Drosophila Stripe is a triple zinc-finger transcription factor that is closely related to mammalian Early Growth Response-1 (EGR-1, also known as Krox24; Frommer et al.; Lee et al., 1995). Although the first reported allele of stripe was a weak hypomorph exhibiting an anterior-to-posterior "stripe" on the adult thorax (Bridges and Morgan, 1923), a number of additional alleles have been reported which affect muscle patterning in the both embryo and adult. Most hypomorphs exhibit defects in the patterning of a specific muscle(s) or muscle group(s), while the most severe (presumed null) mutations result in embryos that cannot hatch due to systemic failure of muscle patterning (Costello and Wyman, 1986; de la Pompa et al., 1989; Lee et al., 1995; Usui, 2004; Soler, 2004; Volk and VijayRaghavan, 1994; Vorbruggen and Jackle, 1997). Interestingly, we now know that stripe is not required in the muscles at all, but in the epidermal tendon cells (Becker et al., 1997; Frommer et al., 1994).
(Kang et al., 2003) mechanism, whereby one regulator controls a second regulator and then both bind a common target gene. It has been shown both in prokaryotes (Shen-Orr et al., 2002) and yeasts (Lee et al., 2002) that this mode of regulation appears relatively frequently and is favored over others, e.g. autoregulation motifs, single input motifs in which one regulator controls several genes, or regulator chain motifs whereby one gene regulates a second which regulates a third, and so on. Such an over-representation of the feed-forward motif is probably due to its potential to provide enhanced sensitivity and temporal control to the transcriptional response. The feed-forward loop is especially suitable for eliciting precise threshold responses of morphogen targets as it allows a strong response of the target gene to small changes in the activity of the regulator that initiates the loop (Dpp), because of the combined action with the second regulator (Zen). In fact Bcd and Dl use mechanisms that are reminiscent of the feed-forward loop to activate their high level targets. Bcd regulates zygotic hunchback (hb) and together Bcd and Hb activate the downstream target even-skipped (eve) stripe 2 (Small et al., 1992), and Dl activates sna with the help of Twi (Ip et al., 1992b). It is striking that the three morphogen gradients involved in specifying the Drosophila embryonic axes use the feed-forward strategy to regulate downstream target genes.
Although Stripe is presumed to have many targets, relatively few have been studied in detail. One such target is the spectraplakin short stop (shot). Spectraplakins are extremely large cytoskeletal proteins with characteristics of both the spectrin and plakin families, and are generally thought to function by linking cytoskeletal filaments to one another or to the membrane (Roper et al., 2002). shot, the only known Drosophila spectraplakin, contains at least four transcriptional start sites and exhibits extensive alternative splicing, producing a highly modular protein with diverse functions (Gregory and Brown, 1998; Lee et al., 2003; Lee et al., 2000; Roper and Brown, 2003; van Vactor et al., 1993). MACF and BPAG1 (dystonin), vertebrate homologs of Shot, are some of the few proteins capable of binding actin filaments and microtubules simultaneously (Karakesisoglou et al., 2000; Lee and Kolodziej, 2002a; Leung et al., 1999). Shot can also associate with junctional proteins, integrins and E-cadherin (Gregory and Brown, 1998; Lee et al., 2003; Lee and Kolodziej, 2002b; Roper and Brown, 2003; Subramanian et al., 2003).
