DEV019547 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Expression patterns of hlh-17::GFP and ptr-10::myrRFP reporter transgenes. (A-D) DIC (A,C) and fluorescence (B,D) images of an L1-stage animal expressing hlh-17::GFP. Expression in the head (A,B) and tail (C,D) is shown. Arrowheads in B indicate expression of GFP in some IL, OL and OLQ sheath and/or socket glia. (E,F) DIC (E) and fluorescence (F) images of an L3-stage animal expressing hlh-17::myrGFP. Arrowheads indicate the expression of myrGFP in motoneuron commissures. (G-L) Merged fluorescence and DIC images (G,I,K) and fluorescence images (H,J,L) of an adult animal expressing ptr-10::myrRFP. Expression in the head (G,H), vulva (I,J) and rectum (K,L) is shown. Arrowheads in H indicate the expression in sheath and socket glia of IL, OL (blue), and OLQ (yellow) sensilla, as well as in CEPsh glia (white).

• Supplemental Figure S2 -

  Fig. S2. AWC and AFD neurons contact CEPsh glia in the nerve ring and ventral ganglion. (A) Left lateral cross-section of the nerve ring. Image reproduced with permission from White et al. (White et al., 1986). Green, CEPshVL glia process; blue, AWCL neuron; purple, AFDL neuron; red, ADFL neuron. (B) Same as A except that a right lateral cross-section is shown. Green, CEPshVR glia process; blue, AWCR neuron; purple, AFDR neuron; red, ADFR neuron. (C) Transverse section through the anterior region of the ventral ganglion. Image reproduced with permission from White et al. (White et al., 1986). Green, CEPshVL/R glia process; blue, AWCL/R neurons; purple, AFDL/R neurons; red, ADFL/R neurons.

• Supplemental Figure S3 -

  Fig. S3. Cell lineage is not altered in mls-2(ns156) and vab-3(ns157) mutant embryos. (A) Fluorescence images of embryos of the indicated genotype containing pie-1::H2B::GFP and his-72::H3.3-GFP, labeling all nuclei. The red, blue, magenta and pink circles indicate the nuclei of CEPshDR, CEPshDL, CEPshVL and CEPshVR glia, respectively, immediately after their birth. (B) Representative lineage trees constructed from tracing the divisions executed by the ABarpa, ABplpaa and ABprpaa precursor cells and their progeny in wild-type (i, iv and vii, respectively), mls-2(ns156) (ii, v and viii, respectively), and vab-3(ns157) (iii, vi and ix, respectively) animals. Note that all three genotypes give rise to essentially identical lineage trees.

• Supplemental Figure S4 -

  Fig. S4. mls-2(ns156) mutants lack ventral CEPsh glia processes. (A) Right, electron micrograph of a section of a wild-type adult showing the ventral ganglion leading into the nerve ring. Left, magnification of the boxed region indicated in the right panel. Arrows demarcate the CEPsh glial process. (B) Same as A except that an mls-2(ns156) mutant is shown. Arrows point to the border of the ventral ganglion showing the absence of a CEPsh glial process. Note the disorganization of the ventral ganglion (right) as compared with the bilaterally symmetric structure of the wild type (A).

• Supplemental Figure S5 -

  Fig. S5. Isolation of hlh-17, hlh-32 and hlh-31 deletion alleles. (A) Alignment of the bHLH domains of the HLH-17, HLH-32 and HLH-31 proteins. Pink indicates identity among all three proteins; blue indicates identity in two of three proteins. (B) Diagrams depicting the wild-type hlh-17/hlh-31 locus (top) and the alterations in the ok487 strain (bottom). Boxes, exons. Λ-shaped lines, introns. The boxed sequences in hlh-31 (colored red) were duplicated and inserted into exon 2 of hlh-17. (C,D) Genomic organization of hlh-32, hlh-17 and hlh-31. (C) Boxes, exons. Λ-shaped lines, introns. Locations of deletion alleles are indicated by red bars, with numbers indicating the precise deletion positions relative to the ATG in the genomic sequences of each gene. Blue areas encode the bHLH domains in each gene. NdeI was used to digest genomic DNA for the Southern blot shown in D. The sizes of predicted NdeI digestion fragments are indicated with red, blue or green numbers. Arrows in D indicate the sizes of each band. Each deletion deletes one NdeI site, yielding only three bands in the triple-knockout strain (D, third lane). The blot in D was probed with ³²P-labeled hlh-17 cDNA, which can detect all the fragments depicted in the panel. The 1959 bp NdeI fragment of hlh-31 and the 2016 bp NdeI fragment of hlh-32 are overlapping. (E) GFP, myrGFP and myrRFP reporter constructs used in this study.

• Supplemental Figure S6 -

  Fig. S6. hlh-17(ns204) mutants do not show defects in CEPsh development. (A) Expression of hlh-17 and ptr-10 in dorsal CEPsh glia of hlh-17(ns204) mutants. (B) Same as A except that ventral CEPsh glia are examined. (C) Axon guidance defects of AWC and ADF neurons in hlh-17(ns204) mutants.

• Supplemental Figure S7 -

  Fig. S7. A triple mutant of hlh-17, hlh-31 and hlh-32 has no defects in ptr-10 reporter expression or CEP dendrite extension. Triple allele, hlh-32(ns223) hlh-17(ns204) hlh-31(ns217). n, number of animals observed. (A,B) ptr-10 expression in dorsal or ventral CEPsh glia, respectively, in the indicated mutant backgrounds. (C,D) hlh-17 and ptr-10 reporter expression in dorsal and ventral CEPsh glia, respectively, in wild-type or hlh triple-mutant animals. (E) CEP neuron dendrite defects in animals of the indicated genotypes.