bluuebunny/crossref_metadata_2025 huggingface dataset:
ventral veinless, a POU domain transcription factor, regulates different transduction pathways required for tracheal branching in Drosophila
Authors: Llimargas Marta , Casanova Jordi
Published: April 2021

Cell migration is an important step in a variety of developmental processes in many multicellular organisms. A particularly appropriate model to address the study of cell migration is the tracheal system of Drosophila, whose formation occurs by migration and fusion from clusters of ectodermal cells specified in each side of ten embryonic segments. Morphogenesis of the tracheal tree requires the activity of many genes, among them breathless (btl) and ventral veinless (vvl) whose mutations abolish tracheal cell migration. Activation of the btl receptor by branchless (bnl), its putative ligand, exerts an instructive role in the process of guiding tracheal cell migration. vvl has been shown to be required for the maintenance of btl expression during tracheal tree formation. Here we show that, in addition, vvl is independently required for the specific expression in the tracheal cells of thick veins (tkv) and rhomboid (rho), two genes whose mutations disrupt only particular branches of the tracheal system. Indeed, we show that expression in the tracheal cells of an activated form of tkv, the putative decapentaplegic (dpp) receptor, is able to induce shifts in their migration, asserting the role of the dpp pathway in establishing the branching pattern of the tracheal tree. In addition, by ubiquitous expression of the btl and tkv genes in vvl mutant embryos we show that both genes contribute to vvl function. These results indicate that through activation of its target genes, vvl makes the tracheal cells competent to further signalling and suggest that the btl transduction pathway could collaborate with other transduction pathways also regulated by vvl to specify the tracheal branching pattern.
Homeotic genes and the regulation of myoblast migration, fusion, and fibre-specific gene expression during adult myogenesis in Drosophila
Authors: Roy Sudipto , VijayRaghavan K.
Published: April 2021

We have investigated the roles of homeotic selector genes in the migration and fusion of myoblasts, and in the differentiation of adult muscle fibres of Drosophila. Altering intrinsic homeotic identities of myoblasts does not affect their segment-specific migration patterns. By transplanting meso – and metathoracic myoblasts into the abdomen, we demonstrate that the fusion abilities of myoblasts are independent of their segmental identities. However, trans-planted thoracic myoblast nuclei are ‘entrained’ by those of the host abdominal muscles to which they fuse and are unable to ‘switch on’ a thoracic muscle-specific reporter gene. This process is likely to be mediated by homeotic repression because mis-expression of an abdominal muscle-specific homeotic gene, Ultrabithorax, in the thoracic muscles results in the repression of the thoracic muscle-specific reporter gene. Finally, we show that removal of Ultrabithorax function specifically from muscle cells of the first abdominal segment, results in the expression of thoracic muscle properties. Many of these functions of homeotic genes in muscle patterning in Drosophila could be conserved during myogenesis in other organisms.
A dominant-negative form of Serrate acts as a general antagonist of Notch activation
Authors: Hukriede Neil A. , Gu Yi , Fleming Robert J.
Published: April 2021

Specification of the dorsal-ventral compartment boundary in the developing Drosophila wing disc requires activation of NOTCH from its dorsal ligand SERRATE and its ventral ligand DELTA. Both NOTCH ligands are required in this process and one cannot be substituted for the other. In the wing disc, expression of a dominant-negative, truncated form of SERRATE called BDG, is capable of inhibiting NOTCH activation in the ventral but not the dorsal compartments. We demonstrate that BDG can act as a general antagonist of both SERRATE and DELTA mediated NOTCH interactions, however, BDG retains the SERRATE protein domain targeted by FRINGE, hence its antagonistic effects are restricted in the dorsal wing disc. Our findings suggest a model in which ligand binding to NOTCH is a necessary but insufficient step toward NOTCH activation.
Sequential expression and role of Hu RNA-binding proteins during neurogenesis
Authors: Wakamatsu Yoshio , Weston James A.
Published: April 2021

We have identified three avian (chicken) Hu/elav family RNA-binding protein genes. cHuD and cHuC are expressed specifically in neurons of both the central and peripheral nervous systems. Although cHuA is expressed in a wide variety of tissues, including neurogenic precursor cells, it is transiently down-regulated, and is then re-expressed in maturing neurons. Misexpression of cHuD in cultured neural crest cells results in a dramatic increase in the proportion of cells exhibiting neuronal morphology, molecular markers for neurons, and neurotrophin dependence. These data confirm that cHuD protein is involved in regulating neuronal differentiation.
Nitric oxide, an endogenous regulator of Dictyostelium discoideum differentiation
Authors: Tao Y. P. , Misko T. P. , Howlett A. C. , Klein C.
Published: April 2021

We have previously demonstrated that nitric oxide (NO)- generating compounds inhibit D. discoideum differentiation by preventing the initiation of cAMP pulses (Tao, Y., Howlett, A. and Klein, C. (1996) Cell. Signal. 8, 37-43). In the present study, we demonstrate that cells produce NO at a relatively constant rate during the initial phase of their developmental cycle. The addition of oxyhemoglobin, an NO scavenger, stimulates cell aggregation, suggesting that NO has a negative effect on the development of aggregation competence. Starvation of cells in the presence of glucose, which has been shown to prevent the initiation of cAMP pulses (Darmon, M. and Klein, C. (1978) Dev. Biol. 63, 377-389), results in an increased production of NO. The inhibition of cell aggregation by glucose treatment can be reversed by oxyhemoglobin. These findings indicate that NO is a signaling molecule for D. discoideum cells and that physiological or environmental conditions that enhance external NO levels will delay the initiation of cAMP pulses, which are essential for cell differentiation.
Argos and Spitz group genes function to regulate midline glial cell number in Drosophila embryos
Authors: Stemerdink Christopher , Jacobs J. Roger
Published: April 2021

