indicate that the vulval induction signal from an anchor cell induces a vulval precursor cell to adopt the 1° fate through LET-23, and then a 1° fate cell induces adjacent cells to adopt the 2° fate, for which LET-23 is not required. Foci of lethality of the let-23 (mn23) mutation were found in ABal and ABplp lineages.
The Hox11 gene is essential for cell survival during spleen development
Authors: Dear T. N. , Colledge W. H. , Carlton M. B. L. , Lavenir I. , Larson T. , Smith A. J. H. , Warren A. J. , Evans M. J. , Sofroniew M. V. , Rabbitts T. H.
Published: April 2021

The HOX11 homeobox gene was identified via the translocation t(10;14) in T cell leukaemia. To determine the function of this gene in mice, null mutations were made using homologous recombination in ES cells to incorporate lacZ into the hox11 transcription unit. Production of β-galactosidase from the recombinant hox11 allele in +/− mutants allowed identification of sites of hox11 expression which included the developing spleen. Newborn hox11 −/− mice exhibit asplenia. Spleen formation commences normally at E11.5 in hox11 −/− mutant embryos but the spleen anlage undergoes rapid and complete resorption between E12.5 and E13.5. Dying spleen cells exhibit molecular features of apoptosis, suggesting that programmed cell death is initiated at this stage of organ development in the absence of hox11 protein. Thus hox11 is not required to initiate spleen development but is essential for the survival of splenic precursors during organogenesis. This function for hox11 suggests that enhanced cell survival may result from the t(10;14) which activates HOX11 in T cell leukaemias, further strengthening the association between oncogene-induced cell survival and tumorigenesis.
A role for the Drosophila Bag-of-marbles protein in the differentiation of cystoblasts from germline stem cells
Authors: McKearin Dennis , Ohlstein Benjamin
Published: April 2021

Cell differentiation commonly dictates a change in the cell cycle of mitotic daughters. Previous investigations have suggested that the Drosophila bag of marbles (bam) gene is required for the differentiation of germline stem cell daughters (cystoblasts) from the mother stem cells, perhaps by altering the cell cycle. In this paper, we report the preparation of antibodies to the Bam protein and the use of those reagents to investigate how Bam is required for germ cell development. We find that Bam exists as both a fusome component and as cytoplasmic protein and that cytoplasmic and fusome Bam might have separable activities. We also show that bam mutant germ cells are blocked in differentiation and are trapped as mitotically active cells like stem cells. A model for how Bam might regulate cystocyte differentiation is presented.
Apoptosis in the developing tooth: association with an embryonic signaling center and suppression by EGF and FGF-4
Authors: Vaahtokari Anne , Åberg Thomas , Thesleff Irma
Published: April 2021

Apoptosis was localized in developing mouse teeth from initiation of morphogenesis to completion of cusp formation by using modified TUNEL method for serial sections and Nile Blue staining for whole mounts. Apoptosis was first detected at bud stage (E12-E13) in the central cells of the invaginating dental epithelium suggesting involvement of cell death in epithelial budding morphogenesis. During cusp development, apoptotic cells were located in the enamel knots, which are transient clusters of dental epithelial cells proposed to act as signaling centers directing the morphogenesis of tooth cusps. Apoptosis was also detected in other restricted epithelial cell populations including the dental lamina, ameloblasts, as well as stratum intermedium and stellate reticulum cells suggesting that the removal of these epithelial cells occurs by apoptosis. Apoptotic cells, presumably osteoclasts, were also located on the surfaces of the developing alveolar bone.

When dissected E13 dental epithelium or mesenchyme were cultured in isolation, apoptotic cells were abundant throughout the tissues, whereas when cultured together, apoptosis was inhibited in both tissues close to their interface indicating that epithelial-mesenchymal tissue interactions prevent apoptosis. Epidermal growth factor (EGF) and fibroblast growth factor-4 (FGF-4) inhibited apoptosis in the dental mesenchyme when applied locally using agarose or heparin-coated acrylic beads, suggesting involvement of these or related growth factors in the prevention of apoptosis in dental tissues in vivo.

The spatially and temporally restricted distribution patterns of apoptotic cells suggest multiple roles for programmed cell death in dental development. Of particular interest is the removal of the enamel knots by apoptosis which may terminate their tasks as regulators of the patterning of the tooth cusps. The apical ectodermal ridge (AER) of the limb bud has similar signaling characteristics as the enamel knot, and it also undergoes apoptosis. Hence, apoptosis may be a general mechanism for the silencing of embryonic signaling centers.
glide directs glial fate commitment and cell fate switch between neurones and glia
Authors: Vincent Stéphane , Vonesch Jean-Luc , Giangrande Angela
Published: April 2021

