bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of wingless in the Drosophila embryo: a conserved cis-acting element lacking conserved Ci-binding sites is required for patched-mediated repression
Authors: Lessing Derek , Nusse Roel
Published: April 2021

Patterning of the Drosophila embryo depends on the accurate expression of wingless (wg), which encodes a secreted signal required for segmentation and many other processes. Early expression of wg is regulated by the nuclear proteins of the gap and pair-rule gene classes but, after gastrulation, wg transcription is also dependent on cell-cell communication. Signaling to the Wg-producing cells is mediated by the secreted protein, Hedgehog (Hh), and by Cubitus interruptus (Ci), a transcriptional effector of the Hh signal transduction pathway. The transmembrane protein Patched (Ptc) acts as a negative regulator of wg expression; ptc− embryos have ectopic wg expression. According to the current models, Ptc is a receptor for Hh. The default activity of Ptc is to inhibit Ci function; when Ptc binds Hh, this inhibition is released and Ci can control wg transcription.

We have investigated cis-acting sequences that regulate wg during the time that wg expression depends on Hh signaling. We show that approximately 4.5 kb immediately upstream of the wg transcription unit can direct expression of the reporter gene lacZ in domains similar to the normal wg pattern in the embryonic ectoderm. Expression of this reporter construct expands in ptc mutants and responds to hh activity. Within this 4.5 kb, a 150 bp element, highly conserved between D. melanogaster and Drosophila virilis, is required to spatially restrict wg transcription. Activity of this element depends on ptc, but it contains no consensus Ci-binding sites. The discovery of an element that is likely to bind a transcriptional repressor was unexpected, since the prevailing model suggests that wg expression is principally controlled by Hh signaling acting through the Ci activator. We show that wg regulatory DNA can drive lacZ in a proper wg-like pattern without any conserved Ci-binding sites and suggest that Ci can not be the sole endpoint of the Hh pathway.
Sex-lethal, the master sex-determining gene in Drosophila, is not sex-specifically regulated in Musca domestica
Authors: Meise Martin , Hilfiker-Kleiner Denise , Dübendorfer Andreas , Nöthiger Claudia Brunner, Rolf , Bopp Daniel
Published: April 2021

Sex-lethal (Sxl) is the master switch gene for somatic sex determination in Drosophila melanogaster. In XX animals, Sxl becomes activated and imposes female development; in X(Y) animals, Sxl remains inactive and male development ensues. A switch gene for sex determination, called F, has also been identified in the housefly, Musca domestica. An active F dictates female development, while male development ensues when F is inactive. To test if the switch functions of Sxl and F are founded on a common molecular basis, we isolated the homologous Sxl gene in the housefly. Though highly conserved in sequence, Musca-Sxl is not sex-specifically regulated: the same transcripts and protein isoforms are expressed in both male and female animals throughout development. Musca-Sxl is apparently not controlled by the primary sex-determining signal and, thus, is unlikely to correspond to the F gene. Ectopic expression of Musca-SXL protein in Drosophila does not exert any noticeable effects on the known target genes of endogenous Sxl. Instead, forced overexpression of the transgene eventually results in lethality of both XY and XX animals and in developmental abnormalities in some escaper XY animals. Similar results were obtained with the Sxl homologue of Ceratitis capitata (Saccone, G., Peluso, I., Artiaco, D., Giodano, E., Bopp, D. and Polito, L. C. (1998) Development 125, 1495–1500) suggesting that, in these non-drosophilid species, Sxl performs a function different from that in sex determination.
The Ceratitis capitata homologue of the Drosophila sex-determining gene Sex-lethal is structurally conserved, but not sex-specifically regulated
Authors: Saccone Giuseppe , Peluso Ivana , Artiaco Dora , Giordano Ennio , Bopp Daniel , Polito Lino C.
Published: April 2021

In Drosophila, Sxl functions as a binary switch in sex determination. Under the control of the primary sex-determining signal, it produces functional protein only in XX animals to implement female development. Here we report that, in contrast to Drosophila, the Sxl homologue in the Medfly, Ceratitis capitata, expresses the same mRNAs and protein isoforms in both XX and XY animals irrespective of the primary sex-determining signal. Also, experiments with two inducible transgenes demonstrate that the corresponding Ceratitis SXL product has no significant sex-transforming effects when expressed in Drosophila. Similar results have been obtained for the Sxl homologue of Musca domestica (Meise, M., Hilfiker-Kleiner, D., Brunner, C., Dübendorfer, A., Nöthiger, R. and Bopp, D. (1998) Development 125, 1487-1494). Our findings suggest that Sxl acquired its master regulatory role in sex determination during evolution of the Acalyptratae group, most probably after phylogenetic divergence of the genus Drosophila from other genera of this group.
The FRUITFULL MADS-box gene mediates cell differentiation during Arabidopsis fruit development
Authors: Gu Qing , Ferrándiz Cristina , Yanofsky Martin F. , Martienssen Robert
Published: April 2021

