Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Mar 11;6(3):e58.
doi: 10.1371/journal.pbio.0060058.

Postmitotic Specification of Drosophila Insulinergic Neurons From Pioneer Neurons

Affiliations
Free PMC article

Postmitotic Specification of Drosophila Insulinergic Neurons From Pioneer Neurons

Irene Miguel-Aliaga et al. PLoS Biol. .
Free PMC article

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.

Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. dMP2 Pioneer Neurons Become Insulinergic Visceral Neurons That Innervate the Larval and Adult Hindgut Muscles
(A) Co-expression of dMP2-GAL4 and Ilp7 in the four posterior pairs of Ilp7-expressing neurons in an air-filled tracheae (AFT) embryo. dMP2-GAL4 expression in A8/A9 is weak or absent. (B–D) Developmental expression of Ilp7. (B) Ilp7 expression in a single dorsal pair and the four ventral, posterior pairs of dMP2 neurons in a first-instar larva. (C) In a second-instar larval CNS, Ilp7 is also expressed in one lateral neuron per hemisegment in two or three abdominal segments. (D) Robust Ilp7 expression in these three cell types in a third-instar larval CNS. (E) Cartoon summarizing the mature Ilp7 expression pattern in a third-instar larva. Besides the eight posterior dMP2 neurons in A6-A9, a dorsal pair (DP) in A1 and the three lateral pairs in A1–A3 typically express Ilp7 (LATs). Weak Ilp7 immunoreactivity is occasionally observed in a fourth lateral neuron per hemisegment in A4, a more anterior dorsal pair, and a neuron located at base of each brain hemisphere (empty circles). dMP2 axons exit in the posterior nerve, whereas DP processes extend anteriorly towards the pars intercerebralis and terminate near the insulin-producing mNSCs (Figure S2). LAT processes are not always apparent, but they seem to project towards the midline. (F) The Ilp7 larval expression pattern is maintained in the adult abdominal ganglion, although some remodelling appears to occur. (G) dMP2 axons (green) on the larval hindgut (muscles in blue). The nerve endings that contact the hindgut have several varicosities, where Ilp7 (red) release may occur. More complex Ilp7-positive dMP2 fibres innervate the adult hindgut (anterior intestine [H] and rectum [I]), as revealed by the presence of oddGAL4-positive axons (green) in close contact with the adult hindgut muscle (blue). (J) From pioneer neuron to insulin-producing visceral neuron. At the end of embryogenesis, anterior dMP2 neurons undergo apoptosis. The expression of the Hox gene Abd-B in posterior segments prevents this death, and posterior dMP2 neurons exit the VNC and become insulin-producing visceral neurons. Genotypes: (A) dMP2-GAL4/UAS-nmEGFP; (B–D and F) y w; (G) UAS-CD8-GFP/+; dMP2-GAL4/+; and (H and I) oddGAL4,UAS-GFP/UAS-CD8-GFP.
Figure 2
Figure 2. Pioneer Neurons Are Not Required to Maintain the Larval Axonal Tracts
(A) Wild-type expression of dMP2-GAL4 in dMP2 neurons. Expression in A8/A9 is weak or absent. (B) No dMP2 neurons remain at the end of embryogenesis following expression of the cell death activators rpr and hid from dMP2-GAL4. (C) Wild-type expression of oddGAL4 in dMP2 and MP1 pioneer neurons in air-filled tracheae embryos at the end of embryogenesis. (D) Complete absence of dMP2 and MP1 pioneer neurons at the end of embryogenesis following expression of rpr and hid from oddGAL4. (E–G) Fasciclin II staining of larval longitudinal connectives is unaffected by the ablation of dMP2 neurons alone (F) or dMP2 and MP1 neurons (G) in late embryos. Genotypes: (A) dMP2-GAL4/UAS-nmEGFP; (B and F) UAS-hid,UAS-rpr/Y; dMP2-GAL4/UAS-nmEGFP; (C) oddGAL4,UAS-GFP/+; (D and G) UAS-hid,UAS-rpr/Y; oddGAL4,UAS-GFP/+; and (E) w1118.
Figure 3
Figure 3. 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.
Figure 4
Figure 4. hb9 Sequentially Regulates dMP2 Apoptosis and Ilp7 Expression