The midline glia of the Drosophila embryonic nerve cord undergo a reduction in cell number after facilitating com-missural tract morphogenesis. The numbers of midline glia entering apoptosis at this stage can be increased by a loss or reduction of function in genes of the spitz group or Drosophila EGF receptor (DER) pathway. Argos, a secreted molecule with an atypical EGF motif, is postulated to function as a DER antagonist. In this work, we assess the role of argos in the determination of midline glia cell number. Although all midline glia express DER, argos expression is restricted to the midline glia which do not enter apoptosis. Fewer midline glia enter apoptosis in embryos lacking argos function. Ectopic expression of argos is sufficient to remove all DER-expressing midline glia from the nerve cord, even those that already express argos. DER expression is not terminated in the midline glia after spitz group signaling triggers changes in gene expression. It is therefore likely that an attenuation of DER signaling by Argos is integrated with the augmentation of DER signaling by Spitz throughout the period of reduction of midline glia number. We suggest that signaling by Spitz but not Argos is restricted to adhesive junctions. In this manner, midline glia not forming signaling junctions remain sensitive to juxtacrine Argos signaling, while an autocrine Argos signal is excluded by the adhesive junction.
An activity-dependent network of interactions links the Rel protein Dorsal with its cytoplasmic regulators
Authors: Edwards David N. , Towb Par , Wasserman Steven A.
Published: April 2021

A signaling pathway active on the ventral side of the Drosophila embryo defines dorsoventral polarity. A cell surface signal relayed by Toll, Tube and Pelle releases the Rel-related protein Dorsal from its cytoplasmic inhibitor Cactus; free Dorsal translocates into nuclei and directs expression of ventral fates. Using the yeast two-hybrid system and immunoprecipitation experiments, we define scaffolding and anchoring interactions among the pathway components. We show that Dorsal binds specifically to Tube, Pelle and Cactus, and that the protein kinase activity of Pelle differentially regulates its interactions with Dorsal and Tube. We also identify Drosophila Filamin as a potential adaptor linking the interaction network, via Tube, to the transmembrane receptor Toll.
Ectopic activation of torpedo/Egfr, a Drosophila receptor tyrosine kinase, dorsalizes both the eggshell and the embryo
Authors: Queenan Anne Marie , Ghabrial Amin , Schüpbach Trudi
Published: April 2021

The Drosophila gene torpedo/Egfr (top/Egfr) encodes a homolog of the vertebrate Epidermal Growth Factor receptor. This receptor is required several times during the life cycle of the fly for the transmission of developmental cues. During oogenesis, Top/Egfr activation is required for the establishment of the dorsal/ventral axis of the egg and the embryo. To examine how ectopic Top/Egfr activation affects cell fate determination, we constructed an activated version of the protein. Expression of this activated form (λtop) in the follicle cells of the ovary induces dorsal cell fates in both the follicular epithelium and the embryo. Different levels of expression resulted in different dorsal follicle cell fates. These dorsal cell fates were expanded in the anterior, but not the posterior, of the egg, even in cases where all the follicle cells covering the oocyte expressed λtop. The expression of genes known to respond to top/Egfr activation, argos (aos), kekkon1 (kek 1) and rhomboid (rho), was also expanded in the presence of the λtop construct. When λtop was expressed in all the follicle cells covering the oocyte, kek 1 and argos expression was induced in follicle cells all along the anterior/posterior axis of the egg chamber. In contrast, rho RNA expression was only activated in the anterior of the egg chamber. These data indicate that the response to Top/Egfr signaling is regulated by an anterior/posterior prepattern in the follicle cells.

Expression of λtop in the entire follicular epithelium resulted in an embryo dorsalized along the entire anterior/posterior axis. Expression of λtop in anterior or posterior subpopulations of follicle cells resulted in regionally autonomous dorsalization of the embryos. This result indicates that subpopulations of follicle cells along the anterior/posterior axis can respond to Top/Egfr activation independently of one another.
Integrins mediate adhesion to agrin and modulate agrin signaling
Authors: Martin Paul T. , Sanes Joshua R.
Published: April 2021

Agrin, a basal lamina-associated proteoglycan, is a crucial nerve-derived organizer of postsynaptic differentiation at the skeletal neuromuscular junction. Because integrins serve as cellular receptors for many basal lamina components, we asked whether agrin interacts with integrins. Agrin-induced aggregation of acetylcholine receptors on cultured myotubes was completely blocked by antibodies to the β1 integrin subunit and partially blocked by antibodies to the αv integrin subunit. Agrin-induced clustering was also inhibited by antisense oligonucleotides to αv and a peptide that blocks the αv binding site. Non-muscle cells that expressed αv and β1 integrin subunits adhered to immobilized agrin, and this adhesion was blocked by anti-αv and anti-β1 antibodies. Integrin αv-negative cells that did not adhere to agrin were rendered adherent by introduction of αv. Together, these results implicate integrins, including αvβ1, as components or modulators of agrin’s signal transduction pathway.
The early blastocyst is bilaterally symmetrical and its axis of symmetry is aligned with the animal-vegetal axis of the zygote in the mouse
Authors: Gardner R. L.
Published: April 2021

