Cut, via CrebA, transcriptionally regulates the COPII secretory pathway to direct dendrite development in Drosophila

Abstract

Dendrite development is crucial in the formation of functional neural networks. Recent studies have provided insights into the involvement of secretory transport in dendritogenesis, raising the question of how the secretory pathway is controlled to direct dendritic elaboration. Here, we identify a functional link between transcriptional regulatory programs and the COPII secretory machinery in driving dendrite morphogenesis in Drosophila dendritic arborization (da) sensory neurons. MARCM analyses and gain-of-function studies reveal cell-autonomous requirements for the COPII coat protein Sec31 in mediating da neuron dendritic homeostasis. We demonstrate that the homeodomain protein Cut transcriptionally regulates Sec31 in addition to other components of COPII secretory transport, to promote dendrite elaboration, accompanied by increased satellite secretory endoplasmic reticulum (ER) and Golgi outposts primarily localized to dendritic branch points. We further establish a novel functional role for the transcription factor CrebA in regulating dendrite development and show that Cut initiates a gene expression cascade through CrebA that coordinately affects the COPII machinery to mediate dendritic morphology.

Keywords: COPII secretory pathway; Dendrite; Transcriptional regulation.

Fig. 1.

sec31 cell-autonomously promotes dendritic branching and growth. (A–H) Wild-type (WT; A,C,E,G) and sec31⁰⁸⁰⁵ mutant (B,D,F,H) third instar larval MARCM clones. (A,B) Class IV ddaC clones; (C,D) class III v'pda clones. (E,F) class II ddaB clones. Scale bars: 50 µm. (A′–F′) Tracings of representative phenotypic regions outlined by dashed boxes in A–F. (G,H) Statistical analyses of the number of dendritic termini and total length, respectively. WT class IV, n = 8; class III, n = 26; class II, n = 18; class I, n = 16; sec31 class IV, n = 9; class III, n = 20; class II, n = 14; class I, n = 16. (I) WT class I vpda neuron labeled by Gal4²²¹,UAS-mCD8::GFP. (J) Sec31 overexpression leads to increased dendritic branching complexity. (K) WT class IV ddaC neuron labeled by Gal4⁴⁷⁷,UAS-mCD8::GFP. (L) Sec31 overexpression leads to an overall reduction in dendritic arbor complexity characterized by decreases in coverage and higher order branching. (M–P) Statistical analyses of the number of dendritic termini (M,O) and total length (N,P) in class I or IV neurons, expressing UAS-sec31 as compared with WT (dark blue bars). n = 8 for all cell types. (Q) Branch order distribution reveals Sec31 overexpression produces a proximal shift towards lower branch orders relative to WT cells. Quantitative data are expressed as means ± s.d., and pairwise comparisons were performed using Student's t-test; *P≤0.05, ***P≤0.001, compared with WT. Scale bars: 100 µm.

Fig. 2.

Sec31 is a target of Cut and is required for Cut-mediated dendritic complexity. (A) WT vpda neuron. (B) Cut ectopic overexpression leads to a dramatic increase in vpda dendritic branching complexity. (C) vpda overexpressing Cut with simultaneous sec31 RNAi knockdown. (D) vpda neuron showing enhancement of the Cut GOF phenotype as a result of simultaneous overexpression of Sec31. (A′–D′) Traces of representative phenotypic regions, indicated by the dashed boxes in A–D. (E,F) Statistical analyses of the number of dendritic terminals and total length, respectively, in the experimental conditions as compared with Cut GOF. (G) qRT-PCR results (n = 4) reveal that Cut overexpression in class I neurons results in significant upregulation of both sec31 and cut relative to controls. (H,H′) GAL4²²¹,UAS-mCD8::GFP third instar larval filets double labeled with HRP and Sec31 antibodies reveal Sec31 expression in class I/III da neurons. (I,I′) UAS-cut;GAL4²²¹,UAS-mCD8::GFP filets double labeled with HRP and Sec31 antibodies reveal specific upregulation of Sec31 in class I ddaD neurons. (J) Statistical analysis of Sec31 fluorescence intensity reveals a 20–25% increase in class I neurons overexpressing Cut relative to WT. (K) Statistical analysis of Sec31 fluorescence intensity in ddaF class III neurons expressing cut^RNAi reveals a ∼35% decrease in expression levels relative to WT, as normalized to intensity levels in ddaC class IV neurons. (L) qRT-PCR results (n = 4) reveal that Cut overexpression in class IV neurons results in significant upregulation of both sec31 and cut relative to controls. In this and all subsequent figures for qRT-PCR studies, WT controls values are indicated by the dashed red line and normalized to GAPDH2 and RpL32. The number of neurons of each genotype is given on the bars in E–G, J–L. Pairwise statistical comparisons were performed using Student's t-test. Quantitative data are expressed as means ± s.d.; **P≤0.01, ***P≤0.001. Scale bars: 100 µm.

