DYF-4 regulates patched-related/DAF-6-mediated sensory compartment formation in C. elegans

Hui Hong; Huicheng Chen; Yuxia Zhang; Zhimao Wu; Yingying Zhang; Yingyi Zhang; Zeng Hu; Jian V Zhang; Kun Ling; Jinghua Hu; Qing Wei

doi:10.1371/journal.pgen.1009618

. 2021 Jun 11;17(6):e1009618. doi: 10.1371/journal.pgen.1009618

DYF-4 regulates patched-related/DAF-6-mediated sensory compartment formation in C. elegans

Hui Hong ^1,^2,^3,^#, Huicheng Chen ^1,^4,^5,^#, Yuxia Zhang ^6,⁷, Zhimao Wu ^1,^4,⁵, Yingying Zhang ^1,^4,⁵, Yingyi Zhang ⁶, Zeng Hu ⁶, Jian V Zhang ⁵, Kun Ling ⁶, Jinghua Hu ⁶, Qing Wei ^5,^*

Editor: Andrew D Chisholm⁸

PMCID: PMC8221789 PMID: 34115759

Abstract

Coordination of neurite extension with surrounding glia development is critical for neuronal function, but the underlying molecular mechanisms remain poorly understood. Through a genome-wide mutagenesis screen in C. elegans, we identified dyf-4 and daf-6 as two mutants sharing similar defects in dendrite extension. DAF-6 encodes a glia-specific patched-related membrane protein that plays vital roles in glial morphogenesis. We cloned dyf-4 and found that DYF-4 encodes a glia-secreted protein. Further investigations revealed that DYF-4 interacts with DAF-6 and functions in a same pathway as DAF-6 to regulate sensory compartment formation. Furthermore, we demonstrated that reported glial suppressors of daf-6 could also restore dendrite elongation and ciliogenesis in both dyf-4 and daf-6 mutants. Collectively, our data reveal that DYF-4 is a regulator for DAF-6 which promotes the proper formation of the glial channel and indirectly affects neurite extension and ciliogenesis.

Author summary

In C. elegans sensory organ, the ciliated neuronal endings are wrapped in a luminal channel formed by glial cells, forming a specialized sensory compartment critical for sensory activity. Coordination of glial channel formation, dendritic tip anchoring and ciliogenesis are critical for the formation of a functional sensory compartment. DAF-6, a patched-related glial membrane protein, was reported to play an important role in glial channel morphogenesis, but the molecular function and regulatory mechanism of DAF-6 remain poorly understood. Here, we found that DYF-4, a glia-secreted protein, interacts and colocalizes with DAF-6, and functions in a same pathway as DAF-6 to regulate sensory compartment formation. We propose that DYF-4 is a novel regulator for DAF-6 to control sensory compartment formation.

Introduction

The sensory organ uses its ending to receive environmental stimuli. In many cases, the ciliated neuronal receptive endings and surrounding glial cells form compartmentalized sensory endings, as observed in C. elegans, Drosophila and the olfactory epithelium of mammals [1–4]. The proper formation of the sensory compartment requires interplay between glial cells and the ciliary endings of sensory neurons. The integrity of the glial compartment and the development of sensory neurons impact each other in a reciprocal manner [5–9]. However, little is known about the underlying mechanism.

C. elegans uses its sensory organs (amphid and phasmid) to sense environmental cues. The amphid sensory organ of the head consists of 12 sensory neurons, whose ciliated endings are wrapped by the ends of a sheath glia cell and a socket glia cell [10]. The cell bodies of sensory neurons are located in the pharyngeal bulb, with the dendrites extending to the anterior end of the nose and terminating with cilia. Both the sheath and socket glial cells extend in parallel along the dendrites of sensory neurons and form discrete tubular channels surrounding amphid cilia. Specifically, the proximal portion of most cilia is surrounded by the membrane of the sheath cell, while the distal portion of most cilia extends through a pore formed by the membrane of the socket cell. The structure of the phasmid of the tail is similar to that of the amphid, except that it contains only 2 sensory neurons.

