Gene expression profiling of Drosophila tracheal fusion cells

Chandran, Rachana R.; Iordanou, Ekaterini; Ajja, Crystal; Wille, Michael; Jiang, Lan

doi:10.1016/j.gep.2014.05.004

Cited by 12 publications

(8 citation statements)

References 45 publications

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“…3, (Lee et al, 2018a). In addition, tracheal expression of CG12206 is consistent with a previous report (Chandran et al, 2014) and the 5HT2B T 2 A-GAL4 expression pattern in the adult brain matches an independently generated T2A-GAL4 (Gnerer et al, 2015) (Supp. Fig.…”

Section: Resultssupporting

confidence: 90%

An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila

Li‐Kroeger

Kanca

Lee

et al. 2018

Preprint

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30We generated new genetic tools to efficiently tag genes in 31 Drosophila. Double Header (DH) utilizes intronic MiMIC/CRIMIC insertions 32 to generate artificial exons for GFP mediated protein trapping or T2A-GAL4 33 gene trapping in vivo based on CRE recombinase to avoid embryo 34 injections. DH significantly increases integration efficiency compared to 35 previous strategies and faithfully reports the expression pattern of genes 36 and proteins. The second technique targets genes lacking coding introns 37 using a two-step cassette exchange. First, we replace the endogenous 38 gene with an excisable compact dominant marker using CRISPR making a 39 null allele. Second, the insertion is replaced with a protein::tag cassette.40 2 This sequential manipulation allows the generation of numerous tagged 41 alleles or insertion of other DNA fragments that facilitates multiple 42 downstream applications. Both techniques allow precise gene manipulation 43 and facilitate detection of gene expression, protein localization and 44 assessment of protein function, as well as numerous other applications. 45 46 47 113 circular plasmid or can be circularized in vivo from an initial insertion locus 114 in the genome through Cre/loxP or Flp/FRT mediated recombination (Diao 115 et al., 2015; Nagarkar-Jaiswal et al., 2015b). Importantly, RMCE cassettes 116 can replace a SIC in either orientation with equal probability due to inverted 117 symmetric attP sequences. Therefore 50% of the insertions are inserted in 118 the opposite orientation of transcription and will not be included in the 119 4 mature mRNA. Hence, only half of all successful exchange events will 120 result in protein or gene trap lines. 121 Here, we show that by combining GFP protein traps and T2A-GAL4 122 gene traps in a single RMCE construct, named Double Header (DH), we 123 significantly increased the number of productive RMCE events for 124 MiMIC/CRIMIC containing genes to generate protein or gene trap alleles. 125 Importantly, we expand the ability to target SICs into genes regardless of 126 the presence of introns to allow access to virtually any gene in the fly 127 genome based on CRISPR/Cas9 mediated HDR. This provides a means to 128 create robust null alleles with simple screening, and to convert the SIC 129 insertion using any DNA, creating scarless modifications to facilitate 130 numerous downstream applications. 131 132 Results 133 134 Double Header (DH) improves the tagging rate of MiMIC containing 135 genes 136 137 SICs in coding introns can be converted into GFP protein traps or 138 T2A-GAL4 gene traps through RMCE. However, because RMCE of SICs in 139 MiMICs and CRIMICs can occur in either orientation, only one out of two 140 events produces a tag that is incorporated in the gene product. Moreover, 141 796 We thank the Bloomington Drosophila Stock Center and the Developmental 797 Studies Hybridoma Bank for antibodies. We thank Zelun Wang for technical 798 help, and Megan Campbell and Shinya Yamamoto for reading the 799 manuscript and providing helpful su...

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Section: Resultssupporting

confidence: 90%

An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila

Li‐Kroeger

Kanca

Lee

et al. 2018

Preprint

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“…However, like Cpr72Ec , its expression is particularly notable in higher order interommatidial pigment cells and may be weakly expressed within photoreceptors. In contrast to the eye-enriched nature of Cpr72Ec , Cpr66d has also been identified in several embryonic tissues, the wing, and the trachea (Chandran et al 2014; Haussmann et al 2008; Ren et al 2005), suggesting a more generalized role for this protein in cuticle formation.…”

Section: Resultsmentioning

confidence: 95%

The cuticular nature of corneal lenses in Drosophila melanogaster

et al. 2017

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The dioptric visual system relies on precisely-focusing lenses that project light onto a neural retina. While the proteins that constitute the lenses of many vertebrates are relatively well-characterized, less is known about the proteins that constitute invertebrate lenses, especially the lens facets in insect compound eyes. To address this question, we used mass spectrophotometry to define the major proteins that comprise the corneal lenses from the adult Drosophila melanogaster compound eye. This led to the identification of four cuticular proteins: two previously identified lens proteins, Drosocrystallin and Retinin, and two newly identified proteins Cpr66D and Cpr72Ec. To determine which ommatidial cells contribute each of these proteins to the lens, we conducted in situ hybridization at 50% pupal development, a key age for lens secretion. Our results confirm previous reports that Drosocrystallin and retinin are expressed in the two primary corneagenous cells - cone cells and primary pigment cells. Cpr72Ec and Cpr66D, on the other hand, are more highly expressed in higher order interommatidial pigment cells. These data suggest that the complementary expression of cuticular proteins give rise to the center vs periphery of the corneal lens facet, possibly facilitating a refractive gradient that is known to reduce spherical aberration. Moreover, these studies provide a framework for future studies aimed at understanding the cuticular basis of corneal lens function in holometabolous insect eyes.

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“…In order to identify novel genes expressed in trachea, we performed an RNA in situ hybridization screening and identified the uncharacterized tracheal expression gene CG15887. CG15887 is expressed throughout entire trachea with high expression in tracheal fusion cells, which guide fusion of the neighboring branches . This gene was named apn recently .…”

Section: Resultsmentioning

confidence: 99%

“…CG15887 is expressed throughout entire trachea with high expression in tracheal fusion cells, which guide fusion of the neighboring branches. 26 This gene was named apn recently. 27 The protein sequence annotation of Apn protein identified three transmembrane (TM) domains ( Figure 1A).…”

Section: Introductionmentioning

confidence: 99%

The novel gene apnoia regulates Drosophila tracheal tube size

Scholl

O’Brien

Chandran

et al. 2019

Developmental Dynamics

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Background Distinct tube size is critical for the function of human tubular organs such as the lung, vascular system, and kidney. Aberrant tube sizes can lead to devastating human illnesses, including polycystic kidney disease. The Drosophila trachea provides a premier genetic system to investigate the fundamental mechanisms that regulate tube size. Results Here we describe the function of a novel gene, apnoia, in tube‐size regulation. apn encodes an apical membrane protein, Apnoia (Apn), with three helical transmembrane domains. Loss‐of‐function apn mutants show shorter‐tube and air‐filling defects in larval trachea, whereas there are no obvious defects in embryonic trachea. Conversely, overexpression of apn in trachea leads to significant tube over‐elongation. We analyzed apical luminal matrix and cell polarity in these longer tubes. It is interesting to note that we observed normal establishment of cell polarity, whereas all luminal matrix components are significantly reduced. In addition, we observed that some matrix components are localized in cytoplasmic vesicles, suggesting secretion defects in apn overexpressing trachea. Conclusion Taken together, these results strongly suggest the possibility that apn is directly or indirectly involved in vesicular trafficking to regulate tube size.

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Gene expression profiling of Drosophila tracheal fusion cells

Cited by 12 publications

References 45 publications

An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila

An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in Drosophila

The cuticular nature of corneal lenses in Drosophila melanogaster

The novel gene apnoia regulates Drosophila tracheal tube size

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