At least one polar body, almost invariably the second, persists intact to the early blastocyst stage in nearly two-thirds of mouse conceptuses of the PO strain. The distribution in early blastocysts of these surviving polar bodies was highly non-random. Most not only lay in the midregion of the embryonic-abembryonic axis but, on discovering that early blastocysts are bilaterally rather than radially symmetrical about this axis, were found to align with the bilateral axis. Cell marking experiments failed to detect movement of polar bodies relative to the surface of the conceptus during either cleavage or blastulation. That the distribution of degenerating polar bodies and their presumed debris was similar to intact ones also argued against their motility, as did the finding that at all stages second polar bodies were attached to conceptuses by a thin, extensible, weakly elastic ‘tether’. Although the transfer of small fluorochromes between them was rarely observed beyond second cleavage, the second polar body and conceptus could remain coupled ionically up to the blastocyst stage. It is concluded that the second polar body normally remains attached to the conceptus through persistence of the intercellular bridge formed during its abstriction, and therefore provides an enduring marker of the animal pole of the zygote. Hence, according to the distribution of polar bodies, the axis of bilateral symmetry of the early blastocysts is normally aligned with the animalvegetal axis of the zygote and its embryonic-abembryonic axis is orthogonal to it. Such relationships suggest that, at least in undisturbed development, specification of the axes of the blastocyst depends on spatial patterning of the zygote.
The Drosophila TGFα homolog Spitz acts in photoreceptor recruitment in the developing retina
Authors: Tio Murni , Moses Kevin
Published: April 2021

In vertebrates and Drosophila, the Epidermal Growth Factor Receptor (EGFR) signal transduction pathway is important in the regulation of cellular development. EGFR is bound by several activating ligands including Trans-forming Growth Factor-α in vertebrates, and its homolog Spitz in Drosophila. It has been shown that Spitz and EGFR act in the development of the Drosophila central nervous system and compound eye. Here we show that spitz function is required in developing ommatidia for the first cell recruitment step, and that Spitz pro-protein is expressed in the retinal neurons as they begin to differentiate. We propose a ‘two-key’ model for additive signal transduction from EGFR and other receptor tyrosine kinases, via the Ras pathway, in the developing eye.
Two novel chick T-box genes related to mouse Brachyury are expressed in different, non-overlapping mesodermal domains during gastrulation
Authors: Knezevic Vladimir , De Santo Ronald , Mackem Susan
Published: April 2021

The mouse Brachyury (T) gene plays critical roles in the genesis of normal mesoderm during gastrulation and in the maintenance of a functioning notochord. Abrogation of Brachyury (T) expression within the chordamesoderm of homozygous null mutants nevertheless spares anterior axis formation. An intriguing possibility to explain the preservation of anterior axis formation in these mutants would be the existence of other genes compensating for the loss of Brachyury. This compensation and the recent demonstration that Brachyury is the prototype for an evolutionarily conserved family, prompted a search for other T-box genes participating in axis formation. The chick Brachyury orthologue and two related chick T-box genes that are expressed at the onset of gastrulation have been isolated. One of these novel genes (Ch-TbxT) becomes restricted to the axial mesoderm lineage and is a potential candidate for complementing or extending Brachyury function in the anterior axis (formation of the head process, prechordal plate). The other gene (Ch-Tbx6L), together with chick T, appears to mark primitive streak progenitors before gastrulation. As cells leave the primitive streak, Ch-Tbx6L becomes restricted to the early paraxial mesoderm lineage and could play a role in regulating somitogenesis.
Spindlin, a major maternal transcript expressed in the mouse during the transition from oocyte to embryo
Authors: Oh Bermseok , Hwang Sue-Yun , Solter Davor , Knowles Barbara B.
Published: April 2021

Timely translation of maternal transcripts and post-translational modification of their gene products control the initial development of preimplantation-stage embryos. We have isolated and characterized a gene encoding a stage-specific embryonic protein. This novel gene, spindlin (Spin), is an abundant maternal transcript present in the unfertilized egg and 2-cell, but not 8-cell, stage embryo. Spin exhibits high homology to a multicopy gene, Y-linked spermiogenesis-specific transcript (Ssty), and together they form a new gene family expressed during gametogenesis. We find that spindlin associates with the meiotic spindle and is modified by phosphorylation in a cell-cycle-dependent fashion. Furthermore, it comigrates with the previously described 30×103Mr metaphase complex which is post-translationally modified during the first mitotic cell cycle. Our data suggest that spindlin plays a role in cell-cycle regulation during the transition from gamete to embryo.
Pax3 is required for cardiac neural crest migration in the mouse: evidence from the splotch (Sp
2H) mutant
Authors: Conway Simon J. , Henderson Deborah J. , Copp Andrew J.
Published: April 2021

