Dorsal-ventral Boundary Research Articles

Vestigial-dependent induction contributes to robust patterning but is not essential for wing-fate recruitment in Drosophila.

Cell recruitment is a process by which a differentiated cell induces neighboring cells to adopt its same cell fate. In Drosophila, cells expressing the protein encoded by the wing selector gene, vestigial (vg), drive a feed-forward recruitment signal that expands the Vg pattern as a wave front. However, previous studies on Vg pattern formation do not reveal these dynamics. Here, we use live imaging to show that multiple cells at the periphery of the wing disc simultaneously activate a fluorescent reporter of the recruitment signal, suggesting that cells may be recruited without the need for their contact neighbors be recruited in advance. In support of this observation, when Vg expression is inhibited either at the dorsal-ventral boundary or away from it, the activation of the recruitment signal still occurs at a distance, suggesting that Vg expression is not absolutely required to send or propagate the recruitment signal. However, the strength and extent of the recruitment signal is clearly compromised. We conclude that a feed-forward, contact-dependent cell recruitment process is not essential for Vg patterning, but it is necessary for robustness. Overall, our findings reveal a previously unidentified role of cell recruitment as a robustness-conferring cell differentiation mechanism.

En1 and Lmx1b do not recapitulate embryonic dorsal-ventral limb patterning functions during mouse digit tip regeneration.

The mouse digit tip regenerates following amputation. How the regenerate is patterned is unknown, but a long-standing hypothesis proposes developmental patterning mechanisms are re-used during regeneration. The digit tip bone exhibits dorsal-ventral (DV) polarity, so we focus on En1 and Lmx1b, two factors necessary for DV patterning during limb development. We investigate whether they are re-expressed during regeneration in a developmental-like pattern and whether they direct DV morphology of the regenerate. We find that both En1 and Lmx1b are expressed in the regenerating digit tip epithelium and mesenchyme, respectively, but without DV polarity. Conditional genetics and quantitative analysis of digit tip bone morphology determine that genetic deletion of En1 or Lmx1b in adult digit tip regeneration modestly reduces bone regeneration but does not affect DV patterning. Collectively, our data suggest that, while En1 and Lmx1b are re-expressed during mouse digit tip regeneration, they do not define the DV axis during regeneration.

Spalt and disco define the dorsal-ventral neuroepithelial compartments of the developing Drosophila medulla.

Spatial patterning of neural stem cell populations is a powerful mechanism by which to generate neuronal diversity. In the developing Drosophila medulla, the symmetrically dividing neuroepithelial cells of the outer proliferation center crescent are spatially patterned by the nonoverlapping expression of 3 transcription factors: Vsx1 in the center, Optix in the adjacent arms, and Rx in the tips. These spatial genes compartmentalize the outer proliferation center and, together with the temporal patterning of neuroblasts, act to diversify medulla neuronal fates. The observation that the dorsal and ventral halves of the outer proliferation center also grow as distinct compartments, together with the fact that a subset of neuronal types is generated from only one half of the crescent, suggests that additional transcription factors spatially pattern the outer proliferation center along the dorsal-ventral axis. Here, we identify the spalt (salm and salr) and disco (disco and disco-r) genes as the dorsal-ventral patterning transcription factors of the outer proliferation center. Spalt and Disco are differentially expressed in the dorsal and ventral outer proliferation center from the embryo through to the third instar larva, where they cross-repress each other to form a sharp dorsal-ventral boundary. We show that hedgehog is necessary for Disco expression in the embryonic optic placode and that disco is subsequently required for the development of the ventral outer proliferation center and its neuronal progeny. We further demonstrate that this dorsal-ventral patterning axis acts independently of Vsx1-Optix-Rx and thus propose that Spalt and Disco represent a third outer proliferation center patterning axis that may act to further diversify medulla fates.

Isl1 Regulation of Nkx2.1 in the Early Foregut Epithelium Is Required for Trachea-Esophageal Separation and Lung Lobation.

