Germ line differentiation factor Bag of Marbles is a regulator of hematopoietic progenitor maintenance during Drosophila hematopoiesis

Abstract

Bag of Marbles (Bam) is a stem cell differentiation factor in the Drosophila germ line. Here, we demonstrate that Bam has a crucial function in the lymph gland, the tissue that orchestrates the second phase of Drosophila hematopoiesis. In bam mutant larvae, depletion of hematopoietic progenitors is observed, coupled with prodigious production of differentiated hemocytes. Conversely, forced expression of Bam in the lymph gland results in expansion of prohemocytes and substantial reduction of differentiated blood cells. These findings identify Bam as a regulatory protein that promotes blood cell precursor maintenance and prevents hemocyte differentiation during larval hematopoiesis. Cell-specific knockdown of bam function via RNAi expression revealed that Bam activity is required cell-autonomously in hematopoietic progenitors for their maintenance. microRNA-7 (mir-7) mutant lymph glands present with phenotypes identical to those seen in bam-null animals and mutants double-heterozygous for bam and mir-7 reveal that the two cooperate to maintain the hematopoietic progenitor population. By contrast, analysis of yan mutant lymph glands revealed that this transcriptional regulator promotes blood cell differentiation and the loss of prohemocyte maintenance. Expression of Bam or mir-7 in hematopoietic progenitors leads to a reduction of Yan protein. Together, these results demonstrate that Bam and mir-7 antagonize the differentiation-promoting function of Yan to maintain the stem-like hematopoietic progenitor state during hematopoiesis.

Fig. 1.

Bam expression and function during hematopoiesis. (A) Immunostaining of wild-type (wt) Drosophila lymph glands with an anti-Bam antibody. (B) dome-lacZ serves as a marker of prohemocytes in wild-type lymph glands. (C) Reduction of dome-lacZ-positive prohemocytes in lymph glands from bam^Δ86 larvae. (D) P1 antibody detects plasmatocytes in lymph glands from wild-type larvae. (E) Increased numbers of plasmatocytes in lymph glands dissected from bam^Δ86 larvae. (F) Expression of BcF6-GFP marks crystal cells (present), whereas activity of MSNF9-mCherry marks lamellocytes (absent) in wild-type lymph glands. (G) Increased crystal cells and induced lamellocytes in lymph glands from bam^Δ86 larvae. (H) Immunostaining with P1 antibody and domeGal4-driven UAS-2XEYFP expression in control lymph glands. (I) Forced expression of the UAS-bamGFP transgene by domeGal4 results in expansion of the medullary zone and reduction of plasmatocytes. CZ, cortical zone; MZ, medullary zone.

Fig. 2.

Bam functions cell-autonomously in hematopoietic progenitors. (A) TepIVGal4-driven UAS-mCD8::GFP marks blood cell precursors whereas P1 marks plasmatocytes within a wild-type Drosophila lymph gland. (B,C) Loss of hematopoietic progenitors and increase in plasmatocytes due to UAS-bam RNAi expression. (D) Control lymph gland stained for prohemocytes and Antp-expressing PSC cells. (E,F) Hematopoietic progenitors are decreased owing to UAS-bam RNAi expression. CZ, cortical zone; MZ, medullary zone; PSC, posterior signaling center.

Fig. 3.

mir-7 expression and function during hematopoiesis. (A) mir-7 enhancer-GFP transgene is expressed in the medullary zone and PSC cells in control Drosophila lymph glands. (B) mir-7 mutant lymph glands possess a diminished medullary zone as marked by dome-lacZ. (C) mir-7 mutant lymph glands present with an overproduction of plasmatocytes (marked by P1) and lamellocytes (marked by MSNF9-mCherry). (D) Immunostaining with P1 antibody and expression pattern of TepIVGal4-driven GFP in a control lymph gland. (E) Forced expression of mir-7 in hematopoietic progenitors results in medullary zone expansion and plasmatocyte reduction. (E′) Expanded pattern of Bam protein accumulation in a mir-7 gain-of-function lymph gland. (F) Double-heterozygous combination of mir-7^Δ1 and bam^Δ86 alleles results in a lymph gland devoid of prohemocytes. MZ, medullary zone; PSC, posterior signaling center.

Fig. 4.

Yan expression and function during hematopoiesis. (A) Immunostaining of wild-type lymph glands with an anti-Yan antibody. Yan-positive (arrow) and Yan-negative (arrowhead) cells are indicated. (B,B′) Yan-positive cells do not express a quiescent hematopoietic progenitor marker. (C,C′) Yan-positive cells do not express a plasmatocyte marker. (D) Overproduction of prohemocytes in lymph glands from yan¹ mutant larvae. (E) Lymph gland expression of domeGal4>UAS-mCD8::GFP and immunostaining with P1 antibody (which marks plasmatocytes) in a control genetic background. (F) Forced expression of yan results in a loss of prohemocytes and increase of plasmatocytes in lymph glands. (G) In mir-7^Δ1 lymph glands, an increase in Yan protein level is observed in a dispersed population of progenitors. (H) Forced expression of mir-7 in medullary zone cells results in reduced Yan expression. (I) In bam^Δ86 lymph glands, an increase in Yan protein level is observed in a dispersed population of progenitors. (J,K) Forced expression of a bamGFP fusion gene results in medullary zone expansion and a loss of Yan-expressing cells. (L) Model for the regulatory interactions between Bam, mir-7 and Yan during lymph gland hematopoiesis.