bluuebunny/crossref_metadata_2025 huggingface dataset:
Neither the homeodomain nor the activation domain of Bicoid is specifically required for its down-regulation by the Torso receptor tyrosine kinase cascade
Authors: Bellaïche Yohanns , Bandyopadhyay Rupa , Desplan Claude , Dostatni Nathalie
Published: April 2021

Bicoid (Bcd) is a maternal morphogen responsible for patterning the head and thorax of the Drosophila embryo. Correct specification of head structure, however, requires the activity of the Torso receptor tyrosine kinase cascade, which also represses expression of Bcd targets at the most anterior tip of the embryo. Here, we investigate the role of both the homeodomain (HD) and the activation domain of Bcd in the anterior repression of its targets. When a Bcd mutant protein whose HD has been replaced by the Gal4 DNA-binding domain is expressed in early embryos, a reporter gene driven by Gal4 DNA-binding sites is first activated in an anterior domain and then repressed from the anterior pole. The down-regulation of Bcd-Gal4 activity requires torso function but does not depend on endogenous bcd activity, indicating that the Bcd protein alone and none of its targets is required to mediate the effect of torso. Functional analysis of a chimeric protein, whose activation domain has been replaced by a generic activation domain, indicates that the activation domain of Bcd is also not specifically required for its downregulation by Torso. We propose that Torso does not affect the ability of Bcd to bind DNA, but instead directs modification of Bcd or of a potential Bcd co-factor, which renders the Bcd protein unable to activate transcription.
Ectopic expression of wingless in imaginal discs interferes with decapentaplegic expression and alters cell determination
Authors: Johnston Laura A. , Schubiger Gerold
Published: April 2021

We have expressed the segment polarity gene wingless (wg) ectopically in imaginal discs to examine its regulation of both ventral patterning and transdetermination. By experimentally manipulating the amount of Wg protein, we show that different thresholds of Wg activity elicit different outcomes, which are mediated by regulation of decapentaplegic (dpp) expression and result in alterations in the expression of homeotic genes. A high level of Wg activity leads to loss of all dorsal pattern elements and the formation of a complete complement of ventral pattern elements on the dorsal side of legs, and is correlated with repression of dpp expression. wg expression in dorsal cells of each disc also leads to dose-dependent transdetermination in those cells in homologous discs such as the labial, antennal and leg, but not in cells of dorsally located discs. When dpp expression is repressed by high levels of Wg, transdetermination does not occur, confirming that dpp participates with wg to induce transdetermination. These and other experiments suggest that dorsal expression of wg alters disc patterning and disc cell determination by modulating the expression of dpp. The dose-dependent effects of wg on dpp expression, ventralization of dorsal cells and transdetermination support a model in which wg functions as a morphogen in imaginal discs.
The Dichaete gene of Drosophila melanogaster encodes a SOX-domain protein required for embryonic segmentation
Authors: Russell Steven R. H. , Sanchez-Soriano Natalia , Wright Charles R. , Ashburner Michael
Published: April 2021

We have cloned and characterised a member of the High Mobility Group superfamily of genes from Drosophila, Sox70D, which is closely related to the mammalian testis determining gene SRY. Sox70D corresponds to the dominant wing mutation Dichaete. Homozygous deletions of the Sox70D gene and recessive lethal Dichaete alleles have a variable embryonic segmentation phenotype. Dichaete is expressed in early embryos in a dynamic pattern reminiscent of gap and pair-rule genes and is required for the appropriate expression of the primary pair-rule genes even skipped, hairy and runt. The molecular nature of Dichaete and its expression pattern during early embryogenesis suggest that the gene plays a key role in early development; the variability in both the segmentation phenotype and the effects on pair-rule gene expression suggests that this role is to support the transcriptional regulation of key developmental genes rather than directly regulate any one of them.
An activated form of type I serine/threonine kinase receptor TARAM-A reveals a specific signalling pathway involved in fish head organiser formation
Authors: Renucci A. , Lemarchandel V. , Rosa F.
Published: April 2021

The role of Transforming Growth Factor β (TGF-β)-related molecules in axis formation and mesoderm patterning in vertebrates has been extensively documented, but the identity and mechanisms of action of the endogenous molecules remained uncertain. In this study, we isolate a novel serine/threonine kinase type I receptor, TARAM-A, expressed during early zebrafish embryogenesis first ubiquitously and then restricted to dorsal mesoderm during gastrulation. A constitutive form of the receptor is able to induce the most anterior dorsal mesoderm rapidly and to confer an anterior organizing activity. By contrast, the wild-type form is only able to induce a local expansion of the dorsal mesoderm. Thus an activated form of TARAM-A is sufficient to induce dorsoanterior structures and TARAM-A may be activated by dorsally localized signals. Our data suggest the existence in fish of a specific TGF-β-related pathway for anterior dorsal mesoderm induction, possibly mediated by TARAM-A and activated at the late blastula stage by localized dorsal determinant.
Zebrafish tinman homolog demarcates the heart field and initiates myocardial differentiation
Authors: Chen Jau-Nian , Fishman Mark C.
Published: April 2021

The fashioning of a vertebrate organ requires integration of decisions of cell fate by individual cells with those that regulate organotypic form. Logical candidates for this role, in an organ such as the heart, are genes that initiate the differentiation process leading to heart muscle and those that define the earliest embryonic heart field, but for neither class are genes defined.

