dissection-lipid-droplet-staining-oenocytes-drosophila

Dissection and Lipid Droplet Staining of Oenocytes in <em>Drosophila</em> Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...

Guangzhou Medical University

the second affiliated hospital of Guangzhou medical university

TALEs (transcription activator-like effectors) are a family of natural transcriptional activators originally isolated from the plant pathogen of Xanthomonas spp. The DNA binding motif of TALEs can be re-designed in such way that they bind specific DNA sequences other than their original targets. Fusion of customized TALEs with an endonuclease, Fok I, generates artificial enzymes that are targeted to specific DNA sites for cutting, allowing gene specific modification of both animal and plant genomes. Previously, we reported the use of TALEN (transcription activator-like effector nuclease) for the highly specific and efficient modification of the two Drosophila loci yellow and CG9797. Here, we describe a detailed protocol for TALEN-mediated genomic modification in Drosophila, with the aim of providing a practical bench guide for the Drosophila research community. 

TALEN-mediated Drosophila genome editing: protocols and applications.

Deregulation of the evolutionarily conserved Hippo pathway has been implicated in abnormal development of animals and in several types of cancer. One mechanism of Hippo pathway regulation is achieved by controlling the stability of its regulatory components. However, the executive E3 ligases that are involved in this process, and how the process is regulated, remain poorly defined. In this study, we identify, through a genetic candidate screen, the SCF(Slmb) E3 ligase as a novel negative regulator of the Hippo pathway in Drosophila imaginal tissues via mediation of the degradation of Expanded (Ex). Mechanistic study shows that Slmb-mediated degradation of Ex is inhibited by the Hippo signaling. Considering the fact that Hippo signaling suppresses the transcription of ex, we propose that the Hippo pathway employs a double security mechanism to ensure fine-tuned homeostasis during development. 

SCF(Slmb) E3 ligase-mediated degradation of Expanded is inhibited by the Hippo pathway in Drosophila.

Tissue expansion and patterning are integral to development; however, it is unknown quantitatively how a mother accumulates molecular resources to invest in the future of instructing robust embryonic patterning. Here we develop a model, Tissue Expansion-Modulated Maternal Morphogen Scaling (TEM(3)S), to study scaled anterior-posterior patterning in Drosophila embryos. Using both ovaries and embryos, we measure a core quantity of the model, the scaling power of the Bicoid (Bcd) morphogen gradient's amplitude nA. We also evaluate directly model-derived predictions about Bcd gradient and patterning properties. Our results show that scaling of the Bcd gradient in the embryo originates from, and is constrained fundamentally by, a dynamic relationship between maternal tissue expansion and bcd gene copy number expansion in the ovary. This delicate connection between the two transitioning stages of a life cycle, stemming from a finite value of nA~3, underscores a key feature of developmental systems depicted by TEM(3)S. 

Fundamental origins and limits for scaling a maternal morphogen gradient.

Body size is one of the features that distinguish one species from another in the biological world. Animals have developed mechanisms to control their body size during normal development. However, how animals cope with genetic alterations and/or environmental stresses to develop into normal-sized adults remain poorly understood. The ability of the animals to develop into a normal-sized adult after the challenges of genetic alterations and/or environmental stresses reveals a robustness of body size control. Here we show that the mutation of dGPAT4, a de novo synthase of lysophosphatidic acid, is a genetic alteration that triggers such a robust response of the animals to body size challenges in Drosophila. Loss of dGPAT4 leads to a severe delay of development, slow growth and resultant small-sized animals during the larval stages, but results in normal-sized adult flies. The robust body size adjustment of the dGPAT4 mutant is likely achieved by corresponding changes in ecdysone and insulin signaling, which is also manifested by compromised food intake. Thus, we propose that a strategy has been evolved by the animals to reach final body size when challenged by genetic alterations, which requires the coordinated ecdysone and insulin signaling. 

Ecdysone and insulin signaling play essential roles in readjusting the altered body size caused by the dGPAT4 mutation in Drosophila.

TALEN (transcription activator-like effector nuclease) is a powerful tool for gene disruption and other genomic modifications. In the past 3 years or so, it has attracted eyes from every corner of the biological world, due to its characteristics of simplicity, high efficiency, low toxicity, and applicability across almost all species. In our lab, we first reported the TALEN-mediated gene disruption in Drosophila, and recently employed this technique in precisely modifying the Drosophila genome, such as in vivo tagging and gene correction. Here, we describe in detail the protocols and experiences in TALEN-mediated genomic modifications to share with the Drosophilists all over the world.

Methods for TALEN-Mediated Genomic Manipulations in Drosophila.

