Heartless and Uninflatable affect the development of wrapping glia.

Confocal images of third instar larval filet preparations stained for CD8::mCherry localization showing segmental nerves posterior to the ventral nerve cord. The genotypes are indicated, arrowheads point to the wrapping glia. (A) Wrapping glial morphology is not changed in control animals expressing a mock dsRNA. (B) Upon expression of a dominant negative form of Htl wrapping glial morphology is impaired. (C) Upon expression of a constitutive active form of Htl, a prominent nerve bulging phenotype is apparent. (D) Wrapping glia specific knockdown of uif using RNA interference dramatically affects glial morphology. (E) Concomitant expression of activated Htl and dsRNA directed against uif results in a uif loss of function phenotype. (F) Wrapping glial morphology is not changed in control animals expressing a mock guide RNA. (G-K) Wrapping glia specific targeting of uif using different guide RNAs targeting different positions along the uif gene (see Figure S1 for details). All uif sgRNAs disrupt wrapping glial cell differentiation. n=5 larvae for all genotypes. Scale bars are 100 µm.

uif affects axonal ensheathment.

(A,B,D-F) Electron microscopic images of cross sections of third instar larval abdominal peripheral nerves. Wrapping glial morphology is indicated, for wrapping indexes see Figure S2. The different genotypes are indicated. (A) Wrapping glial morphology is not changed in animals expressing a mock dsRNA. (B) Upon expression of dsRNA targeting the uif gene, wrapping glial morphology along the nerves is impaired. (C) Single plane of a confocal image stack showing a nerve bulge caused by uif overexpression in wrapping glia nerve stained for CD8::mCherry expression. The dashed white line indicates the section plane of the images shown in (D,E). Expression of uif::GFP in wrapping glial cells causes bulge formation, while outside the bulge wrapping glia appear thin. (D) Upon expression of full length uif tagged with GFP, wrapping glial morphology along the nerves is impaired as observed following knockdown. (E,F) In nerve bulge areas caused by uif overexpression a dramatic increase of folded membrane stacks can be noted. GFPdsRNA n=4 larvae, 4-7 nerves per specimen; uifdsRNA n=3 larvae, 5-9 nerves per specimen; uif::GFP n=3 larvae, 5-6 nerves per specimen. Scale bars are 2 µm, except (C) 50 µm and (F) 1 µm.

Notch is required for wrapping glial development.

Confocal images of third instar larval filet preparations stained for CD8::mCherry localization showing segmental nerves posterior to the ventral nerve cord. The genotypes are indicated, arrowheads point to the wrapping glia. (A) Wrapping glial morphology is not changed in control animals expressing a mock dsRNA. (B,C) Wrapping glia specific knockdown of Notch using RNA interference using two independent dsRNA expressing lines impairs glial morphology. (D) Wrapping glial morphology is not changed in control animals expressing a mock guide RNA. (E) Conditional targeting of Notch using wrapping glia specific Cas9 and guide RNA expression leads to dramatically reduced differentiation of wrapping glial cells. (F) Wild type control for temperature shift experiment. (G) Wrapping glial differentiation is significantly impaired in Nts1 larvae that were cultured at the restrictive temperature from first instar larval stages onwards. n=5 larvae for all genotypes. Scale bars are 100 µm.

Notch function affects axonal wrapping.

(A-C) Electron microscopic images of cross sections of segmental nerves from wandering third instar larvae sectioned 100 µm posterior to the ventral nerve cord. The different genotypes are indicated. (A) Control nerve of an animal expressing dsRNA directed against GFP. Axons are wrapped by processes of the wrapping glia (orange shading). For wrapping index see (D). (B) Upon RNAi based knockdown of Notch glial wrapping is reduced. (C) Upon expression of activated Notch glial wrapping is increased. (D) Quantification of wrapping index. While Notch knockdown significantly decreases the wrapping index (from 0.17 to 0.12, p = 0.00079), activation of Notch signaling significantly increases the wrapping index (from 0.17 to 0.27, p = 0.014). For statistical analysis a t-test was performed for normally distributed data (Shapiro-test), Mann-Whitney-U test was performed for not normally distributed data. GFPdsRNA n=4 larvae with 4-7 nerves each; NdsRNA n=5, larvae with 7-9 nerves each; NICD n=5 larvae with 3-8 nerves each. α=0.05, * p ≤ 0.05, *** p ≤ 0.001. Scale bars 2 µm.

Wrapping glia differentiation requires canonical Notch signaling.

Electron microscopic images of cross sections of segmental nerves from wandering third instar larvae sectioned 100 µm posterior to the ventral nerve cord. The different genotypes are indicated. Glial cell morphology is indicated by orange shading. (A) Wrapping glia specific expression of GFPdsRNA as mock control. (B) Knockdown of Suppressor of Hairless (Su(H)) or (C) mastermind (mam) results in a reduced complexity of wrapping glial cell processes. (D) Neuron specific expression of LacZdsRNA as mock control does not affect glial morphology. (E) Upon neuronal knockdown of Contactin (Cont) glial wrapping of peripheral axons is strongly impaired. Scale bars 2 µm.

Notch counteracts uif and heartless function

Confocal images of third instar larval filet preparations stained for CD8::mCherry localization showing segmental nerves posterior to the ventral nerve cord. All animal express Uif::GFP. The genotypes are indicated, the boxed area is shown in higher magnification as an inlay. (A) Wrapping glial bulging is not affected by expression of a lacZ. (B) Concomitant knockdown of Notch does not affect the nerve bulges. (C) Concomitant expression of activated Notch suppresses the nerve bulging phenotype and restores normal development. (D-G) Whole mount images of living wandering third instar larvae. The segmental nerves posterior to the ventral nerve cord are shown. All larvae express an activated Heartless receptor (λhtl) in the wrapping glia and nuclear GFP in all glial cells. The size of the nerve bulge is indicated by a dashed line. (D) Control larvae co-expressing dsRNA directed against mCherry. The nuclei of the wrapping glial cells are polyploid and thus larger than the perineurial glia nuclei. (E) Upon concomitant knockdown of Notch, the nerve bulging phenotype becomes more pronounced. (D) A similar increase in bulge size is noted when λhtl is expressed in Notchts1 mutant larvae kept at the restrictive temperature from first to third instar larval stages. (G) Concomitant expression of Notch rescues the nerve bulging phenotype caused by λhtl. n=5-7 larvae for each genotype. Scale bars are 100 µm.