Glial cells constitute the second component of the nervous system and are important during neuronal development. In this paper we describe a gene, glial cell deficient, (glide), that is necessary for glial cell fate commitment in Drosophila melanogaster. Mutations at the glide locus prevent glial cell determination in the embryonic central and peripheral nervous system. Moreover, we show that the absence of glial cells is the consequence of a cell fate switch from glia to neurones. This suggests the existence of a multipotent precursor cells in the nervous system. glide mutants also display defects in axonal navigation, which confirms and extends previous results indicating a role for glial cells in these processes.
The eve stripe 2 enhancer employs multiple modes of transcriptional synergy
Authors: Arnosti David N. , Barolo Scott , Levine Michael , Small Stephen
Published: April 2021

Previous studies have provided a detailed model for the regulation of even-skipped (eve) stripe 2 expression in the Drosophila embryo. The bicoid (bcd) regulatory gradient triggers the expression of hunchback (hb); these work synergistically to activate the stripe in the anterior half of the embryo. bcd also coordinates the expression of two repressors, giant (gt) and Kruppel (Kr), which define the anterior and posterior borders of the stripe, respectively. Here, we report the findings of extensive cis- and trans-complementation analyses using a series of defective stripe 2 enhancers in transgenic embryos. This study reaches two primary conclusions. First, the stripe 2 enhancer is inherently ‘sensitized’ for repression by gt. We propose that gt specifies the sharp anterior stripe border by blocking two tiers of transcriptional synergy, cooperative binding to DNA and cooperative contact of bound activators with the transcription complex. Second, we find that the synergistic activity of hb and bcd is ‘promiscuous’. For example, a maternally expressed Gal4-Sp1 fusion protein can functionally replace hb in the stripe 2 enhancer. This finding challenges previous proposals for dedicated hb and bcd interactions in the segmentation process.
In vivo effects of neurotrophin-3 during sensory neurogenesis
Authors: Ockel Michaela , Lewin Gary R. , Barde Yves-Alain
Published: April 2021

The neurotrophins nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 are structurally related proteins regulating the number of neurons in peripheral ganglia of the nervous system. Increased levels of nerve growth factor or of brain-derived neurotrophic factor selectively prevent normally occurring neuronal death, while the targeted elimination of all three genes decreases neuronal numbers. As previous studies indicated that the lack of neurotrophin-3 affects sensory ganglia already during gangliogenesis, the levels of this neurotrophin were increased during selected periods of chick development. We found that early, but not late, applications of neurotrophin-3 lead to a marked decrease in neuronal numbers in peripheral sensory ganglia. This decrease is not seen with BDNF and does not selectively affect subtypes of dorsal root ganglion neurons. It is accompanied by, and might result from, a decrease in the number of proliferating neuroblasts in sensory ganglia of treated embryos.
Distinct subpopulations of enteric neuronal progenitors defined by time of development, sympathoadrenal lineage markers and Mash-1-dependence
Authors: Blaugrund E. , Pham T. D. , Tennyson V. M. , Lo L. , Sommer L. , Anderson D.J. , Gershon M.D.
Published: April 2021

Enteric and sympathetic neurons have previously been proposed to be lineally related. We present independent lines of evidence that suggest that enteric neurons arise from at least two lineages, only one of which expresses markers in common with sympathoadrenal cells. In the rat, sympathoadrenal markers are expressed, in the same order as in sympathetic neurons, by a subset of enteric neuronal precursors, which also transiently express tyrosine hydroxylase. If this precursor pool is eliminated in vitro by complement-mediated lysis, enteric neurons continue to develop; however, none of these are serotonergic. In the mouse, the Mash-1−/− mutation, which eliminates sympathetic neurons, also prevents the development of enteric serotonergic neurons. Other enteric neuronal populations, however, including those that contain calcitonin gene related peptide are present. Enteric tyrosine hydroxylasecontaining cells co-express Mash-1 and are eliminated by the Mash-1−/− mutation, consistent with the idea that in the mouse, as in the rat, these precursors generate serotonergic neurons. Serotonergic neurons are generated early in development, while calcitonin gene related peptide-containing enteric neurons are generated much later. These data suggest that enteric neurons are derived from at least two progenitor lineages. One transiently expresses sympathoadrenal markers, is Mash-1-dependent, and generates early-born enteric neurons, some of which are serotonergic. The other is Mash-1-independent, does not express sympathoadrenal markers, and generates late-born enteric neurons, some of which contain calcitonin gene related peptide.
ERECT WING, the Drosophila member of a family of DNA binding proteins is required in imaginal myoblasts for flight muscle development
Authors: DeSimone Susan , Coelho Carmen , Roy Sudipto , VijayRaghavan K. , White Kalpana
Published: April 2021

