Top PDF Regulation of Gastrulation Through Dynamic Patterning in the Drosophila Embryo

Regulation of Gastrulation Through Dynamic Patterning in the Drosophila Embryo

Regulation of Gastrulation Through Dynamic Patterning in the Drosophila Embryo

In a developing organism, tissues have long been proposed to be patterned by spatially graded signals that specify cell fate in a concentration-dependent manner. Classically, these “morphogens” have been defined as originating from a defined source and forming a graded distribution by diffusion and degradation; however, in recent years it has become clear that morphogens can become spatially organized by a variety of mechanisms. Two of the best-characterized morphogen gradients pattern the anterior-posterior (AP) and dorsal- ventral (DV) axes of the Drosophila early embryo: the Bicoid and Dorsal transcription factor gradients (rev. in Porcher and Dostatni, 2010; Reeves and Stathopoulos, 2009). Their graded distributions are established using very different mechanisms. Bicoid is locally translated because its mRNA contains a localization sequence; whereas, Dorsal is localized to the nucleus more strongly in the ventral regions of the embryo because of localized Toll- receptor associated signaling. Live imaging has revealed significant dynamics in the exact levels of Bicoid (e.g., Gregor et al., 2007b; Little et al., 2011); however, the dynamics of target gene expression examined in fixed embryos suggest that the levels of Bicoid are important, but not the only defining factor in the expression of target genes (e.g., Jaeger et al., 2004; Ochoa-Espinosa et al., 2009). In contrast, no study to date has investigated systematically temporal features of the Dorsal gradient and its relationship to the expression of its target genes.
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Dynamic Decapentaplegic signaling regulates patterning and adhesion in
the Drosophila pupal retina

Dynamic Decapentaplegic signaling regulates patterning and adhesion in the Drosophila pupal retina

Our results in the wing raise the interesting possibility that regulation of DE-cadherin and Rho1-dependent cell shape and cell adhesion might be a characteristic of Dpp pathway activity common to other biological systems. Similar to the pupal retina, epithelial cells in the wing disc with reduced Dpp signaling displayed abnormal morphologies and were unable to maintain their positions. In the case of the wing, these defects were manifested as viable cysts of mutant cells that were basally excluded from the epithelium (Gibson and Perrimon, 2005; Shen and Dahmann, 2005). The mechanisms involved in such cell behaviors remain unknown. Our results suggest that the role of Dpp signaling during wing patterning also involves DE-cadherin and Rho1 (Fig. 9). Our experiments do not distinguish whether the defects in wing cell fates are a direct or a secondary effect of altered cell adhesion, although altering DE-cadherin activity by itself was not sufficient to cause such defects (data not shown). Cell adhesion and cell fate have been related previously: for example, Rho-dependent cell shape changes can influence fate decisions in stem cells (McBeath et al., 2004). Despite the commonalities observed, tissue-specific factors are likely to regulate Dpp-dependent epithelial patterning: for example, Rst does not appear to have a role in wing development, and we did not observe changes in retinal Tubulin distribution reported for the wing (Gibson and Perrimon, 2005) (see Fig. S2E-H in the supplementary material).
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Quantitative Single-Embryo Profile of Drosophila Genome Activation and the Dorsal–Ventral Patterning Network

Quantitative Single-Embryo Profile of Drosophila Genome Activation and the Dorsal–Ventral Patterning Network

