Limits...
Postmitotic specification of Drosophila insulinergic neurons from pioneer neurons.

Miguel-Aliaga I, Thor S, Gould AP - PLoS Biol. (2008)

Bottom Line: We find that the postmitotic transition from pioneer to insulin-producing neuron is a multistep process requiring retrograde bone morphogenetic protein (BMP) signalling and four transcription factors: Abdominal-B, Hb9, Fork Head, and Dimmed.These five inputs contribute in a partially overlapping manner to combinatorial codes for dMP2 apoptosis, survival, and insulinergic differentiation.These studies reveal striking similarities between the transcription factors regulating insulin expression in insect neurons and mammalian pancreatic beta-cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Developmental Neurobiology, Medical Research Council National Institute for Medical Research, London, United Kingdom. i.miguel-aliaga@zoo.cam

ABSTRACT
Insulin and related peptides play important and conserved functions in growth and metabolism. Although Drosophila has proved useful for the genetic analysis of insulin functions, little is known about the transcription factors and cell lineages involved in insulin production. Within the embryonic central nervous system, the MP2 neuroblast divides once to generate a dMP2 neuron that initially functions as a pioneer, guiding the axons of other later-born embryonic neurons. Later during development, dMP2 neurons in anterior segments undergo apoptosis but their posterior counterparts persist. We show here that surviving posterior dMP2 neurons no longer function in axonal scaffolding but differentiate into neuroendocrine cells that express insulin-like peptide 7 (Ilp7) and innervate the hindgut. We find that the postmitotic transition from pioneer to insulin-producing neuron is a multistep process requiring retrograde bone morphogenetic protein (BMP) signalling and four transcription factors: Abdominal-B, Hb9, Fork Head, and Dimmed. These five inputs contribute in a partially overlapping manner to combinatorial codes for dMP2 apoptosis, survival, and insulinergic differentiation. Ectopic reconstitution of this code is sufficient to activate Ilp7 expression in other postmitotic neurons. These studies reveal striking similarities between the transcription factors regulating insulin expression in insect neurons and mammalian pancreatic beta-cells.

Show MeSH

Related in: MedlinePlus

Retrograde Gbb/Wit Signalling and Dimm Regulate Ilp7 Expression(A) Wild-type embryos at the air-filled tracheae (AFT) stage express Ilp7 (red), Odd (blue), and dMP2-GAL4 (green) in A6 to A9 dMP2 neurons. In gbb mutants (B) or wit mutants (C), Ilp7 expression in dMP2 neurons is absent or reduced.(D) In dimm mutant embryos, Ilp7 expression levels are reduced.(E) Blocking retrograde axonal transport cell-autonomously in dMP2 neurons results in absent or reduced Ilp7 expression. dMP2 neurons are otherwise correctly specified in all these mutants, as revealed by their expression of dMP2-GAL4 and Odd (B–E, insets).(F) Postmitotic, cell-autonomous expression of wit in dMP2 neurons restores Ilp7 expression levels.(G) Acquisition of neuropeptidergic fate precedes Ilp7 expression. After their pioneer function, anterior dMP2 neurons die, whereas posterior dMP2 neurons begin to express dimm, exit the VNC, receive a gbb signal that leads to Mad phosphorylation, thus allowing them to express Ilp7.(H) Quantification of phenotypes. The number of posterior dMP2 neurons expressing Ilp7 in wit, gbb, and oddGAL4/UAS-Glued mutants at the end of embryogenesis is significantly reduced when compared to the wild-type (t-test, p < 0.001, n > 9 for all). Ilp7 is more frequently absent from A6/A7 than from A8/A9 segments in wit, gbb, and dimm mutants, probably because a general reduction in expression appears more pronounced in the weaker-expressing segments. dMP2 postmitotic expression of Wit in wit mutants significantly rescues this phenotype (p < 0.0001, n = 10). In first-instar larvae, Ilp7 intensity levels remain low (p < 0.01, n > 6 for all). dMP2 postmitotic expression of Wit in wit mutants or Dimm in dimm mutants significantly rescues this phenotype (p < 0.0001, n > 6 for all). The intensity index for A6/A7 and A8/A9 was quantified separately because the latter segments always express higher levels of Ilp7.Genotypes: (A) UAS-CD8-GFP/+; dMP2-GAL4/+; (B) gbb1/gbb1; UAS-CD8-GFP/dMP2-GAL4; (C) UAS-CD8-GFP/+; dMP2-GAL4,witA12/witB11; (D) dimmP1/dimmrev4; UAS-CD8-GFP/dMP2-GAL4; (E) oddGAL4,UAS-GFP/UAS-GluedDN; (F) UAS-wit,witB11/dMP2-GAL4, witA12; and (H) as above, plus dimm rescue is dimmrev4/dimmP1; UAS-dimm/dMP2-GAL4.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2265769&req=5