Segment-specific properties of the DA3 muscle Col expression in stage 15 embryos showed that the DA3 muscle forms in the thoracic (T) T2-T3 segments and abdominal (A) A1- A7 segments, but not in the T1 segment and was thinner in T2-T3 than in A1-A7 (Fig. 1A). Since the size of Drosophila larval muscles correlates with their number of nuclei (Demontis and Perrimon, 2009), we counted the number of nuclei in the DA3 muscle. Fewer nuclei were present in T2 and T3 (six on average) than in A1-A7 (eight on average) (Fig. 1B), showing that this number is segment specific. Col expression in the somatic mesoderm is first detected at embryonic stage 10, in a cluster of cells at the same dorsal position in all trunk segments, including T1 (Fig. 1C). This cluster gives rise to the DA3/DO5 progenitor in T2 and T3 and to the DA3/DO5 and DO4/DT1 progenitors in A1-A7 (Fig. 1D). Following asymmetric division of the DA3/DO5 progenitor, col transcription is maintained in the DA3 FC but is repressed in the sibling DO5 FC, a repression mediated by N; it is also not maintained in the DO4 and DT1 FCs (Crozatier and Vincent, 1999). Nau is expressed in the same progenitors as Col (Fig. 1C,D). However, Nau and Col co- expression is only transient. col transcription is maintained in the DA3 FC and is activated in the nucleus of each FCM incorporated into the growing DA3 myofiber, whereas nau is transiently transcribed in the DA3 lineage and is activated in FCM nuclei incorporated into the DO5 myofiber (Dubois et al., 2007). This leads to a specific accumulation of Col and Nau in the DA3 and DO5 muscles, respectively (Fig. 1E). Thus, the progenitor/FC stage is the specific step in the DA3/D05 lineage at which Nau and Col are expressed together. One Col- and Nau-expressing progenitor is found in T2 and T3, two progenitors in A1-A7 and none in T1, correlating with the final muscle pattern (Bate and Rushton, 1993; Crozatier and Vincent, 1999). Transient co-transcription of col and nau was nevertheless observed in a cell issued from the Col- expressing cluster in T1, although at a very low level compared with other segments (Fig. 1F). This indicates that a positive input required to upregulate the expression of these two iTFs in progenitor cells is missing in T1. In summary, transient expression of Nau and Col at the progenitor stage is segment specific and foreshadows the segment-specific formation of DA3/DO5 and DO4/DT1 muscles (Fig. 1G).
The atypical cadherin Fat is a conserved regulator of planar cell polarity, but the mechanisms by which Fat controls cell shape and tissue structure are not well understood. Here, we show that Fat is required for the planar polarized organization of actin denticle precursors, adherens junction proteins and microtubules in the epidermis of the late Drosophilaembryo. In wild-type embryos, spatially regulated cell-shape changes and rearrangements organize cells into highly aligned columns. Junctional remodeling is suppressed at dorsal and ventral cell boundaries, where adherens junction proteins accumulate. By contrast, adherens junction proteins fail to accumulate to the wild-type extent and all cell boundaries are equally engaged in junctional remodeling in fat mutants. The effects of loss of Fat on cell shape and junctional localization, but not its role in denticle organization, are recapitulated by mutations in Expanded, an upstream regulator of the conserved Hippo pathway, and mutations in Hippo and Warts, two kinases in the Hippo kinase cascade. However, the cell shape and planar polarity defects in fat mutants are not suppressed by removing the transcriptional co-activator Yorkie, suggesting that these roles of Fat are independent of Yorkie-mediated transcription. The effects of Fat on cell shape, junctional remodeling and microtubule localization are recapitulated by expression of activated Notch. These results demonstrate that cell shape, junctional localization and cytoskeletal planar polarity in the Drosophilaembryo are regulated by a common signal provided by the atypical cadherin Fat and suggest that Fat influences tissue organization through its role in polarized junctional remodeling.
The specific functions of gene products frequently depend on the developmental context in which they are expressed. Thus, studies on gene function will benefit from systems that allow for manipulation of gene expression within model systems where the developmental context is well defined. Here we describe a system that allows for genetically controlled overexpression of any gene of interest under normal physiological conditions in the early Drosophilaembryo. This regulated expression is achieved through the use of Drosophila lines that express a maternal mRNA for the yeast transcription factor GAL4. Embryos derived from females that express GAL4 maternally activate GAL4-dependent UAS transgenes at uniform levels throughout the embryo during the blastoderm stage of embryogenesis. The expression levels can be quantitatively manipulated through the use of lines that have different levels of maternal GAL4 activity. Specific phenotypes are produced by expression of a number of different developmental regulators with this system, including genes that normally do not function during Drosophila embryogenesis. Analysis of the response to overexpression of runt provides evidence that this pair-rule segmentation gene has a direct role in repressing transcription of the segment-polarity gene engrailed. The maternal GAL4 system will have applications both for the measurement of gene activity in reverse genetic experiments as well as for the identification of genetic factors that have quantitative effects on gene function in vivo.
