<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

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 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

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

Prohemocyte-specific knockdown of Dorsal limits crystal cell differentiation.

<p>(<b>A,B</b>) Crystal cell numbers were reduced in the lymph glands of <i>dome-Gal4</i> driven <i>UAS-dl</i>^<i>RNAi</i> (<i>dl</i>^<i>RNAi</i>) compared to controls (+). Lymph glands from mid-third instar larvae. White dotted lines delineate the entire lymph gland. Scale bars: 50 μm. (<b>C</b>) Histogram showing that the percentage of crystal cells decreased in <i>dl</i>^<i>RNAi</i> lymph glands compared to controls. (<b>D</b>) Histogram showing the number of crystal cells decreased in <i>dl</i>^<i>RNAi</i> lymph glands compared to controls. (<b>C,D</b>) Student’s t-test; error bars show standard deviation; control and <i>dl</i>^<i>RNAi</i> (n = 28); P value is as shown.</p

Ush functions in prohemocytes to promote multipotency and block differentiation.

<p>(<b>A-C</b>) Knockdown of Ush (<i>ush</i>^<i>RNAi</i>) significantly increased lamellocyte differentiation compared to controls (+). <i>dome-Gal4</i> females were crossed to control (+) males or males that carry <i>UAS</i>-<i>ush</i>^<i>RNAi</i> transgene. Arrow marks lamellocytes (lm). (<b>C</b>) Histogram showing that lamellocyte differentiation was significantly increased in <i>dome-Gal4</i> driven <i>UAS-ush</i>^<i>RNAi</i> lymph glands compared to controls. Fisher’s Exact test; P value is as shown; controls (n = 18); <i>ush</i>^<i>RNAi</i> (n = 20). (<b>D-F</b>) Over-expression of Ush in prohemocytes significantly increased the size of the MZ. <i>Tep-Gal4</i> females were crossed to control (+) males or males that carry the <i>UAS</i>-<i>ush</i> transgene. (<b>F</b>) Histogram showing the size of the MZ significantly increased in lymph glands in which Ush was over-expressed (<i>ush</i>^<i>GOF</i>). Student’s t-test; error bars show standard deviation; P values are as shown; control and <i>ush</i>^<i>GOF</i> (n = 11). White dotted lines delineate the entire lymph gland; yellow dotted lines delineate the MZ. Scale bars: (<b>A,B</b>) 100 μm; (<b>D,E</b>) 25 μm. (<b>A-C</b>) Lymph glands from late-third instar larvae; (<b>D-F</b>) lymph glands from mid-third instar larvae.</p

Dorsal has no effect on Srp expression.

<p>(<b>A-G</b>) Prohemocyte-specific expression of <i>UAS</i>-<i>dl</i>^<i>RNAi</i>, <i>UAS</i>-<i>dl</i>, or <i>UAS-cact</i>^<i>RNAi</i> transgenes. (<b>A,B,G</b>) <i>dome-Gal4</i> or (<b>C-G</b>) <i>Tep-Gal4</i> females were crossed to control (+) males or males that carry <i>UAS</i>-<i>dl</i>^<i>RNAi</i>, <i>UAS</i>-<i>dl</i>, or <i>UAS-cact</i>^<i>RNAi</i> transgenes. (<b>A,B</b>) knockdown of Dorsal, (<b>C,D</b>) over-expression of Dorsal or (<b>E,F</b>) knockdown of Cact in prohemocytes has no effect on the level of Srp expression in the lymph glands of mid-third instar larvae. White dotted lines delineate the entire lymph gland. Scale bars: 50 μm. (<b>G</b>) Histogram showing that Srp expression is not significantly different in lymph glands with altered levels of Dorsal or Cact compared to controls. Student’s t-test; for both <i>dome>dl</i>^<i>RNAi</i> and <i>Tep>cact</i>^<i>RNAi</i> and their respective controls (n = 12), control and <i>Tep>dl</i> (n = 13); error bars show standard deviation; P values are as shown.</p

E-cadherin blocks paraquat-induced prohemocyte differentiation.