Neural crest cells originating in the occipital region of the avian embryo are known to play a vital role in formation of the septum of the cardiac outflow tract and to contribute cells to the aortic arches, thymus, thyroid and parathyroids. This ‘cardiac’ neural crest sub-population is assumed to exist in mammals, but without direct evidence. In this paper we demonstrate, using RT-PCR and in situ hybridisation, that Pax3 expression can serve as a marker of cardiac neural crest cells in the mouse embryo. Cells of this lineage were traced from the occipital neural tube, via branchial arches 3, 4 and 6, into the aortic sac and aortopulmonary outflow tract. Confirmation that these Pax3-positive cells are indeed cardiac neural crest is provided by experiments in which hearts were deprived of a source of colonising neural crest, by organ culture in vitro, with consequent lack of up-regulation of Pax3. Occipital neural crest cell outgrowths in vitro were also shown to express Pax3. Mutation of Pax3, as occurs in the splotch (Sp2H) mouse, results in development of conotruncal heart defects including persistent truncus arteriosus. Homozygotes also exhibit defects of the aortic arches, thymus, thyroid and parathyroids. Pax3-positive neural crest cells were found to emigrate from the occipital neural tube of Sp2H/Sp2H embryos in a relatively normal fashion, but there was a marked deficiency or absence of neural crest cells traversing branchial arches 3, 4 and 6, and entering the cardiac outflow tract. This decreased expression of Pax3 in Sp2H/Sp2H embryos was not due to down-regulation of Pax3 in neural crest cells, as use of independent neural crest markers, Hoxa-3, CrabpI, Prx1, Prx2 and c-met also revealed a deficiency of migrating cardiac neural crest cells in homozygous embryos. This work demonstrates the essential role of the cardiac neural crest in formation of the heart and great vessels in the mouse and, furthermore, shows that Pax3 function is required for the cardiac neural crest to complete its migration to the developing heart.
Retinoid receptors promote primary neurogenesis in Xenopus
Authors: Sharpe C. R. , Goldstone K.
Published: April 2021

Retinoid receptors, which are members of the nuclear hormone receptor superfamily, act as ligand-dependent transcription factors. They mediate the effects of retinoic acid primarily as heterodimers of retinoic acid receptors (RARs) and retinoid X receptors (RXRs). To analyse their function, xRXR β synthetic mRNA was injected into Xenopus embryos in combination with normal and mutated xRAR α transcripts. Two informative phenotypes are reported here. Firstly, over-expression of xRXR β with xRAR α results in the formation of ectopic primary neurons. Secondly, blocking retinoid signalling with a mutated xRAR α results in a lack of primary neurons. These two phenotypes, from contra-acting manipulations, indicate a role for retinoid signalling during neurogenesis.
Wnt-11 is expressed in early avian mesoderm and required for the differentiation of the quail mesoderm cell line QCE-6
Authors: Eisenberg Carol A. , Gourdie Robert G. , Eisenberg Leonard M.
Published: April 2021

The beginning of mesodermal development involves the aggregation of newly gastrulated cells into epithelial fields, as a prelude to organ formation. To analyze the molecular regulation of this initial patterning, we have focused on the Wnt family of secreted signaling proteins, molecules which have been shown to promote embryonic patterning by regulating cell-cell associations. In this study, we show that the Wnt-11 gene is expressed by newly gastrulated mesoderm cells within avian embryos. The expression pattern of Wnt-11 also suggests that it may be involved in formation of the cardiogenic fields and somites. Subsequently, we utilized the quail mesoderm cell line QCE-6 as a culture model for examining the influence of Wnt-11 on early mesoderm cell differentiation. This cell line has been shown to be representative of early nondifferentiated mesoderm cells and has the potential to differentiate into cardiomyocytes, endohelial or red blood cells. Similar to early mesoderm cells, QCE-6 cells express Wnt-11. We have engineered stable transfectants of these cells that produce either diminished or enhanced levels of Wnt-11 protein. Our studies show that Wnt-11 regulates cellular interactions of QCE-6 cells, as demonstrated by alterations in contact-inhibited growth, tight and gap junction formation and plakoglobin expression. Both the morphology and growth factor-induced differentiation of QCE-6 cells are regulated in a cooperative fashion by Wnt-11 and fibronectin. These results, described in detail below, demonstrate the uniqueness of QCE-6 cells as a culture system for analyzing Wnt activity. In particular, QCE-6 cells are the first cell line that has demonstrated: (1) Wnt-dependent differentiation; (2) concentration-variable responses to Wnt protein; and (3) altered cell phenotypes as a direct response to Wnt-5a class proteins (e.g. Wnt-4 and Wnt-11).
Expression of the Ly-6E.1 (Sca-1) transgene in adult hematopoietic stem cells and the developing mouse embryo
Authors: Miles Colin , Sanchez Maria-Jose , Sinclair Angus , Dzierzak Elaine
Published: April 2021

The mouse hematopoietic marker Sca-1, encoded by the Ly-6E.1 and Ly-6A.2 genes, has been instrumental in the enrichment and characterization of the stem cell for the adult blood system. In the studies reported here, we use Ly-6E.1 genomic fragments to direct expression of a lacZ marker transgene in vivo to study Ly-6E.1 specific regulatory elements in the hematopoietic stem cell and to localize these cells in the developing mouse embryo. We demonstrate that a region approximately 9 kb downstream from the transcriptional start site is required for the distinct, restricted expression pattern of the Ly-6E.1-lacZ transgene within adult hematopoietic stem cells and embryos. We also demonstrate that viable and functional lacZ-expressing hematopoietic stem cells can be enriched by FDG staining and flow cytometric sorting. The Ly-6E.1-lacZ-mediated enrichment of hematopoietic stem cells from adult transgenic bone marrow in combination with the temporal expression pattern of the transgene in the pro/mesonephros suggest an intraembryonic site of development for these cells in the mouse.
The Caenorhabditis elegans NK-2 homeobox gene ceh-22 activates pharyngeal muscle gene expression in combination with pha-1 and is required for normal pharyngeal development
Authors: Okkema Peter G. , Ha Eunju , Haun Christina , Chen Wei , Fire Andrew
Published: April 2021

