Gall Force: Stardust War 720p Torrent EXCLUSIVE

[Yvone Rollman]

<div>pebbled/hindsight DEVELOPMENTAL BIOLOGY Embryonic Initial embryonic expression of Pebbled mRNA and protein occurs in the endoderm (midgut) and extraembryonic membrane (amnioserosa) prior to germ-band extension and continues in these tissues beyond the completion of germ-band retraction. HNT mRNA accumulates beginning at stage 5 in the cellular blastoderm, in the posterior-terminal midgut primordium and dorsally in the presumptive amnioserosa. Hnt protein appears in these cells slightly later (stage 6). During stage 7, dorsal expression expands to cover the entire presumptive amnioserosa from the cephalic furrow to the posterior midgut primordium. Anteroventral staining, corresponding to the anterior midgut primordium, is first detected at stage 8. Accumulation in these tissues continues as gastrulation continues. Commencing at stage 11, expression also occurs in the developing tracheal system, glial cells of the central and peripheral nervous systems, and the ureter of the Malpighian tubules. Strikingly, pebbled is not expressed in the epidermal ectoderm, which is the tissue that undergoes the cell shape changes and movements during germ-band retraction (Yip, 1997). Effects of mutation or deletion The pebbled gene is X-linked. Hemizygous (peb/Y) embryos fail to retract their germ bands. All such embryos have the correct number of thoracic and abdominal segments that are patterned normally. As a consequence of failed-germ-band retraction, the embryos are U-shaped with their posterior region folded into the dorsal side. Additionally, mutant embryos show defects in head involution and often have a severely disrupted cephalopharyngeal skeleton. A viable peb allele is named pebbled because of its rough eye phenotype. peb alleles can be ordered into a phenytypic series (Yip, 1997). A group of genes, referred to as the U-shaped-group (ush-group), is required for maintenance of the amnioserosa tissue once it has differentiated. Using several molecular markers, amnioserosa development was examined in the ush-group mutants: u-shaped (ush), pebbled (peb), serpent (srp) and tail-up (tup). The amnioserosa in these mutants is specified correctly and begins to differentiate as in wild type. However, following germ-band extension, there is a premature loss of the amnioserosa. This cell loss is a consequence of programmed cell death (apoptosis) in ush, peb and srp, but not in tup. The ush-group genes are implicated in the maintainance of the amnioserosa's viability. In light of these mutants' unretracted phenotype, the amnioserosa could be involved in signal reception or the initiation of signal transduction with respect to the adjacent ectoderm (Frank, 1996). Several approaches have been taken to study the relationships between previously identified mutations (u-shaped, serpent, pebbled and tailup) that selectively cause germband retraction defects in homozygous embryos, and a more pleiotropically acting locus, Egfr. The former four loci are elements of at least two parallel and partially redundant cellular pathways that affect germ band retraction by acting in amnioserosal development or maintenance. An additional discrete and unique pathway, represented by Egfr, is likely to function in the germband itself. While the role of the amnioserosa during germband retraction appears to be permissive, the action of Egfr in the germband may be mediated by the cytoskeleton (Goldman-Levi, 1996). The longitudinal glia (LG), progeny of a single glioblast, form a scaffold that presages the formation of longitudinal tracts in the ventral nerve cord (VNC) of the Drosophila embryo. The LG are used as a substrate during the extension of the first axons of the longitudinal tract. The differentiation of the LG has been examined in six mutations in which the longitudinal tracts are absent, displaced, or interrupted to determine whether the axon tract malformations may be attributable to disruptions in the LG scaffold. Embryos mutant for the gene prospero have no longitudinal tracts, and glial differentiation remains arrested at a preaxonogenic state. Two mutants of the Polycomb group also lacked longitudinal tracts; here the glia fail to form an oriented scaffold, but cytological differentiation of the LG is unperturbed. The longitudinal tracts in embryos mutant for slit fuse at the VNC midline and scaffold formation is normal, except that it is medially displaced. Longitudinal tracts have intersegmental interruptions in embryos mutant for pebbled and midline. In pebbled, there are intersegmental gaps in the glial scaffold. In midline, the glial scaffold retracts after initial extension. LG morphogenesis during axonogenesis is abnormal in midline. Commitment to glial identity and glial