A widely appreciated aspect of developmental robustness is pattern formation in proportion to size. But how such scaling features emerge dynamically remains poorly understood. Here we generate a data set of the expression profiles of six gap genes in Drosophila melanogaster embryos that differ significantly in size. Expression patterns exhibit size-dependent dynamics both spatially and temporally. We uncover a dynamic emergence of under-scaling in the posterior, accompanied by reduced expression levels of gap genes near the middle of large embryos. Simulation results show that a size-dependent Bicoid gradient input can lead to reduced Krüppel expression that can have long-range and dynamic effects on gap gene expression in the posterior. Thus, for emergence of scaled patterns, the entire embryo may be viewed as a single unified dynamic system where maternally derived size-dependent information interpreted locally can be propagated in space and time as governed by the dynamics of a gene regulatory network. 

Temporal and spatial dynamics of scaling-specific features of a gene regulatory network in Drosophila.

The CRISPR/Cas9 Genome Editing Revolution.

Components of the SWI/SNF chromatin-remodeling complex are among the most frequently mutated genes in various human cancers, yet only SMARCB1/hSNF5, a core member of the SWI/SNF complex, is mutated in malignant rhabdoid tumors (MRT). How SMARCB1/hSNF5 functions differently from other members of the SWI/SNF complex remains unclear. Here, we use  imaginal epithelial tissues to demonstrate that Snr1, the conserved homolog of human SMARCB1/hSNF5, prevents tumorigenesis by maintaining normal endosomal trafficking-mediated signaling cascades. Removal of Snr1 resulted in neoplastic tumorigenic overgrowth in imaginal epithelial tissues, whereas depletion of any other members of the SWI/SNF complex did not induce similar phenotypes. Unlike other components of the SWI/SNF complex that were detected only in the nucleus, Snr1 was observed in both the nucleus and the cytoplasm. Aberrant regulation of multiple signaling pathways, including Notch, JNK, and JAK/STAT, was responsible for tumor progression upon -depletion. Our results suggest that the cytoplasmic Snr1 may play a tumor suppressive role in  imaginal tissues, offering a foundation for understanding the pivotal role of SMARCB1/hSNF5 in suppressing MRT during early childhood. .

The SWI/SNF Complex Protein Snr1 Is a Tumor Suppressor in  Imaginal Tissues.

Anything impossible with CRISPR/Cas9?

Age-dependent ectopic fat accumulation (EFA) in animals contributes to the progression of tissue aging and diseases such as obesity, diabetes, and cancer. However, the primary causes of age-dependent EFA remain largely elusive. Here, we characterize the occurrence of age-dependent EFA in Drosophila and identify HDAC6, a cytosolic histone deacetylase, as a suppressor of EFA. Loss of HDAC6 leads to significant age-dependent EFA, lipid composition imbalance, and reduced animal longevity on a high-fat diet. The EFA and longevity phenotypes are ameliorated by a reduction of the lipid-droplet-resident protein PLIN2. We show that HDAC6 is associated physically with the chaperone protein dHsc4/Hsc70 to maintain the proteostasis of PLIN2. These findings indicate that proteostasis collapse serves as an intrinsic cue to cause age-dependent EFA. Our study suggests that manipulation of proteostasis could be an alternative approach to the treatment of age-related metabolic diseases such as obesity and diabetes.

HDAC6 Suppresses Age-Dependent Ectopic Fat Accumulation by Maintaining the Proteostasis of PLIN2 in Drosophila.

The process through which multiple types of cell-lineage-restricted progenitor cells are specified from multipotent stem cells is unclear. Here we show that, in intestinal stem cell lineages in adult Drosophila, in which the Delta-Notch-signalling-guided progenitor cell differentiation into enterocytes is the default mode, the specification of enteroendocrine cells (EEs) is initiated by transient Scute activation in a process driven by transcriptional self-stimulation combined with a negative feedback regulation between Scute and Notch targets. Scute activation induces asymmetric intestinal stem cell divisions that generate EE progenitor cells. The mitosis-inducing and fate-inducing activities of Scute guide each EE progenitor cell to divide exactly once prior to its terminal differentiation, yielding a pair of EEs. The transient expression of a fate inducer therefore specifies both type and numbers of committed progenitor cells originating from stem cells, which could represent a general mechanism used for diversifying committed progenitor cells from multipotent stem cells.

Transient Scute activation via a self-stimulatory loop directs enteroendocrine cell pair specification from self-renewing intestinal stem cells.

In the version of this Article originally published, the author had misnumbered the reference citations in the Methods, using numbers 1-14 instead of 46-59. These errors have now been corrected in all online versions of the Article.

Author Correction: Transient Scute activation via a self-stimulatory loop directs enteroendocrine cell pair specification from self-renewing intestinal stem cells.

