Characterization of a Weak Allele of Zebrafish cloche Mutant

Hematopoiesis is a complicated and dynamic process about which the molecular mechanisms remain poorly understood. Danio rerio (zebrafish) is an excellent vertebrate system for studying hematopoiesis and developmental mechanisms. In the previous study, we isolated and identified a cloche172 (clo172) mutant, a novel allele compared to the original cloche (clo) mutant, through using complementation test and initial mapping. Here, according to whole mount in-situ hybridization, we report that the endothelial cells in clo172 mutant embryos, although initially developed, failed to form the functional vascular system eventually. In addition, further characterization indicates that the clo172 mutant exhibited weaker defects instead of completely lost in primitive erythroid cells and definitive hematopoietic cells compared with the clos5 mutant. In contrast, primitive myeloid cells were totally lost in clo172 mutant. Furthermore, these reappeared definitive myeloid cells were demonstrated to initiate from the remaining hematopoietic stem cells (HSCs) in clo172 mutant, confirmed by the dramatic decrease of lyc in clo172runx1w84x double mutant. Collectively, the clo172 mutant is a weak allele compared to the clos5 mutant, therefore providing a model for studying the early development of hematopoietic and vascular system, as well as an opportunity to further understand the function of the cloche gene

Grainy head target genes in epithelial morphogenesis and wound healing

grainy head (grh) genes encode a family of transcription factors conserved from fly to human. Drosophila grh is the founding member of this gene family and has multiple functions, including tracheal tube size control, epidermal barrier formation and reconstruction after wounding. To understand the underlying molecular mechanism of grh functions, we tried to isolate its direct targets and analyze their function. We identified ten grh targets by combining bioinformatics and genetics. Grh directly controls the expression of stitcher (stit), which encodes a Ret family receptor tyrosine kinase (RTK), during both development and wound healing. Stit promotes actin cable assembly and induces extracellular signal-regulated kinase (ERK) phosphorylation around the wound edges upon injury. Stit also activates barrier repair genes and its own expression at the wound sites in a Grh-dependent manner. This positive feedback loop ensures efficient epidermal wound repair. In addition, Grh regulates the expression of multiple genes involved in chitin biosynthesis or modification. Most of the genes are required for tracheal tube size control. Two of them, verm and serp, encode related putative luminal chitin deacetylases. The functional analysis of verm and serp identifies an important role of luminal chitin matrix modification in limiting tracheal tube elongation. Therefore, it is very likely that Grh controls tracheal tube size through regulating multiple targets involved in the assembly or modification of luminal chitin matrix. Grh also directly activates the epidermal expression of Peptidoglycan recognition protein LC (PGRP-LC) gene that is required for the induction of antimicrobial peptides (AMPs) upon infection. Furthermore, ectopically expressing Grh is sufficient to induce AMP Cecropin A lacZ reporter (CecA-LacZ) in the embryonic epidermis. These results suggest a new function of Grh in the local immune responses in Drosophila barrier epithelia.At the time of the doctoral defense, the following papers was unpublished and had a status as follows: Paper 1: Manuscript

Two-tiered control of epithelial growth and autophagy by the insulin receptor and the ret-like receptor, stitcher.

Body size in Drosophila larvae, like in other animals, is controlled by nutrition. Nutrient restriction leads to catabolic responses in the majority of tissues, but the Drosophila mitotic imaginal discs continue growing. The nature of these differential control mechanisms that spare distinct tissues from starvation are poorly understood. Here, we reveal that the Ret-like receptor tyrosine kinase (RTK), Stitcher (Stit), is required for cell growth and proliferation through the PI3K-I/TORC1 pathway in the Drosophila wing disc. Both Stit and insulin receptor (InR) signaling activate PI3K-I and drive cellular proliferation and tissue growth. However, whereas optimal growth requires signaling from both InR and Stit, catabolic changes manifested by autophagy only occur when both signaling pathways are compromised. The combined activities of Stit and InR in ectodermal epithelial tissues provide an RTK-mediated, two-tiered reaction threshold to varying nutritional conditions that promote epithelial organ growth even at low levels of InR signaling

Stit is required for optimal growth of the wing epithelium.