differentiation also occurs before scaffold formation. In all mutants examined, the early distribution of the glycoprotein Neuroglian is perturbed. This is indicative of early alterations in VNC pattern present before LG scaffold formation begins. Therefore, some changes in scaffold formation may reflect changes in the placement and differentiation of other cells of the VNC. In all mutants, alterations in scaffold formation precedes longitudinal axon tract formation (Jacobs, 1993). During animal development, morphogenesis of tissues and organs requires dynamic cell shape changes and movements thatare accomplished without loss of epithelial integrity. Data from vertebrate and invertebrate systems have implicated severalcell surface and cytoskeleton-associated molecules in the establishment and maintenance of epithelial architecture, butthere has been little analysis of the genetic regulatory hierarchies that control epithelial morphogenesis in specific tissues.The Drosophila Hindsight nuclear zinc-finger protein is required during tracheal morphogenesis for themaintenance of epithelial integrity and assembly of apical extracellular structures known as taenidia. In hindsight (hnt)mutants, tracheal placodes form, invaginate, and undergo primary branching as well as early fusion events. However, starting atmidembryogenesis, the tracheal epithelium either collapses or expands to give rise to sacs of tissue. While a subset of hntmutant tracheal cells enters the apoptotic pathway, genetic suppression of apoptosis indicates that this is not the cause ofthe epithelial defects. Surviving hnt mutant tracheal cells retain cell-cell junctions and a normal subcellular distributionof apical markers such as Crumbs and DE-Cadherin. However, taenidia do not form on the lumenal surface of tracheal cells.While loss of epithelial integrity is a common feature of crumbs, stardust, and hnt mutants, defective assembly of taenidiais unique to hnt mutants. These data suggest that Hnt is a tissue-specific factor that regulates maintenance of the trachealepithelium as well as differentiation of taenidia (Wilk, 2000). During embryogenesis hnt expression commencesin the amnioserosa and midgut at stage 5 and,subsequently, initiates in a variety of additional cell types,including the tracheal system. There is nomaternal contribution of HNT RNA or protein to the earlyembryo. There isno detectable accumulation of Hnt protein in the trachealplacodes at stage 10. Hnt accumulates in thenuclei of tracheal cells beginning at early stage 11 andexpression continues in all tracheal cells from stage 12through the remainder of embryogenesis (Wilk, 2000). The epithelial defects in hnt mutants seen in the amnioserosa and in the trachealsystem show striking similarities to otherDrosophila mutants that disrupt epithelial tissues, suggestingthat Hnt has a direct function in regulating themaintenance of epithelial structure. Drosophila genes involvedin epithelial differentiation include shotgun (shg)and crumbs (crb). shg encodesDE-Cadherin, the major epithelial cadherin in Drosophila,which is required for the formation and maintenance ofmost epithelia in the embryo. crb encodes atransmembrane protein that localizes to, and that is requiredfor, maintaining the apical cell surface in ectodermalepithelia. Mutations in crb, shg, and other genes that affect epithelialintegrity (e.g., stardust) cause very similar terminal phenotypes.When an epithelial tissue breaks down, a subset ofthe epithelial cells loses polarity/structural integrity, entersthe programmed cell death pathway, and degenerates. However,the majority of cells in such tissues survive until lateembryogenesis, show normal polarity and junctional differentiation,and form small epithelial (often vesicular) units. In particular, analyses of crb and the phenotypically similar gene stardust (sdt) have revealed that the mechanisms involved in epithelial maintenance change duringdevelopment. crb and sdt are not needed for the formation of the blastoderm epithelium. During gastrulation both genes are required forthe formation of the zonula adherens. In crb and sdtmutants the zonula adherens fails to assemble throughoutthe ectoderm and amnioserosa, a defect that is soon followedby the appearance of gross morphological abnormalitiesin all ectodermal and amnioserosal cells. Surprisingly,recovery of epithelial morphology, including delayeddifferentiation of a junctional complex, is observed in crband sdt mutants during organogenesis. The extent of recoveryof epithelial morphology differs from tissue to tissueand ranges from very little (e.g., epidermis) to almostcomplete (e.g., hindgut). These findingssuggest that tissue-specific factors contribute to maintenanceof epithelial structure and can partially compensatefor loss of crb and