pbio-0060058-g003: Retrograde Gbb/Wit Signalling and Dimm Regulate Ilp7 Expression(A) Wild-type embryos at the air-filled tracheae (AFT) stage express Ilp7 (red), Odd (blue), and dMP2-GAL4 (green) in A6 to A9 dMP2 neurons. In gbb mutants (B) or wit mutants (C), Ilp7 expression in dMP2 neurons is absent or reduced.(D) In dimm mutant embryos, Ilp7 expression levels are reduced.(E) Blocking retrograde axonal transport cell-autonomously in dMP2 neurons results in absent or reduced Ilp7 expression. dMP2 neurons are otherwise correctly specified in all these mutants, as revealed by their expression of dMP2-GAL4 and Odd (B–E, insets).(F) Postmitotic, cell-autonomous expression of wit in dMP2 neurons restores Ilp7 expression levels.(G) Acquisition of neuropeptidergic fate precedes Ilp7 expression. After their pioneer function, anterior dMP2 neurons die, whereas posterior dMP2 neurons begin to express dimm, exit the VNC, receive a gbb signal that leads to Mad phosphorylation, thus allowing them to express Ilp7.(H) Quantification of phenotypes. The number of posterior dMP2 neurons expressing Ilp7 in wit, gbb, and oddGAL4/UAS-Glued mutants at the end of embryogenesis is significantly reduced when compared to the wild-type (t-test, p < 0.001, n > 9 for all). Ilp7 is more frequently absent from A6/A7 than from A8/A9 segments in wit, gbb, and dimm mutants, probably because a general reduction in expression appears more pronounced in the weaker-expressing segments. dMP2 postmitotic expression of Wit in wit mutants significantly rescues this phenotype (p < 0.0001, n = 10). In first-instar larvae, Ilp7 intensity levels remain low (p < 0.01, n > 6 for all). dMP2 postmitotic expression of Wit in wit mutants or Dimm in dimm mutants significantly rescues this phenotype (p < 0.0001, n > 6 for all). The intensity index for A6/A7 and A8/A9 was quantified separately because the latter segments always express higher levels of Ilp7.Genotypes: (A) UAS-CD8-GFP/+; dMP2-GAL4/+; (B) gbb1/gbb1; UAS-CD8-GFP/dMP2-GAL4; (C) UAS-CD8-GFP/+; dMP2-GAL4,witA12/witB11; (D) dimmP1/dimmrev4; UAS-CD8-GFP/dMP2-GAL4; (E) oddGAL4,UAS-GFP/UAS-GluedDN; (F) UAS-wit,witB11/dMP2-GAL4, witA12; and (H) as above, plus dimm rescue is dimmrev4/dimmP1; UAS-dimm/dMP2-GAL4.