Pharyngeal muscle development in the nematode Caenorhabditis elegans appears to share similarities with cardiac muscle development in other species. We have previously described CEH-22, an NK-2 class homeodomain transcription factor similar to Drosophila tinman and vertebrate Nkx2-5, which is expressed exclusively in the pharyngeal muscles. In vitro, CEH-22 binds the enhancer from myo-2, a pharyngeal muscle-specific myosin heavy chain gene. In this paper, we examine the role CEH-22 plays in pharyngeal muscle development and gene activation by (a) ectopically expressing ceh-22 in transgenic C. elegans and (b) examining the phenotype of a ceh-22 loss-of-function mutant. These experiments indicate that CEH-22 is an activator of myo-2 expression and that it is required for normal pharyngeal muscle development. However, ceh-22 is necessary for neither formation of the pharyngeal muscles, nor for myo-2 expression. Our data suggest parallel and potentially compensating pathways contribute to pharyngeal muscle differentiation. We also examine the relationship between ceh-22 and the pharyngeal organ-specific differentiation gene pha-1. Mutations in ceh-22 and pha-1 have strongly synergistic effects on pharyngeal muscle gene expression; in addition, a pha-1 mutation enhances the lethal phenotype caused by a mutation in ceh-22. Wild-type pha-1 is not required for the onset of ceh-22 expression but it appears necessary for maintained expression of ceh-22.
Hyaluronan is a prerequisite for ductal branching morphogenesis
Authors: Gakunga Peter , Frost Gregory , Shuster Svetlana , Cunha Gerald , Formby Bent , Stern Robert
Published: April 2021

Hyaluronan, a macromolecular carbohydrate polymer of the extracellular matrix is prominent early in embryogenesis, coinciding with rapid tissue growth. CD44, the pre-dominant receptor for hyaluronan on vertebrate cells, is a variably expressed transmembrane glycoprotein. Mouse anterior prostate glands obtained at various postnatal time points were examined for the expression of hyaluronan and CD44. Reverse transcriptase polymerase chain reaction analysis was used to map the temporal regulation of specific CD44 variant isoforms. In each age group, hyaluronan was localized exclusively in the stromal matrix. Hyaluronan was greatly reduced in the later ages and was entirely absent around the developmentally quiescent proximal regions of the ducts. Early in prostate development, CD44 was prominent in the mesenchyme. However, in the later phases, CD44 expression became associated with membranes of epithelial cells. The role of hyaluronan-CD44 interactions in ductal branching morphogenesis was studied by serum-free organ culture of mouse anterior prostate. In the presence of optimal levels of testosterone, the organs underwent ductal branching morphogenesis. Treatment with either neutralizing anti-CD44 antibodies, hyaluronan hexasaccharides or the enzyme hyaluronidase inhibited androgen-stimulated ductal branching morphogenesis. These results are suggestive of the significant role played by hyaluronan-CD44 interactions in mediating androgen-induced prostatic growth and morphogenesis.
Polydactyly and ectopic ZPA formation in Alx-4 mutant mice
Authors: Qu Shimian , Niswender Kevin D. , Ji Qunsheng , van der Meer Riet , Keeney Diane , Magnuson Mark A. , Wisdom Ron
Published: April 2021

Correct development of the limb is dependent on coordination between three distinct signaling centers. Recently, fibroblast growth factor-4 has been identified as a crucial determinant of AER function, which directs limb bud outgrowth, and Sonic hedgehog has been identified as a signaling molecule that mediates ZPA function, which specifies anterior-posterior patterning in the developing limb bud. In addition, Shh and FGF-4 reciprocally reinforce each other’s expression via a positive feedback loop, providing a molecular basis for the coordination of limb bud outgrowth and anterior-posterior patterning. The mechanisms by which these signaling centers come to occupy their normal positions in the posterior limb bud during development are not understood. Here we identify and characterize Alx-4, a gene that encodes a paired-type homeodomain protein. Alx-4 is expressed in several populations of mesenchymal cells, including mesenchymal cells in the anterior limb bud, and mice homozygous for targeted disruption of the Alx-4 gene have multiple abnormalities, including preaxial polydactyly. The polydactyly is associated with the formation of an ectopic anterior ZPA, as indicated by anterior expression of Sonic hedgehog, HoxD13 and fibroblast growth factor-4. The expression of other candidate regulators of anterior-posterior positional information in the limb bud, including HoxB8 and Gli3, is not altered in Alx-4 mutant embryos. By chromosomal mapping experiments, Alx-4 is tightly linked to Strong’s luxoid, a polydactylous mouse mutant. The results identify Alx-4 as a determinant of anterior-posterior positional identity in the limb and a component of a regulatory program that restricts ZPA formation to the posterior limb bud mesenchyme.
Involvement of RBP-J in biological functions of mouse Notch1 and its derivatives
Authors: Kato Hisamune , Taniguchi Yoshihito , Kurooka Hisanori , Minoguchi Shigeru , Sakai Takashi , Nomura-Okazaki Saeko , Tamura Kumiko , Honjo Tasuku
Published: April 2021