sdt function. Hnt is likely to representsuch a tissue-specific factor required for epithelial differentiationas suggested by its phenotype and its expressionpattern, which is limited to only a subset of embryonicepithelia. Since Hnt is a zinc-finger nuclear protein that maycontrol gene expression, Hnt could modulate the expressionlevels, or could alter the repertoire, of structuralcomponents that are needed for epithelial differentiation.Such components remain to be identified; Hnt does not appear to be essential for the expression of Crb and Shotgun (Wilk, 2000).The basic structural organization of the tracheal tubes issimilar among insects, consisting of a simple monolayerepithelium with a lamina on the basal (outer) surfaceand cuticle apically (facing the lumen). The cuticle usuallycontains regular folds known as taenidia. These are arrangedin a helical pattern and function to keep the trachealtube open without compromising flexibility. Tracheal cells of hnt embryossecrete an epicuticle as well as other components of thelumen. However, the size of the lumen is variable andit is discontinuous. Moreover, the taenidial folds are absentor are highly disorganized in hnt embryos. Since the tracheaelose their integrity in hnt mutants prior to thepresence of morphologically identifiable taenidia, it is possiblethat the absence of regular taenidial folds is a secondaryeffect. Molecular components of the taenidia have notyet been identified; thus it is not known exactly when thetaenidia begin to form during tracheal morphogenesis. Circumstantialevidence suggests that the taenidia begin to form prior to completion of secretion of the outer epicuticle. It is thus possible that taenidial components are missing from an early stage in hnt mutants and that Hnt plays a direct role in taenidium formation independent of its role in maintenance of tracheal integrity. Consistent with this possibility, regular taenidial folds are present in other mutants, such as sdt and crb, in which the tracheal system disintegrates. Thus abnormal taenidial organization is not an obligatory consequence of loss of integrity of the tracheal epithelium (Wilk, 2000).Together these results suggest that there are three roles forHnt during tracheal morphogenesis: (1) to preserve epithelial integrity; (2) to direct assembly of the extracellularstructures known as taenidia, and (3) to prevent apoptosis.Since Hnt is likely to be a transcription factor, it is presumedthat Hnt resides in a genetic hierarchy that regulates orcoordinates these processes. In one model, Hnt might havea tripartite role, independently regulating epithelial integrity,directing taenidial assembly, and preventing programmedcell death. In a second model, bothapoptosis and defective taenidial assembly may be indirectconsequences of defects in epithelial architecture. A third model suggests that Hnt may independently regulate epithelial architectureand assembly of taenidia, while apoptosis in hnt-mutanttracheae might be an indirect consequence of defectsin the first of these processes (Wilk, 2000). Hindsight and the leading edge The leading edge (LE) is a single row of cells in the Drosophila embryonic epidermis that marks the boundarybetween two fields of cells: the amnioserosa and the dorsal ectoderm. LE cells play a crucial role in the morphogenetic process of dorsal closure and eventually form the dorsal midline of the embryo. Mutations that blockLE differentiation result in a failure of dorsal closure and embryonic lethality. How LE cells are specified remainsunclear. To explore whether LE cells are specified in response to early dorsoventral patterning information or whether they arise secondarily, the extent of amnioserosa and dorsal ectoderm was altered genetically, and LE cell fate was assayed. No expansion of LE fate is observed in dorsalized or ventralized mutants. Furthermore, the LE fate arises as a single row of cells, wherever amnioserosa tissue and dorsal epidermis are physically juxtaposed. Taken together these data indicate that LE formation is a secondary consequence of early zygotic dorsal patterning signals. In particular, proper LE specification requires the function of genes such as u-shaped and hindsight, which are direct transcriptional targets of the early Decapentaplegic/Screw patterning gradient, to establish a competency zone from which LE arises. It is proposed that subsequent inductive signaling between amnioserosa and dorsal ectoderm restricts the formation of LE to a single row of cells (Stronach, 2001). Using mutations that influence DV patterning, it is possible to alter the size and distribution of BMP target gene expression patterns, which indicate the extent of amnioserosa and dorsal ectodermal cell fates. If LE fate was specified directly by a particular threshold