We cloned zebrafish Nkx2.5, a homolog of the tinman homeodomain gene needed for visceral and cardiac mesoderm formation in Drosophila. In the zebrafish, its expression is associated with cardiac precursor cells throughout development, even in the early gastrula, where the level of zebrafish Nkx2.5 is in a gradient which spatially matches the regional propensity of ventral-marginal cells to become heart. Overexpression of Nkx2.5 causes formation of disproportionally larger hearts in otherwise apparently normal embryos. Transplanted cell expressing high levels of Nkx2.5 express cardiac genes even in ectopic locales. Fibroblasts transfected with myc-tagged Nkx2.5 express cardiac genes. These effects require the homeo-domain.

Thus, Nkx2.5 appears to mark the earliest embryonic heart field and to be capable of initiating the cardiogenic differentiation program. Because ectopic cells or transfected fibroblasts do not beat, Nkx2.5 is likely to be but one step in the determination of cardiac myocyte cell fate. Its overexpression increases heart size, perhaps by bringing cells on the edge of the field to a threshold level for initiation of cardiac differentiation.
Bax promotes neuronal survival and antagonises the survival effects of neurotrophic factors
Authors: Middleton Gayle , Nunez Gabriel , Davies Alun M.
Published: April 2021

Bcl-2, Bcl-x and Bax are members of a family of cytoplasmic proteins that influence cell survival. Whereas increased expression of Bcl-2 or Bcl-x promotes cell survival following withdrawal of survival factors, increased expression of Bax is thought to suppress survival. To investigate the potential roles of these proteins in regulating the survival of developing neurons, we compared the effects of overexpressing these proteins in embryonic neurons deprived of different neurotrophic factors in vitro. Surprisingly, overexpression of Bax rescued populations of sensory neurons deprived of nerve growth factor, as did overexpression of Bcl-2 and two Bcl-x variants, Bcl-xL and Bcl-xβ. Bax also enhanced the survival of ciliary neurons deprived of ciliary neurotrophic factor, although this effect was short-lived. Whereas Bcl-2 overexpression did not affect the survival response of neurons to neurotrophic factors, Bax overexpression partially inhibited the action of neurotrophic factors. Co-injection of Bcl-2 and Bax expression vectors promoted the survival of neurotrophic factor-deprived neurons if either was in excess, but failed to rescue neurons if they were injected at a 1:1 ratio. Our findings demonstrate that Bax can promote the survival of neurotrophic factor-deprived neurons and that its effect on survival is dominant to that of neurotrophic factors. Our results also argue that the relative amounts of Bcl-2 and Bax are critical in regulating neuronal survival.
Drosophila Paired regulates late even-skipped expression through a composite binding site for the paired domain and the homeodomain
Authors: Fujioka Miki , Miskiewicz Pawel , Raj Lakshmi , Gulledge Alyssa A. , Weir Michael , Goto Tadaatsu
Published: April 2021

The even-skipped (eve) pair-rule gene plays a key role in the establishment of the anterior-posterior segmental pattern of the Drosophila embryo. The continuously changing pattern of eve expression can be resolved into two phases. Early expression consists of seven broad stripes in the blastoderm embryo, while late expression, which occurs after cellularization, consists of narrow stripes with sharp anterior borders that coincide with the odd-numbered parasegment boundaries. Previous studies have shown that these two phases are controlled by separate classes of cis elements in the eve promoter. Early stripes are expressed by multiple stripe-specific elements under the control of maternal-effect genes and gap genes, while late stripes are expressed by a single regulatory element, the ‘late element’, under the control of pair-rule genes including eve itself. We report here that paired (prd), a pair-rule gene which had been considered to be below eve in the regulatory hierarchy of pair-rule genes, in fact plays a critical role in the regulation of late eve expression. Transgenic analysis shows that this regulation is largely mediated by an evolutionarily conserved sequence within the late element termed PTE (Paired Target Element). In vitro analysis shows that the Prd protein binds strongly to this sequence. Interestingly, PTE contains juxtaposed binding sites for the two DNAbinding domains of the Prd protein, the paired domain and the homeodomain. Mutagenesis of either binding site leads to significant reduction in the activity of the late element, indicating that both DNA-binding domains in the Paired protein are required for regulation.
Functional relationships between Notch, Su(H) and the bHLH genes of the E(spl) complex: the E(spl) genes mediate only a subset of Notch activities during imaginal development
Authors: de Celis Jose F. , Celis Jesus de , Ligoxygakis Petros , Preiss Anette , Delidakis Christos , Bray Sarah
Published: April 2021

The basic helix-loop-helix proteins of the Enhancer of split complex constitute a link between activation of the transmembrane receptor Notch and the resulting effects on transcription of downstream genes. The Suppressor of Hairless protein is the intermediary between Notch activation and expression of all Enhancer of split genes even though individual genes have distinct patterns of expression in imaginal discs. A comparison between the phenotypes produced by Notch, Suppressor of Hairless and Enhancer of split mutations in the wing and thorax indicate that Suppressor of Hairless and Notch requirements are indistinguishable, but that Enhancer of split activity is only essential for a subset of developmental processes involving Notch function. Likewise, the ectopic expression of Enhancer of split proteins does not reproduce all the consequences typical of ectopic Notch activation. We suggest that the Notch pathway bifurcates after the activation of Suppressor of Hairless and that Enhancer of split activity is not required when the consequence of Notch function is the transcriptional activation of downstream genes. Transcriptional activation mediated by Suppressor of Hairless and transcriptional repression mediated by Enhancer of split could provide greater diversity in the response of individual genes to Notch activity.
Evidence that MSL-mediated dosage compensation in Drosophila begins at blastoderm
Authors: Franke Axel , Dernburg Abby , Bashaw Greg J. , Baker Bruce S.
Published: April 2021