<p>(A) Stit inactivation in the dorsal wing compartment using <i>ap</i>-<i>GAL4</i>-induced FRT <i>stit</i> mutant clones or expression of either <i>Stit^KD</i> or <i>stit-IR</i> caused an upwardly bent adult wing. (B) Wing cell area decreased in <i>stit</i> mutant clones compared to wild-type clones. (C) The expression of <i>Stit^KD</i> in the dorsal wing compartment led to a 33% reduction of the dorsal/ventral (D/V) cell number ratio (>15,000 cells from 10 <i>Stit^KD</i> pupal wings counted) relative to <i>GFP</i> expressing controls (8 animals, >15,000 cells counted). Student's <i>t</i> test, <i>p</i><0.005. (D) Expression of <i>stit-IR</i> in the posterior compartment (<i>en-GAL4</i>) caused a backwardly bent wing. (E) The posterior compartment of <i>en>stit-IR</i> wings was reduced in size due to a reduction in both the total cell number and wing cell area relative to GFP-expressing control wings. (F) <i>hs-flp</i>;<i>actin</i>><i>CD2,stop</i>><i>GAL4</i> (AFG4)-generated clones revealed a general reduction of proliferation and an increase in cell doubling time (CDT) when insulin (<i>InR^DN</i>) or amino acid signaling (<i>RagA^DN</i>) was reduced relative to clones expressing GFP alone (48 h after heatshock). A similar effect was observed in <i>Stit^KD</i>-expressing clones, whereas simultaneous reduction of Stit and InR signaling led to a further reduction in proliferation. The number of clones examined (<i>n</i>) for each genotype is indicated. (G) Analysis of Phospho-histone 3 (PH3)-positive mitotic profiles in third instar larval wing discs revealed a general (dorsal, <b>D</b>, and ventral, <b>V</b>, in graph) reduction in the number of mitotically active cells within <i>ap</i>><i>Stit^KD</i> wing discs compared to control, Student's <i>t</i> test, <i>p</i><0.005 when comparing either control compartment with either <i>Stit^KD</i> compartment. The number of discs examined (<i>n</i>) is indicated. Scale bar, 50 µm. (H) 10 min of EdU incorporation did not show a compartment-specific change in cells entering into S-phase in <i>ap</i>><i>Stit^KD</i> discs versus <i>ap>GFP</i> control discs. However, the overall (both dorsal and ventral) labeling was more sparse in <i>Stit^KD</i> wings, although the D/V ratio was close to 1. The number of wing/discs examined (<i>n</i>) is indicated. All error bars indicate standard deviation. The nonautonomous compensatory growth effect is explored further in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001612#pbio.1001612.s001" target="_blank">Figure S1</a>.</p

A model for the different modes of autophagy regulation in tissues that express <i>stit</i>.

<p>(A) A model depicting Stit and its intersection with the InR/PI3K-I/TORC1 pathway. As Stit is required and sufficient for PI3K-I activation, we place it in parallel to the InR. While InR is known to signal through Chico (IRS) and Lnk, the RTK adaptor that couples Stit to PI3K-I signaling is unknown (X). (B) The presence of <i>InR</i> or <i>stit</i> is sufficient to block autophagy in the wing, while both are required for optimal wing growth. When reared on low-energy food, Stit is required for repressing autophagy. These findings predict a two-tiered model for the switch between anabolism to catabolism in epithelial tissues.</p

Stit activates PI3K-I to support growth and suppress starvation-induced autophagy.

<p>(A) Clonal overexpression in the fat body (marked by RFP, asterisk) of <i>PI3K-CaaX</i> led to increased recruitment of GFP-tagged PH probe (<i>tGPH</i>) to the fat body cell plasma membrane and cell enlargement. This was more evident under starvation conditions, where membrane-bound levels of GFP-PH declined in neighboring cells. (B) Clones expressing <i>stit</i> were slightly enlarged, more rounded, and had higher membrane-bound GFP-PH levels than neighbor cells. This persisted upon starvation. (C) Clonal overexpression of <i>PI3K-CaaX</i> or (D) <i>stit</i> (marked by GFP, asterisk) in the fat body of larvae expressing a Cherry-tagged Atg8a reporter expressed under the control of a fat body promoter showed that both PI3K-CaaX and Stit can block the starvation-induced punctate accumulation of Ch::Atg8a. Quantified in (G). (E) Clonal overexpression of either <i>PI3K-CaaX</i> (E) or <i>stit</i> (F) in larvae during programmed autophagy (P.A.) demonstrated that both can block Ch::Atg8a accumulation in the expressing cells. Quantified in (G). Feeding the TORC1 inhibitor rapamycin to larvae expressing <i>PI3K-CaaX</i> (E) or <i>stit</i> (F) in clones reverted the Stit-mediated block of Ch::Atg8a puncta accumulation. Quantified in (G). (G) The intensity of Ch::Atg8a in AFG4-positive cells was measured and compared to the nearest neighbor cells to calculate fold changes where a value of 1 (red hatched line) indicates no difference to the normal autophagy response to each condition (starved, fed, P.A./developmental, or rapamycin induced) observed in wild-type neighbor cells. Stit and PI3K-I could both suppress programmed and starvation-induced Ch::Atg8a accumulation/intensity increase. * indicates Student's <i>t</i> test scores of significance (<i>p</i><0.005) between overexpressing cells and wild-type neighbor cells, while inset Student's <i>t</i> test scores indicate <i>p</i> values of the difference in response of <i>stit</i>-expressing cells/wild-type neighbor cells between conditions—that is, starved and fed. The number of transgene-expressing cells/wild-type neighbor cells where Ch::Atg8a intensity was measured (<i>n</i>) is indicated. Error bars indicate standard deviation. Scale bar, 50 µm. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001612#pbio.1001612.s003" target="_blank">Figure S3</a>.</p

