Epithelia-derived wingless regulates dendrite directional growth of drosophila ddaE neuron through the Fz-Fmi-Dsh-Rac1 pathway

Abstract

Background: Proper dendrite patterning is critical for the receiving and processing of information in the nervous system. Cell-autonomous molecules have been extensively studied in dendrite morphogenesis; however, the regulatory mechanisms of environmental factors in dendrite growth remain to be elucidated.

Results: By evaluating the angle between two primary dendrites (PD-Angle), we found that the directional growth of the primary dendrites of a Drosophila periphery sensory neuron ddaE is regulated by the morphogen molecule Wingless (Wg). During the early stage of dendrite growth, Wg is expressed in a group of epithelial cells posteriorly adjacent to ddaE. When Wg expression is reduced or shifted anteriorly, the PD-Angle is markedly decreased. Furthermore, Wg receptor Frizzled functions together with Flamingo and Dishevelled in transducing the Wg signal into ddaE neuron, and the downstream signal is mediated by non-canonical Wnt pathway through Rac1.

Conclusions: In conclusion, we reveal that epithelia-derived Wg plays a repulsive role in regulating the directional growth of dendrites through the non-canonical Wnt pathway. Thus, our findings provide strong in vivo evidence on how environmental signals serve as spatial cues for dendrite patterning.

Keywords: Dendrite directional growth; Epithelia-derived Wingless; Flamingo; Frizzled; non-canonical Wnt pathway.

Fig. 1

Directional growth of primary dendrites in ddaE neuron is deficient in wg ^l-12 mutant. a Wg is expressed at the posterior side of the dorsal cluster of da neurons in whole mount fly embryos. Wg expression pattern (green) is monitored by UAS- mCD8-eGFP under the control of wg-Gal4. The da neurons (red) are stained by antibody 22C10. White brackets indicate the regions of thorax and abdomen. a ’, Enlarged view of AS4-6 (white square in a). White broken lines 1–3 indicate the positions of X-Z or Y-Z section images 1–3, respectively. Red broken lines indicate the ddaE neurons and green broken lines indicate the Wg-expression cells. b Pseudo-color image shows that Wg-expressing cells are localized mainly in the dorsal-posterior region relative to ddaE neuron in w ¹¹¹⁸ AS. c The initial 20 μm parts of primary dendrites shift towards AP axis in wg ^l-12 mutants. Left panels show the trajectory of primary dendrites of ddaE in the 3^rd instar larvae. Red circles indicate the circle around the soma with a radius of 20 μm. The enlarged view of the intersections of the circle and the trajectory of primary dendrites are shown in right panels. Blue dots and red dots indicate the dorsal intersections and ventral ones, respectively. The Blue arrows and red arrows indicate the average direction of dorsal and ventral primary dendrites, respectively. A, P, D and V indicates the direction of anterior, posterior, dorsal and ventral, respectively. (n = 24–50). d-e PD-Angle significantly decreased in wg ^l-12 mutants. d Representative images of ddaE neurons in wild type w ¹¹¹⁸ and wg ^l-12 mutant at third instar larvae stage. Gal4^2–21, UAS-mCD8-eGFP was used to visualize the entire neuron. e Quantification statistic of PD-Angle in wild type w ¹¹¹⁸ and wg ^l-12 mutant. (n = 24–50). f-g The PD-Angle is decreased in wg ^l-12 mutants at embryonic stage 17. (n = 35-57). Scale bars, 50 μm in (a and d) and 20 μm in (a ’ and f). In all figures, representative images are shown in anterior left and dorsal up; white broken rectangles indicate the position of the inset in the upper-right corner, and red arrows indicate the initial growth direction of the two primary dendrites. *p < 0.05, **p < 0.01, ***p < 0.001, and N.S. indicates no significant changes

Fig. 2

Down regulation of environmental Wg induces the decrease of PD-Angle. a-b At embryonic stage 14, wg RNA is detected by FISH. Normalized to the segment width, both wg RNA distribution and the distance between the center of wg signal and posterior segmental boundary are comparable to wild type control. White arrow heads in (a) indicate the segment boundaries. The insert cartoons in (b) represent the expressing region of Wg with red oval, the segment region with yellow rectangle, and the parameters quantified in corresponding panels. (n = 15–30). c - d Wg expression level is significantly decreased in wg ^l-12 embryos, while its center-to-boundary distance is not changed after normalized to the segment boundary. (n = 28–32). e-f Knockdown of wg, wntless or vps26 in epithelial cells by wg-Gal4 results in the decreased PD-Angle. (n = 22–49). Scale bars, 10μm in (a) and (c) and 25μm in (c)