In Drosophila equalization of the amounts of gene products produced by X-linked genes in the two sexes is achieved by hypertranscription of the single male X chromosome. This process, dosage compensation, is controlled by a set of male-specific lethal (msl) genes, that appear to act at the level of chromatin structure. The properties of the MSL proteins have been extensively studied in the polytene salivary gland chromosomes where they bind to the same set of sites along the male X chromosome in a co-dependent manner. Here we report experiments that show that the MSL proteins first associate with the male X chromosome as early as blastoderm stage, slightly earlier than the histone H4 isoform acetylated at lysine 16 is detected on the X chromosome. MSL binding to the male X chromosome is observed in all somatic tissues of embryos and larvae. Binding of the MSLs to the X chromosome is also interdependent in male embryos and prevented in female embryos by the expression of Sex-lethal (Sxl). A delayed onset of binding of the MSLs in male progeny of homozygous mutant msl-1 or mle mothers coupled with the previous finding that such males have an earlier lethal phase supports the idea that msl-mediated dosage compensation begins early in embryogenesis. Other results show that the maleless (MLE) protein on embryo and larval chromosomes differs in its reactivity with antibodies; the functional significance of this finding remains to be explored.
Identification of a novel cardiac-specific transcript critical for cardiac myocyte differentiation
Authors: Wei Yan , Bader David , Litvin Judith
Published: April 2021

A novel cDNA, pCMF1, which is expressed exclusively and transiently in the myogenic cells of the differentiating chicken heart was isolated and characterized. The fulllength cDNA of pCMF1 has one open reading frame encoding 1538 predicted amino acids. While computer analysis predicts the presence of specific structural motifs, the overall sequence of pCMF1 is unique. The pattern of pCMF1 gene expression during heart formation was determined by whole-mount in situ hybridization. pCMF1 is transiently expressed within the myogenic cells of the primitive heart tube from stages 9 to 18 and is not detected in the heart or any other tissue thereafter. A replicationdeficient retrovirus was used to mediate pCMF1 antisense expression in cardiogenic mesoderm. These analyses determined that the presence of pCMF1 antisense sequences disrupted myosin heavy chain expression during cardiac mesoderm differentiation. pCMF1 antisense had no effect on myosin heavy chain expression in differentiated cardiac myocytes. These data suggest a potential function for pCMF1 during cardiac myogenesis.
wingless signaling in the Drosophila eye and embryonic epidermis
Authors: Cadigan Kenneth M. , Nusse Roel
Published: April 2021

After the onset of pupation, sensory organ precursors, the progenitors of the interommatidial bristles, are selected in the developing Drosophila eye. We have found that wingless, when expressed ectopically in the eye via the sevenless promoter, blocks this process. Transgenic eyes have reduced expression of acheate, suggesting that wingless acts at the level of the proneural genes to block bristle development. This is in contrast to the wing, where wingless positively regulates acheate to promote bristle formation. The sevenless promoter is not active in the acheate-positive cells, indicating that the wingless is acting in a paracrine manner. Clonal analysis revealed a requirement for the genes porcupine, dishevelled and armadillo in mediating the wingless effect. Overexpression of zeste white-3 partially blocks the ability of wingless to inhibit bristle formation, consistent with the notion that wingless acts in opposition to zeste white-3. Thus the wingless signaling pathway in the eye appears to be very similar to that described in the embryo and wing. The Notch gene product has also been suggested to play a role in wingless signaling (J. P. Couso and A. M. Martinez Arias (1994)Cell 79, 259–72). Because Notch has many functions during eye development, including its role in inhibiting bristle formation through the neurogenic pathway, it is difficult to assess the relationship of Notch to wingless in the eye. However, we present evidence that wingless signaling still occurs normally in the complete absence of Notch protein in the embryonic epidermis. Thus, in the simplest model for wingless signalling, a direct role for Notch is unlikely.
Coordination of early neural tube development by BDNF/trkB
Authors: Jungbluth Stefan , Koentges Georgy , Lumsden Andrew
Published: April 2021

Neurotrophins signal through members of the trk family of tyrosine kinase receptors and are known to regulate several neuronal properties. Although initially characterized by their ability to prevent naturally occurring cell death of subsets of neurons during development, neurotrophins can also regulate the proliferation and differentiation of precursor cells. Here we report a novel involvement of neurotrophins in early development of the neural tube. We demonstrate that a functional trkB receptor is expressed by motor neuron progenitors in the ventral neural tube and that treatment of ventral neural tube explants with the trkB ligand Brain-Derived Neurotrophic Factor (BDNF) leads to a significant increase in the number of motor neurons. The only BDNF expression detectable at this stage is by a subset of ventrally projecting interneurons in the dorsal neural tube; ablating this region in vivo leads to a reduction of motor neuron numbers. This loss can be prevented by simultaneous treatment with BDNF. We propose that BDNF produced by dorsal interneurons stimulates proliferation and/or differentiation of motor neuron progenitors after anterograde axonal transport and release in proximity to the trkB-expressing motor neuron precursors, thereby coordinating development between dorsal and ventral regions of the neural tube.
Differential expression of mammalian Numb, Numblike and Notch1 suggests distinct roles during mouse cortical neurogenesis
Authors: Zhong Weimin , Jiang Ming-Ming , Weinmaster Gerry , Jan Lily Yeh , Jan Yuh Nung
Published: April 2021

