Spectra of Mutations Induced by Tritium Beta Radiation or 2-Chloroethyl Methanesulfonate in Drosophila Melanogaster Germ Cells.

Tritium beta radiation (tritium) was used to induce mutations at the alcohol dehydrogenase (Adh) locus in male Drosophila melanogaster post-meiotic germ cells. All 23 mutations recovered were deletions greater than one kilobase in size (multikilobase deletions) as determined by genetic complementation analysis. A statistical difference was observed between the frequency of multikilobase deletions (23/23 or 1.0) induced by tritium and the frequency of multikilobase deletions (19/27 or 0.7) induced by 100-kVp X-radiation (X-rays). The relative frequency of multikilobase deletions induced by tritium with respect to that induced by X-rays was 1.4. This value was compared to the relative biological effectiveness (RBE = 1.4) of these 2 radiation sources, determined from the ratio of the regression coefficients of the respective sex-linked recessive lethal (SLRL) dose-response data. A relationship may exist between the relative frequency of multikilobase deletions induced at the Adh locus and the RBE determined from the SLRL dose-response data. In addition, a genetic map of the multikilobase deletion break-points induced by tritium and by three monofunctional alkylating agents, 1-ethyl-1-nitrosourea, 1-methyl-1-nitrosourea and ethyl methanesulfonate, is presented. Previous in vitro research suggests that 2-chloroethyl methanesulfonate (Cl-EMS) will produce the same DNA adduct as 1,2-dichloroethane or 1,2-dibromoethane. D. melanogaster gonial cells were treated with Cl-EMS, and the sex-linked recessive lethal (SLRL) and alcohol dehydrogenase (Adh) induced mutation frequencies were determined. The results of these studies were the following: First, mutation frequencies 200 times the spontaneous mutation frequency were induced with Cl-EMS. Second, lethal mutations on the X-chromosome were selected against in the hemizygous male. Third, the induced Adh mutation frequency was higher in the male than in the female. Fourth, all of the 83 Adh null mutations recovered were intragenic mutations as determined by complementation analysis. Fifth, treatment of gonial cells resulted in mutations of common origin, verified by DNA sequence analysis. Sixth, all 36 mutations of independent origin were GC to AT transitions. This study was supported by NIEHS PO1-EEO3447 and DE-FG05-86ER60393

Upregulation of the Drosophila Friend of GATA Gene u-shaped by JAK/STAT Signaling Maintains Lymph Gland Prohemocyte Potency▿

Studies using Drosophila melanogaster have contributed significantly to our understanding of the interaction between stem cells and their protective microenvironments or stem cell niches. During lymph gland hematopoiesis, the Drosophila posterior signaling center functions as a stem cell niche to maintain prohemocyte multipotency through Hedgehog and JAK/STAT signaling. In this study, we provide evidence that the Friend of GATA protein U-shaped is an important regulator of lymph gland prohemocyte potency and differentiation. U-shaped expression was determined to be upregulated in third-instar lymph gland prohemocytes and downregulated in a subpopulation of differentiating blood cells. Genetic analyses indicated that U-shaped maintains the prohemocyte population by blocking differentiation. In addition, activated STAT directly regulated ush expression as evidenced by results from loss- and gain-of-function studies and from analyses of the u-shaped hematopoietic cis-regulatory module. Collectively, these findings identify U-shaped as a downstream effector of the posterior signaling center, establishing a novel link between the stem cell niche and the intrinsic regulation of potency and differentiation. Given the functional conservation of Friend of GATA proteins and the role that GATA factors play during cell fate choice, these factors may regulate essential functions of vertebrate hematopoietic stem cells, including processing signals from the stem cell niche

Antioxidants maintain E-cadherin levels to limit Drosophila prohemocyte differentiation.

Mitochondrial reactive oxygen species (ROS) regulate a variety of biological processes by networking with signal transduction pathways to maintain homeostasis and support adaptation to stress. In this capacity, ROS have been shown to promote the differentiation of progenitor cells, including mammalian embryonic and hematopoietic stem cells and Drosophila hematopoietic progenitors (prohemocytes). However, many questions remain about how ROS alter the regulatory machinery to promote progenitor differentiation. Here, we provide evidence for the hypothesis that ROS reduce E-cadherin levels to promote Drosophila prohemocyte differentiation. Specifically, we show that knockdown of the antioxidants, Superoxide dismutatase 2 and Catalase reduce E-cadherin protein levels prior to the loss of Odd-skipped-expressing prohemocytes. Additionally, over-expression of E-cadherin limits prohemocyte differentiation resulting from paraquat-induced oxidative stress. Furthermore, two established targets of ROS, Enhancer of Polycomb and FOS, control the level of E-cadherin protein expression. Finally, we show that knockdown of either Superoxide dismutatase 2 or Catalase leads to an increase in the E-cadherin repressor, Serpent. As a result, antioxidants and targets of ROS can control E-cadherin protein levels, and over-expression of E-cadherin can ameliorate the prohemocyte response to oxidative stress. Collectively, these data strongly suggest that ROS promote differentiation by reducing E-cadherin levels. In mammalian systems, ROS promote embryonic stem cell differentiation, whereas E-cadherin blocks differentiation. However, it is not known if elevated ROS reduce E-cadherin to promote embryonic stem cell differentiation. Thus, our findings may have identified an important mechanism by which ROS promote stem/progenitor cell differentiation