Fig. 3.

The COPII secretory pathway functions downstream of Cut to mediate dendritic complexity. (A) A WT vpda neuron. (B) A vpda neuron overexpressing Cut. (C–F) Images of vpda neurons overexpressing Cut with simultaneous RNAi knockdown of sec23 (C), sec24 (D), sec13 (E) and Sar1 (F). (A′–F′) Traces of representative phenotypic regions, indicated by the dashed boxes in A–F. (G,H) Statistical analyses of the number of dendritic termini and total length, respectively, in experimental conditions compared with Cut GOF. (I) qRT-PCR results (n = 4) reveal that Cut overexpression in class I neurons results in increased expression of the COPII transport machinery relative to WT. The number of neurons of each genotype is given on the bars in G–I. Pairwise statistical comparisons were performed using Student's t-test. Quantitative data are expressed as means ± s.d.; *P≤0.05, **P≤0.01, ***P≤0.001. Scale bars: 100 µm.

Fig. 4.

CrebA is regulated by Cut and functions as a downstream effector of Cut-driven dendritogenesis. (A) A WT vpda neuron. (B) A vpda neuron overexpressing Cut. (C) A vpda neuron showing suppression of the Cut GOF phenotype due to simultaneous CrebA RNAi knockdown. (D) CrebA overexpression in a Cut GOF genetic background enhances the dendritic branching phenotype. (A′–D′) Traces of representative phenotypic regions indicated by the dashed boxes in A–D. (E,F) Statistical analyses of the number of dendritic terminals and total length, respectively, in the experimental conditions as compared with Cut GOF controls. (G) WT ddaF neuron. (H) ddaF neuron expressing cut^RNAi. (I) ddaF neuron expressing UAS-cut^RNAi and UAS-CrebA. (J) qRT-PCR results (n = 4) reveal that Cut overexpression in class I neurons results in significant increases in CrebA, fkh and cut expression relative to the levels in WT. (K) Statistical analysis of CrebA fluorescence intensity in ddaF class III neurons expressing cut^RNAi reveals an ∼35% decrease in expression levels relative to WT levels, normalized to intensity levels in ddaC class IV neurons. (L) Statistical analysis of the number of dendritic terminals of experimental neurons compared with UAS-cut^RNAi expressing neurons. (M) qRT-PCR results (n = 4) reveal that Cut overexpression in class IV neurons results in significant upregulation of CrebA and cut relative to WT levels. (N) Statistical analysis of CrebA fluorescence intensity reveals a 20–25% increase in expression levels in class I ddaD and ddaE neurons overexpressing Cut relative to WT levels. (O,O′) nompC-GAL4 filets double labeled with HRP and CrebA antibodies reveal CrebA expression in class III and IV da neurons. (P,P′) nompC-GAL4 >UAS-cut^RNAi filets double labeled with HRP and CrebA antibodies reveal specific downregulation of CrebA expression in class III ddaF neurons. The number of neurons of each genotype is given on the bars in E,F,J–N. Pairwise statistical comparisons were performed using Student's t-test. Quantitative data are expressed as means ± s.d. **P≤0.01, ***P≤0.001; n.s., not significant. Scale bars: 100 µm.

Fig. 5.

