Structure-Based Analysis of Five Novel Disease-Causing Mutations in 21-Hydroxylase-Deficient Patients

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism, and accounts for 90–95% of CAH cases. The affected enzyme, P450C21, is encoded by the CYP21A2 gene, located together with a 98% nucleotide sequence identity CYP21A1P pseudogene, on chromosome 6p21.3. Even though most patients carry CYP21A1P-derived mutations, an increasing number of novel and rare mutations in disease causing alleles were found in the last years. In the present work, we describe five CYP21A2 novel mutations, p.R132C, p.149C, p.M283V, p.E431K and a frameshift g.2511_2512delGG, in four non-classical and one salt wasting patients from Argentina. All novel point mutations are located in CYP21 protein residues that are conserved throughout mammalian species, and none of them were found in control individuals. The putative pathogenic mechanisms of the novel variants were analyzed in silico. A three-dimensional CYP21 structure was generated by homology modeling and the protein design algorithm FoldX was used to calculate changes in stability of CYP21A2 protein. Our analysis revealed changes in protein stability or in the surface charge of the mutant enzymes, which could be related to the clinical manifestation found in patients

Genomic diagnosis implementation in a pediatric hospital: Preliminary results

The access to new technologies, like Next Generation Sequencing (NGS) and microarray, has allowed the development of effective high-performance diagnosis algorithms for genetic pediatric diseases. The aim of this work was to establish standardized procedures for genomic diagnosis of genetic pediatric diseases in a pediatric hospital. Patients with presumptive diagnoses of genetic diseases (intellectual disability, metabolic, hematological or immune diseases or delay of growth and puberty) were included. DNA from peripheral blood was obtained from the patients and their parents. Genomic diagnosis procedures were performed by NGS (Clinical exome, TruSight One, NextSeq 500 Illumina) and microarray studies (8x60K Platform, Agilent). NGS results were analyzed by own designed bioinformatic pipelines, and B platform (Bitgenia) was used to prioritize variants. All variants found (sequence changes or Copy Number Variations) were classified according to American College of Medical Genetics and Genomics recommendations. This study was approved by the hospital ethical review board. Diagnostic flowchart was implemented according to designed operative protocols. Patients referred by specialized pediatricians were evaluated by the interdisciplinary team to agree on the best diagnostic pathway. From March 2018 to August 2019, 200 probands were included (86 with delay of growth and puberty, 12 hematologic, 4 immunologic and 55 metabolic disorders and 43 with intellectual disability). Among the 36 cases studied by microarray, 5 pathogenic variants (13.9 %), and 3 variants of uncertain significance were found. In 24 of the 60 patients (40 %) studied by NGS, genic variants related to patient?s phenotype were found. Conclusion: Interdisciplinary team work has enabled the successful implementation of these new genomic diagnosis techniques in the hospital. Diagnosis efficiency achieved agrees with international standards.Fil: Scaglia, Paula Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Esnaola Azcoiti, María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Casali, Bárbara María de Los Angeles M. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Valinotto, Laura Elena. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rosenbrock, Solange. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Villegas, Florencia. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Arguelles, Celeste. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Berenstein, Ariel José. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas; ArgentinaFil: Izquierdo, Agustín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Sanguineti, Nora María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Braslavsky, Debora Giselle. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Szlago, Marina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Fernandez, Maria del Carmen. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Armando, Romina Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Brunello, Franco Gino. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Sanso, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Bergadá, Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Arberas, Claudia Liliana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Marti, Marcelo Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Ropelato, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Rey, Rodolfo Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaLXIV Reunión Anual de la Sociedad Argentina de Investigación Clínica; LI Reunión Anual de la Asociación Argentina de Farmacología Experimental; XXI Reunión Anual de la Sociedad Argentina de Biología; XXXI Reunión Anual de la Sociedad Argentina de Protozoología; IX Reunión Anual de la Asociación Argentina de Nanomedicinas y VI Reunión Científica Regional de la Asociación Argentina de Ciencia y Tecnología de Animales de LaboratorioMar del PlataArgentinaSociedad Argentina de Investigación ClínicaSociedad Argentina de ProtozoologíaAsociación Argentina de NanomedicinasAsociación Argentina de Farmacología ExperimentalSociedad Argentina de BiologíaAsociación Argentina de Ciencia y Tecnología de Animales de Laboratori

Extrato etanólico de Casearia sylvestris Sw apresenta atividade antioxidante e antimicrobiana in vitro e efeito hipolipemiante em ratos