Chromatin assembly factor 1 (CAF1), a histone chaperone that mediates the deposition of histone H3/H4 onto newly synthesized DNA, is involved in Notch signaling activation during  wing imaginal disc development. Here, we report another side of CAF1, wherein the subunits CAF1-p105 and CAF1-p180 (also known as CAF1-105 and CAF1-180, respectively) inhibit expression of Notch target genes and show this is required for proliferation of  ovarian follicle cells. Loss-of-function of either CAF1-p105 or CAF1-p180 caused premature activation of Notch signaling reporters and early expression of the Notch target Hindsight (Hnt, also known as Pebbled), leading to Cut downregulation and inhibition of follicle cell mitosis. Our studies further show Notch is functionally responsible for these phenotypes observed in both the CAF1-p105- and CAF1-p180-deficient follicle cells. Moreover, we reveal that CAF1-p105- and CAF1-p180-dependent Cut expression is essential for inhibiting Hnt expression in follicle cells during their mitotic stage. These findings together indicate a novel negative-feedback regulatory loop between Cut and Hnt underlying CAF1-p105 and CAF-p180 regulation, which is crucial for follicle cell differentiation. In conclusion, our studies suggest CAF1 plays a dual role to sustain cell proliferation by positively or negatively regulating  Notch signaling in a tissue-context-dependent manner.

Inhibition of Notch signaling by the p105 and p180 subunits of  chromatin assembly factor 1 is required for follicle cell proliferation.

Autophagy has been evolved as one of the adaptive cellular processes in response to stresses such as nutrient deprivation. Various cellular cargos such as damaged organelles and protein aggregates can be selectively degraded through autophagy. Recently, the lipid storage organelle, lipid droplet (LD), has been reported to be the cargo of starvation-induced autophagy. However, it remains largely unknown how the autophagy machinery recognizes the LDs and whether it can selectively degrade LDs. In this study, we show that Drosophila histone deacetylase 6 (dHDAC6), a key regulator of selective autophagy, is required for the LD turnover in the hepatocyte-like oenocytes in response to starvation. HDAC6 regulates LD turnover via p62/SQSTM1 (sequestosome 1)-mediated aggresome formation, suggesting that the selective autophagy machinery is required for LD recognition and degradation. Furthermore, our results show that the loss of dHDAC6 causes steatosis in response to starvation. Our findings suggest that there is a potential link between selective autophagy and susceptible predisposition to lipid metabolism associated diseases in stress conditions.

Article	Year
TALEN-mediated Drosophila genome editing: protocols and applications. Methods (San Diego, Calif.)\| PubMed ID: 24751823	2014
SCF(Slmb) E3 ligase-mediated degradation of Expanded is inhibited by the Hippo pathway in Drosophila. Cell research\| PubMed ID: 25522691	2015
Fundamental origins and limits for scaling a maternal morphogen gradient. Nature communications\| PubMed ID: 25809405	2015
Ecdysone and insulin signaling play essential roles in readjusting the altered body size caused by the dGPAT4 mutation in Drosophila. Journal of genetics and genomics = Yi chuan xue bao\| PubMed ID: 26408093	2015
Methods for TALEN-Mediated Genomic Manipulations in Drosophila. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 26443222	2016
Temporal and spatial dynamics of scaling-specific features of a gene regulatory network in Drosophila. Nature communications\| PubMed ID: 26644070	2015
The CRISPR/Cas9 Genome Editing Revolution. Journal of genetics and genomics = Yi chuan xue bao\| PubMed ID: 27256542	2016
The SWI/SNF Complex Protein Snr1 Is a Tumor Suppressor in Imaginal Tissues. Cancer research\| PubMed ID: 27923836	2017
Anything impossible with CRISPR/Cas9? Science China. Life sciences\| PubMed ID: 28573353	2017
HDAC6 Suppresses Age-Dependent Ectopic Fat Accumulation by Maintaining the Proteostasis of PLIN2 in Drosophila. Developmental cell\| PubMed ID: 28966044	2017
Transient Scute activation via a self-stimulatory loop directs enteroendocrine cell pair specification from self-renewing intestinal stem cells. Nature cell biology\| PubMed ID: 29335529	2018
Author Correction: Transient Scute activation via a self-stimulatory loop directs enteroendocrine cell pair specification from self-renewing intestinal stem cells. Nature cell biology\| PubMed ID: 29674680	2018
Inhibition of Notch signaling by the p105 and p180 subunits of chromatin assembly factor 1 is required for follicle cell proliferation. Journal of cell science\| PubMed ID: 30630896	2019
HDAC6 regulates lipid droplet turnover in response to nutrient deprivation via p62-mediated selective autophagy. Journal of genetics and genomics = Yi chuan xue bao\| PubMed ID: 31078436	2019

Renjie Jiao

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