ABSTRACT During embryonic development of Drosophila melanogaster, the maternal-to-zygotic transition (MZT) marks a significant and rapid turning point when zygotic transcription begins and control of development is transferred from maternally deposited transcripts. Characterizing the sequential activation of the genome during the MZT requires precise timing and a sensitive assay to measure changes in expression. We utilized the NanoString nCounter instrument, which directly counts messenger RNA transcripts without reverse transcription or amplification, to study .70 genes expressed along the dorsal–ventral (DV) axis of early Drosophila embryos, dividing the MZT into 10 time points. Transcripts were quantified for every gene studied at all time points, providing the first dataset of absolute numbers of transcripts during Drosophila development. We found that gene expression changes quickly during the MZT, with early nuclear cycle 14 (NC14) the most dynamic time for the embryo. twist is one of the most abundant genes in the entire embryo and we use mutants to quantitatively demonstrate how it cooperates with Dorsal to activate transcription and is responsible for some of the rapid changes in transcription observed during early NC14. We also uncovered elements within the gene regulatory network that maintain precise transcript levels for sets of genes that are spatiotemporally cotranscribed within the presumptive mesoderm or dorsal ectoderm. Using these new data, we show that a fine-scale, quantitative analysis of temporal gene expression can provide new insights into developmental biology by uncovering trends in gene networks, including coregulation of target genes and specific temporal input by transcription factors.
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Wollknäuel is required for embryo patterning and encodes the
Drosophila ALG5 UDP glucose:dolichyl phosphate
glucosyltransferase

Wollknäuel is required for embryo patterning and encodes the Drosophila ALG5 UDP glucose:dolichyl phosphate glucosyltransferase

N-linked glycosylation is a prevalent protein modification in eukaryotic cells. Although glycosylation plays an important role in cell signaling during development, a role for N-linked glycosylation in embryonic patterning has not previously been described. In a screen for maternal factors involved in embryo patterning, we isolated mutations in Drosophila ALG5, a UDP-glucose:dolichyl- phosphate glucosyltransferase. Based on the embryonic cuticle phenotype, we designated the ALG5 locus wollknäuel (wol). Mutations in wol result in posterior segmentation phenotypes, reduced Dpp signaling, as well as impaired mesoderm invagination and germband elongation at gastrulation. The segmentation phenotype can be attributed to a post-transcriptional effect on expression of the transcription factor Caudal, whereas wol acts upstream of Dpp signaling by regulating dpp expression. The wol/ALG5 cDNA was able to partially complement the hypoglycosylation phenotype of alg5 mutant S. cerevisiae, whereas the two wol mutant alleles failed to complement. We show that reduced glycosylation in wol mutant embryos triggers endoplasmic reticulum stress and the unfolded protein response (UPR). As a result, phosphorylation of the translation factor eIF2 α is increased. We propose a model in which translation of a few maternal mRNAs, including caudal, are particularly sensitive to increased eIF2 α phosphorylation. According to this view, inappropriate UPR activation can cause specific patterning defects during embryo development.
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Essential roles of a zebrafish prdm1/blimp1 homolog in embryo
patterning and organogenesis

Essential roles of a zebrafish prdm1/blimp1 homolog in embryo patterning and organogenesis

Expression of prdm1 was first detected by whole-mount in situ hybridization in the external YSL at the end of the blastula period (5.5 hpf; Fig. 2A). At the onset of gastrulation (6 hpf), prdm1 expression in the external YSL became more prominent, and shortly thereafter prdm1 transcripts appeared at low levels in the anteriormost part of the prechordal mesoderm (Fig. 2B). By contrast, gsc is expressed in the entire prechordal mesoderm and part of the chordamesoderm throughout gastrulation (Stachel et al., 1993) (Fig. 2F,G). The abundance of prdm1 transcripts in the prechordal mesoderm increased during gastrulation and expression extended more posteriorly compared with the hgg1 expression domain (Fig. 2C,D,H,I). At mid- gastrulation, prdm1 transcripts were identified in the non-neural ventral ectoderm (not shown) and gradually confined to a line of cells marking the boundary between neural and non-neural ectoderm during late gastrula stages (Fig. 2C,D) following the dynamic pattern described for dlx3b (Fig. 2H,I). Furthermore, two stripes of slow muscle precursor cells adjacent to the notochord began to express prdm1 (Fig. 2D). Throughout segmentation, slow muscle expression of prdm1 decreased gradually from anterior to posterior somites but was maintained in the posterior and thus youngest somites (Fig. 2E,J,K). In addition, prdm1 was expressed in a variety of tissue precursors, including the otic vesicle, the branchial arches and unidentified cells or cell groups in the central nervous system (Fig. 2E,J). At
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Quantitative 4D analyses of epithelial folding during Drosophila gastrulation

