Heartless is required for wrapping glia development.

Confocal images of third instar larval filet preparations, stained for CD8::mCherry expression. The segmental nerves posterior to the ventral nerve cord are shown. Wrapping glial morphology is (A) not changed in control animals expressing (nrv2-Gal4) mock GFPdsRNA. (B) Differentiation of wrapping glial cells is affected following expression of a dominant negative form of Htl and thin wrapping glial cells are detected (arrowhead). (C) Expression of a constitutively active form of Htl [nrv2-Gal4; UAS-λhtl] leads to nerve bulges around the wrapping glial nuclei (C, arrowhead). The white dashed lines indicate the level of sections shown in Figure 2.

Heartless controls growth of wrapping glia cells.

Electron microscopic images of segmental nerves from wandering third instar larvae. (A) Nerve of a control larva expressing GFP in wrapping glia sectioned 150 µm posterior to the ventral nerve cord. Normally differentiated wrapping glia can be seen. (B-H) Nerves of larvae expressing activated Heartless in wrapping glia [nrv2-Gal4; UAS-λhtl] sectioned at the positions indicated in Figure 1C. (B) Note the poorly differentiated wrapping glial cells distant from the nerve bulge. (C,D) At the beginning of the nerve bulge, excessive differentiation of wrapping glial cell processes start to be detected that not always grow around axons (magenta). (E-G) Higher magnifications of the boxed areas in (D). Note the formation of wrapping glial cell processes that do not contact axons (ax, magenta) but rather contact glial cell processes. (H) In the central area of the nerve bulge, liquid-filled vacuolar structures (asterisks) can be detected. Thin wrapping glial cell processes (arrows) span the bulged area.

uninflatable affects differentiation of wrapping glia.

Confocal images of third instar larval filet preparations, stained for CD8::mCherry expression. The segmental nerves posterior to the ventral nerve cord are shown. (A) Filet preparation of a control third instar larva. (B) uif knockdown in wrapping glial cells [nrv2-Gal4; UAS-uifdsRNA-HMS01822] impairs their development, which (C) cannot be rescued by co-expression of activated Htl. (D-H) Ubiquitous expression of (D) mock control GFPsgRNA, (E) uifsgRNA 2nd Exon, (F) uifsgRNA CS, (G) uifsgRNA TMD or (H) uifsgRNA CD in Cas9 expressing larvae. All uif sgRNAs disrupt wrapping glial cell development, wrapping glia appear thin and fragmented (arrowheads). n=5 larvae for all genotypes. Scale bars 100 µm.

uif affects axonal ensheathment of wrapping glia.

(A+B,D-F) Electron microscopic cross sections of third instar larval abdominal peripheral nerves with wrapping glia specific expression [nrv2-Gal4; R90C03-Gal80] of (A) GFPdsRNA mock control transgene, (B) uifdsRNA- HMS01822. Upon knockdown of uif, wrapping glial cell complexity is reduced. (C) Single plane of a confocal image of a third instar larval nerve stained for CD8::mCherry expression. Expression of uif::GFP in wrapping glial cells causes bulge formation, while outside the bulge wrapping glia appear thin (arrowheads). The dashed lines indicate the plane of section in relation to the bulge of the images shown in (D,E). (D) Upon uif::GFP overexpression specifically in wrapping glia, glial morphology is reduced outside the bulge region. (E) Within the nerve bulge, wrapping glial membrane increases in size while most axons lack proper wrapping. (F) Close up of region indicated in (E). 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 2 µm unless indicated otherwise.

Notch is required for wrapping glial development.

Filet preparations of wandering third instar larvae stained for CD8::mCherry expression. The segmental nerves just posterior to the ventral nerve cord are shown. (A) Control larvae expressing the mock control GFPdsRNA in wrapping glial cells specifically [nrv2-Gal4; R90C03-Gal80]. Upon expression of dsRNA targeting Notch mRNAs [(B) NGD144, (C) N9G. (D) Control larva expressing sgRNA directed against the GFP open reading frame in [nrv2-Gal4; UAS-Cas9]. (E) Conditional knockout of Notch leads to dramatically altered morphology of wrapping glial cells. (F) Control larva cultured at the same temperature regime as the Nts1 larva shown in (G). Wrapping glial cells appear smaller compared to the control. Scale bars are 100 µm.

Notch function affects axonal wrapping.

Electron microscopic images of segmental nerves from wandering third instar larvae sectioned 100 µm posterior to the ventral nerve cord. (A) Control nerve of an animal expressing dsRNA directed against GFP [nrv2-Gal4; R90C03-Gal80]. Axons are wrapped by processes of the wrapping glia. The entire nerve is engulfed by the perineurial glia and the subperineurial glia. For quantification of glial wrapping see (D). (B) Upon expression of dsRNA targeting Notch mRNA (NGD144) glial wrapping is reduced. (C) Upon expression of an activated form of Notch (Nintra) glial wrapping is increased. (D) Quantification of Wrapping Index (WI). While Notch knockdown significantly decreases the WI (from 0.17 to 0.12, p = 0.00079), activation of Notch signaling significantly increases glial wrapping and the WI (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.

Knockdown of mam, Su(H) and contactin impairs axonal wrapping.

Electron microscopic cross sections of third instar larval abdominal peripheral nerves. Glial cell morphology is indicated in orange color. (A-C) Wrapping glia specific [nrv2-Gal4; R90C03-Gal80] expression of GFPdsRNA as mock control, (B) Suppressor of Hairless (Su(H)) dsRNAHMS05748 or (C) mastermind (mam) dsRNAKK110687. Note reduced complexity of glial cell processes. (D) Neuron specific [nSyb-Gal4] expression of LacZdsRNA as mock control and (E) Contactin (Cont) dsRNAGD12610 inserted on the third Chromosome. Upon neuronal knockdown of Cont, glial wrapping of peripheral axons is impaired. Scale bars 2 µm.

Notch counteracts heartless and uninflatable function.

Squeeze preparations of wandering third instar larvae. The segmental nerves posterior to the ventral nerve cord are shown. (A-C) Larvae expressing uninflatable and CD8::mCherry in the wrapping glia. The nerve bulge shown in the white dashed box is enlarged. (A) Control larvae co-expressing LacZ. Uif expression results in nerve bulges. (B) Upon co-expression of NotchdsRNA (GD144) the nerve bulging phenotype is not suppressed. (C) Upon activation of Notch signaling by expression of NICD, the nerve bulging phenotype is suppressed. (D-G) Larvae expressing an activated Heartless receptor (λhtl) in the wrapping glia [nrv2-Gal4] carrying a repo3.4-stinger::GFP element leading to a panglial nuclear GFP expression. (D) Control larvae co-expressing dsRNA directed against mCherry. Note the prominent nerve bulges. (E) Upon co-expression of NotchdsRNA the nerve bulging phenotype is enhanced. (F) Likewise, the nerve bulging phenotype is enhanced in a Notchts1 mutant background when the larvae are kept at the restrictive temperature. (G) Upon activation of Notch signaling by expression of NICD, the nerve bulging phenotype is significantly rescued. n=7 larvae for all genotypes.

Model

The interplay of FGF-receptor, Uninflatable and Notch signaling controls the switch between glial cell growth and glial cell differentiation leading to extensive neuron-glia contact. For details please see the text.