The proper formation of the sensory compartment in C. elegans includes the compartmentalization of glial endings, neuron dendrite tip anchoring and cilia biogenesis. DAF-6, a patched-related membrane protein, is a key regulator of the compartmentalization of the glial endings in C. elegans [11]. DAF-6 functions in the very early stage to restrict the expansion of the glial compartment [12], and its activity can be antagonized by several suppressors, including the Nemo-like kinase LIT-1, the actin regulator WSP-1, some retromer components (SNX-1, SNX-3, VPS-29) and the Ig/FNIII protein IGDB-2 [12–14]. It is thought that DAF-6 and its suppressors control glia compartment morphogenesis by regulating vesicle dynamics of glial cells in a cell-autonomous manner. In C. elegans, dendrite extension in the amphid and phasmid occurs via a special “retrograde extension” process [7,15,16]. Specifically, glial and neuronal cells are first assembled into a polarized multicellular rosette at the site where the sensory complex will be assembled, then the tips of dendrites stay anchored and the cell bodies grow backward to elongate the dendrites. The tectorin-related proteins DEX-1 and DYF-7 form an anchorage in the extracellular matrix and play essential roles in dendrite tip anchoring [7]. The transition zone (TZ) at the ciliary base is also reported to be involved in dendrite elongation in C. elegans [15]. Combined mutations of individual genes from the Meckel-Gruber Syndrome (MKS) module and Nephronophthisis (NPHP) module result in severe defects in dendrite elongation [15,17–20]. Notably, proper dendritic anchoring and cilia biogenesis are also required for glial compartment morphogenesis. It has been demonstrated that sheath cells cannot extend, and glial channels are disrupted in dyf-7 mutants [7,21]. It was also reported that the glia morphology is altered in cilia mutants [8,22], and the glia compartment formation regulators DAF-6 and its suppressors are mislocalized in ciliogenesis mutants [11,12]. However, little is known about the identity and source of the extracellular signals that coordinate the glial channel formation and dendritic morphogenesis.

In a whole-genome forward genetic screen, we identified a novel gene, dyf-4, whose mutants recapitulate the phenotypes of daf-6, exhibiting truncated dendrites and defective ciliogenesis in both amphid and phasmid neurons. We cloned DYF-4 and found that it encodes a glia-secreted protein that interacts and colocalizes with DAF-6. Interestingly, the dendritic and ciliary defects of dyf-4 could be restored by reported daf-6 suppressors (wsp-1, lit-1 and igdb-2). Our results reveal that DAF-6 is also required for dendrite elongation and ciliogenesis of sensory neurons, DYF-4 and DAF-6 function in the same pathway to regulate the proper formation of the sensory compartment.

Results

dyf-4 encodes C13C12.2 and is required for dendrite extension in C. elegans

In C. elegans, amphid cilia and phasmid cilia are wrapped by sheath and socket cells and extend to the external environment. Defects in environmental exposure or ciliogenesis result in dye-filling defects (dyf) [23]. In a forward genetic screen searching for dyf mutants, we isolated two mutants, jhu431 and jhu500, sharing identical phenotypes, with extremely short phasmid dendrites and slightly truncated amphid dendrites (Figs 1A–1F and 2A–2F). The shorter dendrites suggest that dyf are mainly caused by defects in dendrite elongation in these two mutants. jhu431 was mapped to c13c12.2, a gene of unknown function. C13C12.2 encodes a 383 amino acid protein, predicted to contain a 16 amino acid signal peptide at its N-terminus and a PLAC (protease and lacunin) domain at its C-terminus (Figs 1A and S1A and S1B). The PLAC domain is a six-cysteine region of approximately 40 amino acids that is usually present at the C-terminus of various extracellular proteins [24–26]. jhu431 carries a missense mutation affecting one of the conserved cysteines (C366Y) in the PLAC domain (Fig 1A). A complementation assay indicated that jhu431 failed to complement dyf-4(m158), a mutant isolated 20 years ago that has not yet been cloned. dyf-4 (m158) shows the same phenotype as jhu431, including very short phasmid dendrites and slightly shorter amphid dendrites (Fig 1B–1F). Genomic sequencing showed that dyf-4(m158) possesses a nonsense mutation (Q166stop) in the middle region of C13C12.2. The elongation defects of the dendrites observed in dyf-4(m158) and jhu431 were fully rescued by the transgenic expression of the wild-type (WT) dyf-4 gene under the control of its own promoter but were not rescued by the C13C12.3^C366Y mutant form (Fig 1B–1F), indicating that C13C12.2 is responsible for these defects and that C366 is critical for its function. Therefore, we refer to C13C12.2 as DYF-4 hereafter.