During Drosophila neurogenesis, asymmetric cell divisions are achieved by differential segregation of Numb (d-Numb) into one of the daughter cells to cause a bias in the Notch mediated cell-cell interaction. We have isolated a second mammalian gene with significant sequence similarity tod-numb, mouse numblike. When expressed in dividing neural precursors in Drosophila, Numblike is symmetrically distributed in the cytoplasm, unlike endogenous d-Numb or expressed mouse Numb (m-Numb), both of which are asymmetrically localized to one half of the cell membrane. In d-numb loss-of-function mutant embryos, expression of Numblike allows both daughter cells of a neural precursor to adopt the fate of the cell that normally inherits d-Numb. In mice, numblike mRNA is preferentially expressed in adult and embryonic nervous system. In the developing neocortex, Numblike is expressed in postmitotic neurons in the cortical plate, but not in progenitors within the ventricular zone where m-Numb and Notch1 are expressed. We have also found that, in dividing cortical progenitors, Notch1 is distributed around the entire membrane, unlike m-Numb which is asymmetrically localized to the apical membrane. We propose that an interplay between cell-intrinsic mechanisms (executed by m-numb and numblike) and cell-extrinsic mechanisms (mediated by Notch1) may be involved in both progenitor cell proliferation and neuronal differentiation during mammalian cortical neurogenesis.
Cell fate in the chick limb bud and relationship to gene expression
Authors: Vargesson Neil , Clarke Jonathan D. W. , Vincent Katherine , Coles Clare , Wolpert Lewis , Tickle Cheryll
Published: April 2021

We have produced detailed fate maps for mesenchyme and apical ridge of a stage 20 chick wing bud. The fate maps of the mesenchyme show that most of the wing arises from the posterior half of the bud. Subapical mesenchyme gives rise to digits. Cell populations beneath the ridge in the mid apical region fan out into the anterior tip of the handplate, while posterior cell populations extend right along the posterior margin. Subapical mesenchyme of the leg bud behaves similarly. The absence of anterior bending of posterior cell populations has implications when considering models of vertebrate limb evolution. The fatemaps of the apical ridge show that there is also a marked anterior expansion and cells that were in anterior apical ridge later become incorporated into non-ridge ectoderm along the margin of the bud. Mesenchyme and apical ridge do not expand in concert - the apical ridge extends more anteriorly. We used the fatemaps to investigate the relation-ship between cell lineage and elaboration of Hoxd-13 and Fgf-4 domains. Hoxd-13 and Fgf-4 are initially expressed posteriorly until about the mid-point of the early wing bud in mesenchyme and apical ridge respectively. Later in development, the genes come to be expressed throughout most of the handplate and apical ridge respectively. We found that at the proximal edge of the Hoxd-13 domain, cell populations stopped expressing the gene as development proceeded and found no evidence that the changes in extent of the domains were due to initiation of gene expression in anterior cells. Instead the changes in extent of expression fit with the fate maps and can be attributed to expansion and fanning out of cell populations initially expressing the genes.
Pax-6 functions in boundary formation and axon guidance in the embryonic mouse forebrain
Authors: Mastick Grant S. , Davis Nicole M. , Andrews Gracie L. , Easter Stephen S.
Published: April 2021

The Pax-6 gene encodes a transcription factor that is expressed in regionally restricted patterns in the developing brain and eye. Here we describe Pax-6 expression in the early forebrain (prosencephalon) on embryonic day 9.5 (E9.5) to E10.5 using both whole-mount in situ hybridization and antibody labeling. We find close correlations between Pax-6+ domains and initial neural patterning, and identify corresponding defects in embryos homozygous for the Pax-6 allele, Small eye (Sey). Pax-6 expression defines the prosencephalon-mesencephalon boundary, and mutant embryos lack this morphological boundary. Markers of the caudal prosencephalon are lost (Pax-6, Lim-1, Gsh-1) and a marker for mesencephalon is expanded rostrally into the prosencephalon (Dbx). We conclude that the caudal prosencephalon (prosomere 1) is at least partially transformed to a mesencephalic fate. This transformation results in a specific deficit of posterior commissure axons. Sey/Sey embryos also exhibit an axon pathfinding defect specific to the first longitudinal tract in the prosencephalon (tpoc, tract of the postoptic commissure). In wild type, tpoc axons fan out upon coming in contact with a superficial patch of Pax-6+ neuron cell bodies. In the mutant, the tpoc axons have normal initial projections, but make dramatic errors where they contact the neuron cell bodies, and fail to pioneer this first tract. Thus Pax-6 is required for local navigational information used by axons passing through its domain of expression.