Quantitative 4D analyses of epithelial folding during Drosophila gastrulation

Understanding the cellular and mechanical processes that underlie the shape changes of individual cells and their collective behaviors in a tissue during dynamic and complex morphogenetic events is currently one of the major frontiers in developmental biology. The advent of high- speed time-lapse microscopy and its use in monitoring the cellular events in fluorescently labeled developing organisms demonstrate tremendous promise in establishing detailed descriptions of these events and could potentially provide a foundation for subsequent hypothesis-driven research strategies. However, obtaining quantitative measurements of dynamic shapes and behaviors of cells and tissues in a rapidly developing metazoan embryo using time-lapse 3D microscopy remains technically challenging, with the main hurdle being the shortage of robust imaging processing and analysis tools. We have developed EDGE4D, a software tool for segmenting and tracking membrane-labeled cells using multi-photon microscopy data. Our results demonstrate that EDGE4D enables quantification of the dynamics of cell shape changes, cell interfaces and neighbor relations at single-cell resolution during a complex epithelial folding event in the early Drosophila embryo. We expect this tool to be broadly useful for the analysis of epithelial cell geometries and movements in a wide variety of developmental contexts.
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Response to the BMP gradient requires highly combinatorial inputs from multiple patterning systems in the Drosophila embryo

Response to the BMP gradient requires highly combinatorial inputs from multiple patterning systems in the Drosophila embryo

It remains unclear how target genes respond to low levels of Dpp in the dorsolateral region. Do they also contain multiple clusters of Smad binding sites and/or do they use the affinity threshold mechanism? Here we analyze the regulation of the pannier (pnr) gene, which is expressed in a broad dorsal domain of ~32-35 cells (Jazwinska et al., 1999a; Ashe et al., 2000). To our surprise, the pnr enhancer structure resembled that of Race, containing a similar number of Smad sites with the same relative affinity as those in the Race enhancer, suggesting that the affinity threshold mechanism plays a minimal role, if any, in the response of pnr to low levels of Dpp. Instead, we found that sequences that lie adjacent to a crucial Smad site are required for proper pnr activation, and that the extent of pnr expression is limited by Brinker (Brk)-mediated repression. We also found that the pnr expression pattern is influenced by anteroposterior (AP) genes. Thus, pnr gene regulation relies on a complex combinatorial mechanism that integrates spatial cues from diverse patterning systems.
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Dynamic control of head mesoderm patterning

Dynamic control of head mesoderm patterning

When the head mesoderm is laid down during gastrulation, it is under the influence of RA (Blentic et al., 2003; Bothe and Dietrich, 2006; Hochgreb et al., 2003) (this study). At the time at which Pitx2 and Tbx1 expression commences, RA production has receded posteriorly into the somitic region, the anterior head mesoderm expresses the RA antagonist Cyp26C1, and the RA-responsive Hoxb1 gene is not expressed further anterior than rhombomere 4 (Bel-Vialar et al., 2002; Blentic et al., 2003; Bothe and Dietrich, 2006; Forlani et al., 2003; Hochgreb et al., 2003; Reijntjes et al., 2004). This suggests that the anterior head mesoderm is cleared of RA signalling and the posterior head mesoderm receives low-level and the somites high-level RA signalling. Implantation of RA beads prevented expression of Pitx2 and reduced expression of Tbx1 in a Fig. 6. Influence of Fgf and SU5402. (A-Ri) Dorsal views of chicken embryo heads treated with Fgf8 at HH4-5 (A-F), HH6-7 (G-L) and HH8-9 (M-R) or the Fgf antagonist SU5402 at HH8-9 (Mi-Ri). The BSA control for the Fgf experiment is shown in Fig. 4, the DMSO control for the SU5402 experiment in Fig. 3. The carrier beads are indicated by asterisks. Green arrowheads indicate upregulation, red arrowheads downregulation and blue arrowheads unchanged marker gene expression. Embryos were cultured for 5 hours to reach HH5-6 (early phase 1), HH7-8 (late phase 1) or HH9-10 (phase 2). Markers are indicated on top of the panel. Fgf mildly downregulated Pitx2 and Alx4 (A,G,M,N, red arrowheads) and strongly upregulated and expanded Tbx1 expression (D,J,P, green arrowheads); SU5402 mildly upregulated Pitx2 (Mi, green arrowhead) and suppressed Tbx1(Pi, red arrowhead). This suggests that Fgf initiates and drives the expansion of Tbx1 expression and contributes to the restriction of the two anterior markers. Fgf upregulated and expanded MyoR expression at the time the gene would normally be expressed (O, green arrowhead), SU5402 downregulated MyoR expression (Oi, red arrowhead). Thus, Fgf is necessary but not sufficient to initiate, yet sufficient to expand, MyoR expression. (E,K,Q,Qi) Neither Fgf nor SU5402 treatment affected the expression of Twist (blue arrowheads). (F,L,R,Ri) As expected, Fgf upregulated (green arrowheads) and SU5402 downregulated (red arrowhead) expression of Mkp3 at all times. end, endoderm; hm, head mesoderm; hn, Hensen’s node; lm, lateral mesoderm; ift, inflow tract of the heart; np, neural plate; nt, neural tube; pchme, prechordal mesendoderm; som1, first somite.
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A major role for zygotic hunchback in patterning the
Nasonia embryo