Fruit morphogenesis is a process unique to flowering plants, and yet little is known about its developmental control. Following fertilization, fruits typically undergo a dramatic enlargement that is accompanied by differentiation of numerous distinct cell types. We have identified a mutation in Arabidopsis called fruitfull (ful-1), which abolishes elongation of the silique after fertilization. The ful-1 mutation is caused by the insertion of a DsE transposable enhancer trap element into the 5′ untranslated leader of the AGL8 MADS-box gene. β-glucuronidase (GUS) reporter gene expression in the enhancer trap line is observed specifically in all cell layers of the valve tissue, but not in the replum, the septum or the seeds, and faithfully mimics RNA in situ hybridization data reported previously. The lack of coordinated growth of the fruit tissues leads to crowded seeds, a failure of dehiscence and, frequently, the premature rupture of the carpel valves. The primary defect of ful-1 fruits is within the valves, whose cells fail to elongate and differentiate. Stomata, which are frequent along the epidermis of wild-type valves, are completely eliminated in the ful mutant valves. In addition to the effect on fruit development, ful cauline leaves are broader than those of wild type and show a reduction in the number of internal cell layers. These data suggest that AGL8/FUL regulates the transcription of genes required for cellular differentiation during fruit and leaf development.
Mouse mutant embryos lacking huntingtin are rescued from lethality by wild-type extraembryonic tissues
Authors: Dragatsis Ioannis , Efstratiadis Argiris , Zeitlin Scott
Published: April 2021

Mouse embryos nullizygous for a targeted disruption of the Huntington’s disease gene homologue (Hdh), which encodes a protein (huntingtin) of unknown biochemical function, become developmentally retarded and disorganized, and die early in development. Using chimeric analysis, we demonstrate that extensively chimeric embryos derived by injection of Hdh null ES cells into wild-type host blastocysts are rescued from lethality. In contrast, when wild-type ES cells are injected into Hdh null blastocysts, the chimeric embryos are morphologically indistinguishable from Hdh null mutants derived from natural matings, and die shortly after gastrulation. Therefore, the primary defect in the absence of huntingtin lies in extraembryonic tissues, whereas the epiblast and its derivatives are affected secondarily. It is likely that the mutation results in impairment of the nutritive functions of the visceral endoderm, which otherwise appears to differentiate normally, as evidenced by the expression of several specific marker genes. Consistent with preliminary histochemical analysis indicating that at least the transport of ferric ions is defective in Hdh mutants and in conjunction with the known localization of huntingtin in the membranes of vesicles associated with microtubules, we hypothesize that this protein is involved in the intracellular trafficking of nutrients in early embryos.
Patterned acquisition of skin barrier function during development
Authors: Hardman Matthew J. , Sisi Paraskevi , Banbury David N. , Byrne Carolyn
Published: April 2021

Skin barrier function is conferred by the outer layer of epidermis, the stratum corneum, and is essential for terrestrial life. Quantitative trans-epidermal water loss assays show that barrier forms late in embryogenesis, permitting the foetus to survive a terrestrial environment at birth. Using qualitative in situ assays for skin permeability, we show that barrier forms in a patterned manner late in mouse gestation. Barrier forms at specific epidermal sites, then spreads around the embryo as a moving front. The moving front of permeability change is accompanied by multiple changes in the outer, stratum corneum-precursor cells. We use the permeability assays to show that final stages of cornified envelope assembly are coordinated with initial stages of barrier formation. Hence the whole-mount permeability assays record developmental acquisition of a known, essential component of the adult barrier. We demonstrate the authenticity of the whole-mount assays after maternal glucocorticoid therapy (known to accelerate barrier formation) and in additional species including the rat where barrier formation is well characterized by TEWL assay (Aszterbaum, M., Menon, G. K., Feingold, K. R. and Williams, M. L. Pediatr. Res. 31, 308-317). The demonstration of patterned barrier formation in other species suggests patterned change as the universal mode of embryonic barrier acquisition. These results highlight the importance of patterning as a mode of epidermal maturation during development.
unc-3, a gene required for axonal guidance in Caenorhabditis elegans, encodes a member of the O/E family of transcription factors
Authors: Prasad Brinda C. , Ye Bing , Zackhary Randa , Schrader Karen , Seydoux Geraldine , Reed Randall R.
Published: April 2021

The expression of specialized signal transduction components in mammalian olfactory neurons is thought to be regulated by the O/E (Olf-1/EBF) family of transcription factors. The O/E proteins are expressed in cells of the olfactory neuronal lineage throughout development and are also expressed transiently in neurons in the developing nervous system during embryogenesis. We have identified a C. elegans homologue of the mammalian O/E proteins, which displays greater than 80% similarity over 350 amino acids. Like its mammalian homologues, CeO/E is expressed in certain chemosensory neurons (ASI amphid neurons) throughout development and is also expressed transiently in developing motor neurons when these cells undergo axonal outgrowth. We demonstrate that CeO/E is the product of the unc-3 gene, mutations in which cause defects in the axonal outgrowth of motor neurons, as well as defects in dauer formation, a process requiring chemosensory inputs. These observations suggest that the O/E family of transcription factors play a central and evolutionarily conserved role in the expression of proteins essential for axonal pathfinding and/or neuronal differentiation in both sensory and motor neurons.
Homeotic gene action in embryonic brain development of Drosophila
Authors: Hirth Frank , Hartmann Beate , Reichert Heinrich
Published: April 2021