(A) Schematic diagrams of the genomic and protein structures of *dyf-4* (*c13c12*.2). DYF-4 has a predicted signal peptide at its N-terminus and a PLAC domain at its C-terminus. *dyf-4* (*jhu431)* carries a point mutation (c.1097G>A) that leads to a missense mutation in one of the conserved cysteines (C366Y) in the PLAC domain. *dyf-4*(*m158)* possesses a point mutation (c.466C>T) that results in a nonsense mutation (Q166stop) in the middle region of the DYF-4 protein. As *dyf-4*(*jhu431)* and *dyf-4*(*m158)* exhibit the same mutant phenotypes, we used the *dyf-4*(*m158)* mutant in subsequent experiments if not otherwise specified. (B) Upper panels: schematic representation of C. *elegans* amphid and phasmid structures. Lower panels: Fluorescence micrographs of sensory neurons in the amphid and phasmid in adult WT, *dyf-4*(*jhu431)*, *jhu431*; *Ex [DYF-4]*, *dyf-4*(*m158*), *m158*; *Ex [DYF-4]* and *m158*; *Ex [DYF-4*^C366Y] worms. The IFT-B component OSM-6::GFP was used to label cell bodies, dendrites and cilia. The white vertical line at left represents the nose tip position. Scale bars: 5 μm. (C) Statistics of the dye-filling percentages in the amphids of the indicated worm lines. (D) Statistics of the distance between basal bodies and the nose tip at the head of the indicated worm lines. (E) Statistics of the dye-filling percentages in the phasmids of the indicated worm lines. (F) Statistics of the ratio of phasmids with severely collapsed dendrites in the indicated worm lines. Dendrites with cilia located near the cell bodies are defined as severely collapsed dendrites. Data are presented as the mean ± SEM from three independent experiments (n ≥ 80 per experiment). ***P < 0.001 (Fisher’s Exact test for Fig 1C, 1E and 1F, Mann-Whitney test for Fig 1D).

(A) Schematic diagram of *daf-6* alleles. *daf-6(e1377)* was previously reported [11]. *daf-6(jhu500)* has a 1-bp deletion (c.2272delC) at its C-terminus, leading to a frameshift. (B) Fluorescence micrographs of sensory neurons in the amphids and phasmids of adult WT, *daf-6(e1377)*, *daf-6(jhu500)* and *jhu500*; *Ex [DAF-6]* worms expressing OSM-6::GFP. The white vertical line at left represents the nose tip position. Scale bars: 5 μm. (C) Statistics of the dye-filling percentages in the amphids of the indicated worm lines. (D) Statistics of the distance between basal bodies and the nose tip at the head in the indicated worm lines. (E) Statistics of the dye-filling percentages in the phasmids of the indicated worm lines. (F) Statistics of the phasmid dendrite defect ratio in the indicated worm lines. Dendrites with cilia located near the cell bodies are defined as severely collapsed dendrites. Data are presented as the mean ± SEM from three independent experiments (n ≥ 80 per experiment). (G) Analysis of the defects of dendrites at L1 and L2 stages in the indicated worm lines. ***P < 0.001 (Fisher’s Exact test for Fig 2C, 2E and 2F, Mann-Whitney test for Fig 2D and 2G).

daf-6 mimics the phenotype of dyf-4

Through a SNP mapping strategy and whole-genome sequencing, the jhu500 allele was mapped to daf-6 as a 1-bp deletion (c.2272delC) that leads to a frameshift and loss of the C-terminal region (Fig 2A). A null allele of daf-6 (e1377) showed the same phenotype as jhu500 and failed to complement jhu500. The introduction of the WT daf-6 cDNA fully rescued the dendrite extension defects of jhu500, revealing that jhu500 is a new allele of daf-6 (Fig 2B–2F).

