Journal: Neural Development

Article Title: Microtubules are organized independently of the centrosome in Drosophila neurons

doi: 10.1186/1749-8104-6-38

Figure Lengend Snippet: Larval sensory neurons contain centrioles but have compromised centriole function . yw (control) and DSas-4 embryos were fixed and stained with antibodies against either 22C10 to label stable microtubules or fluorescein isothiocyanate (FITC)-conjugated HRP to label neurons, β-galactosidase (to label heterozygote animals with a lacZ-marked balancer), and centriole-associated proteins. Stage 16 embryos were examined except where noted. (A) yw flies have punctate DSas-4 staining in ciliated neurons of the dorsal da cluster (arrows) and chordotonal neurons (box). DSas-4 embryos do not have any punctate DSas-4 staining. (B) Embryos were stained with FITC-conjugated HRP and γ-tubulin antibody. The γ-tubulin antibody localized at the base of each cilium in chordotonal neurons (boxes) of DSas-4 and yw animals. (C) Embryos were stained with antibodies against futsch (22C10) and the centriole-associated Drosophila pericentrin-like protein (D-PLP). D-PLP puncta were present in the chordotonal neurons of both control and DSas-4 mutants (boxes). Other centriolar proteins examined are seen in Additional file . (D) Cilia of chordotonal neurons were analyzed in DSas-4 and control animals. Top: embryos were stained with FITC-conjugated HRP. Middle: larvae expressing EB1-GFP in all neurons were examined by live imaging. Bottom: quantification of ciliary defects was performed. Neurons with abnormal cilia were classified as such by displaying either no cilium (arrow in (B), and arrow in the middle image of (D)) or a truncated cilium (asterisks, top image). DSas-4 mutants had a greater percentage of neurons with abnormal cilia compared to control samples. (E) Embryos were stained with antibodies against futsch and NompC. In control neurons, NompC localized to the distal tip of of mechanosensory cilia (arrow), while in DSas-4 mutants, NompC localization was seen at the base of the cilium (arrowhead).

Article Snippet: Primary antibodies used were: mouse anti-γ-tubulin (Sigma), rabbit anti-γ-tubulin (Sigma), 22C10 (Developmental Studies Hybridoma Bank), FITC-conjugated rabbit anti-HRP (Jackson Immunoresearch, West Grove, PA, USA), chicken anti-β-galactosidase (Gallus Immunotech, Fergus, ON, Canada), rabbit anti-DSas-4 (gift from J Raff), rabbit anti-Asterless (gift from J Raff), rabbit anti-Bld10 (gift from T Megraw), rabbit anti-D-PLP (gift from Greg Rogers), and rabbit anti-NompC (gift from Li Cheng and Yuh Nung Jan).

Techniques: Staining, Expressing, Imaging

Journal: PLoS Genetics

Article Title: Retinal Expression of the Drosophila eyes absent Gene Is Controlled by Several Cooperatively Acting Cis-regulatory Elements

doi: 10.1371/journal.pgen.1006462

Figure Lengend Snippet: (A) Illustration of the genomic map of the eya locus—representation is not to scale. Sequences and genomic location of each fragment are provided in supplementary materials and methods S1-3. Size of each fragment (bp) is indicated within each bar. Blue bars = individual retinal enhancers. PSE = photoreceptor specific enhancer previously identified and named by Graeme Mardon’s group in . E = 319bp extant enhancer previously identified in . Enhancer 1 (IAM) = immediately ahead of morphogenetic furrow enhancer was previously identified and named by Graeme Mardon’s group in . We refer to this enhancer as 1 as it shows a different expression pattern than previously reported. 2–4 represent newly identified enhancer elements. Orange bar = composite enhancer, purple bar = enhancer 1+E, grey bars indicate regions that do not drive expression in the retina including the fragment used as the 319bp spacer, asterisks = So binding sites, red bars = regions of So ChIP peaks. eya 1 and eya 2 deletions are indicated by red lines immediately ahead of exon 1 (B-G) Light microscope images of third instar eye-antennal discs. All images represent lacZ reporter expression in a wild type genetic background. LacZ reporter activation is indicated by antibody staining against β-galactosidase. White arrowheads mark the position of the morphogenetic furrow. (B) The PSE enhancer drives expression of the reporter only in cells that lie posterior to the morphogenetic furrow. (C) Enhancer 1 (also called IAM) drives expression in cells ahead and behind the morphogenetic furrow. (D) The 319bp extant enhancer drives weak reporter expression in cells ahead and posterior to the morphogenetic furrow. (E) Enhancer 2 drives expression of the reporter in cells anterior and posterior to furrow. (F) Enhancer 3 drives expression in cells ahead and behind the morphogenetic furrow. (G) Enhancer 4 drives expression only in cells posterior to the morphogenetic furrow. No single enhancer element fully recapitulates endogenous Eya expression. Anterior is to the right in imaginal disc images. At least 30 imaginal discs were examined for each genotype. Scale bar, 100μm