<i>Drosophila</i> E-Cadherin Functions in Hematopoietic Progenitors to Maintain Multipotency and Block Differentiation

<div><p>A fundamental question in stem cell biology concerns the regulatory strategies that control the choice between multipotency and differentiation. <i>Drosophila</i> blood progenitors or prohemocytes exhibit key stem cell characteristics, including multipotency, quiescence, and niche dependence. As a result, studies of <i>Drosophila</i> hematopoiesis have provided important insights into the molecular mechanisms that control these processes. Here, we show that E-cadherin is an important regulator of prohemocyte fate choice, maintaining prohemocyte multipotency and blocking differentiation. These functions are reminiscent of the role of E-cadherin in mammalian embryonic stem cells. We also show that mis-expression of E-cadherin in differentiating hemocytes disrupts the boundary between these cells and undifferentiated prohemocytes. Additionally, upregulation of E-cadherin in differentiating hemocytes increases the number of intermediate cell types expressing the prohemocyte marker, Patched. Furthermore, our studies indicate that the <i>Drosophila</i> GATA transcriptional co-factor, U-shaped, is required for E-cadherin expression. Consequently, E-cadherin is a downstream target of U-shaped in the maintenance of prohemocyte multipotency. In contrast, we showed that forced expression of the U-shaped GATA-binding partner, Serpent, repressed E-cadherin expression and promoted lamellocyte differentiation. Thus, U-shaped may maintain E-cadherin expression by blocking the inhibitory activity of Serpent. Collectively, these observations suggest that GATA:FOG complex formation regulates E-cadherin levels and, thereby, the choice between multipotency and differentiation. The work presented in this report further defines the molecular basis of prohemocyte cell fate choice, which will provide important insights into the mechanisms that govern stem cell biology.</p></div

E-cadherin mis-expression in the cortical zone upregulates Patched expression and perturbs zonal boundaries.

<p>(<b>A–E</b>) Mis-expression of E-cadherin (Ecad) in cortical zone hemocytes expands the Ptc expression domain, but has no effect on plasmatocyte differentiation. <i>Eater-Gal4</i> females were crossed to (<b>A,C</b>) control (+) or (<b>B,D</b>) <i>UAS-Ecad</i> males. (<b>B</b>) <i>Eater-Gal4</i> driven Ecad significantly increased the Ptc expression domain compared to (<b>A</b>) the control. (<b>C,D</b>) However, <i>Eater-Gal4</i> driven Ecad had no effect on plasmatocyte differentiation. Plasmatocytes were identified using the cell-specific marker, P1. Yellow dotted lines delineate the entire lymph gland; white dotted lines delineate the Ptc expression domain. Scale bars: 50 µm. (<b>E</b>) Histogram showing the percentage of Ptc labeled cells per primary lymph gland lobe in <i>Eater-Gal4</i> or <i>hml-Gal4</i> driven Ecad lymph glands compared to controls (+). Two tailed Student’s t-test; error bars show standard deviation; P values are as shown; n = 12. (<b>F–G”</b>) <i>hml-Gal4</i> driven Ecad expression produces an increase in the following: 1) the Ptc expression domain; 2) the number of cells that are positive for both Ptc and <i>hml-Gal4</i> driven GFP (Ptc⁺; hml>GFP⁺); and 3) the number of hml>GFP⁺ hemocytes within the medial region of the lymph gland. (<b>F,G</b>) Lymph glands co-stained for Ptc and GFP. (<b>F’,F”,G’,G”</b>) The same lymph glands showing only (<b>F’,G’</b>) Ptc staining and (<b>F”,G”</b>) GFP staining. (<b>F–G”</b>) Insets are an enlarged region of medullary zone showing co-expression of Ptc and GFP. (<b>H–I”</b>) <i>Eater-Gal4</i> driven Ecad expression has no effect on Odd or Pxn expression, but produces an increase in the number of Pxn⁺ hemocytes within the medial region of the lymph gland. (<b>H,I</b>) Lymph glands co-stained for Odd and Pxn. (<b>H’,H”,I’,I”</b>) The same lymph glands showing only (<b>H’,I’</b>) Odd staining and (<b>H”,I”</b>) Pxn staining. (<b>H–I”</b>) Insets are an enlarged region showing expression of Pxn⁺ hemocytes or Odd⁺ prohemocytes. Scale bars: 50 µm.</p

The Friend of GATA Transcriptional Co-Regulator, U-Shaped, Is a Downstream Antagonist of Dorsal-Driven Prohemocyte Differentiation in Drosophila.