DAF-6 is a glial protein similar to the hedgehog membrane receptor Patched. It has been proved that DAF-6 regulates glial compartment morphogenesis in C. elegans [11,12]. The dendrite elongation defect described here is a new phenotype of daf-6 mutants, indicating that DAF-6 is also involved in normal dendrite extension of sensory neurons. Previous studies have shown that the glial compartment defect in daf-6 mutants progressively becomes more severe throughout the larval development and becomes extremely disorganized in adult [11,12,27]. Interestingly, we observed that the dendrite length of daf-6 and dyf-4 was comparable to that in WT at the L1 stage, but was significantly shorter than that of WT at the L2 stage (Fig 2G), indicating that the dendrite defect is also gradually severe during development and likely to be a secondary effect.

Dendrite elongation in C. elegans is mediated by “retrograde extension”, with dendritic tips first being anchored to the extracellular matrix at the destination in the embryonic stage, after which the cell bodies travel backward during growth, trailing the elongating dendrites behind them [7]. Since the localization of neuron cell bodies is normal in both daf-6 (jhu500) and dyf-4 (jhu431), their short dendrites should be caused by the abnormal position of their dendritic tips. It is likely that the abnormal morphology of glial channels in daf-6 [11] or dyf-4 (see the data below) changes the anchoring position of the tips of dendrites, which indirectly leads to the shortening of dendrites.

DYF-4 is a glial secretory protein required for DAF-6 localization

Due to the identical mutant phenotypes shared by dyf-4 and daf-6, we speculate that DYF-4 may function in the same pathway as DAF-6 to regulate sensory compartment formation. To test our hypothesis, we first determined where dyf-4 gene is expressed. To this end, a transcriptional reporter for dyf-4 (Pdyf-4::GFP) was generated. Interestingly, as expected, we found that, similar to daf-6, Pdyf-4::GFP was expressed in sheath and socket glial cells in both the amphid and phasmid (Fig 3A and 3B). To further confirm that glial cells are the functional sites of dyf-4, we expressed dyf-4 cDNA driven by the daf-6 promoter or the neuron-specific dyf-7 promoter. As expected, only the daf-6 promoter-driven expression of DYF-4 rescued the dyf-4 mutant phenotype (Fig 3C).

Fig 3 — (A) Illustration of the C. *elegans* amphid. Right: Each amphid consists of 12 sensory neurons, a socket glial cell and a sheath glial cell. Left: Detail of the sensory compartment in the amphid. (B) Fluorescence micrographs of the *dyf-4* expression pattern in the head and tail region in worms expressing *Pdyf-4*::GFP. Sheath glial cells and socket glial cells are indicated by arrowheads. Scale bar: 5 μm. (C) The percentages of dye filling in amphid and phasmid in WT and *dyf-4(m158)* mutants expressing the WT *dyf-4* gene driven by different promoters. Data are presented as the mean ± SEM from three independent experiments (n ≥ 80 per experiment). ***P < 0.001 (Fisher’s Exact test). (D) Representative images of DYF-4::GFP localization in the amphids and phasmids of young adult of WT. DYF-4::GFP localizes to the sensory compartment region. n = 30. Scale bar: 5 μm. (E) Colocalization of DYF-4 and DAF-6 in the amphid and phasmid of young adult of WT. n = 10. (F) The localization pattern of DYF-4::OPT-GFP in the amphid and phasmid of young adult of WT. Scale bar: 5 μm. (G) Colocalization of DYF-4::OPT-GFP and OSM-6::mCherry in the amphid and phasmid of young adult of WT. DYF-4::OPT-GFP showed punctate localization and tended to accumulate at the middle of sensory compartment in amphids. Scale bar: 5 μm.