Studies in vertebrates show that homeotic genes are involved in axial patterning and in specifying segmental identity of the embryonic hindbrain and spinal cord. To gain further insights into homeotic gene action during CNS development, we here characterize the role of the homeotic genes in embryonic brain development of Drosophila. We first use neuroanatomical techniques to map the entire anteroposterior order of homeotic gene expression in the Drosophila CNS, and demonstrate that this order is virtually identical in the CNS of Drosophila and mammals. We then carry out a genetic analysis of the labial gene in embryonic brain development. Our analysis shows that loss-of-function mutation and ubiquitous overexpression of labial results in ectopic expression of neighboring regulatory genes. Furthermore, this analysis demonstrates that mutational inactivation of labial results in regionalized axonal patterning defects which are due to both cell-autonomous and cell-nonautonomous effects. Thus, in the absence of labial, mutant cells are generated and positioned correctly in the brain, but these cells do not extend axons. Additionally, extending axons of neighboring wild-type neurons stop at the mutant domains or project ectopically, and defective commissural and longitudinal pathways result. Immunocytochemical analysis demonstrates that cells in the mutant domains do not express neuronal markers, indicating a complete lack of neuronal identity. An alternative glial identity is not adopted by these mutant cells. Comparable effects are seen in Deformed mutants but not in other homeotic gene mutants. Our findings demonstrate that the action of the homeotic genes labial and Deformed are required for neuronal differentiation in the developing brain of Drosophila.
A common mechanism controls the life cycle and architecture of plants
Authors: Ratcliffe Oliver J. , Amaya Iraida , Vincent Coral A. , Rothstein Steven , Carpenter Rosemary , Coen Enrico S. , Bradley Desmond J.
Published: April 2021

The overall aerial architecture of flowering plants depends on a group of meristematic cells in the shoot apex. We demonstrate that the Arabidopsis TERMINAL FLOWER 1 gene has a unified effect on the rate of progression of the shoot apex through different developmental phases. In transgenic Arabidopsis plants which ectopically express TERMINAL FLOWER 1, both the vegetative and reproductive phases are greatly extended. As a consequence, these plants exhibit dramatic changes in their overall morphology, producing an enlarged vegetative rosette of leaves, followed by a highly branched inflorescence which eventually forms normal flowers. Activity of the floral meristem identity genes LEAFY and APETALA 1 is not directly inhibited by TERMINAL FLOWER 1, but their upregulation is markedly delayed compared to wild-type controls. These phenotypic and molecular effects complement those observed in the tfl1 mutant, where all phases are shortened. The results suggest that TERMINAL FLOWER 1 participates in a common mechanism underlying major shoot apical phase transitions, rather than there being unrelated mechanisms which regulate each specific transition during the life cycle.
CHR3: a Caenorhabditis elegans orphan nuclear hormone receptor required for proper epidermal development and molting
Authors: Kostrouchova Marta , Krause Michael , Kostrouch Zdenek , Rall Joseph Edward
Published: April 2021

CHR3 is a Caenorhabditis elegans orphan nuclear hormone receptor highly homologous to Drosophila DHR3, an ecdysone-inducible gene product involved in metamorphosis. Related vertebrate factors include RORalpha/RZRalpha, RZRbeta and RevErb. Gel-shift studies show that CHR3 can bind the DR5-type hormone response sequence. CHR3 is a nuclear protein present in all blastomeres during early embryogenesis. During morphogenesis, both CHR3 protein and zygotically active reporter genes are detectable in epidermal cells and their precursors. Inhibition of the gene encoding CHR3 results in several larval defects associated with abnormal epidermal cell function, including molting and body size regulation, suggesting that CHR3 is an essential epidermal factor required for proper postembryonic development.
prdl-a, a gene marker for hydra apical differentiation related to triploblastic paired-like head-specific genes
Authors: Gauchat Dominique , Kreger Sylvia , Holstein Thomas , Galliot Brigitte
Published: April 2021

Two homeobox genes, prdl-a and prdl-b, which were isolated from a Hydra vulgaris cDNA library, encode paired-like class homeodomains highly related to that of the aristaless-related genes. In adult polyps, prdl-b is a marker for synchronously dividing nematoblasts while prdl-a displays an expression restricted to the the nerve cell lineage of the head region. During budding and apical regeneration, an early and transient prdl-a expression was observed in endodermal cells of the stump at a time when the head organizer is established. When apical regeneration was delayed upon concomittant budding, prdl-a expression was found to be altered in the stump. Furthermore, a specific anti-prdl-a protein immunoserum revealed that prdl-a was overexpressed in adult polyps of the Chlorohydra viridissima multiheaded mutant, with an expression domain extending below the tentacle ring towards the body column. Accordingly, prdl-a DNA-binding activity was enhanced in nuclear extracts from this mutant. These results suggest that prdl-a responds to apical forming signals and might thus be involved in apical specification. When a marine hydrozoan (Podocorynae carnea) was used, the anti-prdl-a antibody showed cross-reactivity with cells located around the oral region, indicating that prdl-a function is shared by other cnidaria. The ancestral role for prdl-a-related genes in the molecular definition of the head (or oral-surrounding region) is discussed.
Runt determines cell fates in the Drosophila embryonic CNS
Authors: Dormand Emma-Louise , Brand Andrea H.
Published: April 2021

The segmentation gene, runt, is expressed by a subset of the 30 neuroblasts that give rise to each neuromere of the Drosophila embryo. Runt activity in the neuroblasts is necessary for expression of even-skipped in the EL neurons. runt is therefore a good candidate for a gene specifying neuroblast identities. We have ectopically expressed Runt in restricted subsets of neuroblasts and show that Runt is sufficient to activate even-skipped expression in the progeny of specific neuroblasts. Using the marker Tau-green fluorescent protein to highlight the axons, we have found that the extra Even-skipped-expressing neurons project axons along the same pathway as the EL neurons. We find that Runt is expressed in neuroblast 3-3, supporting an autonomous role for runt during neuroblast specification.
Genetic analysis of a Hoxd-12 regulatory element reveals global versus local modes of controls in the HoxD complex
Authors: Hérault Yann , Beckers Johannes , Kondo Takashi , Fraudeau Nadine , Duboule Denis
Published: April 2021