Notch is involved in the cell fate determination of many cell lineages. The intracellular region (RAMIC) of Notch1 trans-activates genes by interaction with a DNA binding protein RBP-J. We have compared the activities of mouse RAMIC and its derivatives in transactivation and differentiation sup-pression of myogenic precursor cells. RAMIC comprises two separate domains, IC for transactivation and RAM for RBP-J binding. Although the physical interaction of IC with RBP-J was much weaker than with RAM, transactivation activity of IC was shown to involve RBP-J by using an RBP-J null mutant cell line. IC showed differentiation suppression activity that was generally comparable to its transactivation activity. The RBP-J-VP16 fusion protein, which has strong transactivation activity, also suppressed myogenesis of C2C12. The RAM domain, which has no other activities than binding to RBP-J, synergistically stimulated transactivation activity of IC to the level of RAMIC. The RAM domain was proposed to compete with a putative co-repressor for binding to RBP-J because the RAM domain can also stimulate the activity of RBP-J-VP16. These results taken together, indicate that differentiation suppression of myogenic precursor cells by Notch signalling is due to transactivation of genes carrying RBP-J binding motifs.
Homophilic synaptic target recognition mediated by immunoglobulin-like cell adhesion molecule Fasciclin III
Authors: Kose Hiroyuki , Rose Demian , Zhu Xiaomao , Chiba Akira
Published: April 2021

We demonstrate that the cell adhesion molecule Fasciclin III (FAS3) mediates synaptic target recognition through homophilic interaction. FAS3 is expressed by the RP3 motoneuron and its target muscles during synaptic target recognition. The RP3 growth cone can form synapses on muscles that ectopically express FAS3. This mistargeting is dependent on FAS3 expression in the motoneurons. In addition, when the FAS3-negative aCC and SNa motoneuron growth cones ectopically express FAS3, they gain the ability to recognize FAS3-expressing muscles as alternative targets. We propose that homophilic synaptic target recog-nition serves as a basic mechanism of neural network formation.
The Drosophila 14-3-3 protein Leonardo enhances Torso signaling through D-Raf in a Ras1-dependent manner
Authors: Li Willis , Skoulakis Efthimios M. C. , Davis Ronald L. , Perrimon Norbert
Published: April 2021

14-3-3 proteins have been shown to interact with Raf-1 and cause its activation when overexpressed. However, their precise role in Raf-1 activation is still enigmatic, as they are ubiquitously present in cells and found to associate with Raf-1 in vivo regardless of its activation state. We have analyzed the function of the Drosophila 14-3-3 gene leonardo (leo) in the Torso (Tor) receptor tyrosine kinase (RTK) pathway. In the syncytial blastoderm embryo, acti-vation of Tor triggers the Ras/Raf/MEK pathway that controls the transcription of tailless (tll). We find that, in the absence of Tor, overexpression of leo is sufficient to activate tll expression. The effect of leo requires D-Raf and Ras1 activities but not KSR or DOS, two recently identi-fied essential components of Drosophila RTK signaling pathways. Tor signaling is impaired in embryos derived from females lacking maternal expression of leo. We propose that binding to 14-3-3 by Raf is necessary but not sufficient for the activation of Raf and that overexpressed Drosophila 14-3-3 requires Ras1 to activate D-Raf.
Hoxb-8 has a role in establishing early anterior-posterior polarity in chick forelimb but not hindlimb
Authors: Stratford Thomas H. , Kostakopoulou Konstadina , Maden Malcolm
Published: April 2021

The distribution of Hoxb-8 transcripts through the chick flank and early forelimb mirrors the distribution of polarizing activity in the flank at these early stages. Polarizing activity displayed by Hoxb-8-expressing tissue is only realised when placed adjacent to the AER and appears to be mediated through Shh induction, suggesting that Hoxb-8 may lie genetically upstream of Shh. Accordingly, Hoxb-8 expression is rapidly induced by retinoic acid (RA) treatment in the anterior of the forelimb in a spatial and temporal manner that is consistent with the induction of Shh and formation of the ZPA. Furthermore, inhibition of RA synthesis in the flank downregulates the expression of endogenous Hoxb-8 and results in the loss of Shh expression. However, once the ZPA has become established the posterior limb mesoderm displays resistance to the induction of Hoxb-8 expression. Grafting of ZPA cells to the anterior of a host limb renders the host anterior tissue resistant to RA-induced Hoxb-8 expression. These results indicate that Hoxb-8 expression may be regulated by the established ZPA through a negative feedback loop. The anterior AER also secretes an inhibitory factor, preventing RA-induced or already established Hoxb-8 expression in the cells immediately underneath the AER.

Consistent with a role for Hoxb-8 in positioning of the forelimb ZPA, Hoxb-8 expression is not seen in RA-induced duplications at the anterior of the hindlimb. However, grafting of Hoxb-8-expressing tissue to the hindlimb can lead to Shh expression and similar duplications, suggesting that factors mediating ZPA formation are very similar in both wing and leg.
Epidermal induction and inhibition of neural fate by translation initiation factor 4AIII
Authors: Weinstein Daniel C. , Honoré Eric , Hemmati-Brivanlou Ali
Published: April 2021