To observe the subcellular localization of DYF-4 protein, we created transgenic worms expressing GFP-tagged DYF-4 fusion proteins. Unfortunately, the DYF-4::GFP signal cannot be directly observed. To improve the signal of DYF-4::GFP, we first employed the indirect immunofluorescence method to enhance the signal by using the anti-GFP antibody. Interestingly, through this method, we observed that DYF-4::GFP was specifically enriched and partially co-localized with DAF-6 in the sensory compartment region in both amphids and phasmids (Fig 3D and 3E). Compared with DYF-4::GFP, the localization pattern of DYF-4^C366Y::GFP was changed and tended to accumulate in glia cells, suggesting that defects in dyf-4 (jhu431) mutants may arise from the mislocalization of DYF-4 (S2A Fig). Notably, without anti-GFP staining, DYF-4^Δ(1–16)::GFP (the signal peptide deletion mutant of DYF-4) was directly observed to accumulate in vesicular structures in both amphid and phasmid (S2B Fig).

On the other hand, we replaced the GFP tag with an OPT-GFP tag which improves fluorescence and folding efficiency [28]. Interestingly, the DYF-4::OPT-GFP signal could be observed directly. Consistently, we observed that DYF-4::OPT-GFP signal was enriched in sensory compartment region in both amphids and phasmids (Fig 3F), showing puncta/granule patterns, suggesting that they are in vesicular structures (Fig 3G). Notably, DYF-4::OPT-GFP was expressed in the early developing glial cells of the embryo and tended to accumulate between the embryo and the eggshell, likely to be secreted (S2C Fig).

Next, we investigated the interdependence of the subcellular localization of the DYF-4 and DAF-6 proteins. In daf-6 mutants, the localization of DYF-4::OPT-GFP was basically normal, only a fraction (~10%) of amphids had abnormal DYF-4 signals (Fig 4A). However, compared to WT in which DAF-6::GFP localized along the socket glial channel and showed weak signals in the sheath lumen, DYF-6::GFP accumulated strongly in both cell bodies and sensory compartment region of glial cells in dyf-4 mutants (Figs 4B and S2D), indicating that DYF-4 is required for the proper localization of DAF-6.

Fig 4 — (A) Subcellular localization of DYF-4::OPT-GFP in young adults of WT and *daf-6* worms. The localization of DYF-4 was basically normal in *daf-6* mutants. n>100 worms. (B) Subcellular localization of DAF-6::GFP in young adults of WT and *dyf-4* worms. DYF-4 is required for the glial channel localization of DAF-6. n>100 worms. Scale bars: 5 μm. (C) DIC images of heads of WT and *dyf-4* mutants. Vacuolar structures were observed in *dyf-4* mutants. Scale bars: 5 μm. (D) The amphid channel expressing *T02B11*.3pro::GFP (Green, sheath glia) and *gcy-5pro*::mCherry (Red, ASE neuron) in indicated genetic background. The ASE cilium extended through sheath cell lumen in WT, while it often bended and failed to extend through sheath cell lumen in both *dyf-4* and *daf-6* mutants. (E) TEM images of cross-sections of amphid channel in WT and *dyf-4* mutants. Blue arrow: cilia; White asterisk: vesicular accumulations. Magnified red square region in *dyf-4* mutants is shown on the right.

