(A) A second instar larva showing class IV da neurons that completely and non-redundantly cover the entire body wall with dendritic arbors. The somas of the class IV da neurons in abdominal segment 3 (A3) are boxed. Scale bar, 100 μm. (B) Diagram of a cross section of the epidermis, showing relative positions of the cuticle, an epidermal cell, the ECM, and a terminal dendrite of a class IV da neuron. The tip of the terminal dendrite is depicted to extend into the epidermal cell. (C–E′) Dendritic fields of ddaC (C and C′), v’ada (B and B′), and vdaB (E and E′) labeled by ppk-CD4-tdTom. Live images of the neurons are inverted to show the dendritic patterns in (C), (D), and (E). The dendritic arbors attached to ECM (green) and those enclosed in the epidermal layer (magenta) are shown in (C′), (D′), and (E′). Three representative regions from each dendritic field are selected (boxed with broken outlines) to show enclosed dendrites that are within primary branches (1), are parts of terminal branches (2), or form crossings with other dendrites attached to the ECM (3). High magnification views of the selected regions are in small panels on the right, with both projections on the Z axis (upper panels) and cross sections along the blue lines (lower panels). Scale bars, 50 μm in (C), (D), and (E) or 5 μm in small panels. (F–F″) A 2D view (F) and 3D renderings (F′ and F″) of two crossing dendrites that are not in direct contact. The dendrites attached to the ECM are in green and the enclosed dendrites are in magenta. The surface rendering of the ECM (F″) is in blue. Part of the dendrites appear to be inside of the ECM due to the resolution limit of confocal microscopy. Scale bar, 10 μm. See also Movie S1.

(A–B) A ventral ddaC dendritic field imaged at 72 hr AEL (A) and 84 hr AEL (B), with dendrites attached to the ECM in green and enclosed dendrites in magenta. The arrowheads point to a segment of primary dendrites that become enclosed during the 12 hr period. The open arrowheads point to a terminal branch that extended a new segment into the epidermal cell layer. The arrows point to an enclosed terminal branch that continued to grow in the epidermal cell layer. Scale bars, 30 μm. (C) Quantitative analysis of terminal dendrites that were enclosed (open bars) or at the basal surface of epidermal cells (filled bars) at both time points, showing the change of terminal dendrites over the 12 hr period. Rerouted denotes dendrites that changed direction after retraction (D–J) TEM images showing class IV da dendrites in cross sections of the larval body wall. Dendrites labeled by the dark DAB staining are either attached to the ECM (D–F) or enclosed in the epidermal cell layer (G–J). The arrow in (G) points to the channel formed by the basal cell membrane of the epidermal cell. The arrows in (H) point to the junction of two neighboring epidermal cells. The arrowheads in (I) and (J) point to the dendrites that are completely wrapped in the middle of epidermal cytoplasm. Scale bars, 500 nm.

(A–C) Dendritic patterns of wildtype (A), mys¹ (B), and mew^M6 (C) ddaC neurons generated with MARCM. Some dendritic crossings in (B) and (C) are indicated by blue arrows. Scale bars, 100 μm. (D–E) Quantification of crossing points in wildtype, mys¹ and mew^M6 mutant neurons. (D) Number of crossing points normalized to total dendritic length. (E) Percentages of contacting (open bars) and non-contacting (filled bars) dendritic crossings. (F–H) Epidermal enclosing of dendrites at the dorsal midline of a control ddaC neuron (F), and neurons that express UAS-mys-RNAi (G) or UAS-mew-RNAi (H). See also Movie S2. (I) Quantification of enclosed dendrites in control and RNAi animals. (J–M) Epidermal enclosing of ddaC dendrites at the dorsal midline in wildtype (J), mys¹ hemizygote (K), mew^M6 hemizygote (L), and mys¹/mew^M6 transheterozygote animals (M). (N) Quantification of enclosed dendrites in wildtype, heterozygous and transheterozygous integrin mutant animals. The dendrites attached to the ECM are in green and enclosed dendrites are in magenta (F–H and J–M). Scale bars, 30 μm (F–H and J–M). Error bars represent standard deviations (D, I, and N). ***P<0.001; ns, not significant; one-way analysis of variance and Dunnett’s test.

(A–B) Epidermal enclosing of dendrites at the ventral midline, showing dendrites of wildtype vdaB neurons (A) and vdaB neurons expressing both UAS-mys and UAS-mew driven by ppk-Gal4 (B). The dendrites attached to ECM are in green and enclosed dendrites are in magenta. The dark blue arrows point to non-contacting dendritic crossings. Scale bars, 10 μm. (C) Quantification of enclosed dendrites in wildtype and integrin-overexpressing animals. Error bars represent standard deviations. ***P<0.001, Student’s t test. (D–E″) Immunostaining of Mys (D–D″) and Mew (E–E″) in animals expressing both UAS-mys and UAS-mew driven by ppk-Gal4. The light blue arrows (D–D″) point to Mys staining on the dendrites. Scale bars, 10 μm. See also Figure S1.