A major role for zygotic hunchback in patterning the Nasonia embryo

Collection and fixation of Nasonia embryos and ovaries When Nasonia embryos are collected from virgin females, all embryos are precisely staged – there are no older embryos from previously fertlized eggs as in Drosophila. Embryos after gastrulation were fixed as described in Pultz et al. (Pultz et al., 1999). Most of the blastoderm embryos were shaken in heptane for 2 minutes, then an equal volume of methanol was added and they were shaken for an additional 2-3 minutes at room temperature. Later, we found that sufficiently dry blastoderm embryos can also be fixed in 1:1 heptane: 4% formaldehyde in 1 ⫻ PBS, improving morphology. Very early embryos (0-3 hours old) cannot be effectively devitellinated with methanol. These were fixed for 1 hour in heptane pre-saturated with 37% formaldehyde, then hand-peeled on double-stick tape in 1 ⫻ PBS. Older hand-peeled embryos with a known expression pattern were included as a positive control. All embryos were males, collected from virgin mothers. To avoid cross reactivity of the anti-Nasonia hunchback antibody with endosymbiotic bacteria, we used wild-type Nasonia vitripennis cured of Wolbachia (a gift from Jack Werren), and we cured the hunchback hl stock of Wolbachia by treating the mothers
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Maternal Torso-Like Coordinates Tissue Folding During Drosophila Gastrulation

Maternal Torso-Like Coordinates Tissue Folding During Drosophila Gastrulation

How might polar localized Tsl influence ventral Fog activ- ity? Recent data on the role of Tsl in terminal patterning suggest that it mediates the extracellular accumulation of the ligand for the Tor receptor, Trk (Johnson et al. 2015). Thus, one possibility is that Tsl acts to directly mediate the secretion or activity of Fog. However, this idea seems unlikely as the overlap between the ventral cells that produce Fog and polar localized Tsl would be small or nonexistent. Furthermore, mosaic analyses by Costa et al. (1994) estimated that Fog could induce apical constriction only two to three cells away from its cell of origin. In addition, since immunos- taining experiments by Mineo et al. (2015) have shown that Tsl remains localized to the embryo termini plasma membrane, we therefore reason that it is improbable that Tsl could directly influence Fog produced at the center of the embryo. Accordingly, an alternative idea is that Tsl is responsible for the extracellular accumulation of a hitherto unidentified molecule that can diffuse to the ventral region and control local Fog activity. A mechanism such as this might aid in coordinating the timing of apical constriction and subsequent furrow formation by controlling extracel- lular Fog activity uniformly across the cells of the ventral domain.
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microRNA 31 modulates skeletal patterning in the sea urchin embryo