ABSTRACT The Drosophila embryonic Central Nervous System (CNS) develops from the ventro- lateral region of the embryo, the neuroectoderm. Neuroblasts arise from the neuroectoderm and acquire unique fates based on the positions in which they are formed. Previous work has identified six genes that pattern the dorsoventral axis of the neuroectoderm: Drosophila epidermal growth factor receptor (Egfr), ventral nerve cord defective (vnd), intermediate neuroblast defective (ind), muscle segment homeobox (msh), Dichaete and Sox-Neuro (SoxN). The activities of these genes partition the early neuroectoderm into three parallel longitudinal columns (medial, intermediate, lateral) from which three distinct columns of neural stem cells arise. Most of our knowledge of the regulatory relationships among these genes derives from classical loss of function analyses. To gain a more in depth understanding of Egfr-mediated regulation of vnd, ind and msh and investigate potential cross-regulatory interactions among these genes, we combined loss of function with ectopic activation of Egfr activity. We observe that ubiquitous activation of Egfr expands the expression of vnd and ind into the lateral column and reduces that of msh in the lateral column. Through this work, we identified the genetic criteria required for the development of the medial and intermediate column cell fates. We also show that ind appears to repress vnd, adding an additional layer of complexity to the genetic regulatory hierarchy that patterns the dorsoven- tral axis of the CNS. Finally, we demonstrate that Egfr and the genes of the achaete-scute complex act in parallel to regulate the individual fate of neural stem cells.
The first signs of junction establishment are already observ- able during cellularization and define different membrane do- mains along the baso-apical axis. These early polarisation cues are rapidly reinforced through the activity of embryonic genes. A key step is the formation, from the earlier dispersed spot junc- tions, of a continuous ring of apical adherens junctions connected with actin filaments, the zonula adherens. Zonula adherens contribute to epithelium cohesion and prevent diffusion of mol- ecules along lateral membranes and between adjacent cells (Knust and Bossinger, 2002). Inactivation of shotgun (Tepass, 1996) or armadillo (Muller and Wieschaus, 1996) , which encode major components of adherens junctions (DE-cadherin and β - catenin, respectively), results in the loss of epithelial features. In addition, adherens junctions are a major element of epithelial polarity and, together with other protein complexes, define suc- cessive basal-apical regions (Fig. 2). Schematically, the apical- most membrane domain can be characterised by the localisation of a membrane protein, Stranded-at-second (Sas) (Schonbaum et al. , 1992) and the secretion of Yellow (Kornezos and Chia, 1992), a protein putatively involved in catecholamine synthesis (see below). The apical domain is specified by a protein complex that is composed of two PDZ proteins, Bazooka (Par3) (Benton and St Johnston, 2003) and DmPar6 (Hurd et al. , 2003) and the Drosophila atypical protein kinase (DaPKC) (Rolls et al. , 2003). A subapical region (SAR or Marginal Zone) lies between the apical cell face and the adherens junctions and is determined by a second protein complex. The SAR domain is organised by Crumbs, a transmembrane protein thought to participate in tissue cohesion through homotypic extracellular interactions (Tepass et al. , 1990). Consistent with this interpretation, embryos mutant for crumbs (like those lacking bazooka ) display a dramatic phenotype, with the absence of most of the cuticle resulting from a highly disorganised epidermis (Tepass et al. , 1990). A short cytoplasmic Fig. 1. Epidermal cells of the Drosophilaembryo: origin and formation
The ventral epidermis of Drosophila embryos is a well-established system for studying cell fate specification. At the end of embryogenesis, epidermal cells secrete a patterned array of cuticular structures that reflect the cell identities acquired in the epidermis at earlier stages of development. On the ventral surface of the larval abdomen, eight segmental belts of hook-shaped denticles alternate with expanses of flat, or naked, cuticle (Campos-Ortega and Hartenstein, 1985). Each belt contains roughly six rows of denticles, with each row displaying a characteristic size, shape and polarity (Bejsovec and Wieschaus, 1993). These distinct morphologies indicate unique positional values, at least some of which are imparted by signal transduction from the highly conserved Wg/Wnt growth factor pathway (reviewed by Bejsovec, 2006). During early embryogenesis, a cascade of transcription factors (reviewed by Akam, 1987) leads to activation of wg gene expression in segmental stripes that lie within the zone of cells that will secrete naked cuticle (Payre et al., 1999). Ectopic overexpression of wg across the segment (Noordermeer et al., 1992), or hyperactivation of downstream components in the Wg signaling pathway (Pai et al., 1997), eliminates the denticle belts. Conversely, loss of wg activity causes all ventral epidermal cells to secrete denticles (Nüsslein-Volhard et al., 1984). The diversity of denticles is also reduced in wg null mutants, with most resembling the large denticles typical of the fifth row of the wild-type belt (Bejsovec and Wieschaus, 1993). Thus Wg signaling controls not only the segmental specification of naked cuticle expanses, but also generates the diversity of cell fates that give rise to the uniquely shaped denticles within the denticle belt.