Recent studies suggest that mammalian hematopoietic stem and progenitor cells (HSPCs) respond directly to infection and inflammatory signaling. These signaling pathways also regulate HSPCs during steady-state conditions (absence of infection), and dysregulation may lead to cancer or age-related loss of progenitor repopulation capacity. Toll-like receptors (TLRs) are a major class of pathogen recognition receptors, and are expressed on the surface of immune effector cells and HSPCs. TLR/NF-κB activation promotes HSPCs differentiation; however, the mechanisms by which this signaling pathway alters the intrinsic transcriptional landscape are not well understood. Although Drosophila prohemocytes are the functional equivalent of mammalian HSPCs, a prohemocyte-specific function for Toll signaling has not been reported. Using Drosophila transgenics, we identified prohemocyte-specific roles for Toll pathway members, Dorsal and Cactus. We showed that Dorsal is required to limit the size of the progenitor pool. Additionally, we showed that activation of Toll signaling in prohemocytes drives differentiation in a manner that is analogous to TLR/NF-κB-driven HSPC differentiation. This was accomplished by showing that over-expression of Dorsal, or knockdown of Cactus, promotes differentiation. We also investigated whether Dorsal and Cactus control prohemocyte differentiation by regulating a key intrinsic prohemocyte factor, U-shaped (Ush), which is known to promote multipotency and block differentiation. We showed that Dorsal repressed Ush expression levels to promote differentiation, whereas Cactus maintained Ush levels to block differentiation. Additionally, we showed that another Toll antagonist, Lesswright, also maintained the level of Ush to block differentiation and promote proliferative quiescence. Collectively, these results identify a novel role for Ush as a downstream target of Toll signaling

Serpent represses E-cadherin and promotes lamellocyte differentiation.

<p>Forced expression of SrpNC or the Ush non-binding mutant form of SrpNC (SrpNC^V421G) represses E-cadherin (Ecad) expression and promotes lamellocyte differentiation. (<b>A–D</b>) Forced expression of SrpNC or SrpNC^V421G represses Ecad expression differentiation in lymph glands from early-third instar (L3) larvae. (<b>E,F</b>) Forced expression of SrpNC^V421G promotes lamellocyte differentiation during mid-L3. <i>Tep4-Gal4</i> females were crossed to (<b>A,C,E</b>) control (+), (<b>B</b>) <i>UAS-srpNC</i> (SrpNC) or (<b>D,F</b>) <i>UAS-srpNC^V421G</i> (SrpNC^V421G) males. Ecad expression and lamellocyte (lm) production were assessed using immunofluorescent staining. Lamellocytes were identified with the specific marker, L1. Yellow dotted lines delineate the entire lymph gland; white dotted lines delineate the prohemocyte pool. Scale bars: A,B 10 µm; C–F 20 µm. (<b>G</b>) Histogram showing the percentage of Ecad labeled cells per primary lymph gland lobe in control (+), SrpNC (n = 16), and SrpNC^V421G (n = 15) lymph glands. Two-tailed Student’s t-test; error bars show standard deviation; P values are as shown. (<b>H</b>) Histogram showing the number of primary lymph gland lobes exhibiting aberrant lamellocyte differentiation in control (+), SrpNC (n = 21), and SrpNC^V421G (n = 26) lymph glands. Two-tailed Fisher’s exact test; P values are as shown. (<b>I</b>) Lamellocyte differentation is not observed until late-L3 in <i>Tep4-Gal4</i> driven <i>UAS-srpNC</i> (SrpNC). In <i>Tep4-Gal4</i> driven <i>UAS-srpNC^V421G</i> (SrpNC^V421G), lamellocyte differentiation is observed in mid-L3, which is earlier than <i>Tep4-Gal4</i> driven <i>UAS-srpNC</i> (SrpNC). Lamellocytes are identified with the specific marker, L1. Scale bars: 50 µm.</p

E-cadherin is a downstream effector of U-shaped.