Vertebrate Hoxd genes are essential determinants of limb morphogenesis. In order to understand the genetic control of their complex expression patterns, we have used a combined approach involving interspecies sequence alignments in parallel with transgenic analyses, followed by in vivo mutagenesis. Here, we report on the identification of a regulatory element that is located in the vicinity of the Hoxd-12 gene. While this element is well conserved in tetrapods, little sequence similarity was scored when compared to the cognate fish DNA. The regulatory potential of this region XI (RXI) was first assayed in the context of a Hoxd-12/lacZ reporter transgene and shown to direct reporter gene expression in posterior limb buds. A deletion of this region was generated by targeted mutagenesis in ES cells and introduced into mice. Analyses of animals homozygous for the HoxDRXI mutant allele revealed the function of this region in controlling Hoxd-12 expression in the presumptive posterior zeugopod where it genetically interacts with Hoxa-11. Downregulation of Hoxd-12 expression was also detected in the trunk suggesting that RXI may mediate a rather general function in the activation of Hoxd-12. These results support a model whereby global as well as local regulatory influences are necessary to build up the complex expression patterns of Hoxd genes during limb development.
The PS2 integrin ligand tiggrin is required for proper muscle function in Drosophila
Authors: Bunch Thomas A. , Graner Michael W. , Fessler Liselotte I. , Fessler John H. , Schneider Kimberly D. , Kerschen Arthur , Choy Lynna P. , Burgess Bradford W. , Brower Danny L.
Published: April 2021

Tiggrin is a novel extracellular matrix ligand for the Drosophila PS2 integrins. We have used flanking P elements to generate a precise deletion of tiggrin. Most flies lacking tiggrin die as larvae or pupae. A few adults do emerge and these appear to be relatively normal, displaying only misshapen abdomens and a low frequency of wing defects. Examination of larvae shows that muscle connections, function and morphology are defective in tiggrin mutants. Muscle contraction waves that extend the length of the larvae are much slower in tiggrin mutants. Direct examination of bodywall muscles shows defects in muscle attachment sites, where tiggrin is specifically localized, and muscles appear thinner. Transgenes expressing tiggrin are capable of rescuing tiggrin mutant phenotypes. Transgenes expressing a mutant tiggrin, whose Arg-Gly-Asp (RGD) integrin recognition sequence has been mutated to Leu-Gly-Ala (LGA) show much reduced, but significant, rescuing ability. Cell spreading assays detect no interactions of this mutant tiggrin with PS2 integrins. Therefore, while the RGD sequence is critical for PS2 interactions and full activity in the whole fly, the mutant tiggrin retains some function(s) that are probably mediated by interactions with other ECM molecules or cell surface receptors
Progressive lineage analysis by cell sorting and culture identiﬁes FLK1+VEcadherin+ cells at a diverging point of endothelial and hemopoietic lineages
Authors: Nishikawa Shin-Ichi , Nishikawa Satomi , Hirashima Masanori , Matsuyoshi Norihisa , Kodama Hiroaki
Published: April 2021

Totipotent murine ES cells h ave an enormous potential for the study of cell speciﬁcation. He re we demonstrate that ES cells can diffe rentiate to hemopoietic cells th rough the proximal lateral mesoderm, me rely upon culturing in type IV collagen-coated dishes. Separation of the Flk1+ mesoderm f rom other cell lineages was critical for hemopoietic cell diffe rentiation, whe reas formation of the embryoid body was not. Since the two-dimensionally spreading cells can be monito red easily in real time, this culture system will g reatly facilitate the study of the mechanisms i nvolved in the cell speciﬁcation to mesoderm, endothelial, and hemopoietic cells. In the cultu re of ES cells, how ever, lineages and stages of diffe rentiating cells can only be deﬁned by their own characteristics. We showed that a combination of monoclonal antibodies against E-cadherin, Flk1/KDR, PDGF recepto rα, VEcadherin, CD45 and Ter119 was suf ficient to deﬁne most intermediate stages during diffe rentiation of ES cells to blood cells. Using this cultu re system and surface markers, we determined the following order for blood cell differentiation: ES cell (E-cadherin+Flk1™PDGFRα™), proximal lateral mesoderm (E-cadherin™ Flk1+VEcadherin™ ), progenitor with hemoangiogenic potential (Flk1+VE-cadherin+CD45™ ), hemopoietic p rogenitor (CD45+c-Kit+) and mature blood cells (c-Kit™ CD45+ or Ter119+), though direct differentiation of blood cells f rom the Flk1+VE-cadherin™ stage cannot be ruled out. Not only the VE-cadherin+CD45™ population generated f rom ES cells but also those di rectly sorted f rom the yolk sac of 9.5 dpc embryos h ave a potential to give rise to hemopoietic cells. P rogenitors with hemoangiogenic potential were identiﬁed in both the Flk1+VE-cadherin™and Flk1+VEcadherin+populations by the single cell deposition experiment. This line of evidence implicates Flk1+VEcadherin+ cells as a diverging point of hemopoietic and endothelial cell lineages.
Ectodermal patterning in the avian embryo: epidermis versus neural plate
Authors: Pera Edgar , Stein Stefan , Kessel Michael
Published: April 2021