Article Snippet: The following antibodies were used: (1) mouse anti-Eya (1:5, DSHB), (2) mouse anti-β-galactosidase (1:250, Promega), (3) chicken anti-β-galactosidase (1:800, Promega), (4) rat anti-Elav (1:100, DSHB). (5) rabbit anti-Dcp-1 (1:100, Cell Signaling Technologies).

Techniques: Expressing, Binding Assay, Light Microscopy, Activation Assay, Staining

Journal: PLoS Genetics

Article Title: Retinal Expression of the Drosophila eyes absent Gene Is Controlled by Several Cooperatively Acting Cis-regulatory Elements

doi: 10.1371/journal.pgen.1006462

Figure Lengend Snippet: (A-U) Light microscope images of developing eye-antennal discs. Images of imaginal discs at 48hrs and 72hrs AEL were taken at 20X while images of wandering third instar larvae were taken at 10X. AEL = after egg laying. Red = Eya protein, green = β-galactosidase, yellow = positions of co-localization between Eya and β-galactosidase. Arrowhead marks the position of the morphogenetic furrow. All enhancer-lacZ reporters are placed in a wild type genetic background. (A-C) Localization of Eya protein in developing wild type retinas at different developmental time points. (D-F) Enhancer 1 dependent expression is activated in a few Eya expressing cells early in development and co-localizes with Eya posterior to the morphogenetic furrow late in development. (G-I) Extant enhancer dependent expression co-localizes with Eya and is robust early in development but becomes weaker and sparse as development proceeds. (J-L) Enhancer 2-dependent expression is largely present in non- eya expressing cells early in development. Co-localization with Eya can be seen in cells anterior and posterior to the furrow later in development but a significant portion of reporter expression still present in non- eya expressing cells. (M-O) Composite enhancer-dependent expression shows co-localization with Eya protein throughout all stages of larval eye development. This is the only construct to fully recapitulate temporal and spatial eya expression. (P-R) Enhancer 3-dependent expression is largely present in non- eya expressing cells throughout development. Some co-localization with Eya protein is seen at later stages in cells anterior and posterior to the furrow. (S-U) Enhancer 4-dependent expression co-localizes with a few Eya expressing cells posterior to the furrow late in development. Anterior is to the right in imaginal disc images. At least 30 imaginal discs were examined for each genotype and developmental time point. Scale bar, 50μm

Article Snippet: The following antibodies were used: (1) mouse anti-Eya (1:5, DSHB), (2) mouse anti-β-galactosidase (1:250, Promega), (3) chicken anti-β-galactosidase (1:800, Promega), (4) rat anti-Elav (1:100, DSHB). (5) rabbit anti-Dcp-1 (1:100, Cell Signaling Technologies).

Techniques: Light Microscopy, Expressing, Construct

Journal: PLoS Genetics

Article Title: Retinal Expression of the Drosophila eyes absent Gene Is Controlled by Several Cooperatively Acting Cis-regulatory Elements