(A) Hb9 expression in anterior and posterior dMP2 neurons before anterior apoptosis. (B) Hb9 persists in A8 and A9 larval dMP2 neurons, but it is downregulated from A6 and A7 dMP2s. (C) Wild-type, Ilp7-expressing dMP2 neurons in posterior segments in an early L1 CNS. (D) In hb9 mutants, anterior dMP2 neurons fail to undergo apoptosis. Neither posterior dMP2 neurons nor ectopic anterior dMP2s express Ilp7. (E) Postmitotic, cell-autonomous expression of hb9 from stage 16 in dMP2 neurons using dMP2-GAL4 restores Ilp7 expression levels, but not apoptosis. A9 expression is not rescued, as dMP2-GAL4 is rarely expressed in this segment. (F) Expression of hb9 in dMP2 neurons from stage 10 rescues both Ilp7 expression and anterior apoptosis. (G–L and N) Other aspects of dMP2 specification are unaffected in hb9 mutants. Anterior and posterior dMP2 neurons express early markers, such as Odd and Fkh (G and H, respectively). Unusually, Odd expression was weaker in anterior segments and was often absent from the S3, T1, and T2 anterior-most segments at stage 16, even though dMP2-GAL4 and Fkh were present (unpublished data). Posterior dMP2 neurons express Abd-B (I). In late embryos, posterior dMP2 neurons express pMad (J) and Dimm (K), and exit the VNC in the correct nerve (L). Abnormal, lateral projections were only rarely observed (5% of posterior segments, unpublished data). (N) dMP2 axons innervate the hindgut correctly. (M) Quantification of hb9 phenotypes. In the rescue of Ilp7 with dMP2-GAL4, Ilp7 expression was only assessed in GAL4+ dMP2s, as assessed by UAS-CD8-GFP expression, given that dMP2-GAL4 is not always expressed in A8, and it is rarely expressed in A9. For clarity, the total percentage of Ilp7-expressing/dMP2-GAL4-expressing posterior neurons per VNC has been recalculated for a final number of eight neurons, in order to compare it to the wild-type Ilp7 expression. n > 10 VNCs for each marker. Asterisks denote significance (t-test p < 10−5 or less) when hb9 mutants were compared to wild-type CNSs, or when rescued CNSs were compared to hb9 mutants. Genotypes: (A–C) UAS-CD8-GFP/+; dMP2-GAL4/+; (D, G–L, and N) UAS-CD8-GFP; hb9JJ154/dMP2-GAL4,hb9KK30; (E) UAS-CD8-GFP/+; UAS-hb9,hb9 JJ154/dMP2-GAL4,hb9 KK30; and (F) oddGAL4,UAS-GFP; UAS-hb9,hb9 JJ154 /hb9 KK30.
Figure 5
Figure 5. fkh Regulates All Aspects of Late dMP2 Identity
(A) Fkh expression in both anterior and posterior dMP2 neurons precedes apoptosis and Ilp7 expression. (B) Fkh expression persists in larval dMP2 neurons. (C) Wild-type, Ilp7-expressing dMP2 neurons in posterior segments in late embryo. (D) In fkh mutants, almost all anterior dMP2 neurons fail to undergo apoptosis. Neither posterior dMP2 neurons nor these ectopic anterior dMP2s express Ilp7. (E) Cell-autonomous expression of fkh in dMP2 neurons restores anterior apoptosis and posterior Ilp7 expression. (F–K) In fkh mutants, the transition from dMP2 pioneer to Ilp7 neuropeptidergic neuron is stalled. dMP2 neurons express early markers in every segment, such as Odd and Hb9 (F and G). Posterior dMP2 neurons also express Abd-B (H). Later, activation of Mad occurs, but is often delayed (I) and Dimm expression is delayed or absent (arrows). (K) Some late pathfinding defects are apparent, such as premature exit of dMP2 axons laterally. Gut innervation could not be assessed because of the absence of hindgut in fkh mutants [71]. (L) Quantification of observed phenotypes in posterior dMP2 neurons. Left graph: expression of dMP2 markers in posterior dMP2 neurons in mutant and rescued CNSs. Middle graph: Number of segments with pathfinding defects per mutant VNC. Right graph: ectopic anterior survival in mutant and rescued CNSs. Asterisks indicate that mutant numbers are significantly different from wild-type, or that rescue numbers are significantly different from mutant ones (p < 0.001 or less for all t-tests except for midline crossing, where p = 0.05, n > 10 for each genotype). (M and N). Rescue of Dimm expression and pathfinding defects upon cell-autonomous expression of fkh in dMP2 neurons in fkh mutants. Genotypes: (A and B) UAS-CD8-GFP/+; dMP2-GAL4/+; (C) oddGAL4,UAS-GFP/+; (D) oddGAL4,UAS-GFP/+; fkh6/fkh6 ; (E) oddGAL4,UAS-GFP/+; UAS-fkh,fkh6/fkh6; (F–K) UAS-CD8-GFP; CY27-GAL4,fkh6/fkh6; and (M and N) UAS-CD8-GFP; CY27-GAL4,fkh6/UAS-fkh,fkh6. The routinely used dMP2-GAL4 driver is not expressed in fkh mutants, hence the choice of CY27-GAL4 to identify dMP2 neurons up to stage 17 or oddGAL4 in late embryos with air-filled tracheae where CY27-GAL4 expression is no longer restricted to dMP2 neurons.
Figure 6
Figure 6. Abd-B, but Not Other Hox Genes, Is Required for Ilp7 Expression