Bone Morphogenetic Protein-4 (BMP-4) is a potent epidermal inducer and inhibitor of neural fate. We have used differential screening to identify genes involved in epidermal induction downstream of BMP-4 and report here evidence of a novel translational mechanism that regulates the division of the vertebrate ectoderm into regions of neural and epidermal fate. In dissociated Xenopus ectoderm, addition of ectopic BMP-4 leads to an increase in the expression of translation initiation factor 4AIII (eIF-4AIII), a divergent member of the eIF-4A gene family until now characterized only in plants. In the gastrula embryo XenopuseIF-4AIII (XeIF-4AIII) expression is elevated in the ventral ectoderm, a site of active BMP signal transduction. Moreover, overexpression of XeIF-4AIII induces epidermis in dissociated cells that would otherwise adopt a neural fate, mimicking the effects of BMP-4. Epidermal induction by XeIF-4AIII requires both an active BMP signaling pathway and an extracellular intermediate. Our results suggest that XeIF-4AIII can regulate changes in cell fate through selective mRNA translation. We propose that BMPs and XeIF-4AIII interact through a positive feedback loop in the ventral ectoderm of the vertebrate gastrula.
Notochord to endoderm signaling is required for pancreas development
Authors: Kim Seung K. , Hebrok Matthias , Melton Douglas A.
Published: April 2021

The role of the notochord in inducing and patterning adjacent neural and mesodermal tissues is well established. We provide evidence that the notochord is also required for one of the earliest known steps in the development of the pancreas, an endodermally derived organ. At a developmental stage in chick embryos when the notochord touches the endoderm, removal of notochord eliminates subsequent expression of several markers of dorsal pancreas bud development, including insulin, glucagon and carboxypeptidase A. Pancreatic gene expression can be initiated and maintained in prepancreatic chick endoderm grown in vitro with notochord. Non-pancreatic endoderm, however, does not express pancreatic genes when recombined with the same notochord. The results suggest that the notochord provides a permissive signal to endoderm to specify pan-creatic fate in a stepwise manner.
A role for FGF-8 in the dorsoventral patterning of the zebrafish gastrula
Authors: Fürthauer Maximilian , Thisse Christine , Thisse Bernard
Published: April 2021

Signals released from Spemann’s organizer, together with ventralizing factors such as BMPs, are necessary to pattern the dorsoventral axis of the vertebrate embryo. We report that a member of the FGF family, fgf-8, not secreted by the axial mesoderm but expressed in a dorsoventral gradient at the margin of the zebrafish gastrula, also contributes to the establishment of the dorsoventral axis of the embryo. Ectopic expression of FGF-8 leads to the expansion of dorsolateral derivatives at the expense of ventral and posterior domains. Moreover, FGF-8 displays some organizer properties as it induces the formation of a partial secondary axis in the absence of factors released from Spemann’s organizer territory. Analysis of its interaction with the ventralizing factors, BMPs, reveals that overexpression of FGF-8 inhibits the expression of these factors in the ventral part of the embryo as early as blastula stage, suggesting that FGF8 acts upstream of BMP2 and BMP4. We conclude that FGF-8 is involved in defining dorsoventral identity and is an important organizing factor responsible for specification of mesodermal and ectodermal dorsolateral territories of the zebrafish gastrula.
Suppressor of Hairless-independent events in Notch signaling imply novel pathway elements
Authors: Matsuno Kenji , Go Masahiro J. , Sun Xin , Eastman Deborah S. , Artavanis-Tsakonas Spyros
Published: April 2021

The Notch (N) pathway defines an evolutionarily conserved cell signaling mechanism that governs cell fate choices through local cell interactions. The ankyrin repeat region of the Notch receptor is essential for signaling and has been implicated in the interactions between Notch and two intra-cellular elements of the pathway: Deltex (Dx) and Sup-pressor of Hairless (Su(H)). Here we examine directly the function of the Notch cdc10/ankyrin repeats (ANK repeats) by transgenic and biochemical analysis. We present evidence implicating the ANK repeats in the regulation of Notch signaling through homotypic interactions. In vivo expression of the Notch ANK repeats reveals a cell non-autonomous effect and elicits mutant phenotypes that indicate the existence of novel downstream events in Notch signaling. These signaling activities are independent of the known effector Su(H) and suggest the existence of yet unidentified Notch pathway components.
The terminal differentiation factor LIN-29 is required for proper vulval morphogenesis and egg laying in Caenorhabditis elegans
Authors: Bettinger Jill C. , Euling Susan , Rougvie Ann E.
Published: April 2021

Caenorhabditis elegans vulval development culminates during exit from the L4-to-adult molt with the formation of an opening through the adult hypodermis and cuticle that is used for egg laying and mating. Vulva formation requires the heterochronic gene lin-29, which triggers hypodermal cell terminal differentiation during the final molt. lin-29 mutants are unable to lay eggs or mate because no vulval opening forms; instead, a protrusion forms at the site of the vulva. We demonstrate through analysis of genetic mosaics that lin-29 is absolutely required in a small subset of lateral hypodermal seam cells, adjacent to the vulva, for wild-type vulva formation and egg laying. However, lin-29 function is not strictly limited to the lateral hypodermis. First, LIN-29 accumulates in many non-hypodermal cells with known roles in vulva formation or egg laying. Second, animals homozygous for one lin-29 allele, ga94, have the vulval defect and cannot lay eggs, despite having a terminally differentiated adult lateral hypodermis. Finally, vulval morphogenesis and egg laying requires lin-29 activity within the EMS lineage, a lineage that does not generate hypodermal cells.
A silencer is required for maintenance of transcriptional repression throughout Drosophila development
Authors: Busturia Ana , Wightman Christopher D. , Sakonju Shigeru
Published: April 2021