microRNA 31 modulates skeletal patterning in the sea urchin embryo

To test the impact of miR-31 regulation of key genes within the PMC GRN in the dynamic environment of a developing embryo, we designed miR-31 target protector morpholino antisense oligonucleotides (miR-31 TPs) to competitively block endogenous miR-31 suppression of Alx1 or VegfR7 (Staton and Giraldez, 2011; Stepicheva et al., 2015). BLASTN of the miR-31 TP sequences indicated that they are uniquely complementary to the functional miR-31 sites identified by the luciferase assays. We microinjected Alx1 miR-31 TP, VegfR7 miR-31 TP or both into newly fertilized eggs and observed a significant decrease in the skeleton spicule length compared with control embryos (Fig. 6A,B). A small but significant decrease in skeleton spicule length persisted in Alx1, but not in VegfR7, miR-31 TP-injected larvae (72 hpf ) (Fig. S4). We found that 2%, 7.5% and 4.6% of Alx1, VegfR7 and Alx1+VegfR7 miR-31 TP-injected gastrulae developed extra tri-radiate rudiments, respectively (Fig. 6A). We also observed that some PMCs were mislocalized in the miR-31 TP-injected embryos compared with the control using the PMC-specific antibody 1D5 (McClay et al., 1983) (Fig. 6C,D). The decrease in skeleton spicule length, formation of the extra tri-radiate rudiments and the defects in PMC patterning (Fig. 6) in the Alx1 and VegfR7 miR-31 TP-injected embryos partially mimicked the miR-31 KD phenotypes (Figs 2 and 3), indicating that miR-31 KD phenotypes are in part caused by the lack of miR-31 regulation within PMCs.
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Quantitative Analysis of Gene Function in the Drosophila Embryo

Quantitative Analysis of Gene Function in the Drosophila Embryo

framework for functional studies on the transcription We have been using the Drosophila embryo as a model factors that participate in these processes. Indeed, ex- to investigate the function of the pair-rule segmentation periments with the Drosophila embryo provide several gene runt. Runt is the founding member of the Runt of the most elegant and well-understood examples of domain family of transcriptional regulators (Kago- transcriptional regulation in developmental biology. Al- shima et al. 1993) and has pivotal roles in sex determina- though the roles of many genes in the embryo were tion, segmentation, and neurogenesis in the Drosophila initially deduced from loss-of-function phenotypes, stud- embryo (Gergen and Wieschaus 1986; Duffy and ies on gene function have also benefited from analysis Gergen 1991; Duffy et al. 1991; Torres and Sanchez of gain-of-function phenotypes produced by overexpres- 1992; Dormand and Brand 1998; Kramer et al. 1999). sion. For example, numerous investigations on the regu- Lozenge, a second Runt domain protein in Drosophila, latory interactions and mechanisms of transcriptional has postembryonic roles in patterning in the antenna regulation that are involved in the segmentation path- and eye (Daga et al. 1996; Flores et al. 1998; Gupta way have used the Drosophila heat-shock promoter to et al. 1998). In vertebrates, Runt domain genes have induce ectopic gene expression (Struhl 1985; Ish- important roles in the normal development of blood and bone, and mutations in these genes are associated with defects in these processes in humans (Okuda et
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Dorsal Ventral Patterning and Gene Regulation in the Early Embryo of Drosophila melanogaster

Dorsal Ventral Patterning and Gene Regulation in the Early Embryo of Drosophila melanogaster

Egfr signaling is present in the presumptive neurogenic ectoderm, and it is essential for proper patterning. The Egfr receptor is ubiquitously expressed at this stage and the ligand vein is activated by Dorsal in lateral regions of the embryo (Schnepp et al., 1996). rho, which activates Egfr signaling by cleaving the Egfr ligand Spitz, is also activated by Dorsal in a domain similar to vein (Bier et al., 1990; Ip et al., 1992a). During later stages of development neuroblasts develop a unique identity based on where they are located and differentiate into specialized neuroblast accordingly. Their location is determined based on which proneural gene they express and whether they receive Egfr signaling. In Egfr mutants expression of ind is lost and consequently no intermediate neuroblast are formed (Skeath, 1998). The medial neuroblast, are still specified and express vnd, but the lack of Egfr signaling causes them to display some traits specific to lateral neuroblast. The lateral neuroblasts, which are specified by msh, expand into regions where intermediate neuroblasts would normally form. Thus it is clear that Egfr signaling plays and important role in patterning the dorsal-ventral axis of the neurogenic ectoderm. Even though Egfr signaling is only necessary for the activation of ind, it has a dramatic affect on the patterning of the neurogenic ectoderm; as loss of just one of the neurogenic ectoderm genes results in patterning defects of the entire tissue.
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Twisted gastrulation promotes BMP signaling in zebrafish dorsal ventral axial patterning