Ectodermal patterning of the chick embryo begins in the uterus and continues during gastrulation, when cells with a neural fate become restricted to the neural plate around the primitive streak, and cells fated to become the epidermis to the periphery. The prospective epidermis at early stages is characterized by the expression of the homeobox gene DLX5, which remains an epidermal marker during gastrulation and neurulation. Later, some DLX5-expressing cells become internalized into the ventral forebrain and the neural crest at the hindbrain level. We studied the mechanism of ectodermal patterning by transplantation of Hensen’s nodes and prechordal plates. The DLX5 marker indicates that not only a neural plate, but also a surrounding epidermis is induced in such operations. Similar effects can be obtained with neural plate grafts. These experiments demonstrate that the induction of a DLX5-positive epidermis is triggered by the midline, and the effect is transferred via the neural plate to the periphery. By repeated extirpations of the endoderm we suppressed the formation of an endoderm/mesoderm layer under the epiblast. This led to the generation of epidermis, and to the inhibition of neuroepithelium in the naked ectoderm. This suggests a signal necessary for neural, but inhibitory for epidermal development, normally coming from the lower layers. Finally, we demonstrate that BMP4, as well as BMP2, is capable of inducing epidermal fate by distorting the epidermis-neural plate boundary. This, however, does not happen independently within the neural plate or outside the normal DLX5 domain. In the area opaca, the co-transplantation of a BMP4 bead with a node graft leads to the induction of DLX5, thus indicating the cooperation of two factors. We conclude that ectodermal patterning is achieved by signalling both from the midline and from the periphery, within the upper but also from the lower layers.
CRABS CLAW and SPATULA, two Arabidopsis genes that control carpel development in parallel with AGAMOUS
Authors: Alvarez John , Smyth David R.
Published: April 2021

To help understand the process of carpel morphogenesis, the roles of three carpel development genes have been partitioned genetically. Mutants of CRABS CLAW cause the gynoecium to develop into a wider but shorter structure, and the two carpels are unfused at the apex. Mutants of a second gene, SPATULA, show reduced growth of the style, stigma, and septum, and the transmitting tract is absent. Double mutants of crabs claw and spatula with homeotic mutants that develop ectopic carpels demonstrate that CRABS CLAW and SPATULA are necessary for, and inseparable from, carpel development, and that their action is negatively regulated by A and B organ identity genes. The third carpel gene studied, AGAMOUS, encodes C function that has been proposed to fully specify carpel identity. When AGAMOUS function is removed together with the A class gene APETALA2, however, the organs retain many carpelloid properties, suggesting that other genes are also involved. We show here that further mutant disruption of both CRABS CLAW and SPATULA function removes remaining carpelloid properties, revealing that the three genes together are necessary to generate the mature gynoecium. In particular, AGAMOUS is required to specify the identity of the carpel wall and to promote the stylar outgrowth at the apex, CRABS CLAW suppresses radial growth of the developing gynoecium but promotes its longitudinal growth, and SPATULA supports development of the carpel margins and tissues derived from them. The three genes mostly act independently, although there is genetic evidence that CRABS CLAW enhances AGAMOUS and SPATULA function.
CRABS CLAW, a gene that regulates carpel and nectary development in Arabidopsis, encodes a novel protein with zinc finger and helix-loop-helix domains
Authors: Bowman John L. , Smyth David R.
Published: April 2021

Studies of plants with mutations in the CRABS CLAW gene indicate that it is involved in suppressing early radial growth of the gynoecium and in promoting its later elongation. It is also required for the initiation of nectary development. To gain further insight, the gene was cloned by chromosome walking. CRABS CLAW encodes a putative transcription factor containing a zinc finger and a helix-loop-helix domain. The latter resembles the first two helices of the HMG box, known to bind DNA. At least five other genes of Arabidopsis carry the same combination of domains, and we have named them the yabby family. The new helix-loop-helix domain itself we call the yabby domain. Consistent with the mutant phenotype, CRABS CLAW expression is mostly limited to carpels and nectaries. It is expressed in gynoecial primordia from their inception, firstly in lateral sectors where it may inhibit radial growth, and later in the epidermis and in four internal strips. The internal expression may be sufficient to support longitudinal growth, as carpels are longer in a crabs claw promoter mutant where expression is now confined to these regions. The patterns of expression of CRABS CLAW in ectopic carpels of floral homeotic mutants suggest that it is negatively regulated by the A and B organ identity functions, but largely independent of C function. CRABS CLAW expression occurs in nectaries throughout their growth and maturation. It is also expressed in their presumptive anlagen so it may specify cells that will later develop as nectaries. Nectaries arise from the floral receptacle at normal positions in all A, B and C organ identity mutants examined, and CRABS CLAW is always expressed within them. Thus CRABS CLAW expression is regulated independently in carpels and nectaries.
Reconstitution of the organizer is both sufficient and required to re-establish a fully patterned body plan in avian embryos
Authors: Yuan Shipeng , Schoenwolf Gary C.
Published: April 2021