Transcriptional silencing by the Polycomb Group of genes maintains the position-specific repression of homeotic genes throughout Drosophila development. The Polycomb Group of genes characterized to date encode chromatinassociated proteins that have been suggested to form heterochromatin-like structures. By studying the expression of reporter genes, we have identified a 725 bp fragment, called MCP725, in the homeotic gene Abdominal-B, that accurately maintains position-specific silencing during proliferation of imaginal cells. Silencing by MCP725 requires the Polycomb and the Polycomblike genes, indicating that it contains a Polycomb response element. To investigate the mechanisms of transcriptional silencing by MCP725, we have studied its temporal requirements by removing MCP725 from the transgene at various times during development. We have discovered that excision of MCP725 during larval stages leads to loss of silencing. Our findings indicate that the silencer is required for the maintenance of the repressed state throughout cell proliferation. They also suggest that propagation of the silenced state does not occur merely by templating of a heterochromatin structure by virtue of protein-protein interactions. Rather, they suggest that silencers play an active role in the maintenance of the position-specific repression throughout development.
bag-of-marbles and benign gonial cell neoplasm act in the germline to restrict proliferation during Drosophila spermatogenesis
Authors: Gönczy Pierre , Matunis Erika , DiNardo Stephen
Published: April 2021

Stem cells divide asymmetrically, regenerating a parental stem cell and giving rise to a daughter cell with a distinct fate. In many stem cell lineages, this daughter cell undergoes several amplificatory mitoses, thus generating more cells that embark on the differentiation program specific for the given lineage. Spermatogenesis in Drosophila is a model system to identify molecules regulating stem cell lineages. Mutations at two previously identified loci, bag-of-marbles (bam) and benign gonial cell neoplasm (bgcn), prevent progression through spermatogenesis and oogenesis, resulting in the overproliferation of undifferentiated germ cells. Here we investigate how bam and bgcn regulate the male germline stem cell lineage. By generating FLP-mediated clones, we demonstrate that both bam and bgcn act autonomously in the germline to restrict proliferation during spermatogenesis. By using enhancer trap lines, we find that the overproliferating germ cells express markers specific to amplifying germ cells, while at the same time retaining the expression of some markers of stem cell and primary spermatogonial cell fate. However, we find that germ cells accumulating in bam or bgcn mutant testes most resemble amplifying germ cells, because they undergo incomplete cytokinesis and progress through the cell cycle in synchrony within a cyst, which are two characteristics of amplifying germ cells, but not of stem cells. Taken together, our results suggest that bam and bgcn regulate progression through the male germline stem cell lineage by cell-intrinsically restricting the proliferation of amplifying germ cells.
Targeted disruption of Hoxd-10 affects mouse hindlimb development
Authors: Carpenter Ellen M. , Goddard Judy M. , Davis Allan P. , Nguyen T. Paul , Capecchi Mario R.
Published: April 2021

Targeted disruption of the Hoxd-10 gene, a 5′ member of the mouse HoxD linkage group, produces mice with hindlimb-specific defects in gait and adduction. To determine the underlying causes of this locomotor defect, mutant mice were examined for skeletal, muscular and neural abnormalities. Mutant mice exhibit alterations in the vertebral column and in the bones of the hindlimb. Sacral vertebrae beginning at the level of S2 exhibit homeotic transformations to adopt the morphology of the next most anterior vertebra. In the hindlimb, there is an anterior shift in the position of the patella, an occasional production of an anterior sesamoid bone, and an outward rotation of the lower part of the leg, all of which contribute to the defects in locomotion. No major alterations in hindlimb musculature were observed, but defects in the nervous system were evident. There was a decrease in the number of spinal segments projecting nerve fibers through the sacral plexus to innervate the musculature of the hindlimb. Deletion of a hindlimb nerve was seen in some animals, and a shift was evident in the position of the lumbar lateral motor column. These observations suggest a role for the Hoxd-10 gene in establishing regional identity within the spinal cord and imply that patterning of the spinal cord may have intrinsic components and is not completely imposed by the surrounding mesoderm.
Linking Frizzled and Wnt signaling in Drosophila development
Authors: Tomlinson Andrew , Strapps Walter R. , Heemskerk Jill
Published: April 2021

Drosophila Frizzled-2 (Dfz2) has been identified as a putative fly Wingless (Wg) receptor. Although Dfz2 shows significant homology with Fz, a protein that operates in the mechanisms that establish planar polarity in Drosophila epithelia, any clear evidence for an involvement by Fz in a Wnt signaling pathway has hitherto been absent. Here we describe the planar polarity phenotypes of loss-of-function and overexpression of Fz in the developing Drosophila eye and find it almost identical to the loss-of-function or over-expression of Dishevelled (Dsh – a protein operating in Wnt second messenger systems). In addition, we show that over-expression of Shaggy (Sgg – another component of the Wnt pathway) in the eye also causes a phenotype similar to Fz and Dsh. To test further the link between planar polarity and Wnt signaling we misexpressed Wg in the developing eye and found it had a potent polarizing effect in the retinal epithelium. Since the overexpression of Fz in the developing eye gave a phenotype consistent with activating the Wnt pathway, we tested overexpression of Fz in the developing embryonic ectoderm and found that it phenocopied over-expression of Wg. To check that Fz was indeed able to activate a Wnt pathway we overexpressed it in Drosophila tissue culture cells and observed the characteristic phosphorylation of Dsh that occurs in response to Wnt signaling. Taken together our results significantly strengthen the case for Fz acting in a Wnt signaling pathway in Drosophila.