Mentions: We then explored whether cell extrinsic factors might regulate the dMP2 insulinergic programme. Expression of the neuropeptide FMRFamide (FMRFa) requires a retrograde signal from Glass-bottom boat (Gbb), a bone morphogenetic protein (BMP) family growth factor [34,35]. This activates the Wishful-thinking (Wit) receptor in neurons, leading to the phosphorylation and consequent activation of Mothers against dpp (Mad). Although pMad activation is an absolute requirement for expressing FMRFa, no other Drosophila neuropeptides or neurotransmitters have yet been shown to be regulated in this manner. As Mad is also activated in posterior dMP2 neurons around the time when Ilp7 is first expressed (Figure 3G) [28], we examined Ilp7 expression in BMP pathway mutants. Reduced or absent Ilp7 expression was observed in gbb mutants (Figure 3B), Mad mutants (Figure S3E and S3F), saxophone (sax) mutants (Figure S4), and upon cell-autonomous interference with retrograde axonal transport using the dominant-negative dynactin, P150/Glued (UAS-GluedDN, Figure 3E). In wit mutants, we observed that Ilp7 expression is consistently reduced or absent in late embryos and early L1 larvae (Figure 3A, 3C, and 3H, p < 0.001). Even by the end of L1, Ilp7 intensity levels remain significantly lower than those of stage-matched wild-type larvae (Figure 3H, p < 0.01). Ilp7 expression is likely to be regulated at the transcriptional level by wit, gbb, and Mad as loss-of-function mutants show altered expression of Ilp7 RNA (Figure S3A–S3F). Cell-autonomous reintroduction of wit in dMP2 neurons rescues Mad activation and Ilp7 expression, thus demonstrating that the insulinergic programme requires BMP signal transduction in a cell-autonomous and postmitotic manner (Figure 3F and 3H, p < 0.001). In contrast to Ilp7 expression, dMP2 early specification and segment-specific survival are not affected in P150/Glued or BMP pathway mutants (Figures 3A–3E,insets, S5, and S6). Hence, retrograde BMP signalling does not provide a general dMP2 specification or survival signal but is required, at late postmitotic stages, to promote Ilp7 expression.


Postmitotic specification of Drosophila insulinergic neurons from pioneer neurons.

Miguel-Aliaga I, Thor S, Gould AP - PLoS Biol. (2008)

Retrograde Gbb/Wit Signalling and Dimm Regulate Ilp7 Expression(A) Wild-type embryos at the air-filled tracheae (AFT) stage express Ilp7 (red), Odd (blue), and dMP2-GAL4 (green) in A6 to A9 dMP2 neurons. In gbb mutants (B) or wit mutants (C), Ilp7 expression in dMP2 neurons is absent or reduced.(D) In dimm mutant embryos, Ilp7 expression levels are reduced.(E) Blocking retrograde axonal transport cell-autonomously in dMP2 neurons results in absent or reduced Ilp7 expression. dMP2 neurons are otherwise correctly specified in all these mutants, as revealed by their expression of dMP2-GAL4 and Odd (B–E, insets).(F) Postmitotic, cell-autonomous expression of wit in dMP2 neurons restores Ilp7 expression levels.(G) Acquisition of neuropeptidergic fate precedes Ilp7 expression. After their pioneer function, anterior dMP2 neurons die, whereas posterior dMP2 neurons begin to express dimm, exit the VNC, receive a gbb signal that leads to Mad phosphorylation, thus allowing them to express Ilp7.(H) Quantification of phenotypes. The number of posterior dMP2 neurons expressing Ilp7 in wit, gbb, and oddGAL4/UAS-Glued mutants at the end of embryogenesis is significantly reduced when compared to the wild-type (t-test, p < 0.001, n > 9 for all). Ilp7 is more frequently absent from A6/A7 than from A8/A9 segments in wit, gbb, and dimm mutants, probably because a general reduction in expression appears more pronounced in the weaker-expressing segments. dMP2 postmitotic expression of Wit in wit mutants significantly rescues this phenotype (p < 0.0001, n = 10). In first-instar larvae, Ilp7 intensity levels remain low (p < 0.01, n > 6 for all). dMP2 postmitotic expression of Wit in wit mutants or Dimm in dimm mutants significantly rescues this phenotype (p < 0.0001, n > 6 for all). The intensity index for A6/A7 and A8/A9 was quantified separately because the latter segments always express higher levels of Ilp7.Genotypes: (A) UAS-CD8-GFP/+; dMP2-GAL4/+; (B) gbb1/gbb1; UAS-CD8-GFP/dMP2-GAL4; (C) UAS-CD8-GFP/+; dMP2-GAL4,witA12/witB11; (D) dimmP1/dimmrev4; UAS-CD8-GFP/dMP2-GAL4; (E) oddGAL4,UAS-GFP/UAS-GluedDN; (F) UAS-wit,witB11/dMP2-GAL4, witA12; and (H) as above, plus dimm rescue is dimmrev4/dimmP1; UAS-dimm/dMP2-GAL4.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2265769&req=5