The erect wing locus of the fruit fly Drosophila melanogaster encodes a protein, EWG, that shares extensive homology with the P3A2 DNA binding protein of sea urchin and a recently identified mammalian transcription factor. Loss-of-function erect wing alleles result in embryonic lethality. Viable alleles of erect wing cause severe abnormalities of the indirect flight muscles. We have analyzed the spatial pattern of erect wing expression in the developing indirect flight muscles during postembryonic development. EWG is detected, 10 hours after puparium formation, in myoblasts that will form the indirect flight muscles. The early events of muscle development are normal in ewg mutants. However, a few hours after the onset of erect wing expression in myoblasts, defects are seen in the developing indirect flight muscles which subsequently degenerate. We present results that show that the normal development of the indirect flight muscles requires erect wing expression in the progenitor myoblasts themselves. Finally, we examine the role of target muscles in the arborization of motor axons by studying the developing innervation to the flight muscle in erect wing mutants. Our study demonstrates, for the first time, a role for a regulatory gene expressed in imaginal myoblasts in Drosophila.
Spatial and temporal information processing in the sea urchin embryo: modular and intramodular organization of the CyIIIa gene cis-regulatory system
Authors: Kirchhamer Carmen V. , Davidson Eric H.
Published: April 2021

The CyIIIa cytoskeletal actin gene of Strongylocentrotus purpuratus is expressed specifically in the aboral ectoderm. In earlier work we identified a 2.3 kb cis-regulatory region that is necessary and sufficient for correct spatial and temporal expression of a CyIIIa•CAT gene. This region includes about 20 sites of specific protein-DNA interaction, at which at least nine different transcription factors may be bound. All except two of these factors have been cloned. In this work we have analyzed by deletion or mutagenesis each specific interaction. A specific function was identified for every binding site examined. These individual functions include control of amplitude and timing of expression at different phases of embryogenesis, and control of spatial expression. We show that particular negative regulatory interactions are required to repress expression of the CyIIIa•CAT construct in oral ectoderm and in skeletogenic mesenchyme at different stages. In further experiments we determined the overall functional organization of the CyIIIa cis-regulatory system, and we show that this system is modular in its regulatory structure. The ‘proximal module’ (with respect to the transcription start site) extends upstream for about 800 base pairs, and includes nine target sites serviced by six different transcription factors. Its major role is to establish CyIIIa expression in the aboral ectoderm territory as the blastomere founder cells are specified and the oral-aboral axis is determined, and to activate the CyIIIa gene late in cleavage. The ‘middle module,’ which lies upstream of the proximal module, acquires major control of CyIIIa function after the blastula stage. It includes six target sites, serviced by four different factors. The middle module is responsible for a sharp increase in expression occurring during gastrulation, mediated by the positively acting factors that bind within it. The middle module also includes sites at which two different negatively acting spatial control factors bind, the functions of which are required for correct spatial expression late in embryogenesis. The ‘distal module’ contains a number of sites at which a positively acting factor binds, but this module exercises no spatial regula-tory function. Interactions within the distal module are required for the normal levels of function of both the proximal and middle modules.
Maternal β-catenin establishes a ‘dorsal signal’ in early Xenopus embryos
Authors: Wylie C. , Kofron M. , Payne C. , Anderson R. , Hosobuchi M. , Joseph E. , Heasman J.
Published: April 2021

In previous work, we demonstrated that maternally encoded β-catenin, the vertebrate homolog of armadillo, is required for formation of dorsal axial structures in early Xenopus embryos (Heasman, J., Crawford, A., Goldstone, K., Garner-Hamrick, P., Gumbiner, B., Kintner, C., Yoshida-Noro, C. and Wylie, C. (1994). Cell 79, 791-803). Here we investigated, firstly, the role(s) of β-catenin in spatial terms, in different regions of the embryo, by injecting β-catenin mRNA into individual blastomeres of β-catenin-depleted embryos at the 32 cell stage. The results indicate that β-catenin can rescue the dorsal axial structures in a non-cell-autonomous way and without changing the fates of the injected cells. This suggests that cells overexpressing β-catenin send a ‘dorsal signal’ to other cells. This was confirmed by showing that β-catenin overexpressing animal caps did not cause wild-type caps to form mesoderm, but did cause isolated β-catenin-deficient marginal zones to form dorsal mesoderm. Furthermore β-catenin-deficient vegetal masses treated with overexpressing caps regained their ability to act as Nieuwkoop Centers.

Secondly, we studied the temporal activity of β-catenin. We showed that zygotic transcription of β-catenin starts after the midblastula transition (MBT), but does not rescue dorsal axial structures. We further demonstrated that the vegetal mass does not release a dorsal signal until after the onset of transcription, at the midblastula stage, suggesting that maternal β-catenin protein is required at or before this time. Thirdly we investigated where, in relationship to other gene products known to be active in axis formation,β-catenin is placed. We find that BVg1, bFGF, tBR (the truncated form of BMP2/4R), siamois and noggin activities are all downstream of β-catenin, as shown by the fact that injection of their mRNAs rescues the effect of depleting maternally encoded β-catenin. Interference with the action of glycogen synthase kinase (GSK), a vertebrate homolog of the Drosophila gene product, zeste white 3 kinase, does not rescue the effect, suggesting that it is upstream.