The Casearia sylvestris Sw (Flacourtiaceae) is a shrub that occurs in forests of Southern Brazil; its leaves are widely used in folk medicine as a depurative, analgesic, anti-inflammatory and antiulcerogenic agent. The objective of this study was to perform the phytochemical description and to evaluate the pharmacological activities (antimicrobial, antifungal, antioxidant and toxicity) of the ethanolic extract (EE) of C. sylvestris Sw. In addition, we also evaluated the effect of the EE of C. sylvestris Sw on the glucose levels and lipid profile in blood serum of rats submitted to a model of streptozotocin-induced diabetes. Material and Methods: In vitro assay: the detection of chemical groups was done through chemical reactions with the development of color or precipitate and by chromatographic profile; the antioxidant activity was measured by the method of reduction of DPPH free radical (2,2-diphenyl-1-picrylhydrazyl); the Minimum Inhibitory Concentration was evaluated by the broth microdilution method, and the Minimum Bactericide Concentration and the Minimum Fungicide Concentration were performed in Petri dishes; the cytotoxic activity was measured by the Artemia salina test. In vivo assay: diabetic and non-diabetic rats were treated with EE of C. sylvestris Sw (300 mg/kg) for 45 days, and the glycaemia and lipid profile were analyzed. Results: The EE showed a Lethal Dose50 of 724.76 μg.mL-1 and important antioxidant, fungicide and fungistatic activities. The EE showed better antimicrobial activity regarding the microorganisms Staphylococcus aureus, Escherichia coli and Salmonella setubal. Conclusion: The EE of C. sylvestris Sw produces a significant decrease in triglycerides, total cholesterol and VLDL levels without any significant alteration in the glycaemia. The EE of C. sylvestris Sw presents antioxidant and antimicrobial activities and it exhibits a potent hypolipidemic effect.Casearia sylvestris Sw (Flacourtiaceae) é uma planta comumente encontrada em florestas do sul do Brasil; suas folhas são amplamente utilizadas na medicina popular como depurativa, analgésica, anti-inflamatória e anti ulcerogênica. O objetivo deste estudo foi apresentar uma descrição fitoquímica e da atividade farmacológica (antimicrobiana, antifúngica, antioxidante e toxicidade) do extrato etanólico (EE) da C. Sylvestris Sw. Adicionalmente, procurou-se avaliar o efeito do EE da C. Sylvestris Sw sobre os níveis séricos de glicose e perfil lipídico de ratos submetidos a um modelo de diabetes induzida por estreptozotocina. A detecção de grupos químicos foi realizada por reações químicas de coloração ou precipitação, e também por cromatografia; a atividade antioxidante foi mensurada pelo método de redução do DPPH (2,2-difenil-1-picril-hidrazil); a concentração mínima inibitória foi realizada pela técnica de micro-diluição, e concentração mínima bactericida e concentração mínima fungicida foram realizadas em placa de Petri; enquanto a atividade citotóxica foi conduzida pelo teste da Artemia salina. Nos ensaios in vivo, ratos diabéticos e não-diabéticos foram tratado com EE da C. Sylvestris Sw (300mg/kg) por 45 dias, e os níveis glicêmico e perfil lipídico foram medidos. A dose Letal50 do EE foi de 724.76 μg.mL-1; mostrando importante atividades antioxidante, fungicida e fungistática e melhor atividade antimicrobiana contra Staphylococcus aureus, Escherichia coli e Salmonella setubal. O EE da C. Sylvestris Sw promoveu diminuição significativa nos níveis de triglicerídeos, colesterol total e VLDL; porém sem efeito significativo nos níveis glicêmicos. O EE da C. Sylvestris Sw, além de apresentar atividade antioxidante e antimicrobiana; possui também potente efeito hipolipidêmico

Deciphering the Genetic Programme Triggering Timely and Spatially-Regulated Chitin Deposition

<div><p>Organ and tissue formation requires a finely tuned temporal and spatial regulation of differentiation programmes. This is necessary to balance sufficient plasticity to undergo morphogenesis with the acquisition of the mature traits needed for physiological activity. Here we addressed this issue by analysing the deposition of the chitinous extracellular matrix of <i>Drosophila</i>, an essential element of the cuticle (skin) and respiratory system (tracheae) in this insect. Chitin deposition requires the activity of the chitin synthase Krotzkopf verkehrt (Kkv). Our data demonstrate that this process equally requires the activity of two other genes, namely <i>expansion</i> (<i>exp</i>) and <i>rebuf</i> (<i>reb</i>). We found that Exp and Reb have interchangeable functions, and in their absence no chitin is produced, in spite of the presence of Kkv. Conversely, when Kkv and Exp/Reb are co-expressed in the ectoderm, they promote chitin deposition, even in tissues normally devoid of this polysaccharide. Therefore, our results indicate that both functions are not only required but also sufficient to trigger chitin accumulation. We show that this mechanism is highly regulated in time and space, ensuring chitin accumulation in the correct tissues and developmental stages. Accordingly, we observed that unregulated chitin deposition disturbs morphogenesis, thus highlighting the need for tight regulation of this process. In summary, here we identify the genetic programme that triggers the timely and spatially regulated deposition of chitin and thus provide new insights into the extracellular matrix maturation required for physiological activity.</p></div

<i>reb</i> and <i>kkv</i> overexpression effects.