Fig. 3

Anterior-shifted Wg expression results in a reduced PD-Angle. a-b The PD-Angle is significantly decreased in TS and wg ^spd-fg in both late embryos and third instar larvae. (n =26- 57). c-d wg expression region is significantly wider in both wild type TS2-3 and wg ^spd-fg AS4-6, when compared to wild type AS4-6. The distance between the center of wg signal and posterior segmental boundary is also significantly increased when normalized to segment width. (n = 20–29). e-f Wg expression is markedly increased in TS2-3 of wild-type (w ¹¹¹⁸) and in AS4-6 of wg ^spd-fg mutants, and the center of Wg signal is also significantly anteriorly shifted. (n = 23-36). g Schematic diagram of Wg expression pattern and dendrite orientation. The ddaE neuron is located at the anterior side and adjacent to Wg-expressing cells, and the two primary dendrites are orientated towards the posterior when Wg expression is reduced (as in wg ^l-12 and wg-Gal4 > wg ^RNAi), or when Wg is ectopically expressed in the anterior region (as in wg ^spd-fg and wild-type TS). Scale bar: 50 μm in (a) and 10 μm in (c and e)

Fig. 4

Frizzled and flamingo are required by the ddaE neuron for dendrite directional growth. a Fz is expressed in ddaE neuron in the 3^rd instar larva. Fz is labeled by Fz antibody (red) and neurons are labeled by elav-Gal4 (green). White dot lines show the soma of ddaE neuron. b fz expression level is decreased in fz ^EY03114 mutant. (n = 3). c-d The PD-Angle is affected by Frizzled. The PD-Angle is significantly decreased in fz ^EY03114 hypomorph mutant and neural fz ^RNAi expression larvae. Overexpression of Fz in ddaE neuron does not result in an increase of the PD-Angle. Overexpression of Fz in ddaE neuron in fz ^EY03114 mutant background slightly increased the PD-Angle. (n = 50–79). e-f The PD-Angle is affected by Fmi. Knockdown of fmi in ddaE leads to decreased PD-Angle, and overexpressing Fmi increases it. Neuronal expressing Fmi rescues the decreased PD-Angle in stan ^f00907 mutant. (n = 26–50). g Knockdown of fz in a stan ^f00907 mutant background does not further decrease the PD-Angle when compared to stan ^f00907 mutant or neural knocking down of fz. (n = 39–42). h Overexpression of Fz in a stan ^f00907 mutant background rescues the decreased PD-Angle in stan ^f00907 mutant to the wild type degree. (n = 32–49). i PD-Angle of ddaE neuron with neural overexpression of Fmi in fz ^EY03114 mutant background is comparable to the wild type control. (n = 26–32). Red arrows indicate the initial parts of primary dendrites. Scale bar: 10 μm in (a) and 50 μm in (c)

Fig. 5

Frizzled and flamingo mediates Wg signal to regulate dendrite directional growth in ddaE neuron. a-d Knockdown of fz in wg ^spd-fg mutant background rescues the PD-Angle to wild type level, while knockdown of fz in wg ^l-12 mutant background does not further decrease the PD-Angle. (n = 25-45). e-h Knockdown of fmi in a wg ^spd-fg or wg ^l-12 mutant background does not further decrease the PD-Angle. (n = 26-44). Red arrows indicate the initial parts of primary dendrites. Scale bar: 50 μm

Fig. 6

Dishevelled and Racl are involved in regulating dendrite orientation. a-b Knockdown of dsh results in a significant reduction in the PD-Angle, which is rescued by expressing Dsh in the neuron. (n = 24–46). c-d The PD-Angle is decreased when Rac1 is suppressed by expressing either rac1 ^RNAi or Rac1^T17N (a dominant negative form), whereas overexpressing Rac1 shows no effect on the PD-Angle. (n = 38–53). e-f Knockdown of rac1 partially rescues the reduced PD-Angle in wg ^spd-fg mutant background. (n = 59–77). g-h Knockdown rac1 does not further decrease the PD-Angle in wg ^l-12 mutant background. (n = 24–36). i-j Knockdown of rac1 does not lead to further change of the PD-Angle in either fmi mutant stan ^f00907 or fz Knockdown of background. (n = 37–76). k Schematic diagram of molecules in Wg signal pathway that regulates dendrite orientation. Fz, Fmi, Dsh, and Rac1 are essential for mediating Wg signal to regulate directional growth of dendrites, while G proteins, β-catenin pathway, and Cdc42/Rho1 are dispensable for the dendrite directional growth of ddaE neuron. Red arrows indicate the initial parts of primary dendrites. Scale bar: 50 μm