Twisted gastrulation promotes BMP signaling in zebrafish dorsal ventral axial patterning

In Drosophila, Tsg also modulates BMP signaling during DV axial patterning. Tsg functions similarly to the Chordin ortholog Short gastrulation (Sog) in specifying the dorsal-most tissue, the amnioserosa, which requires the highest levels of BMP signaling in the fly embryo (Ashe and Levine, 1999; Francois et al., 1994; Mason et al., 1994; Ross et al., 2001; Zusman and Wieschaus, 1985). In addition to this pro-BMP activity, both Tsg and Sog exhibit anti-BMP activity in dorsolateral regions of the embryo (Ross et al., 2001). Current models suggest that Sog and Tsg bind to and transport BMP ligands toward dorsal regions of the embryo, where they are released from Sog by the activity of Tolloid, thereby generating the highest levels of BMP signal dorsally (Decotto and Ferguson, 2001; Eldar et al., 2002; Shimmi and O’Connor, 2003). In this model, the activity of Tsg relies on the presence of Sog and Tolloid. In vertebrates, there is no evidence for a role for Chordin in promoting gastrula BMP signaling, although it is possible it plays such a role at later stages in tail patterning (Hammerschmidt and Mullins, 2002; Wagner and Mullins, 2002). Thus, all aspects of how Sog/Chordin and Tsg function in DV patterning in vertebrates and invertebrates may not be conserved.
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Role of knot (kn) in Wing Patterning in Drosophila

Role of knot (kn) in Wing Patterning in Drosophila

Furthermore, the double mutant ptc kn clones induced near the anterior margin of the wing are fully capable of inducing a com- plete duplication of the anterior wing blade[r]

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Compositional and structural requirements for laminin and basement membranes during mouse embryo implantation and gastrulation

Compositional and structural requirements for laminin and basement membranes during mouse embryo implantation and gastrulation

laminin 1 into an organized RM (Williamson et al., 1997). In these embryos, the resulting RM has its normal cellular components (PE and trophoblast giant cells) and architecture, but loses its barrier capacity. The second function of laminin 1 in RM is informational: interaction of both PE and trophoblast with laminin 1 influences their differentiation and behavior. When there is a complete lack of laminin, as demonstrated in the Lama1, Lamb1 and Lamc1 mutants, both the cellular and molecular components of RM are lacking. The PE cells fail to differentiate and fail to migrate properly to the periphery of the embryo (Smyth et al., 1999), while the trophoblast cells do not differentiate to giant cells and do not form the blood sinuses. Dystroglycan is, thus, the crucial receptor for assembly of RM, but not for transmitting specific signals from laminin 1 to the overlying trophoblast cells, or for promoting PE migration. It will be interesting to determine the mechanism of RM assembly in Myd (Large – Mouse Genome Informatics) mutant mice, which have defects in glycosylation and produce a dystroglycan molecule unable to interact with laminin in adults (Michele et al., 2002).
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Sequential allocation and global pattern of movement of the definitive endoderm in the mouse embryo during gastrulation

Sequential allocation and global pattern of movement of the definitive endoderm in the mouse embryo during gastrulation

Cells in the endoderm of MS- and M-LS-stage ARC/s, Mixl1 +/GFP and Mixl1 GFP/GFP embryos were labeled by painting the with carbocyanine dyes: DiO (D275), CM-DiI (C-7001) or DiI (D282, D3911, Molecular Probes). While holding the embryo by suction against a wide-bored pipette with polished tip, the endoderm was painted by touching the cells with a bolus of dye that was partially extruded from the tip of another micropipette. A broad area of the endoderm of the ARC/s embryos was painted in single color with either DiI (red) or DiO (green), or in both of these colors by painting consecutively with DiI and DiO. Painting was performed using DiI on the Mixl1 +/GFP and Mixl1 GFP/GFP embryos to contrast labeled cells with the green fluorescent host tissues. Painted embryos were imaged by fluorescence microscopy and digital photography 1 hour after labeling to determine the paint pattern; and at 6-, 12- or 24-hours in vitro to visualize the distribution of labeled endoderm cells in the embryonic gut and the yolk sac.
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Control of Denticle Diversity in the Drosophila Embryo