The esophagus and trachea arise from the dorsal and ventral aspects of the anterior foregut, respectively. Abnormal trachea-esophageal separation leads to the common birth defect esophageal atresia with or without trachea-esophageal fistula (EA/TEF). Yet the underlying cellular mechanisms remain unknown. Here, we combine Xenopus and mouse genetic models to identify that the transcription factor Isl1 orchestrates trachea-esophageal separation through modulating a specific epithelial progenitor cell population (midline epithelial cells [MECs], Isl1+ Nkx2.1+ Sox2+) located at the dorsal-ventral boundary of the foregut. Lineage tracing experiments show that MECs contribute to both tracheal and esophageal epithelium, and Isl1 is required for Nkx2.1 transcription in MECs. Deletion of the chromosomal region spanning the ISL1 gene has been found in patients with abnormal trachea-esophageal separation. Our studies thus provide definitive evidence that ISL1 is a critical player in the process of foregut morphogenesis, acting in a small progenitor population of boundary cells.

Exocyst-mediated apical Wg secretion activates signaling in the Drosophila wing epithelium.

Wnt proteins are secreted signaling factors that regulate cell fate specification and patterning decisions throughout the animal kingdom. In the Drosophila wing epithelium, Wingless (Wg, the homolog of Wnt1) is secreted from a narrow strip of cells at the dorsal-ventral boundary. However, the route of Wg secretion in polarized epithelial cells remains poorly understood and key proteins involved in this process are still unknown. Here, we performed an in vivo RNAi screen and identified members of the exocyst complex to be required for apical but not basolateral Wg secretion. Specifically blocking the apical Wg secretion leads to reduced downstream signaling. Using an in vivo ‘temporal-rescue’ assay, our results further indicate that apically secreted Wg activates target genes that require high signaling activity. In conclusion, our results demonstrate that the exocyst is required for an apical route of Wg secretion from polarized wing epithelial cells.

Isl1 Regulation of Nkx2.1 in the Early Foregut Epithelium Is Required for Trachea-Esophageal Separation and Lung Lobation

The esophagus and trachea arise from the dorsal and ventral aspects of the anterior foregut, respectively. Abnormal separation of the esophagus and trachea leads to the formation of the common birth defect esophageal atresia with/without tracheoesophageal fistula (EA/TEF). Although loss of multiple signaling molecules and transcription factors has been implicated in EA/TEF formation, the underlying cellular mechanisms remain unknown. Here, we combine Xenopus and mouse genetic models to identify the transcription factor Isl1 as a new player regulating trachea-esophageal separation. Significantly, we find that Isl1 orchestrates the separation process through modulating a specific epithelial progenitor cell population (Isl1+ Nkx2.1+ Sox2+) located at the dorsal-ventral boundary of the foregut. Our lineage tracing experiments show that this population contributes to both tracheal and esophageal epithelium. Moreover, Isl1 is required for the transcription of Nkx2.1 in this unique population. Finally, deletion of the chromosomal region spanning the ISL1 gene has been found in patients with abnormal trachea-esophageal separation. Our studies thus provide definitive evidence that ISL1 is a new player in the process of foregut morphogenesis, acting in a small progenitor population of boundary cells.

Landmarks in Existing Tissue at Wounds Are Utilized to Generate Pattern in Regenerating Tissue