DYF-4 is required for sensory compartment formation

DAF-6 is critical for the formation of amphid glia channels. Due to the abnormal glial channel, daf-6 mutants often have curved cilia and vacuole accumulation in amphids [11]. Similarly, curved cilia and vacuolar structures were often observed in amphids of dyf-4 mutants (Figs 4C and S3A). To visualize the sheath glia morphology, we labeled the amphid sheath glia with GFP driven by the T02B11.3 promoter and the ASER neuron with mCherry driven by the gcy-5 promoter. The ASER cilium extended through sheath glia in WT, but it was bent and fails to extend through sheath glia in dyf-4 mutants (Fig 4D), similar results have been observed in daf-6 mutants [12]. Dendrite defects are much more severe in phasmids of dyf-4 mutants. we observed that sheath glia cells of dyf-4 mutants fail to extend as they did in WT (S3B Fig). GFP::WSP-1A and ARX-2::GFP localize along the distal portion of the glial compartment channels (formed by socket glia) in WT worms [12,29]. Compared to WT, the signal length of WSP-1 and ARX-2 was significantly shortened in dyf-4 mutants (S3C–S3H Fig). To directly examine the glia channel defects, we performed TEM on the amphid channel of dyf-4 adult mutants. As showed in Fig 4E, enlarged pore lumen and accumulation of granule vesicles were observed in dyf-4 mutants. All these results indicate that the glia channel is abnormal in dyf-4 mutants.

DYF-4 acts in a same pathway as DAF-6

We then wondered whether DYF-4 physically interacts with DAF-6. DAF-6 is a protein with 12 transmembrane domains, and both its N- and C-termini are predicted to reside in the cytoplasm. On the extracellular side, there are two longer extracellular loops (ECLs), ECL1 and ECL4. By using GST pull-down assay, we demonstrated that DYF-4 could specifically interact directly with both ECL1 and ECL4 of DAF-6 (Fig 5A), but not with ECL1 and ECL4 of CHE-14 (another 12 transmembrane glial protein containing sterol-sensing domain (SSD)) (Fig 5B), supporting the existence of an intriguing mechanism in which a complex formed by DYF-4 and DAF-6 is vital for the proper function of glial cells.

Fig 5 — (A) DYF-4 interacted with DAF-6. Upper panel: Schematic representation of DAF-6 transmembrane protein. Lower panel: DYF-4 interacted with both DAF-6 ECL1 and DAF-6 ECL4 in the GST pull-down assay. (B) DYF-4 did not interact with ECLs of CHE-14 in the GST pull-down assay. (C) *dyf-4; daf-6* double mutants showed similar phenotypes with *dyf-4* or *daf-6* single mutants. The white vertical line at left represents the nose tip position. Statistics of the distance between basal bodies and the nose tip were showed on the right. All data are presented as the mean ± SEM (n ≥ 63 for each genotype). ***P < 0.001 (Mann-Whitney test).

If DYF-4 acts in a same pathway as DAF-6, then the phenotype of daf-4; daf-6 double mutant should be similar as either single mutant. Indeed, we found that co-deletion of dyf-4 and daf-6 did not aggravate dendrite elongation defects compared with either single mutants (Fig 5C).

Taken together, all these results support our hypothesis that DYF-4 and DAF-6 act in the same pathway to regulate sensory compartment formation.

daf-6 suppressors effectively restore dyf-4 mutant phenotypes

Previous studies demonstrated that the Nemo-like kinase LIT-1, the actin regulator WSP-1, some retromer components and the Ig/FNIII protein IGDB-2 antagonize DAF-6 to promote glial compartment formation. The deletion of these genes also partially restores dye filling defects in the amphids of daf-6 mutants [12–14]. We reasoned that if DYF-4 and DAF-6 function together to regulate glial function, daf-6 suppressors should rescue phenotypes of dyf-4 single mutants and dyf-4; daf-6 double mutants. As expected, wsp-1, lit-1 or igdb-2 mutants all restored the dye-filling defects of dyf-4 single mutants and dyf-4; daf-6 double mutants, as they did in daf-6 mutants (Fig 6A).