Stit and the insulin receptor cooperate to activate the PI3K-I pathway.

<p>(A) Clones of cells (marked with RFP) expressing <i>PI3K-CaaX</i> in the larval wing discs led to an increased recruitment of the tubulin::GFP-PH probe (tGPH) to the plasma membrane detectable following 24-h starvation. Inset X–Z section (3–4 µm) and line intensity graph is through/over the indicated region in merged panels. RFP (red) and GFP (green) intensities are represented as line intensity graphs. Peaks represent membrane-localized GFP, while troughs correspond to cytoplasmic signal. GFP-PH membrane-associated intensity in the PI3K-CaaX-expressing region is higher than the nonexpressing region. (B) MS1096 expression of <i>stit</i> (red) causes increased GFP-PH membrane recruitment in the expressing cells. (C) Clones of cells expressing <i>Stit^KD</i> or (D) <i>InR^DN</i> alone did not decrease membrane GFP-PH localization. (E) Clones expressing both <i>Stit^KD</i> and <i>InR^DN</i> had lower levels of membrane-localized GFP-PH than wild-type neighbors. Arrowheads denote clone boundaries. Insets show X–Z projections spanning the clones. All images are thin (8 µm) confocal projections. Scale bars, 10 µm.</p

The PI3K-I/TORC1signalling cassette is required for Stit-dependent protection against starvation.

<p>Clonal expression (cells marked by GFP/hatched line) of <i>stit</i> and/or transgenes interfering with TORC1 signaling components in the fat body of starved (5 h) larvae expressing Ch::Atg8a under the control of a fat body promoter (A) <i>PI3K-I</i>-expressing cells have larger nuclei, are protected from starvation-induced Ch::Atg8a puncta formation, and maintain high p-4E-BP levels compared to wild-type neighbors. (B) <i>stit</i>-expressing cells behave like <i>PI3K-I</i>-expressing cells in (A). (C) <i>PI3K-I^DN</i>-expressing cells are smaller and show Ch::Atg8a autophagic puncta and low levels of p-4E-BP (indistinguishable from neighbor cells). (D) <i>stit</i> and <i>PI3K-I^DN</i> co-expression resulted in smaller cells with autophagic puncta and low levels of p-4E-BP following starvation, indicating PI3K-I signaling lies downstream of Stit. (E) <i>Akt-IR</i>, (G) <i>RagA^DN</i>, and (I) <i>TOR^TED</i>-expressing cells are smaller, readily form autophagic puncta, and had lower or similar p-4E-BP levels to neighbor cells. (F) <i>Akt-IR</i>, (H) <i>RagA^DN</i>, or (J) <i>TOR^TED</i> co-expression in cells expressing <i>stit</i> inhibited the increase in cell size, resistance to starvation-induced autophagy (Ch::Atg8a puncta), or maintenance of p-4E-BP levels, indicating that these members of the PI3K-I/TORC1 signaling cassette are required for Stit-dependent starvation resistance. Scale bars, 25 µm. (K) Plot of the ratios of p-4E-BP labeling intensities in cells expressing the transgene to wild-type neighbor cells. The number of overexpressing cells/wild-type nearest neighbor cells counted (<i>n</i>) is given. * indicates Student's <i>t</i> test <i>p</i> values <0.005 between transgene-expressing and wild-type neighbor cells. Co-expression of <i>RagA^DN</i> together with <i>stit</i> blocked <i>stit</i>-supported growth under starvation. This effect was so strong on p-4E-BP levels that it reduced levels far beyond the starved wild-type cell levels, giving significant differences. Inset <i>p</i> values compare differences in preservation of p-4E-BP signal between <i>stit</i>-expressing cells/wild-type neighbor cells and <i>stit</i> and transgene co-expressing cells/wild-type cells. Error bars indicate standard deviation.</p

Stit and InR cooperatively govern TORC1 activity.