<p>All images are projections of confocal sections, except G, U and V, which are single sections. <i>reb</i> and <i>kkvGFP</i> overexpression in the <i>btl</i> pattern (in green in A) brings about early chitin deposition in the trachea (A’,B), which leads to a defective branching pattern (C) and defects of intercalation (arrows in D). In addition, it promotes chitin deposition in midline (A,E) and proventriculus (arrow in F). Misexpression of the two genes in imaginal discs (G) or salivary glands (H,I) also induces chitin deposition. <i>reb</i> and <i>kkvGFP</i> misexpression in the intestinal tract identified clear differences between the midgut (of endodermal origin) and the proventriculus (of ectodermal origin) (J). In the proventriculus, KkvGFP and Reb localised apically and chitin deposited in the lumen (J’,K,L). In the midgut, which is continuous with the proventriculus, Reb accumulation was cytoplasmic and KkvGFP localised in the entire cortical region (K,L). Chitin was not deposited extracellularly but few chitin particles we often detected (J’). Chitin precursors accumulate apically (arrow in M) and in the lumen (arrowhead in M). In <i>exp reb</i> mutants they accumulate apically (arrow in N). KkvGFP is found apically in control and <i>exp reb</i> mutants (O,P). In <i>kkv</i> mutants, Exp and Reb are expressed and accumulated apically (Q-T). Reb (U) colocalises with KkvGFP (U, U’) at the apical membrane when misexpressed in SGs, and chitin fibers (red in U,U’; white in U’’) are deposited extracellularly in the lumen. (V) When only <i>kkvGFP</i> is misexpressed in the SGs, no luminal chitin fibers are found but instead chitin particles (red in V, white in V’) are enriched in the apical and membrane region. Scale bars A,D,G,J,O 25 μm, B,E,H,Q 10 μm, C 50 μm, F 75 μm, M,U 7.5 μm, V 5 μm.</p

Effects of <i>reb</i> tracheal overexpression.

<p>(B-J and L) Projections of confocal sections of embryos at the indicated stages. (A,K) Bright field images. Tracheal <i>reb</i> overexpression (using LA00733) leads to a strong accumulation of the protein, mainly apically (arrowheads in B,C,G,H). It also brings about early and increased chitin deposition (G,H). Consequently, tracheal morphogenesis in terms of branching (I) and intercalation (J) is affected, and the larval tracheal system does not inflate (K). These defects are reverted when <i>reb</i> is down-regulated (L). Scale bars B,C 25 μm, F 50 μm.</p

Tracheal expression of <i>exp</i> and <i>reb</i>.

<p>(A-E, H-K) Projections of confocal sections of embryos at the indicated stages. (F) Bright field image of a whole mount ISH in a dorsolateral view. (A-C) Exp is expressed in the tracheal system in a dynamic pattern, being first present in the VB, LT and TC and later in the DT and DB. (D-F) <i>reb</i> is expressed only in the DT region (arrow in F). The protein localises mainly apically and more strongly in the branch fusion region (arrowheads in D,E). (G) Scheme of the genomic region and of the deficiencies and transgenes used. The genes uncovered by the deficiencies are indicated. (H-K) Sal negatively regulates <i>exp</i> expression, as in its absence Exp is expressed in the DT-DB region already at stage 13 (arrowheads in I). In contrast, in <i>sal</i> mutants, Reb is not expressed (arrows in J,K). (L) Evolutionary tree of CG13188 (short and long isoforms) and CG13183 obtained using the MEGA 5.2.2 software from a Clustal O-alignment of homologous sequences from insects and non-insects (shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004939#pgen.1004939.s007" target="_blank">S1 Text</a>). The human Smad protein “Mothers against Dpp homolog 3 (isoform X1)”, which also has an N-terminal MH2 domain, is the closest CG13183-similar human protein and was included as an out-group. The <i>Apis mellifera</i> and <i>Tribolium castaneum</i> sequences were added to the alignment as examples of expectedly distant orthologues [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004939#pgen.1004939.ref043" target="_blank">43</a>]. The sequence of the dipteran <i>Anopheles gambiae</i> was included as an example of an expectedly close orthologue. The scale bar in the figure indicates changes per site (residue), thereby implying the evolutionary distance. Scale bars A,D 10 μm, B,H,J 25 μm.</p

Structure-based analysis of five novel disease-causing mutations in 21-hydroxylase-deficient patients