Control of Denticle Diversity in the Drosophila Embryo

Segment polarity mutants such as ptc and wg disrupt additional aspects of epithelial patterning that stripe does not. In wild-type, the actin-based protrusions that lead to denticle formation emanate from the posterior edge of each cell (Fig 2-2F; Price et al., 2006; Walters et al., 2006). This signifies that cells of the prospective denticle field are polarized in the plane of the epithelium, a process known as planar cell polarity (PCP). While the signals that define PCP in this epithelium are unknown, mutations affecting the organizing signals Wg and Hh disrupt tissue polarity globally. As a result, denticles are not only disordered at the level of hook orientation but now arise at variable positions on the cell surface (Fig 2-4B,D; Alexandre et al., 1999; Price et al., 2006; Colosimo and Tolwinski, 2006). In addition, mutations in the Wg or Hh pathway lead to massive cell death and global disruptions in epidermal cell shape. In contrast, stripe mutants do not exhibit obvious disruptions in tissue polarity or rectilinear organization (Fig 2-5B). Thus, stripe is the only known gene that affects denticle hook orientation without affecting tissue polarity or other global cellular properties.
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Anteroposterior patterning of the zebrafish ear through Fgf- and Hh-dependent regulation of hmx3a expression

Anteroposterior patterning of the zebrafish ear through Fgf- and Hh-dependent regulation of hmx3a expression

The otic placode—precursor of the vertebrate inner ear—has the remarkable ability to generate a mirror-image organ with duplicate structures under some experimental conditions in fish and amphibians, as originally described by R. G. Harrison over eighty years ago (reviewed in [1]). Understanding the generation of such duplicated structures can give us fundamental insights into mechanisms of organ patterning, tissue polarity and symmetry-breaking during embryogenesis. During normal development in the zebrafish, anteroposterior asymmetries in otic gene expression are evident as early as the 4-somite stage (11.5 hours post fertilisation (hpf)), when expression of the transcription factor gene hmx3a appears at the anterior of the otic placode [2]. Additional genes with predominantly anterior patterns of expression in the otic placode or vesicle begin to be expressed over the next 10 hours, including the transcription factor genes hmx2 and pax5 [2, 3], together with the fibroblast growth factor (Fgf) family genes fgf3, fgf8a and fgf10a [4, 5]. Later, at otic vesicle stages (24 hpf onwards), the size and position of the otoliths, together with the position, shape and planar polarity patterns of the sensory maculae, provide landmarks for distinguishing anterior and posterior structures in the ear [6]. In addition, a few markers begin to be expressed specifically in posterior otic tissue (pou3f3b, bmp7a and fsta) at otic vesicle stages [3, 7, 8], but these are not reliable posterior markers at earlier otic placode stages.
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Patterning function of homothorax/extradenticle in the thorax of
Drosophila

Patterning function of homothorax/extradenticle in the thorax of Drosophila

Crude Drosophila discs extracts were prepared by homogenizing 0.2 ml of third instar larvae in 0.4 ml of lysis buffer (1 PBS, 1% NP-40, 1 mM PMSF and 20 µ g/ml each of peptastin and leupeptin). The homogenates were centrifuged, and the aqueous phase was mixed with the His-tagged, full-length Hth protein extracted under native conditions. The complexes formed were purified using the Ni-NTA Agarose (Quiagen), washed five times in 1 PBS, 0.5 M NaCl and one final time in 50 mM Tris (pH 6.8). They were boiled in loading buffer and resolved by SDS-PAGE. After blotting to nitrocellulose, the filter was incubated with the anti-Eyg antibody and the signal was detected using the Amersham ECL western blotting analysis system.
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