Regeneration in many organisms involves the formation of a blastema, which differentiates and organizes into the appropriate missing tissues. How blastema pattern is generated and integrated with pre-existing tissues is a central question in the field of regeneration. Planarians are free-living flatworms capable of rapidly regenerating from small body fragments [1]. A cell cluster at the anterior tip of planarian head blastemas (the anterior pole) is required foranterior-posterior (AP) and medial-lateral (ML) blastema patterning [2-4]. Transplantation of the head tip into tails induced host tissues to grow patterned head-like outgrowths containing a midline. Given the important patterning role of the anterior pole, understanding how it becomes localized during regeneration would help explain how wounds establish pattern in new tissue. Anterior pole progenitors were specified at the pre-existing midline of regenerating fragments, even when this location deviated from the ML median plane of the wound face. Anterior pole progenitors were specified broadly on the dorsal-ventral (DV) axis and subsequently formed a cluster at the DV boundary of the animal. We propose that three landmarks of pre-existing tissue at wounds set the location of anterior pole formation: a polarized AP axis, the pre-existing midline, and the dorsal-ventral median plane. Subsequently, blastema pattern is organized around the anterior pole. This process, utilizing positional information in existing tissue at unpredictably shaped wounds, can influence the patterning of new tissue in a manner thatfacilitates integration with pre-existing tissue in regeneration.

The Ecdysone and Notch Pathways Synergistically Regulate Cut at the Dorsal–Ventral Boundary in Drosophila Wing Discs

Metazoan development requires coordination of signaling pathways to regulate patterns of gene expression. In Drosophila, the wing imaginal disc provides an excellent model for the study of how signaling pathways interact to regulate pattern formation. The determination of the dorsal–ventral (DV) boundary of the wing disc depends on the Notch pathway, which is activated along the DV boundary and induces the expression of the homeobox transcription factor, Cut. Here, we show that Broad (Br), a zinc-finger transcription factor, is also involved in regulating Cut expression in the DV boundary region. However, Br expression is not regulated by Notch signaling in wing discs, while ecdysone signaling is the upstream signal that induces Br for Cut upregulation. Also, we find that the ecdysone-Br cascade upregulates cut-lacZ expression, a reporter containing a 2.7 kb cut enhancer region, implying that ecdysone signaling, similar to Notch, regulates cut at the transcriptional level. Collectively, our findings reveal that the Notch and ecdysone signaling pathways synergistically regulate Cut expression for proper DV boundary formation in the wing disc. Additionally, we show br promotes Delta, a Notch ligand, near the DV boundary to suppress aberrant high Notch activity, indicating further interaction between the two pathways for DV patterning of the wing disc.

The Drosophila melanogaster Mutants apblot and apXasta Affect an Essential apterous Wing Enhancer.

The selector gene apterous (ap) plays a key role during the development of the Drosophila melanogaster wing because it governs the establishment of the dorsal-ventral (D-V) compartment boundary. The D-V compartment boundary is known to serve as an important signaling center that is essential for the growth of the wing. The role of Ap and its downstream effectors have been studied extensively. However, very little is known about the transcriptional regulation of ap during wing disc development. In this study, we present a first characterization of an essential wing-specific ap enhancer. First, we defined an 874-bp fragment about 10 kb upstream of the ap transcription start that faithfully recapitulates the expression pattern of ap in the wing imaginal disc. Analysis of deletions in the ap locus covering this element demonstrated that it is essential for proper regulation of ap and formation of the wing. Moreover, we showed that the mutations apblot and apXasta directly affect the integrity of this enhancer, leading to characteristic wing phenotypes. Furthermore, we engineered an in situ rescue system at the endogenous ap gene locus, allowing us to investigate the role of enhancer fragments in their native environment. Using this system, we were able to demonstrate that the essential wing enhancer alone is not sufficient for normal wing development. The in situ rescue system will allow us to characterize the ap regulatory sequences in great detail at the endogenous locus.

Fringe proteins modulate Notch-ligand cis and trans interactions to specify signaling states.

The Notch signaling pathway consists of multiple types of receptors and ligands, whose interactions can be tuned by Fringe glycosyltransferases. A major challenge is to determine how these components control the specificity and directionality of Notch signaling in developmental contexts. Here, we analyzed same-cell (cis) Notch-ligand interactions for Notch1, Dll1, and Jag1, and their dependence on Fringe protein expression in mammalian cells. We found that Dll1 and Jag1 can cis-inhibit Notch1, and Fringe proteins modulate these interactions in a way that parallels their effects on trans interactions. Fringe similarly modulated Notch-ligand cis interactions during Drosophila development. Based on these and previously identified interactions, we show how the design of the Notch signaling pathway leads to a restricted repertoire of signaling states that promote heterotypic signaling between distinct cell types, providing insight into the design principles of the Notch signaling system, and the specific developmental process of Drosophila dorsal-ventral boundary formation.