pbio-0060058-g003: Retrograde Gbb/Wit Signalling and Dimm Regulate Ilp7 Expression(A) Wild-type embryos at the air-filled tracheae (AFT) stage express Ilp7 (red), Odd (blue), and dMP2-GAL4 (green) in A6 to A9 dMP2 neurons. In gbb mutants (B) or wit mutants (C), Ilp7 expression in dMP2 neurons is absent or reduced.(D) In dimm mutant embryos, Ilp7 expression levels are reduced.(E) Blocking retrograde axonal transport cell-autonomously in dMP2 neurons results in absent or reduced Ilp7 expression. dMP2 neurons are otherwise correctly specified in all these mutants, as revealed by their expression of dMP2-GAL4 and Odd (B–E, insets).(F) Postmitotic, cell-autonomous expression of wit in dMP2 neurons restores Ilp7 expression levels.(G) Acquisition of neuropeptidergic fate precedes Ilp7 expression. After their pioneer function, anterior dMP2 neurons die, whereas posterior dMP2 neurons begin to express dimm, exit the VNC, receive a gbb signal that leads to Mad phosphorylation, thus allowing them to express Ilp7.(H) Quantification of phenotypes. The number of posterior dMP2 neurons expressing Ilp7 in wit, gbb, and oddGAL4/UAS-Glued mutants at the end of embryogenesis is significantly reduced when compared to the wild-type (t-test, p < 0.001, n > 9 for all). Ilp7 is more frequently absent from A6/A7 than from A8/A9 segments in wit, gbb, and dimm mutants, probably because a general reduction in expression appears more pronounced in the weaker-expressing segments. dMP2 postmitotic expression of Wit in wit mutants significantly rescues this phenotype (p < 0.0001, n = 10). In first-instar larvae, Ilp7 intensity levels remain low (p < 0.01, n > 6 for all). dMP2 postmitotic expression of Wit in wit mutants or Dimm in dimm mutants significantly rescues this phenotype (p < 0.0001, n > 6 for all). The intensity index for A6/A7 and A8/A9 was quantified separately because the latter segments always express higher levels of Ilp7.Genotypes: (A) UAS-CD8-GFP/+; dMP2-GAL4/+; (B) gbb1/gbb1; UAS-CD8-GFP/dMP2-GAL4; (C) UAS-CD8-GFP/+; dMP2-GAL4,witA12/witB11; (D) dimmP1/dimmrev4; UAS-CD8-GFP/dMP2-GAL4; (E) oddGAL4,UAS-GFP/UAS-GluedDN; (F) UAS-wit,witB11/dMP2-GAL4, witA12; and (H) as above, plus dimm rescue is dimmrev4/dimmP1; UAS-dimm/dMP2-GAL4.
Mentions: We then explored whether cell extrinsic factors might regulate the dMP2 insulinergic programme. Expression of the neuropeptide FMRFamide (FMRFa) requires a retrograde signal from Glass-bottom boat (Gbb), a bone morphogenetic protein (BMP) family growth factor [34,35]. This activates the Wishful-thinking (Wit) receptor in neurons, leading to the phosphorylation and consequent activation of Mothers against dpp (Mad). Although pMad activation is an absolute requirement for expressing FMRFa, no other Drosophila neuropeptides or neurotransmitters have yet been shown to be regulated in this manner. As Mad is also activated in posterior dMP2 neurons around the time when Ilp7 is first expressed (Figure 3G) [28], we examined Ilp7 expression in BMP pathway mutants. Reduced or absent Ilp7 expression was observed in gbb mutants (Figure 3B), Mad mutants (Figure S3E and S3F), saxophone (sax) mutants (Figure S4), and upon cell-autonomous interference with retrograde axonal transport using the dominant-negative dynactin, P150/Glued (UAS-GluedDN, Figure 3E). In wit mutants, we observed that Ilp7 expression is consistently reduced or absent in late embryos and early L1 larvae (Figure 3A, 3C, and 3H, p < 0.001). Even by the end of L1, Ilp7 intensity levels remain significantly lower than those of stage-matched wild-type larvae (Figure 3H, p < 0.01). Ilp7 expression is likely to be regulated at the transcriptional level by wit, gbb, and Mad as loss-of-function mutants show altered expression of Ilp7 RNA (Figure S3A–S3F). Cell-autonomous reintroduction of wit in dMP2 neurons rescues Mad activation and Ilp7 expression, thus demonstrating that the insulinergic programme requires BMP signal transduction in a cell-autonomous and postmitotic manner (Figure 3F and 3H, p < 0.001). In contrast to Ilp7 expression, dMP2 early specification and segment-specific survival are not affected in P150/Glued or BMP pathway mutants (Figures 3A–3E,insets, S5, and S6). Hence, retrograde BMP signalling does not provide a general dMP2 specification or survival signal but is required, at late postmitotic stages, to promote Ilp7 expression.

