Dual Role of Jun N-Terminal Kinase Activity in Bone Morphogenetic Protein-Mediated Drosophila Ventral Head Development

Abstract

The Drosophila bone morphogenetic protein encoded by decapentaplegic (dpp) controls ventral head morphogenesis by expression in the head primordia, eye-antennal imaginal discs. These are epithelial sacs made of two layers: columnar disc proper cells and squamous cells of the peripodial epithelium. dpp expression related to head formation occurs in the peripodial epithelium; cis-regulatory mutations disrupting this expression display defects in sensory vibrissae, rostral membrane, gena, and maxillary palps. Here we document that disruption of this dpp expression causes apoptosis in peripodial cells and underlying disc proper cells. We further show that peripodial Dpp acts directly on the disc proper, indicating that Dpp must cross the disc lumen to act. We demonstrate that palp defects are mechanistically separable from the other mutant phenotypes; both are affected by the c-Jun N-terminal kinase pathway but in opposite ways. Slight reduction of both Jun N-terminal kinase and Dpp activity in peripodial cells causes stronger vibrissae, rostral membrane, and gena defects than Dpp alone; additionally, strong reduction of Jun N-terminal kinase activity alone causes identical defects. A more severe reduction of dpp results in similar vibrissae, rostral membrane, and gena defects, but also causes mutant maxillary palps. This latter defect is correlated with increased peripodial Jun N-terminal kinase activity and can be caused solely by ectopic activation of Jun N-terminal kinase. We conclude that formation of sensory vibrissae, rostral membrane, and gena tissue in head morphogenesis requires the action of Jun N-terminal kinase in peripodial cells, while excessive Jun N-terminal kinase signaling in these same cells inhibits the formation of maxillary palps.

Keywords: Drosophila; Jun N-terminal kinase (JNK); apoptosis; bone morphogenetic protein (BMP); decapentaplegic (dpp); head morphogenesis.

Figure 1

Quantitation of head capsule mutant phenotypes of a dpp^s-hc allelic series. (A) Fate map of third instar eye-antennal imaginal disc with relevant adult structures arising from the disc proper marked: PAL, maxillary palp; ANT, antenna; GE, gena; VI, vibrissae (Ouweneel 1970; Haynie and Bryant 1986). Expression of dpp^s-hc-LacZ (Stultz et al. 2006), driven by the dpp hc enhancer in overlying PE, is indicated in blue. Left is lateral, and right is medial. (B) Schematic diagram of the adult head, with relevant structures labeled. Areas affected in dpp^s-hc mutants are shaded in blue. (C) Mutant phenotypes displayed by an allelic series of dpp^s-hc mutations: dpp^TgR46.1/dpp^s-hc1 (mild) (n = 168), dpp^s11/dpp^s11 (mild/moderate) (n = 100), Df(2L)DTD2, P20/dpp^s-hc1 (moderate) (n = 76), and Df(2L)DTD2, P20/dpp^Tg46.1 (strong) (n = 20) at 25°. Symbols indicate wt, normal head; v, vibrissae defects including reduction in eye size, rostral membrane, and gena tissues (2v = symmetric, 1v = asymmetric); and p, maxillary palp loss (1p = loss of one palp, 0p = loss of both palps). (D–H) Adult fly heads from (D) wild type and (E–H) dpp^s-hc mutations (E) dpp^TgR46.1/dpp^s-hc1, (F) dpp^s11/dpp^s11, (G) Df(2L)DTD2, P20/dpp^s-hc1, and (H) Df(2L)DTD2, P20/dpp^Tg46.1 at 25°. Solid arrow indicates wild-type vibrissae, and solid arrowhead indicates a wild-type palp. Open arrows indicate mutant vibrissae, and open arrowheads indicate missing palps.