level of BMP signal, then one would expect LE fate to be perturbed in concert with amnioserosa and dorsal ectoderm fates in DV mutants. Mutations in genes such as dorsal, Toll, brinker and short gastrulation alter the size of BMP target gene expression domains; however, these mutants failed to alter specification of LE fate. Among these genotypes, brk and sog specifically modulate the shape of the BMP signaling gradient in a region where LE fate might arise, yet LE formation in these mutants is fundamentally normal. Furthermore, in dorsalized embryos, LE cells were observed regularly at the boundary between amnioserosa and dorsal ectoderm even when the morphology of these tissues was severely disrupted. Islands of amnioserosa cells within a field of ectoderm were consistently surrounded with a single row of LE cells, independent of the number of amnioserosa cells constituting the island. The converse situation also occurred; again, a single row of LE cells formed at the boundary between the ectoderm and amnioserosa (Stronach, 2001). DV mutants were also analyzed to determine whether a decrease in BMP signaling activity converts amnioserosa to LE as predicted by a gradient patterning model. A range of ventralizing mutations (cactus, sog, screw, dpp) displaying progressive loss of amnioserosa tissue did not give rise to embryos with an expanded domain of LE cells. In fact, LE cells were not detected in the absence of amnioserosa. No situation was found in which an altered BMP gradient was associated with expanded LE fate, thus the prediction of a direct gradient response model does not explain LE fate specification (Stronach, 2001). Notably, DV mutant embryos that perturb the BMP gradient, also perturb the expression domains of target genes, including u-shaped and hindsight, without altering LE specification. However, loss of ush and hnt function results in specific and distinct perturbations in LE formation. Thus, the interpretation that LE fate specification is not a direct early response to the BMP gradient is favored, but rather is a secondary consequence of the specification of dorsal fates through the action of BMP target genes like ush and hnt (Stronach, 2001). Taken together, these results raise the possibility that amnioserosa may be required for LE formation. To address the function of amnioserosa for LE specification, puc enhancer expression was examined in several mutants of the U-shaped class, including u-shaped (ush) and hindsight (hnt). Incidentally, the dorsal expression domains of these genes are directly regulated by DV patterning signals. In these mutant embryos, the amnioserosa tissue is fated normally and begins to differentiate up to stage 11, but then degenerates prematurely. In both ush and hnt mutants, programmed cell death takes place over the course of a few hours, with elimination of amnioserosa cells by stage 13 -- the time when dorsal closure would normally commence. Unexpectedly, different patterns of expression were observed with the puc enhancer in the two mutants. In ush embryos, ß-gal-positive cells were not detected. In contrast, hnt mutant embryos displayed Puc-positive LE cells at the edge of the dorsal ectoderm, albeit with less uniform expression than normally observed. To confirm these observations, the accumulation of dpp mRNA in the LE was examined. Similar to puc enhancer expression, differential expression of dpp was observed in ush versus hnt mutant embryos. ush mutant embryos show a consistent and significant reduction in LE dpp expression, although residual dpp transcripts are seen. dpp expression appears relatively normal in hnt mutant embryos (Stronach, 2001). In addition to the differential expression of two LE markers in the U-shaped mutants, ectopic expression of LE markers is observed only in hnt mutant embryos. ß-gal-positive cells were observed in the region of the amnioserosa in hnt mutants as early as stage 11, raising the possibility that this could be an example of expanded LE cell fates. These cells adopt only partial LE cell fate, for the following reasons. These cells do not express the LE marker Fasciclin III, but do express two other LE molecules, albeit aberrantly. puc, for example, is expressed precociously in these cells, preceding Fasciclin III expression in the ectoderm, and dpp is rarely but reproducibly expressed. Additionally, cells in this region express amnioserosa fate markers such as race, through stage 11. Thus, based on the possibility that these cells may co-express LE and amnioserosa markers during stage 11, their identity cannot be unequivocally determined. These results may indicate that these cells are of mixed fate. The presence of ectopic LE-like cells in hnt mutant embryos, coupled with the severe reduction of LE fate markers in ush</</div>