Deadpan Contributes to the Robustness of the Notch Response

Notch signaling regulates many fundamental events including lateral inhibition and boundary formation to generate very reproducible patterns in developing tissues. Its targets include genes of the bHLH hairy and Enhancer of split [E(spl)] family, which contribute to many of these developmental decisions. One member of this family in Drosophila, deadpan (dpn), was originally found to have functions independent of Notch in promoting neural development. Employing genome-wide chromatin-immunoprecipitation we have identified several Notch responsive enhancers in dpn, demonstrating its direct regulation by Notch in a range of contexts including the Drosophila wing and eye. dpn expression largely overlaps that of several E(spl) genes and the combined knock-down leads to more severe phenotypes than either alone. In addition, Dpn contributes to the establishment of Cut expression at the wing dorsal-ventral (D/V) boundary; in its absence Cut expression is delayed. Furthermore, over-expression of Dpn inhibits expression from E(spl) gene enhancers, but not vice versa, suggesting that dpn contributes to a feed-back mechanism that limits E(spl) gene expression following Notch activation. Thus the combined actions of dpn and E(spl) appear to provide a mechanism that confers an initial rapid output from Notch activity which becomes self-limited via feedback between the targets.

Multiscale modeling goes out on a limb: in silico simulations of developmental mechanisms shared between somitogenesis and the developing embryonic avian limb bud

Avian limb buds form primarily from a mixture of ectodermal and mesenchymal cells. The mesenchymal cells derive from two sources, the lateral plate mesoderm and the somites. Somite‐derived structures in mature limbs include muscles, tendons, and vascular endothelium. Limb development potentially shares many regulatory pathways and developmental mechanisms with somitogenesis, such as WNT and FGF8 regulation of cell proliferation, MET transitions, and differentiation. Although some mechanisms are likely to be conserved, tissue‐specific contextual cues are likely to introduce differences both significant and subtle. We present three‐dimensional, multi‐scale, multicell models using the open‐source CompuCell3D modeling environment to simulate these experimentally‐observed developmental mechanisms. We combine sub‐cellular (genetic and signaling networks), cellular (cell growth, death, division, differentiation and motility) and tissue (extracellular matrix and morphogen production) level scales to explore if combining WNT, FGF8 and Delta/Notch signaling (active during somitogenesis) with contact inhibition of growth in the ectoderm and the formation and maintenance of the Dorsal‐Ventral boundary at the apical ecotodermal ridge suffices to generate the unique patterning and growth dynamics of the limb bud. Supported by NIH 2R01GM076692–07and EPA R‐10–0049A3.Grant Funding Source: N/A

Anisotropy of Crumbs and aPKC Drives Myosin Cable Assembly during Tube Formation

SummaryThe formation of tubular structures from epithelial sheets is a key process of organ formation in all animals, but the cytoskeletal rearrangements that cause the cell shape changes that drive tubulogenesis are not well understood. Using live imaging and super-resolution microscopy to analyze the tubulogenesis of the Drosophila salivary glands, I find that an anisotropic plasma membrane distribution of the protein Crumbs, mediated by its large extracellular domain, determines the subcellular localization of a supracellular actomyosin cable in the cells at the placode border, with myosin II accumulating at edges where Crumbs is lowest. Laser ablation shows that the cable is under increased tension, implying an active involvement in the invagination process. Crumbs anisotropy leads to anisotropic distribution of aPKC, which in turn can negatively regulate Rok, thus preventing the formation of a cable where Crumbs and aPKC are localized.