<p>(<b>A–D</b>) Over-expression of E-cadherin blocks paraquat-induced prohemocyte loss. (<b>A,B</b>) The percentage of Odd-expressing prohemocytes was significantly reduced in lymph glands of paraquat-treated (10 mM) <i>Tep-Gal4</i> heterozygotes (+) compared to untreated (0 mM) <i>Tep-Gal4</i> heterozygotes (+). (<b>C</b>) The percentage of Odd-expressing prohemocytes was significantly increased in paraquat-treated animals with <i>Tep-Gal4</i> driving <i>UAS-E-cadherin</i> (Ecad) compared to (<b>B</b>) treated <i>Tep-Gal4</i> heterozygotes. White dotted lines delineate the entire lymph gland; yellow dotted lines delineate the prohemocyte pool. (<b>D</b>) Histogram showing the percentage of Odd-expressing prohemocytes was significantly greater in untreated <i>Tep-Gal4</i> heterozygotes (+) compared to treated <i>Tep-Gal4</i> heterozygotes (+). In addition, the percentage of Odd-expressing prohemocyte was significantly greater in treated animals with <i>Tep-Gal4</i> driving <i>UAS-E-cadherin</i> (Ecad) compared to treated <i>Tep-Gal4</i> heterozygotes (+). Student's t-test; error bars show standard deviation; P values are as shown; n = 14. (<b>E–G</b>) E-cadherin limits paraquat-induced lamellocyte differentiation. (<b>E,F</b>) The number of lymph gland lobes showing aberrant lamellocyte differentiation was significantly greater in treated <i>Tep-Gal4</i> heterozygotes (+) compared to treated animals with <i>Tep-Gal4</i> driving <i>UAS-E-cadherin</i> (Ecad). White dotted lines delineate the entire lymph gland. (<b>G</b>) Histogram showing that the number of primary lymph gland lobes with aberrant lamellocyte differentiation was significantly greater in <i>Tep-Gal4</i> heterozygotes (+) compared to animals with <i>Tep-Gal4</i> driving <i>UAS-E-cadherin</i> (Ecad). Fisher's Exact test; P value is as shown; +, n = 21; Ecad, n = 23.</p

Ush acts with Cact to limit Dorsal-driven prohemocyte loss and differentiation.

<p>(<b>A-F</b>) Over-expression of Ush rescues Cact knockdown-induced prohemocyte loss and lamellocyte differentiation. <i>Tep-Gal4</i> females were crossed to males that carry (<b>A,D</b>) the <i>UAS-cact</i>^<i>RNAi</i> (<i>cact</i>^<i>RNAi</i>) transgene or (<b>B,E</b>) both the <i>UAS-ush</i> (<i>ush</i>^<i>GOF</i>) and <i>UAS-cact</i>^<i>RNAi</i> transgenes. (<b>B</b>) Co-expression of <i>ush</i> and <i>cact</i>^<i>RNAi</i> transgenes significantly increased the size of the MZ compared to (<b>A</b>) expression of the <i>cact</i>^<i>RNAi</i> transgene alone. (<b>C</b>) Histogram showing the size of the MZ significantly increased in lymph glands with <i>Tep-Gal4</i> driven co-expression of <i>UAS</i>-<i>ush</i> and <i>UAS-cact</i>^<i>RNAi</i> compared to <i>Tep-Gal4</i> driven expression of <i>UAS</i>-<i>cact</i>^<i>RNAi</i> alone. Student’s t-test; error bars show standard deviation; P values are as shown; <i>ush</i> with <i>cact</i>^<i>RNAi</i> and <i>cact</i>^<i>RNAi</i> alone (n = 22). (<b>E</b>) Co-expression of <i>ush</i> and <i>cact</i>^<i>RNAi</i> transgenes significantly reduced lamellocyte differentiation compared to (<b>D</b>) expression of the <i>cact</i>^<i>RNAi</i> transgene alone. (<b>F</b>) Histogram showing the number of lymph gland lobes with aberrant lamellocyte differentiation was significantly greater in lymph glands with <i>Tep-Gal4</i> driven <i>UAS</i>-<i>cact</i>^<i>RNAi</i> compared to <i>Tep-Gal4</i> driven co-expression of <i>UAS</i>-<i>ush</i> and <i>UAS-cact</i>^<i>RNAi</i>. Fisher’s exact test; P values are as shown; <i>cact</i>^<i>RNAi</i> alone (n = 21), <i>ush</i> with <i>cact</i>^<i>RNAi</i> (n = 22). (<b>G-I</b>) The size of the MZ was significantly larger in (<b>H</b>) <i>dl/ush</i> trans-heterozygous lymph glands compared to (<b>G</b>) <i>ush/+</i> lymph glands. (<b>I</b>) Histogram showing the size of the MZ in <i>ush/+</i> and <i>dl/ush</i> lymph glands. Student’s t-test; error bars show standard deviation; P values are as shown; <i>ush/+</i> and <i>dl/ush</i> (n = 18). <b>(J-M)</b> Ush and Cactus act together to block lamellocyte differentiation. (<b>L</b>) Lamellocyte differentiation in <i>cact/ush</i> trans-heterozygous lymph glands was significantly increased compared to either (<b>J</b>) <i>cact/+</i> or (<b>K</b>) <i>ush/+</i> lymph glands. (<b>M</b>) Histogram showing that the number of primary lymph gland lobes with aberrant lamellocyte differentiation was significantly greater in <i>cact/ush</i> trans-heterozygous (n = 30) lymph glands compared to either <i>cact/+</i> (n = 28) or <i>ush/+</i> (n = 26) lymph glands. Chi-square test; P value is as shown. White dotted lines delineate the entire lymph gland; yellow dotted lines delineate the prohemocyte pool. Arrows mark lamellocytes (lm). (<b>A-C,G-I</b>) Mid-third instar larvae; <b>(D-F,J-M</b>) late-third instar larvae. Scale bars: (<b>A-C,G-I</b>) 50 μm; (<b>D-F,J-M</b>) 100 μm.</p