doi: 10.1371/journal.pgen.1006462

Figure Lengend Snippet: (A-D, F-I, K-N,Q,R) Light microscope images of developing eye-antennal discs. Images of imaginal discs at 48hrs and 72hrs AEL were taken at 20X while images of wandering third instar larvae were taken at 10X. Red = Eya, green = β-galactosidase, yellow = regions where Eya and β-galactosidase co-localize. White arrowheads mark the position of the morphogenetic furrow. Each enhancer is driving expression of lacZ within wild type eye-antennal discs. AEL = after egg laying. (E,J,O,S) SEM images of adult Drosophila compound eyes and heads from enhancer— eya RB cDNA rescue experiments. Each enhancer is driving expression of the eya RB isoform within the developing eye of either eya 1 or eya 2 mutants. (P) SEM image of adult Drosophila compound eyes and heads from enhancer 1+spacer+2—eya RB cDNA rescue experiments of eya 1 mutants. (A-C) Expression driven by enhancer 1+2 is only activated late in eye development in some eya expressing cells posterior to the furrow. (D) Expression driven by enhancer 1+2 is activated in very few cells in eya 2 mutant discs. (E) The enhancer 1+2—eya RB cDNA does not rescue eya 2 mutants. (F-H) Expression driven by enhancer 1+5bp+2 is activated mostly in non- eya expressing cells early in development while later activation is seen in eya expressing cells both anterior and posterior to the furrow. (I) Expression driven by enhancer 1+5bp+2 is activated weakly throughout the eye disc of eya 2 mutants. (J) The enhancer 1+5bp+2—eya RB cDNA does not rescue eya 2 mutants. (K-M) Expression driven by enhancer 1+spacer+2 restores some early expression in eya expressing cells but does not fully recapitulate eya expression at all stages of development. (N) Expression driven by enhancer 1+spacer+2 is strongly activated throughout the eye disc of eya 2 mutants. (O) Enhancer 1+spacer+2—eya RB cDNA partially rescues 100% of eya 2 mutants suggesting a restoration of function. (P) Enhancer 1+spacer+2—eya RB cDNA partially rescues 100% of eya 1 mutants suggesting a restoration of function. (Q-R) The neutral 319bp of DNA that was used to construct 1+spacer+2 does not drive reporter activation on its own in either wild type or eya 2 discs. (S) The 319bp spacer does not rescue eya 2 mutants. Anterior is to the right in adult head and imaginal disc images. At least 100 adult flies and 30 imaginal discs were qualitatively examined for each genotype and at each developmental time point. Panel A-D, F-I, K-N, Q-R Scale bar, 50μm. Panel E,J, O,P,S Scale bar, 100μm

Article Snippet: The following antibodies were used: (1) mouse anti-Eya (1:5, DSHB), (2) mouse anti-β-galactosidase (1:250, Promega), (3) chicken anti-β-galactosidase (1:800, Promega), (4) rat anti-Elav (1:100, DSHB). (5) rabbit anti-Dcp-1 (1:100, Cell Signaling Technologies).

Techniques: Light Microscopy, Expressing, Mutagenesis, Activation Assay, Construct

Journal: PLoS Genetics

Article Title: Retinal Expression of the Drosophila eyes absent Gene Is Controlled by Several Cooperatively Acting Cis-regulatory Elements

doi: 10.1371/journal.pgen.1006462

Figure Lengend Snippet: (A-F) Light microscope images of wandering third instar so 1 mutant eye-antennal discs. Each enhancer is driving expression of lacZ within so 1 eye-antennal discs. LacZ reporter activation is detected with an antibody that recognizes the β-galactosidase enzyme. (A) so 1 ; enhancer 1—lacZ . Reporter expression driven by enhancer 1 is activated only in cells at the far posterior edge of the eye disc. (B) so 1 ; enhancer E–lacZ . Reporter expression driven by the extant enhancer E is weakly activated throughout the eye disc. (C) so 1 ; enhancer 2 –lacZ . Reporter expression driven by enhancer 2 is strongly activated throughout the remaining eye disc. (D) so 1 ; enhancer 1+E+2 –lacZ . Reporter expression driven by the composite enhancer is strongly activated throughout the eye disc. (E) so 1 ; enhancer 3 –lacZ . Reporter expression driven by enhancer 3 is activated broadly throughout the entire eye-antennal disc. (F) so 1 ; enhancer 4 –lacZ . Reporter expression driven by enhancer 4 is not activated in the eye disc. (G-J) Light microscope images of wandering third instar eye antennal discs in which so 3 null clones have been generated. The absence of GFP marks the position of clones lacking so . Green = GFP, red = Eya protein, blue = lacZ. Yellow arrows mark the position of so 3 mutant clones in which Eya protein is present and the composite enhancer lacZ reporter is activated. White arrowheads mark the position of the morphogenetic furrow. Anterior is to the right in all imaginal disc images. At least 30 discs were examined for each genotype. Scale bar, 100μm.

Article Snippet: The following antibodies were used: (1) mouse anti-Eya (1:5, DSHB), (2) mouse anti-β-galactosidase (1:250, Promega), (3) chicken anti-β-galactosidase (1:800, Promega), (4) rat anti-Elav (1:100, DSHB). (5) rabbit anti-Dcp-1 (1:100, Cell Signaling Technologies).

Techniques: Light Microscopy, Mutagenesis, Expressing, Activation Assay, Clone Assay, Generated