(A) Hox code in wild-type posterior dMP2 neurons at the onset of Ilp7 expression. While A8 and A9 only express Abd-B, A6 and A7 co-express Abd-A and Abd-B. Ubx expression is occasionally detected in A6. Note no Hox expression in A8 other than Abd-B. (B) Wild-type Ilp7 expression in A6–A9. (C) Hox code of posterior dMP2 neurons in Abd-Bm, rpr, skl mutants: A6 and A7 express Hox genes other than Abd-B (namely Abd-A and, occasionally, Ubx), A8 is a Hox-free dMP2, and A9 expresses Abd-B, like in the wild-type. (D) Ilp7 immunoreactivity in Abd-Bm, rpr, skl mutants is only apparent in A9: the only segment with Abd-B expression. (E–J) Other aspects of dMP2 identity are generally unaffected by the absence of Hox expression. A8 dMP2 neurons express early markers, such as Odd (E), Fkh (F), and Hb9 (G). Expression of pMad and Dimm is occasionally affected (H and I, arrows). No pathfinding defects are apparent (J). (K) Postmitotic, cell-autonomous expression of Abd-B in dMP2 neurons rescues Ilp7 expression in Abd-B, rpr, skl mutants. By contrast, expression of other Hox genes does not rescue (L and M). (N) No Ilp7 rescue by restoring wild-type Dimm and pMad expression in Abd-B, rpr, skl mutants. Genotypes: (A and B) dMP2-GAL4/UAS-nmEGFP; (C and D) UAS-nmEGFP/+; dMP2-GAL4,H99,AbdBM5/XR38,AbdBM5; (E–J) UAS-CD8-GFP/+; dMP2-GAL4,H99,AbdBM5/XR38,AbdBM5; (K) UAS-Abd-B/+; dMP2-GAL4,H99,AbdBM5/XR38,AbdBM5; (L) dMP2-GAL4,H99,AbdBM5/UAS-abd-A,XR38,AbdBM5; (M) UAS-Ubx/+; dMP2-GAL4,H99,AbdBM5/XR38,AbdBM5; and (N) UAS-tkvA,UAS-saxA/+; dMP2-GAL4,H99,AbdBM5/UAS-Dimm,XR38,AbdBM5.
Figure 7
Figure 7. Coexpression of dimm, fkh and Abd-B Triggers Ectopic Ilp7 Activation in a Subset of Motor Neurons
(A–C) Single misexpression of Abd-B (A), Fkh (B) or Dimm (C) in motor neurons has no or very limited effect. (D) Misexpression of Dimm, Fkh and Abd-B in motor neurons leads to strong ectopic Ilp7 activation in one motor neuron per hemisegment in A1–A7 (arrowhead) in a first-instar VNC. Weak Ilp7 activation is occasionally observed in several other motor neurons (arrows). (E) Quantification of Ilp7 activation. The identified Ilp7 regulators act in a combinatorial manner: the triple comisexpression triggers the highest number of Ilp7 ectopic neurons. This number is significantly higher than that of any double co-expression (p < 10−8 when compared to any of them). Co-expression of Dimm, Fkh significantly triggers more ectopic Ilp7 than Dimm (p < 10−6). The ectopic activation obtained with Dimm, Abd-B is not significantly different from that of Dimm alone (p = 0.2). n > 20 for all genotypes. (F) Misexpression of Dimm, Fkh and Abd-B in motor neurons does not trigger ectopic Ilp2 activation. Only endogenous Ilp2 is apparent in the brain mNSCs. Genotypes: (A) OK371-GAL4/UAS-Abd-B; (B) OK371-GAL4/+; UAS-fkh/+; (C) OK371-GAL4/+; UAS-dimm/+; and (D and F) OK371-GAL4/UAS-Abd-B; UAS-dimm,UAS-fkh.
Figure 8
Figure 8. A Multi-Step Transcriptional Programme for Insulinergic Identity
Cartoon summarizing the genes and transitions from the generation of the MP2 neuroblast to the late specification of Ilp7 visceral neurons. Lateral inhibition singles out the MP2 neuroblast, which also requires Vnd for its formation. Asymmetric localization of Numb to dMP2 neurons during MP2 mitosis represses Notch signalling and specifies dMP2 identity. Nerfin-1 is required for pioneer function. The later death/survival decision and expression of Ilp7 require the intrinsic and extrinsic factors identified in this study (gene names in colour). See text for details.

Similar articles

See all similar articles

Cited by 42 articles

See all "Cited by" articles

References

    1. Saltiel AR, Kahn CR. Insulin signalling and the regulation of glucose and lipid metabolism. Nature. 2001;414:799–806. - PubMed
    1. Biddinger SB, Kahn CR. From mice to men: insights into the insulin resistance syndromes. Annu Rev Physiol. 2006;68:123–158. - PubMed
    1. Marshall S. Role of insulin, adipocyte hormones, and nutrient-sensing pathways in regulating fuel metabolism and energy homeostasis: a nutritional perspective of diabetes, obesity, and cancer. Sci STKE. 2006;2006:re7. - PubMed
    1. Murtaugh LC. Pancreas and beta-cell development: from the actual to the possible. Development. 2007;134:427–438. - PubMed
    1. Murtaugh LC, Melton DA. Genes, signals, and lineages in pancreas development. Annu Rev Cell Dev Biol. 2003;19:71–89. - PubMed

Publication types

MeSH terms

Feedback