The generation of cell diversity depends on a series of events which occur during embryonic development. This set of events have to be coordinated in order to achieve the spatial organization which will allow normal morphogenesis. In most cases, the final size and shape of the organism are species-specific features, based in ordered cell proliferation and spatial cell differentiation. These two closely linked processes are under simultaneous con- trol by signaling systems. The local activation of these signaling pathways might be controlled by cell-cell interactions through a global program of communication. "Entelechia" (see García-Bellido and de Celis, 1992 for a detailed description) is reached when this program of communication and the morphogenetic process are completed. In this sense, Driesch’s "entelechia" as "a resultant action of many complicated elemental interactions" (Driesch, 1908), becomes implemented by discrete molecular and cellular opera- tions. In a developmental landscape, as postulated by Antonio García-Bellido, local differences in the activity of "martial" genes, nuclear factors in charge of the developmental operations affecting spatial organization and differentiation, will drive the expression of control ligands or "emissors" which will affect neighboring cell proliferation. "Entelechia" would be attained when the level of activity of these "martial" genes turns equal in the total cell population and cell division stops.
Fig. 2. (Right) Sense organs of wild type, bxd and abx embryos stained with antibody 22C10. (A) Wild type larva showing the full complement of sensory neurons on one side of the body. In this and all other figures, anterior is to the left and dorsal is up. (B) Segments T3 and A1 of a wild type larva showing the dch3 of T3 (oblique arrow) and lch5 of A1 (horizontal arrow). The dbd neurons are indicated with oblique arrowheads. (C) Segments A1 and A2 of a bxd mutant embryo. Typically in bxd embryos vchA/B (arrow)) and v’ch1 (arrowhead) are missing in A1. (D) Segments T2 and T3 of an abx mutant embryo showing an additional ch sensillum comprising dch3 in T3. T2 dch3 is normal. Individual ch sensilla are indicated with asterisks.
Tests of simple eﬀ ects are presented in Tab. IV. Relative to control, the presence of embryo still exerts decelerating inﬂ uence on mycelium growth in the direction opposite from embryo ( 0 ), as well as on the vertical size of the growth curve (). The embryo appears to inhibit the growth on the side of the embryo, especially in late stages of the growth experiment, which is reﬂ ected by signiﬁ cant increase of 1 and indicates slower growth in respective dishes. The restrictive eﬀ ects of embryo placement on mycelium growth were also evident in the direction opposite from embryo, but to a lesser extent. This observation could be explained by release of substances inhibiting mycelium growth by the plant embryo and successive spread of the inhibitors by diﬀ usion through the cultivation medium.
The SDD model is possible under artificial conditions The drug treatment data of  revealed that the inner part of the egg, i. e. the yolk became permissive for Bcd if embryos were bathed in substances affecting the cyto- architecture. In vinblastine-treated embryos, the Bcd protein movement behaved exactly as the SDD model would have predicted, i. e. it moved to the posterior in a broad front (Fig. 2e) and the yolk became permissive for Bcd. However, these are artificial conditions since em- bryos are not exposed to vinblastine in nature. This ob- servation also revealed several apparent weaknesses of the SDD model that were never before discussed. If the model was correct, why should the embryo translate a protein at the tip while the majority remains in the inter- ior of the embryo, never reaching the blastoderm nuclei? Secondly, how would an insect egg three times the size of Drosophila, e. g. that of the blow fly Lucilia sericata [18, 23] solve the problem of Bcd movement through the yolk where the expansive distance in this large em- bryo creates further physical constraints? Thirdly, why
)"t J De\' BioI 35 63 67 (1991) 63 Shurl CUIII rilil/liull The role of the polarizing zone in the pattern of experimental chondrogenesis in the chick embryo interdigital space DOMINGO MACiAS' and YOLA[.]