Developmental Biology: A Growing Role for Computer Simulations

Keeping cells separated in well-defined domains is essential for development. A new computational-experimental study elucidates the physical mechanisms that establish and maintain the dorsal-ventral compartment boundary in the Drosophila wing disc and demonstrates the increasing value of computer simulations in developmental biology.

Notch-mediated repression of bantam miRNA contributes to boundary formation in the Drosophila wing

Subdivision of proliferating tissues into adjacent compartments that do not mix plays a key role in animal development. The Actin cytoskeleton has recently been shown to mediate cell sorting at compartment boundaries, and reduced cell proliferation in boundary cells has been proposed as a way of stabilizing compartment boundaries. Cell interactions mediated by the receptor Notch have been implicated in the specification of compartment boundaries in vertebrates and in Drosophila, but the molecular effectors remain largely unidentified. Here, we present evidence that Notch mediates boundary formation in the Drosophila wing in part through repression of bantam miRNA. bantam induces cell proliferation and we have identified the Actin regulator Enabled as a new target of bantam. Increased levels of Enabled and reduced proliferation rates contribute to the maintenance of the dorsal-ventral affinity boundary. The activity of Notch also defines, through the homeobox-containing gene cut, a distinct population of boundary cells at the dorsal-ventral (DV) interface that helps to segregate boundary from non-boundary cells and contributes to the maintenance of the DV affinity boundary.

Requirements for mediator complex subunits distinguish three classes of notch target genes at the Drosophila wing margin

Spatial and temporal gene regulation relies on a combinatorial code of sequence-specific transcription factors that must be integrated by the general transcriptional machinery. A key link between the two is the mediator complex, which consists of a core complex that reversibly associates with the accessory kinase module. We show here that genes activated by Notch signaling at the dorsal-ventral boundary of the Drosophila wing disc fall into three classes that are affected differently by the loss of kinase module subunits. One class requires all four kinase module subunits for activation, while the others require only Med12 and Med13, either for activation or for repression. These distinctions do not result from different requirements for the Notch coactivator Mastermind or the corepressors Hairless and Groucho. We propose that interactions with the kinase module through distinct cofactors allow the DNA-binding protein Suppressor of Hairless to carry out both its activator and repressor functions.

Enhancer-PRE communication contributes to the expansion of gene expression domains in proliferating primordia

Trithorax-group and Polycomb-group proteins interact with chromosomal elements, termed PRE/TREs, to ensure stable heritable maintenance of the transcriptional state of nearby genes. Regulatory elements that bind both groups of proteins are termed maintenance elements (MEs). Some of these MEs maintain the initial activated transcriptional state of a nearby reporter gene through several rounds of mitosis during development. Here, we show that expression of hedgehog in the posterior compartment of the Drosophila wing results from the communication between a previously defined ME and a nearby cis-regulatory element termed the C enhancer. The C enhancer integrates the activities of the Notch and Hedgehog signalling pathways and, from the early wing primordium stage, drives expression to a thin stripe in the posterior compartment that corresponds to the dorsal-ventral compartment boundary. The ME maintains the initial activated transcriptional state conferred by the C enhancer and contributes to the expansion, by growth, of its expression domain throughout the posterior compartment. Communication between the ME and the C enhancer also contributes to repression of gene expression in anterior cells. Most interestingly, we present evidence that enhancers and MEs of different genes are interchangeable modules whose communication is involved in restricting and expanding the domains of gene expression. Our results emphasize the modular role of MEs in regulation of gene expression within growing tissues.