<p>Anti-p-dS6K labeling of third instar larval wing discs revealed discrete puncta lying basally within cells. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001612#pbio.1001612.s005" target="_blank">Figure S5</a> for control experiments addressing labeling specificity. Transgenes were expressed by <i>ap-GAL4</i> and expressing cells were identified by GFP (A–H and J) or by Stit detection (I, K). Arrows or arrowheads mark the D/V compartment boundary. D/V ratios of p-dS6K intensities are quantified in (L). (A) Expression of <i>RagA^DN</i> led to a reduction in basal p-dS6K puncta in the dorsal compartment. X–Z sections of the boxed area are shown in (B). (B–F) X–Z sections of discs expressing <i>RagA^DN</i>, <i>raptor-IR</i>, <i>InR^DN</i>, <i>stit-IR</i>, or <i>Stit^KD</i> in the dorsal compartment. The basal p-dS6K signal of interest is boxed in (B) and the dorsal (D) and ventral (V) compartments indicated. Dividing cells at the top of the panel also label strongly (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001612#pbio.1001612.s006" target="_blank">Figure S6</a>). The dorsal compartment lies to the right in all X–Z sections and arrowheads mark the compartment borders. (G) Expression of <i>stit</i> caused overgrowth of the dorsal region of the disc accompanied by a change in disc morphology, preventing analysis of basal p-dS6K levels. (H) Co-expression of <i>Stit^KD</i> and <i>InR^DN</i> reduced the p-dS6K signal, to a similar extent as either inactivation alone; see (L) for quantification. (I) Co-expression of <i>stit</i> with <i>Stit^KD</i> leads to a reduction of the <i>stit</i> overgrowth phenotype but gave excessively high p-dS6K levels precluding quantification. (J) <i>Rheb</i> expression increased p-dS6K levels within the dorsal region. (K) Co-expression of <i>Rheb</i> with <i>Stit^KD</i> reverted the decrease in p-dS6K levels resulting from Stit inactivation (see L). Scale bar, 50 µm. (L) The intensity of basal p-dS6K within both dorsal and ventral compartments was measured and the D/V ratio for each genotype calculated. Student's <i>t</i> test showed <i>p</i><0,001 when wild-type D/V ratios were compared with all genotypes except when compared to <i>ap>Stit^KD>Rheb</i> (<i>p</i><0.01). <i>ap>Stit^KD>Rheb</i> was significantly different from <i>ap>Stit^KD</i> (<i>p</i> = 0.0015). (M) Immunoblots of total lysates prepared from third instar larval wing discs of wild-type, wild-type starved, and <i>stit</i> mutant larvae. The ratio of p-dS6k/dS6K was reproducibly lower (40% average reduction (45% in M), 15% standard error, 5 independent experiments, 7 samples) in <i>stit</i> mutant discs compared to wild-type fed animals. Arrowhead indicates the band recognized by the anti p-dS6K antibody.</p

Electrolyte and Additive Engineering for Zn Anode Interfacial Regulation in Aqueous Zinc Batteries

Aqueous Zn-metal batteries (AZMBs) have gained great interest due to their low cost, eco-friendliness, and inherent safety, which serve as a promising complement to the existing metal-based batteries, e.g., lithium-metal batteries and sodium-metal batteries. Although the utilization of aqueous electrolytes and Zn metal anode in AZMBs ensures their improved safety over other metal batteries meanwhile guaranteeing their decent energy density at the cell level, plenty of challenges involved with metallic Zn anode still await to be addressed, including dendrite growth, hydrogen evolution reaction, and zinc corrosion and passivation. In the past years, several attempts have been adopted to address these problems, among which engineering the aqueous electrolytes and additives is regarded as a facile and promising approach. In this review, a comprehensive summary of aqueous electrolytes and electrolyte additives will be given based on the recent literature, aiming at providing a fundamental understanding of the challenges associated with the metallic Zn anode in aqueous electrolytes, meanwhile offering a guideline for the electrolytes and additives engineering strategies toward stable AZMBs in the future