<p>(<b>A,B</b>) U-shaped (Ush) is required for E-cadherin (Ecad) expression in early-third instar larvae. (<b>B</b>) Ecad expression is reduced in <i>ush^vx22/r24</i> trans-heterozygotes compared to (<b>A</b>) controls (+). Yellow dotted lines delineate the entire lymph gland; white dotted lines delineate the Ecad expression domain. (<b>C</b>) Histogram showing the percentage of Ecad-expressing cells per primary lobe was significantly reduced in <i>ush</i>^vx22/r24 lymph glands compared to controls (+). Two-tailed Student’s t-test; error bars show standard deviation; P value is as shown; n = 16. (<b>D–G</b>) Ush and Ecad function in the same pathway. (<b>D–F</b>) Loss of one copy of either <i>ush</i> or <i>shg</i> does not produce increased lamellocyte (lm) differentiation. (<b>G</b>) In contrast, <i>ush/shg</i> transheterozygotes show a dramatic increase in lamellocyte differentiation. (<b>H</b>) Histogram showing the number of primary lymph gland lobes exhibiting lamellocyte differentiation in wild-type (+), <i>yw;shg²</i>/+, <i>yw;shg^E17B</i>/+, <i>yw;ush^vx22</i>/+, <i>yw;ush^vx22/shg²</i>, <i>yw;ush^vx22/shg^E17B</i>, <i>yw;ush^vx22/shg²</i> recombinants and <i>yw;odd⁰¹⁸⁶³</i>/<i>shg²</i> larvae. Two-tailed Fisher’s exact test; P values are as shown; n = 20. (<b>I–N</b>) Ecad rescues loss of Ush function. <i>dome-Gal4</i> females were crossed to <i>UAS-Ush^RNAi</i> (Ush^RNAi) or <i>UAS-Ush^RNAi</i>;<i>UAS-Ecad^RNAi</i> (Ush^RNAi;Ecad) males. Odd expression was reduced in (<b>I</b>) lymph glands that expressed Ush^RNAi alone compared to (<b>J</b>) lymph glands that expressed both Ush^RNAi and Ecad. Yellow dotted lines delineate the entire lymph gland; white dotted lines delineate the Odd expression domain. (<b>K</b>) Histogram showing the percentage of Odd labeled cells was significantly reduced in Ush^RNAi lymph glands compared to Ush^RNAi;Ecad lymph glands. Two tailed Student’s t-test; error bars show standard deviation; P values are as shown; n = 14. (<b>L</b>) Lamellocyte (lm) differentiation increased in lymph glands that expressed Ush^RNAi compared to (<b>M</b>) lymph glands that expressed both Ush^RNAi and Ecad. (<b>N</b>) Histogram showing lamellocyte differentiation significantly increased in Ush^RNAi lymph glands compared to Ush^RNAi;Ecad lymph glands. Two-tailed Fisher’s exact test; P value is as shown; n = 20. Scale bars: A,B 10 µm; D–G,I,J,L,M 50 µm.</p

E-cadherin maintains the prohemocyte pool.

<p>(<b>A–F</b>) Loss of E-cadherin (Ecad) function reduces the prohemocyte population. <i>dome-Gal4</i> females were crossed to (<b>A,C,E</b>) control (+) or (<b>B,D,F</b>) <i>UAS-Ecad^RNAi</i> (Ecad^RNAi) males to knockdown Ecad expression in prohemocytes. (<b>A,B</b>) <i>dome-Gal4</i> driven Ecad^RNAi dramatically reduced the number of Ecad-expressing cells. (<b>C–F</b>) Targeted knockdown of Ecad reduced expression of the prohemocyte markers (<b>D</b>) Odd and (<b>F</b>) Ptc compared to (<b>C,E</b>) controls. (<b>G–L</b>) Gain of Ecad function expands the prohemocyte population. <i>dome-Gal4</i> females were crossed to (<b>G,I,K</b>) control (+) or (<b>H,J,L</b>) <i>UAS-Ecad</i> males. (<b>G–L</b>) Over-expression of the Ecad wild-type transgene increased the number of cells expressing (<b>H</b>) Ecad, (<b>J</b>) Odd and (<b>L</b>) Ptc compared to (<b>G,I,K</b>) controls. Yellow dotted lines delineate the entire lymph gland; white dotted lines delineate the prohemocyte pool. Scale bars: 50 µm. (<b>M</b>) Histogram showing the percentage of labeled cells per primary lymph gland lobe; Ecad (n = 12), Odd (n = 15), and Ptc (n = 12) in control (+) and Ecad knockdown (Ecad^RNAi) lymph glands. (<b>N</b>) Histogram showing the percentage of labeled cells per primary lymph gland lobe; (Ecad; n = 12), (Odd; n = 16), and (Ptc; n = 12) in control (+) and Ecad over-expression genetic backgrounds. Two tailed Student’s t-test; error bars show standard deviation; P values are as shown.</p