We conclude that Pax-6 plays multiple roles in forebrain patterning, including boundary formation, regional patterning, neuron specification and axon guidance.
Reassessing embryogenesis in the Ctenophora: the inductive role of e1 micromeres in organizing ctene row formation in the ‘mosaic’ embryo, Mnemiopsis leidyi
Authors: Martindale Mark Q. , Henry Jonathan Q.
Published: April 2021

Ctenophores are a phylum of diploblastic marine animals displaying biradial symmetry organized along an oral aboral axis. One of the apomorphic sets of adult structures in ctenophores are the eight external comb rows, which run along the oral-aboral axis. Comb rows consist of serial arrays of individual comb plates of cilia, which beat in a coordinated fashion for locomotory behavior. Classical cell lineage experiments using chalk particles indicated that comb rows are derived exclusively from the four e1 micromeres at the 16-cell stage. This conclusion was also supported by the fact that no ctene rows (or their underly ing endodermal canals) form when all four e1 micromeres were deleted. We have used intracellular diI cell lineage tracing to determine that, in addition to e1 micromeres, the four m1 micromeres also make significant contributions to the ctene rows. Thus, e1 micromere derivatives not only generate comb plates but are required for ctene row formation by m1 derivatives. These results demonstrate that inductive interactions are an important component of early development in ctenophores and indicate that e1 micromeres influence the development of adjacent cell lineages (both m1 and endodermal lineages) during ctenophore embryogenesis. In addition, intracellular labeling has revealed that there are subtle variations in the composition of clones derived from identified embryonic blastomeres. Together these findings reveal a picture of ctenophore embryogenesis, which is in marked contrast to the former rigid ‘mosaic’ reputation of ctenophore devel opment, and invite speculation as to the role of the cleavage program in embryonic patterning in the lower Metazoa.
Tight junction assembly during mouse blastocyst formation is regulated by late expression of ZO-1 α+ isoform
Authors: Sheth Bhavwanti , Fesenko Ira , Collins Jane E. , Moran Breda , Wild Arthur E. , Anderson James M. , Fleming Tom P.
Published: April 2021

The mouse preimplantation embryo has been used to investigate the de novo synthesis of tight junctions during trophectoderm epithelial differentiation. We have shown previously that individual components of the tight junction assemble in a temporal sequence, with membrane assembly of the cytoplasmic plaque protein ZO-1 occurring 12 hours before that of cingulin. Subsequently, two alternatively spliced isoforms of ZO-1 (α+ and α−), differing in the presence or absence of an 80 residue α domain were reported. Here, the temporal and spatial expression of these ZO-1 isoforms has been investigated at different stages of preimplantation development. ZO-1α−mRNA was present in oocytes and all preimplantation stages, whilst ZO-1α +transcripts were first detected in embryos at the morula stage, close to the time of blastocoele formation. mRNAs for both isoforms were detected in trophectoderm and ICM cells. Immunoprecipitation of 33S-labelled embryos also showed synthesis of ZO-1α − throughout cleavage, whereas synthesis of ZO-1α +was only apparent from the blastocyst stage. In addition, 33P-labelling showed both isoforms to be phosphorylated at the early blastocyst stage. The pattern and timing of membrane assembly of the two isoforms was also distinct. ZO-1α− was initially seen as punctate sites at the cell-cell contacts of compact 8-cell embryos. These sites then coalesced laterally along the

membrane until they completely surrounded each cell with a zonular belt by the late morula stage. ZO-1α +however, was first seen as perinuclear foci in late morulae before assembling at the tight junction. Membrane assembly of ZO-1α +first occurred during the 32-cell stage and was zonular just prior to the early blastocyst stage. Immunostaining indicative of both isoforms was restricted to the trophectoderm lineage. Membrane assembly of ZO-1α +and blastocoele formation were sensitive to brefeldin A, an inhibitor of intracellular trafficking beyond the Golgi complex. In addition, the tight junction transmembrane protein occludin co-localised with ZO-1α +at the perinuclear sites in late morulae and at the newly assembled cell junctions. These results provide direct evidence from a native epithelium that ZO-1 isoforms perform distinct roles in tight junction assembly. Moreover, the late expression of ZO-1α +and its apparent intracellular interaction with occludin may act as a final rate-limiting step in the synthesis of the tight junction, thereby regulating the time of junction sealing and blastocoele formation in the early embryo.
Müller-cell-derived leukaemia inhibitory factor arrests rod photoreceptor differentiation at a postmitotic pre-rod stage of development
Authors: Neophytou Costas , Vernallis Ann B. , Smith Austin , Raff Martin C.
Published: April 2021

In the present study, we examine rod photoreceptor development in dissociated-cell cultures of neonatal mouse retina. We show that, although very few rhodopsin+ rods develop in the presence of 10% foetal calf serum (FCS), large numbers develop in the absence of serum, but only if the cell density in the cultures is high. The rods all develop from nondividing rhodopsin−cells, and new rods continue to develop from rhodopsin− cells for at least 6-8 days, indicating that there can be a long delay between when a precursor cell withdraws from the cell cycle and when it becomes a rhodopsin+ rod. We show that FCS arrests rod development in these cultures at a postmitotic, rhodopsin−, pre-rod stage. We present evidence that FCS acts indirectly by stimulating the proliferation of Müller cells, which arrest rod differentiation by releasing leukaemia inhibitory factor (LIF). These findings identify an inhibitory cell-cell interaction, which may help to explain the long delay that can occur both in vitro and in vivo between cell-cycle withdrawal and rhodopsin expression during rod development.
The Drosophila kinesin-like protein KLP3A is required for proper behavior of male and female pronuclei at fertilization
Authors: Williams Byron C. , Dernburgs Abby F. , Puro Jaakko , Nokkala Seppo , Goldberg Michael L.
Published: April 2021