Figure 2

Inhibition of apoptosis suppresses the vibrissae defect. dpp^TgR46.1/dpp^s-hc1 produces asymmetrical and symmetrical vibrissae defects (n = 157). This phenotype can be suppressed by the provision of P35 driven by dpp^s-hc-Gal4(w) at 29° (n = 118). *P = 5.3 × 10⁻¹⁴. dpp^RNAi driven by dpp^s-hc-Gal4(s) at 25° produces a high percentage of asymmetrical and symmetrical vibrissae defects (n = 54), but the simultaneous provision of P35 produces rescue of this phenotype (n = 28). **P = 4.8 × 10⁻¹¹.

Figure 3

The effect of peripodial Dpp expression on the disc proper is direct and not through a secondary signal. Constitutively activated receptor protein (tkv^QD) was expressed in the lateral peripodial region with dpp^s-hc-Gal4(w) at 29° in a Df(2L)DTD2, P20/dpp^s-hc1 mutant background. UAS-dpp was expressed in parallel, as a positive control. dpp^s-hc-Gal4 > Dpp rescued all aspects of the head capsule mutant phenotype (both vibrissae and palp, which were not scored separately in this experiment) almost completely (98% rescue) (n = 216), while dpp^s-hc-Gal4 > tkv^QD failed to rescue head capsule mutant phenotypes (n = 99).

Figure 4

JNK activity exerts a positive effect on ventral head development. The vibrissae defect is rescued by increasing JNK activity in backgrounds with reduced peripodial Dpp, while decreasing JNK activity in wild-type flies phenocopies the vibrissae defect. (A) dpp^RNAi driven by the dpp^s-hc-Gal4(m) driver at 29° produces strongly penetrant vibrissae defects (n = 73). Simultaneously expressing Hep (n = 29) or Bsk (n = 38) in this background rescues these vibrissae defects. *P = 3.4 × 10⁻¹⁸, **P = 8.6 × 10⁻²⁰, respectively. (B) Targeted expression of Bsk^DN (n = 80), Rho1^DN/Bsk^DN (n = 11), slpr^RNAi/Bsk^DN (n = 38), Dfos^bzip/Bsk^DN (n = 6), or Puc/Bsk^DN (n = 10) by dpp^s-hc-Gal4(s) at 29° produces vibrissae defects even in the presence of normal levels of BMP.

Figure 5

Loss of dpp expression induces JNK activity. (A and B) JNK activity is detected by puc^E69-lacZ expression on the lateral side of early, mid-, and late third larval instar eye-antennal discs in Df(2L)DTD2, P20/dpp^TgR46.1: puc^E69/+ (B), but not in wild-type discs from the same stages (A). Lateral peripodial expression is indicated by black arrows. (C) Detection of puc-lacZ expression by anti-β-galactosidase and cell death by an antibody to activated Caspase-3 in the same genotype as in B. Note that puc-lacZ expression does not greatly colocalize with apoptotic cells. Lateral peripodial expression is indicated by a white arrow. Box indicates some colocalization of puc-lacZ and Caspase-3 expression in the DP.

Figure 6

(A–D) Induction of the JNK pathway in the lateral PE is correlated with loss of maxillary palps in dpp^s-hc mutations. Shown is expression of puc^E69-lacZ (red) and activated Caspase-3 (green) in third instar larval eye-antennal discs in (A) puc^E69/+, (B) dpp^s11/dpp^s11 ; puc^E69/+, (C) Df(2L)DTD2, P20/dpp^s-hc1 ; puc^E69/+, and (D) Df(2L)DTD2, P20/dpp^TgR46.1; puc^E69/+. Genotypes in C and D are missing palps, while that in B has fully penetrant vibrissae defects, but normal palps (see also Figure 1). The red channel is shown alone in A′–D′. (E–J) Peripodial cells do not express cell death markers to the same extent as disc proper cells. Expression of puc^E69-lacZ in GFP⁻ tkv⁸ LOF clones in the DP (E) and the PE (H) shows increased JNK activity. puc^E69-lacZ is shown in red and GFP in green in these merged images. The red channel is shown alone in E′ and H′. Some expression of puc^E69-lacZ seen outside a clone is wild-type peripodial JNK activity as seen on the medial side of third instar discs (see Figure 5A). Activated Caspase-3 detected by antibody in GFP⁻ tkv⁸ LOF clones in the DP (F) and the PE (I) and reaper expression detected via rpr11-lacZ in the DP (G) and the PE (J) are shown. Antibody to Caspase-3 is shown in magenta, rpr11-lacZ is red, and GFP is green. Both are poorly expressed in PE tkv LOF clones compared to clones induced in the DP from the same experiment. Magenta and red channels are shown alone in F′, I′, G′, and J′. (K and L) Cell death is observed in the lateral PE when dpp^RNAi and the anti-apoptotic gene p35 are coexpressed. (K) Few Caspase-3-positive cells (red) are seen in dpp^s-hc-Gal4(s)>dpp^RNAi (at 25°), while (L) a significant amount of Caspase-3-positive cells are seen in the lateral PE in dpp^s-hc-Gal4(s)>dpp^RNAi ; P35 (at 25°).