Identification of 11-amino acid peptides that disrupt Notch-mediated processes in Drosophila

BackgroundThe conserved Notch signaling pathway regulates cell fate decisions and maintains stem cells in multicellular organisms. Up-regulation of Notch signaling is observed in several types of cancer and is causally involved in proliferation and survival of cancer cells. Thus, it is of great interest to look for anti-Notch reagents for therapeutic purposes. In model animal Drosophila, Notch signaling restricts selection of sensory organ precursors (SOPs) during external sensory (ES) organ development. To look for novel genes that can suppress Notch signaling, we performed a gain-of-function modifier screen to look for genes that enhance the phenotype of ectopic ES organs induced by overexpression of phyllopod, a gene required for SOP specification.ResultsFrom the gain-of-function screen, we discovered that overexpression of polished rice/tarsal-less (pri/tal) increases the numbers of ES organs as well as SOPs. pri/tal is a polycistronic gene that contains four short open reading frames encoding three 11-amino acid and one 32-amino acid peptides. Ectopic expression of the 11 amino-acid peptides recapitulates the pri/tal misexpression phenotype in ectopic ES organ formation. In situ hybridization experiment reveals that pri/tal mRNA is expressed in the SOPs of the chemosensory organs and the stretch-sensing chordotonal organs.In Drosophila wing development, the Notch signaling pathway mediates the formation of the dorsal-ventral (DV) compartmental boundary and the restriction of the vein width from the primordial veins, the proveins. We also found that pri/tal mRNA is expressed in the DV boundary and the longitudinal proveins, and overexpression of Pri/Tal peptides disrupts the DV boundary formation and helps to expand the width of the wing vein. Genetic analyses further show that a Notch loss-of-function allele strongly enhances these two phenotypes. Cut and E(spl)mβ are target genes of the Notch pathway in DV boundary formation and vein specification, respectively. We also found that overexpression of Pri/Tal peptides abolishes Cut expression and co-expression of Pri/Tal peptides with phyl strongly reduces E(spl)mβ expression.ConclusionsWe show for the first time that the overexpression of Pri/Tal 11-amino acid peptides disrupts multiple Notch-mediated processes and reduces Notch target gene expression in Drosophila, suggesting that these peptides have novel antagonistic activity to the Notch pathway. Thus, our discovery might provide insights into designing new therapeutic reagents for Notch-related diseases.

Robustness and Stability of the Gene Regulatory Network Involved in DV Boundary Formation in the Drosophila Wing

Gene regulatory networks have been conserved during evolution. The Drosophila wing and the vertebrate hindbrain share the gene network involved in the establishment of the boundary between dorsal and ventral compartments in the wing and adjacent rhombomeres in the hindbrain. A positive feedback-loop between boundary and non-boundary cells and mediated by the activities of Notch and Wingless/Wnt-1 leads to the establishment of a Notch dependent organizer at the boundary. By means of a Systems Biology approach that combines mathematical modeling and both in silico and in vivo experiments in the Drosophila wing primordium, we modeled and tested this regulatory network and present evidence that a novel property, namely refractoriness to the Wingless signaling molecule, is required in boundary cells for the formation of a stable dorsal-ventral boundary. This new property has been validated in vivo, promotes mutually exclusive domains of Notch and Wingless activities and confers stability to the dorsal-ventral boundary. A robustness analysis of the regulatory network complements our results and ensures its biological plausibility.

Son of Notch, a Winged‐helix Gene Involved in Boundary Formation in the Drosophila Wing

A P element enhancer trap screen was conducted to identify genes involved in dorsal-ventral boundary formation in Drosophila. The son of Notch (son) gene was identified by the son(2205) enhancer trap insertion, which is a partial loss-of-function mutation. Based on son(2205) mutant phenotypes and genetic interactions with Notch and wingless mutations, we conclude that son participates in wing development, and functions in the Notch signaling pathway at the dorsal-ventral boundary in the wing. Notch signaling pathway components activate son enhancer trap expression in wing cells. son enhancer trap expression is regulated positively by wingless, and negatively by cut in boundary cells. Ectopic Son protein induces wingless and cut expression in wing discs. We hypothesize that there is positive feedback regulation of son by wingless, and negative regulation by cut at the dorsal-ventral boundary during wing development.