The culture of the grapevine in the north Morocco is practiced in a traditional way. The vegetable material used corresponds to very old varieties or local denominations. There are no introductions or uses of new or selected varieties. Although the prospecting was made on a limited study they showed the existence of a great varietal diversity. Indeed, Twenty one “local varieties” were listed in this study. However, in the absence of a pomological and morphological characterization, the value of the
There are both slow and fast heat shock response genes. The fast heat shock response genes increased their level of expression rapidly within 30 minutes of exposure to an inducing temperature. Strikingly, these genes have restored their level of expression to almost normal levels after 1 h heat shock followed by a 30 minutes recovery period. Such a behaviour is observed for examples for the genes hsp22, alpha tubulin at 84D, posterior sex combs, frizzled but also for twist and many uncharacterised genes such as CG I8282. Their relative levels of detected transcript in response to heat shock decreases quickly within 30 minutes (fig.6.2). In contrast, the slow heat shock response genes, such as Hsc70Cb or P h a s f the latter encodes a eukaryotic-initiation- factor-4E binding protein or the unknown genes CG4484, CG2676, CG2708 (and many others) seemed to reach significantly increased levels of expression only during the recovery period (fig.6.3.). This observation is consistent with a recent study, which investigated the tissue and development specific turnover of hsp70 transcripts after heat shock and during recovery in Drosophila. A remarkable two hours delay in the transcriptional activation of the hsp70 gene was observed in cyst cells and others unidentified cells of the adult testes (Lakhotia and Prasanth, 2002). The larval Malpighian tubules are also known to synthesize hsp70 only during recovery but not immediately after heat shock (Krebs and Feder, 1997). It is interesting to note that not
To examine the expression pattern of cIFABP , we dissected the small intestine of embryos of various developmental stages and examined it with in situ hybridization. The expression of cIFABP was not detected before day 6 (Fig. 1B), and began to be detected at day 7 of incubation (Fig. 1C), and was maintained during embryonic period (Fig. 1 D,E). The expression of cIFABP was seen specifically in the small intestinal epithelium, not in the esophagus, proventriculus and gizzard (data not shown). We also re-investigated the expression of sucrase by the immunohis- tochemical technique and of CdxA by in situ hybridization tech- nique. As is reported previously (Matsushita, 1985), sucrase was first expressed in 10 day embryo and was maintained after this stage up to post-hatch stages except that it disappeared just before hatching (Fig. 1 F,G). Expression pattern of CdxA in the chicken embryo was previously reported (Ishii et al., 1997). Expression begins in the gut endoderm at stage 10 (Hamburger and Hamilton, 1951), and continues throughout the embryonic stage (Fig. 1 H-J). Thus cIFABP , as sucrase and CdxA , is a specific marker of the small intestinal epithelium.
T HE central nervous system (CNS) of the Drosophila stein and Posakony 1990; Bhat and Schedl 1994; Bhat embryo provides an important paradigm for investi- et al. 1995; Hirata et al. 1995; Knoblich et al. 1995; gating the problem of asymmetric division of neural Spana and Doe 1995, 1996; Buescher et al. 1998; Dye precursor cells during development. In the ventral et al. 1998; Skeath and Doe 1998; Lear et al. 1999; Wai nerve cord of the Drosophilaembryo, ⵑ30 neuroblast et al. 1999; Mehta and Bhat 2001). One of the earliest (NB) cells in each hemi-segment delaminate in about evidences comes from a study on the development of five successive waves along the mediolateral and anterior- the adult sensilla in which the neurogenic gene Notch posterior axes in rows and columns in a stereotyped plays a role in generating asymmetric division of second- and spatio-temporal pattern (Hartenstein and Campos- ary precursor cells (Hartenstein and Posakony 1990). Ortega 1984; Doe 1992). Each of these NBs has ac- Using the temperature-sensitive allele of Notch, it was quired a unique fate by the time it is formed, and the shown that eliminating Notch activity in sensillum pre- NB that forms in a given position at a given time always cursors leads to hyperplasia of the sensory neurons at acquires the same fate (reviewed in Bhat 1999). A neu- the expense of accessory cells (i.e., shaft, socket cells). roblast then functions as a stem cell and divides by asym- Notch, together with Numb (Nb), also regulates asym- metric mitosis, renewing itself with each division and pro- metric fate specification to progeny of GMC in the ven- ducing a chain of ganglion mother cells (GMCs). A GMC tral nerve cord (Buescher et al. 1998; Skeath and Doe does not self-renew; instead it divides to generate two 1998; Lear et al. 1999; Wai et al. 1999). In the distinct neurons. These postmitotic neurons then un- GMC-1 → RP2/sib lineage, loss of Notch or nb leads to dergo cyto-differentiation. At the end of neurogenesis, the symmetric division of GMC-1. While both progeny each of the hemi-neuromeres has ⵑ320 neurons and assume RP2 fate in Notch mutants, they assume a sib ⵑ30 glia, the other principal cell type in the CNS (Boss- fate in nb mutants (Buescher et al. 1998; Lear et al. ing et al. 1996; Schmidt et al. 1997). Thus, a complex 1999; Schuldt and Brand 1999; Wai et al. 1999). Nb array of different cell types is formed from relatively few appears to block the intracellular domain of Notch from
Int J De' BioI 35 191 195 (1991) 191 Morphogenetic features in the tail region of the rat embryo LJILJANA KOSTOVIC KNEZEVIC', SRECKO GAJOVIC and ANTON 5VAJGER Department of Histology and Embryology, F[.]