Lateral blastoderm isolates (LBIs) at the late gastrula/early neurula stage (i.e., stage 3d/4) that lack Hensen’s node (organizer) and primitive streak can reconstitute a functional organizer and primitive streak within 10-12 hours in culture. We used LBIs to study the initiation and regionalization of the body plan. A complete body plan forms in each LBI by 36 hours in culture, and normal craniocaudal, dorsoventral, and mediolateral axes are re-established. Thus, reconstitution of the organizer is sufficient to re-establish a fully patterned body plan. LBIs can be modified so that reconstitution of the organizer does not occur. In such modified LBIs, tissue-type specific differentiation (with the exception of heart differentiation) and reconstitution of the body plan fail to occur. Thus, the reconstitution of the organizer is not only sufficient to re-establish a fully patterned body plan, it is also required. Finally, our results show that formation and patterning of the heart is under the control of the organizer, and that such control is exerted during the early to mid-gastrula stages (i.e., stages 2-3a), prior to formation of the fully elongated primitive streak.
rhomboid and Star interact synergistically to promote EGFR/MAPK signaling during Drosophila wing vein development
Authors: Guichard Annabel , Biehs Brian , Sturtevant Mark A. , Wickline Laura , Chacko Julie , Howard Katherine , Bier Ethan
Published: April 2021

Genes of the ventrolateral group in Drosophila are dedicated to developmental regulation of Egfr signaling in multiple processes including wing vein development. Among these genes, Egfr encodes the Drosophila EGF-Receptor, spitz (spi) and vein (vn) encode EGF-related ligands, and rhomboid (rho) and Star (S) encode membrane proteins. In this study, we show that rho-mediated hyperactivation of the EGFR/MAPK pathway is required for vein formation throughout late larval and early pupal development. Consistent with this observation, rho activity is necessary and sufficient to activate MAPK in vein primordium during late larval and early pupal stages. Epistasis studies using a dominant negative version of Egfr and a ligand-independent activated form of Egfr suggest that rho acts upstream of the receptor. We show that rho and S function in a common aspect of vein development since loss-of-function clones of rho or S result in nearly identical non-autonomous loss-of-vein phenotypes. Furthermore, mis-expression of rho and S in wild-type and mutant backgrounds reveals that these genes function in a synergistic and co-dependent manner. In contrast, spi does not play an essential role in the wing. These data indicate that rho and S act in concert, but independently of spi, to promote vein development through the EGFR/MAPK signaling pathway.
Targeted inactivation of the EGF and amphiregulin genes reveals distinct roles for EGF receptor ligands in mouse mammary gland development
Authors: Luetteke Noreen C. , Qiu Ting Hu , Fssssenton Suzanne E. , Troyer Kelly L. , Riedel Richard F. , Chang Aileen , Lee David C.
Published: April 2021

Targeted mice lacking functional EGF or amphiregulin (AR) were derived and bred to the TGFα-knockout to generate mice lacking various combinations of the three ligands. In contrast to EGF receptor (EGFR) knockout mice, triple null mice lacking half of the EGFR ligand family were healthy and fertile, indicative of overlapping or compensatory functions among EGF family members. Nevertheless, pups born to triple null dams frequently died or were runted, suggesting a mammary gland defect. Comparison of individual and combinatorial knockouts established that specific loss of AR severely stunted ductal outgrowth during puberty, consistent with dramatic expression of AR transcripts in normal developing ducts. Surprisingly, loss of all three ligands did not significantly affect cellular proliferation, apoptosis, or ERK activation within terminal end buds. Following pregnancy, most AR single null females, but few triple null females could nurse their young, revealing collaborative roles for EGF and TGFα in mammopoiesis and lactogenesis. In triple null glands, alveoli were poorly organized and differentiated, and milk protein gene expression was decreased. Additionally, Stat5a activation was frequently reduced in AR single and combinatorial nulls in association with impaired lactation. Collectively, our results provide genetic confirmation of a requirement for EGFR signaling throughout the development of the mouse mammary gland, and reveal stage-dependent activities for different EGFR ligands. Finally, the additional loss of growth factors from pups nursed by triple null dams further worsened their survival and growth, establishing functions for both maternal- and neonatal-derived growth factors.
Signalling by the RET receptor tyrosine kinase and its role in the development of the mammalian enteric nervous system
Authors: Taraviras Stavros , Marcos-Gutierrez Camelia V. , Durbec Pascale , Jani Harsha , Grigoriou Maria , Sukumaran Madhu , Wang Li-Chong , Hynes Mary , Raisman Geoffrey , Pachnis Vassilis
Published: April 2021

RET is a member of the receptor tyrosine kinase (RTK) superfamily, which can transduce signalling by glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) in cultured cells. In order to determine whether in addition to being sufficient, RET is also necessary for signalling by these growth factors, we studied the response to GDNF and NTN of primary neuronal cultures (peripheral sensory and central dopaminergic neurons) derived from wild-type and RET-deficient mice. Our experiments show that absence of a functional RET receptor abrogates the biological responses of neuronal cells to both GDNF and NTN.