(A–B) Enclosure of ddaC dendrites in hh-expressing epidermal cells in a control animal (A) and an animal expressing hh-Gal4 UAS-wb-RNAi(B). Scale bars, 20 μm. See also Figure S2 and Movies S3 and S4. (C) Quantification of enclosed dendrites in wildtype and wb knock-down animals. Error bars represent standard deviations. ***P<0.001, Student’s t test. (D–I) Epidermal enclosing of ddaC dendrites at the dorsal midline in transheterozygous mutants of integrin and laminin genes. Scale bars, 30 μm. (J) Quantification of enclosed dendrites in hemizygous integrin mutants and transheterozygous mutants of integrin and laminin genes. Error bars represent standard deviations. *P<0.05, **P<0.01, ***P<0.001, one-way analysis of variance and Dunnett’s test. In all images of dendritic fields, dendrites attached to the ECM are in green and enclosed dendrites are in magenta.

(A–D) Enclosure of ddaC dendrites in the epidermis at the dorsal midline in fry¹/fry⁶ (A), trc¹/Df(3L)BSC445 (B), Sin1^e03756 (C), and Dscam²¹/Dscam^B17-1 (D) mutant larvae. The dendrites attached to the ECM are in green and enclosed dendrites are in magenta. Scale bars, 30 μm. Blue arrowheads in (A)–(C) point to non-contacting crossings. Arrows in (D) point to crossings formed by contacting dendrites attached to the ECM. (E) Quantification of enclosed dendrites in fry, trc, Sin1, and Dscam mutants. Error bars represent standard deviations. **P<0.01, ***P<0.001, one-way analysis of variance and Dunnett’s test compared to the wildtype. See also Figure S3 and Movie S5.

(A–C) Timelapse imaging of class IV da dendrites in fry¹ mutant animals. These images show the retraction of two approaching dendrites that are both attached to the ECM (A), turning of an extending arbor when coming in close proximity to another branch, with both branches attached to the ECM (B), and passing of an enclosed terminal branch over another dendrite attached to the ECM (C). The time points relative to the first frame in each series are indicated. Scale bars, 5 μm. (D) Quantification of behaviors of approaching dendrites in wildtype and fry¹ mutant animals. The dendrite pairs were classified as “both attached to the ECM” (2D) or “located on different focal planes” (3D). In each group, the dendrites either avoid each other (Turn/Retract) or continue extending to form crossing (Cross). (E–F′) Dorsal midline of a wildtype (E and E′) and a fry¹ (F and F′) larvae imaged at 72 hr and 96 hr AEL. Contacting crossings (black arrows) and non-contacting crossings (blue arrowheads) are indicated for the dendritic fields at 72 hr AEL and the corresponding positions are also indicated for 96 hr AEL. Scale bars, 30 μm. (G) Quantification of changes of dendritic crossings in wildtype and fry¹ mutant animals during 72–96 hr AEL. At 72 hr AEL, dendritic crossings were identified as either non-contacting (NC) or contacting (C). At 96 hr AEL, they either disappear (X) or remain as crossings (NC or C). In all images of dendritic fields, dendrites attached to the ECM are in green and enclosed dendrites are in magenta.

(A–B) Dendrite enclosure of ddaC neurons overexpressing both UAS-mys and UAS-mew (UAS-integrins) driven by ppk-Gal4 in fry¹/fry⁶ (A) and Sin1^e03756 (B) mutants at the dorsal midline. The dendrites attached to the ECM are in green and enclosed dendrites are in magenta. Scale bars, 30 μm. (C) Quantification of enclosed dendrites. (D–H) Representative dendritic fields of ddaC neurons in wildtype, fry, and Sin1 mutants without and with overexpression of UAS-mys and UAS-mew driven by ppk-Gal4. Scale bars, 30 μm. (I–J) Quantification of crossings of ddaC dendrites, showing the number of dendritic crossings normalized to total dendritic length (I) and percentages of contacting and non-contacting dendritic crossings (J). (K–O) Dendritic patterns at the interface between v’ada (green) and vdaB (magenta) dendritic fields in wildtype and fry, Sin1 mutants without and with overexpression of UAS-mys and UAS-mew driven by ppk-Gal4. Scale bars, 30 μm. (P–Q) Quantification of heteroneuronal crossings between v’ada and vdaB dendrites, showing the number of dendritic crossings normalized to the width of v’ada-vdaB interface (P) and percentages of contacting and non-contacting dendritic crossings (Q). The error bars represent standard deviations (C, I, and P). ***P<0.001, one-way analysis of variance and Bonferroni test for selected pairs of columns.