Drosophila melanogaster females homozygous for mutations in the gene encoding the kinesin-like protein KLP3A are sterile (Williams et al., 1995). We have investi-gated the basis of this sterility. The eggs produced by KLP3A mutant mothers are fertilized by sperm, and female meiosis appears to occur normally. However, the large majority of these embryos arrest their development soon thereafter with a characteristic phenotype. The four nuclei produced by female meiosis associate together in a polar body-like structure, while a bipolar spindle is established around the metaphase-arrested male pronucleus. Thus, the major defect caused by depletion of the KLP3A protein is either in specification of the female pronucleus, or in migration of the male and female pronuclei toward each other. We have also found that the KLP3A protein is located throughout the metaphase spindle during meiosis and the early embryonic mitotic divisions, but later accu-mulates specifically at the midzone of these same spindles during telophase. The protein is also present on two other microtubule structures: the sperm aster; and the radial, monastral array of microtubules established between the two meiosis II spindles. We discuss these results in light of possible functions of the KLP3A protein in pronuclear specification and migration.
A role for Ultraspiracle, the Drosophila RXR, in morphogenetic furrow movement and photoreceptor cluster formation
Authors: Zelhof Andrew C. , Ghbeish Nora , Tsai Chihcheng , Evans Ronald M. , McKeown Michael
Published: April 2021

Many of the same genes needed for proper eye and limb development in vertebrates, such as hairy, hedgehog, patched and cyclic AMP-dependent protein kinase A, are responsible for patterning Drosophila imaginal discs, the tissues that will give rise to the adult cuticle structures. This is well demonstrated in the control of morphogenetic furrow movement and differentiation in the eye imaginal disc. We report that ultraspiracle, the gene encoding the Drosophila cognate of the Retinoid X Receptor, is required for normal morphogenetic furrow movement and ommatidial cluster formation. Examination of the expression of genes involved in regulating the furrow suggests that ultraspiracle defines a novel regulatory pathway in eye differentiation.
The differentiation of the serotonergic neurons in the Drosophila ventral nerve cord depends on the combined function of the zinc finger proteins Eagle and Huckebein
Authors: Dittrich R. , Bossing T. , Gould A. P. , Technau G. M. , Urban J.
Published: April 2021

The Drosophila ventral nerve cord (vNC) derives from a stereotyped population of neural stem cells, neuroblasts (NBs), each of which gives rise to a characteristic cell lineage. The mechanisms leading to the specification and differentiation of these lineages are largely unknown. Here we analyse mechanisms leading to cell differentiation within the NB 7-3 lineage. Analogous to the grasshopper, NB 7-3 is the progenitor of the Drosophila vNC serotonergic neurons. The zinc finger protein Eagle (Eg) is expressed in NB 7-3 just after delamination and is present in all NB 7-3 progeny until late stage 17. DiI cell lineage tracing and immunocytochemistry reveal that eg is required for normal pathfinding of interneuronal projections and for restricting the cell number in the thoracic NB 7-3 lineage. Moreover, eg is required for serotonin expression. Ectopic expression of Eg protein forces specific additional CNS cells to enter the serotonergic differentiation pathway. Like NB 7-3, the progenitor(s) of these ectopic cells express Huckebein (Hkb), another zinc finger protein. However, their progenitors do not express engrailed (en) as opposed to the NB 7-3 lineage, where en acts upstream of eg. We conclude that eg and hkb act in concert to determine serotonergic cell fate, while en is more distantly involved in this process by activating eg expression. Thus, we provide the first functional evidence for a combinatorial code of transcription factors acting early but downstream of segment polarity genes to specify a unique neuronal cell fate.
Gli1 is a target of Sonic hedgehog that induces ventral neural tube development
Authors: Lee Jeffrey , Platt Kenneth A. , Censullo Patricia , Altaba Ariel Ruizi
Published: April 2021

The vertebrate zinc finger genes of the Gli family are homologs of the Drosophila gene cubitus interruptus. In frog embryos, Gli1 is expressed transiently in the prospective floor plate during gastrulation and in cells lateral to the midline during late gastrula and neurula stages. In contrast, Gli2 and Gli3 are absent from the neural plate midline with Gli2 expressed widely and Gli3 in a graded fashion with highest levels in lateral regions. In mouse embryos, the three Gli genes show a similar pattern of expression in the neural tube but are coexpressed throughout the early neural plate. Because Gli1 is the only Gli gene expressed in prospective floor plate cells of frog embryos, we have investigated a possible involvement of this gene in ventral neural tube development. Here we show that Shh signaling activates Gli1 transcription and that widespread expression of endogenous frog or human glioma Gli1, but not Gli3, in developing frog embryos results in the ectopic differentiation of floor plate cells and ventral neurons within the neural tube. Floor-plate-inducing ability is retained when cytoplasmic Gli1 proteins are forced into the nucleus or are fused to the VP16 transactivating domain. Thus, our results identify Gli1 as a midline target of Shh and suggest that it mediates the induction of floor plate cells and ventral neurons by Shh acting as a transcriptional regulator.
Reciprocal signaling between Drosophila epidermal muscle attachment cells and their corresponding muscles
Authors: Becker Shirly , Pasca Giora , Strumpf Dan , Min Li , Volk Talila
Published: April 2021

Directed intercellular interactions between distinct cell types underlie the basis for organogenesis during embryonic development. This paper focuses on the establishment of the final somatic muscle pattern in Drosophila, and on the possible cross-talk between the myotubes and the epidermal muscle attachment cells, occurring while both cell types undergo distinct developmental programs.