Fil: Minutolo, Carolina. ANLIS Dr.C.G.Malbrán. Centro Nacional de Genética Médica; Argentina.Fil: Nadra, Alejandro D. Universidad de Buenos Aires. Departamento de Fisiología Biología Molecular y Celular; Argentina.Fil: Fernández, Cecilia. ANLIS Dr.C.G.Malbrán. Centro Nacional de Genética Médica; Argentina.Fil: Taboas, Melisa. ANLIS Dr.C.G.Malbrán. Centro Nacional de Genética Médica; Argentina.Fil: Buzzalino, Noemí. ANLIS Dr.C.G.Malbrán. Centro Nacional de Genética Médica; Argentina.Fil: Casali, Bárbara. ANLIS Dr.C.G.Malbrán. Centro Nacional de Genética Médica; Argentina.Fil: Belli, Susana. Hospital Durand. División Endocrinología; Argentina.Fil: Charreau, Eduardo H. Instituto de Biología y Medicina Experimental; Argentina.Fil: Alba, Liliana. ANLIS Dr.C.G.Malbrán. Centro Nacional de Genética Médica; Argentina.Fil: Dain, Liliana. ANLIS Dr.C.G.Malbrán. Centro Nacional de Genética Médica; Argentina.Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism, and accounts for 90–95% of CAH cases. The affected enzyme, P450C21, is encoded by the CYP21A2 gene, located together with a 98% nucleotide sequence identity CYP21A1P pseudogene, on chromosome 6p21.3. Even though most patients carry CYP21A1P-derived mutations, an increasing number of novel and rare mutations in disease causing alleles were found in the last years. In the present work, we describe five CYP21A2 novel mutations, p.R132C, p.149C, p.M283V, p.E431K and a frameshift g.2511_2512delGG, in four non-classical and one salt wasting patients from Argentina. All novel point mutations are located in CYP21 protein residues that are conserved throughout mammalian species, and none of them were found in control individuals. The putative pathogenic mechanisms of the novel variants were analyzed in silico. A three-dimensional CYP21 structure was generated by homology modeling and the protein design algorithm FoldX was used to calculate changes in stability of CYP21A2 protein. Our analysis revealed changes in protein stability or in the surface charge of the mutant enzymes, which could be related to the clinical manifestation found in patients

Phenotypes of <i>exp reb</i> mutants.

<p>(A-C and E-L) Projections of confocal sections of embryos at stage 14 to 16. (D,O,P) bright field and (M,N) dark field images of early larvae. (Q-T) TEM micrographs. Lack of tracheal chitin filament in deficiency combination mutants (A) can be rescued by adding back either <i>exp</i> (B) or <i>reb</i> (C) in the trachea. The absence of <i>exp</i> and <i>reb</i> prevents luminal (F) and cuticular (H) chitin deposition. Apical markers show a normal apicobasal polarity and adhesion, but also the presence of dilations and expansions in the DT (arrowheads in J,L) and apical expansions in lateral branches (arrowheads in J,L). Mutants show inflated larval cuticles (N) and defects in the mouth cuticular structures (arrows in P). At the ultrastructural level, the wild-type mature cuticle (Q) consists of three composite layers: envelope (env), epicuticle (epi), and the inner procuticle (pro, filled with fibrous chitin). In <i>exp reb</i> mutants (R) the envelope and epicuticle are thin, and the procuticle is devoid of any fibrous structure and is thinner than that of the wild-type. In the tracheal cuticle, the lumen (lum) is stabilised by the spiral cuticle. The ridges of the cuticle are the taenidia (tae), formed by a thick procuticle (pro), a thin and electron-dense epicuticle, and the envelope (S). The cuticle of <i>exp reb</i> mutants (T) detaches from the surface of tracheal cells. The procuticle is bloated and contains little material. The lumen collapses. Scale bars, A,E,I 25 μm, G, 10 μm, Q,S,T 500 nm.</p

Analysis of <i>exp reb</i> and <i>kkv</i> phenotypes, epistatic interactions, and subcellular accumulation.

<p>(A-F and M-Q) Projections of confocal sections of embryos at stage 15. (G-I ) Dark field images. (J-L) TEM micrographs. (A-I) Note the similar tracheal defects of <i>kkv</i> and <i>exp reb</i> mutants. (J-L) In <i>exp reb</i> and <i>kkv</i> mutant embryos the procuticle is amorph and thin. Probably as a consequence, the envelope and the epicuticle appear to be disorganised. (M-O) Epistatic experiments show that <i>kkv</i> overexpression does not rescue the absence of <i>exp/reb</i> and <i>reb</i> does not rescue <i>kkv</i> defects. (P-Q) Analysis of subcellular accumulations shows localisation of KkvGFP (arrow in P’,Q’), Exp (arrow in P’’) and Reb (arrow in Q’) in the apical membrane of tracheal cells. There, we detect a partial colocalisation of KkvGFP and Exp (arrow in P) or Reb (arrow in Q). Scale bars A,M 25 μm, D 10 μm, P 7.5 μm, J 500 nm.</p