Despite the established role of the RET signal transduction pathway in the development of the mammalian enteric nervous system (ENS), very little is known regarding its cellular mechanism(s) of action. Here, we have studied the effects of GDNF and NTN on cultures of neural crest (NC)-derived cells isolated from the gut of rat embryos. Our findings suggest that GDNF and NTN promote the survival of enteric neurons as well as the survival, proliferation and differentiation of multipotential ENS progenitors present in the gut of E12.5-13.5 rat embryos. However, the effects of these growth factors are stage-specific, since similar ENS cultures established from later stage embryos (E14.5-15.5), show markedly diminished response to GDNF and NTN. To examine whether the in vitro effects of RET activation reflect the in vivo function(s) of this receptor, the extent of programmed cell death was examined in the gut of wild-type and RET-deficient mouse embryos by TUNEL histochemistry. Our experiments show that a subpopulation of enteric NC undergoes apoptotic cell death specifically in the foregut of embryos lacking the RET receptor. We suggest that normal function of the RET RTK is required in vivo during early stages of ENS histogenesis for the survival of undifferentiated enteric NC and their derivatives.
Different sequence requirements for expression in erythroid and megakaryocytic cells within a regulatory element upstream of the GATA-1 gene
Authors: Vyas Paresh , McDevitt Micheal A. , Cantor Alan B. , Katz Samuel G. , Fujiwara Yuko , Orkin Stuart H.
Published: April 2021

The lineage-restricted transcription factor GATA-1 is required for differentiation of erythroid and megakaryocytic cells. We have localized a 317 base pair cis-acting regulatory element, HS I, associated with a hematopoietic-specific DNase I hypersensitive site, which lies approx. 3.7 kilobases upstream of the murine hematopoietic-specific GATA-1 IE promoter. HS I directs high-level expression of reporter GATA-1/lacZ genes to primitive and definitive erythroid cells and megakaryocytes in transgenic mice. Comparative sequence analysis of HS I between human and mouse shows approx. 63% nucleotide identity with a more conserved core of 169 base pairs (86% identity). This core contains a GATA site separated by 10 base pairs from an E-box motif. The composite motif binds a multi-protein hematopoietic-specific transcription factor complex which includes GATA-1, SCL/tal-1, E2A, Lmo2 and Ldb-1. Point mutations of the GATA site abolishes HS I function, whereas mutation of the E-box motif still allows reporter gene expression in both lineages. Strict dependence of HS I activity on a GATA site implies that assembly of a protein complex containing a GATA-factor, presumably GATA-1 or GATA-2, is critical to activating or maintaining its function. Further dissection of the 317 base pair region demonstrates that, whereas all 317 base pairs are required for expression in megakaryocytes, only the 5′ 62 base pairs are needed for erythroid-specific reporter expression. These findings demonstrate differential lineage requirements for expression within the HS I element.
The N-terminal domain of Sxl protein disrupts Sxl autoregulation in females and promotes female-specific splicing of tra in males
Authors: Deshpande Girish , Calhoun Gretchen , Schedl Paul D.
Published: April 2021

Sex determination in Drosophila depends upon the post-transcriptional regulatory activities of the Sex-lethal (Sxl) gene. Sxl maintains the female determined state and activates female differentiation pathways by directing the female-specific splicing of Sxl and tra pre-mRNAs. While there is compelling evidence that Sxl proteins regulate splicing by directly binding to target RNAs, previous studies indicate that the two Sxl RNA-binding domains are not in themselves sufficient for biological activity and that an intact N-terminal domain is also critical for splicing function. To further investigate the functions of the Sxl N terminus, we ectopically expressed a chimeric protein consisting of the N-terminal 99 amino acids fused to β-galactosidase. The Nβ-gal fusion protein behaves like a dominant negative, interfering with the Sxl autoregulatory feedback loop and killing females. This dominant negative activity can be attributed to the recruitment of the fusion protein into the large Sxl:Snf splicing complexes that are found in vivo and the consequent disruption of these complexes. In addition to the dominant negative activity, the Nβ-gal fusion protein has a novel gain-of-function activity in males: it promotes the female-specific processing of tra pre-mRNAs. This novel activity is discussed in light of the blockage model for the tra splicing regulation.
High Bicoid levels render the terminal system dispensable for Drosophila head development
Authors: Schaeffer Valérie , Killian Darrell , Desplan Claude , Wimmer Ernst A.
Published: April 2021

In Drosophila, the gradient of the Bicoid (Bcd) morphogen organizes the anteroposterior axis while the ends of the embryo are patterned by the maternal terminal system. At the posterior pole, expression of terminal gap genes is mediated by the local activation of the Torso receptor tyrosine kinase (Tor). At the anterior, terminal gap genes are also activated by the Tor pathway but Bcd contributes to their activation. Here we present evidence that Tor and Bcd act independently on common target genes in an additive manner. Furthermore, we show that the terminal maternal system is not required for proper head development, since high levels of Bcd activity can functionally rescue the lack of terminal system activity at the anterior pole. This observation is consistent with a recent evolution of an anterior morphogenetic center consisting of Bcd and anterior Tor function.
Drosophila grain encodes a GATA transcription factor required for cell rearrangement during morphogenesis
Authors: Brown Stephen , Castelli-Gair Hombría James
Published: April 2021

The genetic mechanisms controlling organ shape are largely unknown. We show that the Drosophila grain gene is required during development for shaping the adult legs and the larval posterior spiracles. Mutant legs are short and wide rather than long and thin, while the spiracles are flat instead of dome-shaped. We demonstrate that grain encodes the GATAc transcription factor. Analysis of loss- of-function mutations at the cellular level indicates that grain affects organ shape by locally controlling cell rearrangement. Ectopic grain expression causes major morphogenetic movements, resulting in the invagination of the posterior segments into the embryo. This is the first gene that has been shown to affect epithelial morphogenesis by controlling cell rearrangements, and suggests a novel function for GATA transcription factors.
The role of Xenopus dickkopf1 in prechordal plate specification and neural patterning
Authors: Kazanskaya Olga , Glinka Andrei , Niehrs Christof
Published: April 2021