Our findings suggest that the stripe gene is necessary and sufficient to initiate the developmental program of epidermal muscle attachment cells. In stripe mutant embryos, these cells do not differentiate correctly. Ectopic expression of Stripe in various epidermal cells transforms these cells into muscle-attachment cells expressing an array of epidermal muscle attachment cell-specific markers. Moreover, these ectopic epidermal muscle attachment cells are capable of attracting somatic myotubes from a limited distance, providing that the myotube has not yet been attached to or been influenced by a closer wild-type attachment cell.

Analysis of the relationships between muscle binding and differentiation of the epidermal muscle attachment cell was performed in mutant embryos in which loss of muscles, or ectopic muscles were induced. This analysis indicated that, although the initial expression of epidermal muscle-attachment cell-specific genes including stripe and groovin is muscle independent, their continuous expression is maintained only in epidermal muscle attachment cells that are connected to muscles. These results suggest that the binding of a somatic muscle to an epidermal muscle attachment cell triggers a signal affecting gene expression in the attachment cell.

Taken together, our results suggest the presence of a reciprocal signaling mechanism between the approaching muscles and the epidermal muscle attachment cells. First the epidermal muscle attachment cells signal the myotubes and induce myotube attraction and adhesion to their target cells. Following this binding, the muscle cells send a reciprocal signal to the epidermal muscle attachment cells inducing their terminal differentiation into tendon-like cells.
Dorsal hindbrain ablation results in rerouting of neural crest migration and changes in gene expression, but normal hyoid development
Authors: Saldivar Jose R. , Sechrist John W. , Krull Catherine E. , Ruffins Seth , Bronner-Fraser Marianne
Published: April 2021

Our previous studies have shown that hindbrain neural tube cells can regulate to form neural crest cells for a limited time after neural fold removal (Scherson, T., Serbedzija, G., Fraser, S. E. and Bronner-Fraser, M. (1993). Development 188, 1049-1061; Sechrist, J., Nieto, M. A., Zamanian, R. T. and Bronner-Fraser, M. (1995). Development 121, 4103-4115). In the present study, we ablated the dorsal hindbrain at later stages to examine possible alterations in migratory behavior and/or gene expression in neural crest populations rostral and caudal to the operated region. The results were compared with those obtained by misdirecting neural crest cells via rhombomere rotation. Following surgical ablation of dorsal r5 and r6 prior to the 10 somite stage, r4 neural crest cells migrate along normal pathways toward the second branchial arch. Similarly, r7 neural crest cells migrate primarily to the fourth branchial arch. When analogous ablations are performed at the 1012 somite stage, however, a marked increase in the numbers of DiI/Hoxa-3-positive cells from r7 are observed within the third branchial arch. In addition, some DiI-labeled r4 cells migrate into the depleted hindbrain region and the third branchial arch. During their migration, a subset of these r4 cells up-regulate Hoxa-3, a transcript they do not normally express. Krox20 transcript levels were augmented after ablation in a population of neural crest cells migrating from r4, caudal r3 and rostral r3. Long-term survivors of bilateral ablations possess normal neural crest-derived cartilage of the hyoid complex, suggesting that misrouted r4 and r7 cells contribute to cranial derivatives appropriate for their new location. In contrast, misdirecting of the neural crest by rostrocaudal rotation of r4 through r6 results in a reduction of Hoxa-3 expression in the third branchial arch and corresponding deficits in third archderived structures of the hyoid apparatus. These results demonstrate that neural crest/tube progenitors in the hindbrain can compensate by altering migratory trajectories and patterns of gene expression when the adjacent neural crest is removed, but fail to compensate appropriately when the existing neural crest is misrouted by neural tube rotation.
Control of cell fates and segmentation in the Drosophila mesoderm
Authors: Riechmann Veit , Irion Uwe , Wilson Robert , Grosskortenhaus Ruth , Leptin Maria
Published: April 2021

The primordia for heart, fat body, and visceral and somatic muscles arise in specific areas of each segment in the Drosophila mesoderm. We show that the primordium of the somatic muscles, which expresses high levels of twist, a crucial factor of somatic muscle determination, is lost in sloppy-paired mutants. Simultaneously, the primordium of the visceral muscles is expanded. The visceral muscle and fat body primordia require even-skipped for their development and the mesoderm is thought to be unsegmented in even-skipped mutants. However, we find that even-skipped mutants retain the segmental modulation of the expression of twist. Both the domain of even-skipped function and the level of twist expression are regulated by sloppy-paired. sloppy-paired thus controls segmental allocation of mesodermal cells to different fates.
Musculoskeletal patterning in the pharyngeal segments of the zebrafish embryo
Authors: Schilling Thomas F. , Kimmel Charles B.
Published: April 2021