CrebA promotes higher order dendritic branching complexity. (A) A WT class III v'pda neuron. (B) A v'pda expressing UAS-CrebA^RNAi displays decreased dendritic branching complexity. (C) A WT class IV ddaC neuron. (D) A ddaC neuron expressing UAS-CrebA^RNAi is characterized by an overall reduction in dendritic arbor complexity with reduced coverage and higher order branching, compared with control neurons. (A′–D′) Traces of representative phenotypic regions indicated by the dashed boxes in A–D. (E,F) Statistical analyses of the number of dendritic termini and total length, respectively, of class III/IV neurons of CrebA^RNAi compared with WT neurons. (G) WT vpda neuron. (H) CrebA overexpression results in increased dendritic branching complexity as compared with WT. (I) WT ddaC neuron. (J) CrebA overexpression leads to an overall reduction in dendritic arbor complexity with reduced dendritic coverage and terminal branching as compared with WT. (K,L) Statistical analyses of number of dendritic termini and total length, respectively, in class I and IV da neurons expressing UAS-CrebA as compared with WT. (M–O) GAL4²¹⁷,UAS-mCD8::GFP filet double labeled with HRP (N) and CrebA (O) antibodies showing nuclear and cytoplasmic CrebA expression. White arrows indicate nuclear staining. (P–R) Dendritic processes double labeled (P), with HRP (Q) and CrebA (R) reveal CrebA puncta along dendrites. (S–U) CrebA⁰³⁷⁵⁶ mutant filet double labeled (S) with HRP (T) and CrebA (U) reveals no detectable CrebA expression. The number of neurons of each genotype is given on the bars in E,F,K,L. Pairwise statistical comparisons were performed using Student's t-test. Quantitative data are expressed as means ± s.d.; *P≤0.05, **P≤0.01, ***P≤0.001. Scale bars: 100 µm.

Fig. 6.

CrebA mediates dendrite morphogenesis through regulation of COPII component genes. (A) A WT vpda neuron. (B) CrebA overexpression. (C–G) Representative images of vpda neurons showing suppression of the CrebA GOF phenotype due to simultaneous RNAi knockdown of sec31 (C), sec13 (D), sec23 (E), sec24 (F) or Sar1 (G). (H) Co-overexpression of Sec31 and CrebA enhances dendritic branching complexity. (A′–H′) Traces of representative phenotypic regions indicated by the dashed boxes in A–H. (I,J) Statistical analyses of the number of dendritic terminals and total length, respectively, in the experimental conditions as compared with CrebA GOF control. (K) qRT-PCR (n = 6) of class I neurons overexpressing Cut with simultaneous knockdown of CrebA show significant percentage reductions in expression levels of all COPII genes as compared with class I neurons expressing Cut alone as control. The CrebA levels are also reduced, confirming the efficacy of CrebA^RNAi. (L) qRT-PCR (n = 4) reveals that CrebA overexpression in da neurons results in significant increases in COPII secretory pathway component expression relative to WT. (M) Statistical analyses of number of dendritic terminals in the experimental conditions as compared with CrebA^RNAi. The number of neurons of each genotype is given on the bars in I–M. Pairwise statistical comparisons were performed using Student's t-test. Quantitative data are expressed as means ± s.d.; *P≤0.05, **P≤0.01, ***P≤0.001.

Fig. 7.

Cut-mediated dendritic complexity is accompanied by an increased number of satellite secretory outposts localized to branch points. (A–C) WT and (D–F) Cut overexpressing vpda dendritic arbors labeled with UAS-mCD8::RFP to mark the membrane (C,F) and UAS-Sten-GFP to mark dendritic ER exit sites (B,E). (A,D) Merged images indicate punctate localization of dendritic ER exit sites at branch points and along the dendrite (arrowheads in A). Note the increase in the number of dendritic ER exit sites that also coincide with branch points in the Cut overexpression background (arrowheads in D). (G–I) WT and (J–L) Cut ectopic overexpressing vpda dendritic arbors labeled with UAS-mCD8::RFP to mark the membrane (I,L) and UAS-ManII-eGFP to mark dendritic Golgi outposts (H,K). (G,J) Merged images indicate punctate localization of dendritic Golgi outposts at branch points and along the dendrite (arrowheads in G). As with ER exit sites, Golgi outpost puncta are highly coincident with branch points (arrowheads in J). (M) Statistical analysis of the number of dendritic ER exit sites reveals a strong increase in class I neurons overexpressing Cut, relative to WT. (N,O) Percentage colocalization of satellite secretory outposts with branch points in Cut-overexpressing and WT class I neurons. Greater than 90% of ER (N) and Golgi outposts (O) were detected at branch points in both WT and class I neurons expressing Cut. The number of ER outposts analyzed is given on the bars in M–O. Pairwise statistical comparisons were performed using Student's t-test. Quantitative data are expressed as means ± s.d.; ***P≤0.001.