Dickkopf1 (dkk1) encodes a secreted WNT inhibitor expressed in Spemann’s organizer, which has been implicated in head induction in Xenopus. Here we have analyzed the role of dkk1 in endomesoderm specification and neural patterning by gain-and loss-of-function approaches. We find that dkk1, unlike other WNT inhibitors, is able to induce functional prechordal plate, which explains its ability to induce secondary heads with bilateral eyes. This may be due to differential WNT inhibition since dkk1, unlike frzb, inhibits Wnt3a signalling. Injection of inhibitory antiDkk1 antibodies reveals that dkk1 is not only sufficient but also required for prechordal plate formation but not for notochord formation. In the neural plate dkk1 is required for anteroposterior and dorsoventral patterning between mes-and telencephalon, where dkk1 promotes anterior and ventral fates. Both the requirement of anterior explants for dkk1 function and their ability to respond to dkk1 terminate at late gastrula stage. Xenopus embryos posteriorized with bFGF, BMP4 and Smads are rescued by dkk1. dkk1 does not interfere with the ability of bFGF to induce its immediate early target gene Xbra, indicating that its effect is indirect. In contrast, there is cross-talk between BMP and WNT signalling, since induction of BMP target genes is sensitive to WNT inhibitors until the early gastrula stage. Embryos treated with retinoic acid (RA) are not rescued by dkk1 and RA affects the central nervous system (CNS) more posterior than dkk1, suggesting that WNTs and retinoids may act to pattern anterior and posterior CNS, respectively, during gastrulation.
BMP and FGF regulate the development of EGF-responsive neural progenitor cells
Authors: Lillien Laura , Raphael Heather
Published: April 2021

Temporal changes in progenitor cell responses to extrinsic signals play an important role in development, but little is known about the mechanisms that determine how these changes occur. In the rodent CNS, expression of epidermal growth factor receptors (EGFRs) increases during embryonic development, conferring mitotic responsiveness to EGF among multipotent stem cells. Here we show that cell-cell signaling controls this change. Whereas EGF-responsive stem cells develop on schedule in explant and aggregate cultures of embryonic cortex, co-culture with younger cortical cells delays their development. Exogenous BMP4 mimics the effect of younger cells, reversibly inhibiting changes in EGFR expression and responsiveness. Moreover, blocking endogenous BMP receptors in progenitors with a virus transducing dnBMPR1B accelerates changes in EGFR signaling. This involves a non-cell-autonomous mechanism, suggesting that BMP negatively regulates signal(s) that promote the development of EGF-responsive stem cells. FGF2 is a good candidate for such a signal, as we find that it antagonizes the inhibitory effects of younger cortical cells and exogenous BMP4. These findings suggest that a balance between antagonistic extrinsic signals regulates temporal changes in an intrinsic property of neural progenitor cells.
The WNT antagonist cSFRP2 modulates programmed cell death in the developing hindbrain
Authors: Ellies Debra L. , Church Vicki , Francis-West Philippa , Lumsden Andrew
Published: April 2021

In the avian hindbrain, the loss of premigratory neural crest cells from rhombomeres 3 and 5 (r3, r5) through programmed cell death contributes to the patterning of emigrant crest cells into three discrete streams. Programmed cell death is induced by the upregulation of Bmp4 and Msx2 in r3 and r5. We show that cSFRP2, a WNT antagonist, is expressed in the even-numbered rhombomeres and that over-expression of cSfrp2 inhibits Bmp4 expression in r3 and r5, preventing programmed cell death. By contrast, depleting cSFRP2 function in r4 results in elevated levels of Msx2 expression and ectopic programmed cell death, as does overexpression of Wnt1. We propose that programmed cell death in the rhombencephalic neural crest is modulated by pre-patterned cSfrp2 expression and a WNT-BMP signalling loop.
Control of the migratory pathway of facial branchiomotor neurones
Authors: Garel Sonia , Garcia-Dominguez Mario , Charnay Patrick
Published: April 2021

Facial branchiomotor (fbm) neurones undergo a complex migration in the segmented mouse hindbrain. They are born in the basal plate of rhombomere (r) 4, migrate caudally through r5, and then dorsally and radially in r6. To study how migrating cells adapt to their changing environment and control their pathway, we have analysed this stereotyped migration in wild-type and mutant backgrounds. We show that during their migration, fbm neurones regulate the expression of genes encoding the cell membrane proteins TAG-1, Ret and cadherin 8. Specific combinations of these markers are associated with each migratory phase in r4, r5 and r6. In Krox20 and kreisler mutant mouse embryos, both of which lack r5, fbm neurones migrate dorsally into the anteriorly positioned r6 and adopt an r6-specific expression pattern. In embryos deficient for Ebf1, a gene normally expressed in fbm neurones, part of the fbm neurones migrate dorsally within r5. Accordingly, fbm neurones prematurely express a combination of markers characteristic of an r6 location. These data suggest that fbm neurones adapt to their changing environment by switching on and off specific genes, and that Ebf1 is involved in the control of these responses. In addition, they establish a close correlation between the expression pattern of fbm neurones and their migratory behaviour, suggesting that modifications in gene expression participate in the selection of the local migratory pathway.