The head skeleton and muscles of the zebrafish develop in a stereotyped pattern in the embryo, including seven pharyngeal arches and a basicranium underlying the brain and sense organs. To investigate how individual cartilages and muscles are specified and organized within each head segment, we have examined their early differentiation using Alcian labeling of cartilage and expression of several molecular markers of muscle cells. Zebrafish larvae begin feeding by four days after fertilization, but cartilage and muscle precursors develop in the pharyngeal arches up to 2 days earlier. These chondroblasts and myoblasts lie close together within each segment and differentiate in synchrony, perhaps reflecting the interdependent nature of their patterning. Initially, cells within a segment condense and gradually become subdivided into individual dorsal and ventral structures of the differentiated arch. Cartilages or muscles in one segment show similar patterns of condensation and differentiation as their homologues in another, but vary in size and shape in the most anterior (mandibular and hyoid) and posterior (tooth-bearing) arches, possibly as a consequence of changes in the timing of their development. Our results reveal a segmental scaffold of early cartilage and muscle precursors and suggest that interactions between them coordinate their patterning in the embryo. These data provide a descriptive basis for genetic analyses of craniofacial patterning.
Pax-6 is involved in the specification of hindbrain motor neuron subtype
Authors: Osumi Noriko , Hirota Arisa , Ohuchi Hideyo , Nakafuku Masato , Iimura Tadahiro , Kuratani Shigeru , Fujiwara Michio , Noji Sumihare , Eto Kazuhiro
Published: April 2021

Pax-6 is a member of the vertebrate Pax gene family, which is structurally related to the Drosophila pair-rule gene, paired. In mammals, Pax-6 is expressed in several discrete domains of the developing CNS and has been implicated in neural development, although its precise role remains elusive. We found a novel Small eye rat strain (rSey2) with phenotypes similar to mouse and rat Small eye. Analyses of the Pax-6 gene revealed one base (C) insertion in an exon encoding the region downstream of the paired box of the Pax-6 gene, resulting in generation of truncated protein due to the frame shift. To explore the roles of Pax-6 in neural development, we searched for abnormalities in the nervous system in rSey2 homozygous embryos. rSey2/rSey2 exhibited abnormal development of motor neurons in the hindbrain. The Islet-1-positive motor neurons were generated just ventral to the Pax-6-expressing domain both in the wild-type and mutant embryos. However, two somatic motor (SM) nerves, the abducent and hypoglossal nerves, were missing in homozygous embryos. By retrograde and anterograde labeling, we found no SM-type axonogenesis (ventrally growing) in the mutant postotic hindbrain, though branchiomotor and visceral motor (BM/VM)-type axons (dorsally growing) were observed within the neural tube. To discover whether the identity of these motor neuron subtypes was changed in the mutant, we examined expression of LIM homeobox genes, Islet-1, Islet-2 and Lim-3. At the postotic levels of the hindbrain, SM neurons expressed all the three LIM genes, whereas BM/VM-type neurons were marked by Islet-1 only. In the Pax-6 mutant hindbrain, Islet-2 expression was specifically missing, which resulted in the loss of the cells harboring the postotic hindbrain SM-type LIM code (Islet-1 + Islet-2 + Lim-3). Furthermore, we found that expression of Wnt-7b, which overlapped with Pax-6 in the ventrolateral domain of the neural tube, was also specifically missing in the mutant hindbrain, while it remained intact in the dorsal non-over-lapping domain. These results strongly suggest that Pax-6 is involved in the specification of subtypes of hindbrain motor neurons, presumably through the regulation of Islet-2 and Wnt-7b expression.
Antagonism between EGFR and Wingless signalling in the larval cuticle of Drosophila
Authors: Szüts Dávid , Freeman Matthew , Bienz Mariann
Published: April 2021

Signalling by the epidermal growth factor receptor (EGFR) plays a critical role in the segmental patterning of the ventral larval cuticle in Drosophila: by expressing a dominant-negative EGFR molecule or Spitz, an activating ligand of EGFR, we show that EGFR signalling specifies the anterior denticles in each segment of the larval abdomen. We provide evidence that these denticles derive from a segmental zone of embryonic cells in which EGFR signalling activity is maximal. Within each segment, there is a competition between the denticle fate specified by EGFR signalling and the naked cuticle fate specified by Wingless signalling. The final pattern of the denticle belts is the product of this antagonism between the two signalling pathways. Finally, we show that the segmental zones of high EGFR signalling activity depend on bithorax gene function and that they account for the main difference in shape between abdominal and thoracic denticle belts.
Ectopic expression of the maize kn1 gene phenocopies the Hooded mutant of barley
Authors: Williams-Carrier Rosalind E. , Lie Yung S. , Hake Sarah , Lemaux Peggy G.
Published: April 2021

The homeobox gene, knotted1, (kn1) is expressed in shoot meristems and is required for maintaining indeterminacy and preventing cellular differentiation. Awns, extensions of the bract-like lemma found in all grass inflorescences, are normally determinate structures. We show that ectopic expression of kn1 in the barley awn is sufficient to direct the development of ectopic meristems, forming inflorescence-like structures. This homeotic transformation is similar to the phenotype produced by misexpression of the barley hvknox3 gene, associated with the dominant Hooded mutant (Müller, K. J., Romano, N., Gerstner, O., Garcia-Maroto, F., Pozzi, C., Salamini, F. and Rohde, W. (1995) Nature 374, 727-730). We suggest that the inverse polarity of the ectopic flowers seen in Hooded and transgenic kn1 plants results from the transformation of the awn into reiterative inflorescence axes. We observed that the protein and mRNA localization of the transgene, driven by a constitutive promoter, is similar to the expression pattern of hvknox3 in awns of Hooded mutants, suggesting posttranscriptional regulation.