Figure 7

JNK activity exerts a negative effect on maxillary palp development. Modulation of JNK activity influences the palp defect caused by loss of peripodial Dpp. (A) Overexpression of Slpr (JNKKK) with the eyeless-Gal4 driver (n = 53) at 25° in a Df(2L)DTD2, P20/dpp^s-hc1 mutant background strongly enhances the palp defect over that observed in the control (n = 84), while the overexpression of Puc, a negative regulator of JNK, can partially rescue the palp defect (n = 84). *P = 4.6 × 10⁻¹⁵, **P = 0.001, respectively. (B) dpp^RNAi driven by the dpp^s-hc-Gal4(m) driver at 29° produces penetrant palp defects (n = 73). Simultaneously expressing Bsk (n = 38) in this background enhances these palp defects. ^‡P = 0.0011. Note that this is the identical experiment to that described in Figure 4, except that the palp defect is being monitored. (C) dpp^RNAi driven by dpp^s-hc-Gal4(s) at 25° produced adult flies with highly penetrant palp loss (n = 80). Simultaneous expression of hep^RNAi (n = 69) or Bsk^DN (n = 48) in this genotype resulted in partial, statistically significant rescue of the palp defect. ^§P = 1.5 × 10⁻⁶, ^§§P = 0.00055, respectively.

Figure 8

Increased JNK activity in the lateral PE causes palp loss and apoptosis even in the presence of normal BMP. (A) Slpr, the combined expression of Bsk and Slpr, or Egr driven by dpp^s-hc-Gal4(s) at 29°. The combined expression of Bsk and Slpr (n = 37) or Egr alone (n =40) caused significant palp loss over that seen in the control (dpp^s-hc-Gal4 with no UAS construct) (n = 50). *P = 6.5 × 10⁻¹⁰, **P = 4.5 × 10⁻²⁹, and ***P = 4.9 × 10⁻⁴¹. (B and C) Third instar eye-antennal discs from dpp^s-hc-Gal4(s)>Bsk:Slpr at 29° (B) and dpp^s-hc-Gal4(s)>Eiger at 29° (C) showed lateral PE expression of puc-lacZ (red) and antibody to activated Caspase-3 (green). (D) An adult head from dpp^s-hc-Gal4(s)>Eiger at 29° with no maxillary palps. Arrowheads indicate the position of missing palps (see Figure 1D).

Figure 9

Coordination of Dpp and JNK in the formation of the ventral head. (A) Expression from dpp^s-hc-GFP in a pupal head 27 hr (25°) after the formation of white prepupae. (B) Schematic diagram of the adult head, showing location of the expression from the dpp hc enhancer seen at the pupal stage. Expression of dpp^s-hc-GFP is indicated in green. (C) Schematic diagram modeling Dpp signaling between the PE and the DP in the eye-antennal disc. Dpp expression is in blue, and JNK activity is in green. Black text in the DP indicates position of mapped primordia (PAL, maxillary palp; GE, gena; VI, vibrissae), and red text indicates the layer where cell death is observed. (D) Chart outlining the relationship between Dpp signaling activity and JNK signaling activity in cells of the lateral peripodial epithelium. Mutant phenotypes observed are indicated by + or − at the bottom of the diagram. Scales are relative and absolute amounts are not implied.