Bottom Line: We find that the postmitotic transition from pioneer to insulin-producing neuron is a multistep process requiring retrograde bone morphogenetic protein (BMP) signalling and four transcription factors: Abdominal-B, Hb9, Fork Head, and Dimmed.These five inputs contribute in a partially overlapping manner to combinatorial codes for dMP2 apoptosis, survival, and insulinergic differentiation.These studies reveal striking similarities between the transcription factors regulating insulin expression in insect neurons and mammalian pancreatic beta-cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Developmental Neurobiology, Medical Research Council National Institute for Medical Research, London, United Kingdom. i.miguel-aliaga@zoo.cam

ABSTRACT
Insulin and related peptides play important and conserved functions in growth and metabolism. Although Drosophila has proved useful for the genetic analysis of insulin functions, little is known about the transcription factors and cell lineages involved in insulin production. Within the embryonic central nervous system, the MP2 neuroblast divides once to generate a dMP2 neuron that initially functions as a pioneer, guiding the axons of other later-born embryonic neurons. Later during development, dMP2 neurons in anterior segments undergo apoptosis but their posterior counterparts persist. We show here that surviving posterior dMP2 neurons no longer function in axonal scaffolding but differentiate into neuroendocrine cells that express insulin-like peptide 7 (Ilp7) and innervate the hindgut. We find that the postmitotic transition from pioneer to insulin-producing neuron is a multistep process requiring retrograde bone morphogenetic protein (BMP) signalling and four transcription factors: Abdominal-B, Hb9, Fork Head, and Dimmed. These five inputs contribute in a partially overlapping manner to combinatorial codes for dMP2 apoptosis, survival, and insulinergic differentiation. Ectopic reconstitution of this code is sufficient to activate Ilp7 expression in other postmitotic neurons. These studies reveal striking similarities between the transcription factors regulating insulin expression in insect neurons and mammalian pancreatic beta-cells.

Show MeSH
Related in: MedlinePlus