Fig 6 — (A) Statistics of the dye-filling defect percentages in the amphid neurons of the indicated worm lines. Suppressors of *daf-6*, *wsp-1a (gm324)*, *lit-1 (or131)* and *igdb-2* inhibited the defects in dendrite elongation in *dyf-4* and *daf-6* mutants. (B) Fluorescence micrographs of phasmid dendrites in the indicated genetic background. Scale bar: 5 μm. (C) The percentages of severely collapsed dendrites of the phasmid in the indicated worm lines. Dendrites with cilia located near the cell bodies are defined as severely collapsed dendrites. Data are presented as the mean ± SEM from three independent experiments (n ≥ 80 per experiment). ***P≤0.001 (Fisher’s Exact test).

To confirm that the restoration of dye filling resulted from the rescue of the shortened dendrites in dyf-4 and daf-6 mutants, we focused on phasmids in which dendrites were severely shortened. In dyf-4 or daf-6 mutants, all the phasmid neuron dendrites are collapsed into the cell bodies. In contrast, in dyf-4; wsp-1a and daf-6; wsp-1a double mutants, only 3.19% and 5.55% of phasmid dendrites, respectively, were severely collapsed (Fig 6B and 6C). Consistent with these findings, the deletion of lit-1(or131) or igdb-2(vc21022) partially inhibited the dendrite elongation defects of dyf-4 or daf-6 single mutants (Figs 5B and 6C).

Genetic interaction between glia compartment formation regulators, DYF-7 and the transition zone (TZ) in dendrite elongation

In addition to dyf-4 and daf-6 mutants, the phenomenon of shortened amphid or phasmid dendrites was previously reported in mutants of tectorin-related genes dex-1 and dyf-7, and mutants of certain combinations of TZ genes. DEX-1 and DYF-7 act in extracellular matrixes to anchor the dendritic tips or prevent the rupture of the connection between the sheath glia and the socket glia, deletion of either protein causes dendrite collapse [7,21]. Combined mutation of individual gene from CEP290, MKS module or NPHP module results in severe dendrite elongation defects in phasmid [15,17–20]. Although DYF-7/DEX-1 module and TZ proteins function at different developmental stages, and probably regulate the dendrite elongation through different mechanisms, the genetic interaction between them in phasmid dendrite length has been observed [15]. Therefore, we wondered whether there are genetic interactions between glia compartment formation regulators and luminal ECM components or the ciliary TZ in dendrite extension. Since the phasmid dendrites in dyf-4 or daf-6 single mutants collapsed completely, they are not suitable for the synergetic interaction assay. Then we asked whether suppressors of dyf-4 and daf-6 could suppress the phenotype of dyf-7(ns117) or nphp-1; mks-6 double mutants. To this end, we generated wsp-1; dyf-7 double mutants and wsp-1; nphp-1; mks-6 triple mutants. Interestingly, we did observe that wsp-1 significantly suppressed the dendrite collapse in phasmid in either dyf-7 or nphp-1; mks-6 double mutants (S4A Fig), suggesting that there are genetic interactions (even probably indirectly) between glia compartment morphogenesis, dendrite anchoring complex and the TZ in dendrite extension.

The ciliogenesis defects of dyf-4 and daf-6 mutants can be rescued by suppressors

In addition to the phenotype of short dendrites, ciliogenesis is also defective in dyf-4 and daf-6 mutants. The cilia are significantly shorter and usually lack distal segments in both types of mutants (Fig 7A). The length of cilia is approximately 7 μm on average in WT, while it is only approximately 3 μm in both mutants (Fig 7B). The intraflagellar transport (IFT) machinery is essential for cilia biogenesis [30]. The examination of various IFT components, including the IFT-B components OSM-6 (the homolog of IFT52) and OSM-5 (the homolog of IFT88), the IFT-A component CHE-11 (the homolog of IFT14) and the BBSome component BBS-7, showed that the ciliary fluorescence levels of all these IFT components were dramatically decreased, suggesting that the ciliary entry of IFT was compromised in dyf-4 and daf-6 mutants (S5A Fig). Notably, the observed ciliogenesis defects could be fully rescued by the transgenic expression of the wild-type dyf-4 or daf-6 genomic sequence (Fig 7A and 7B). How glial dysfunction indirectly impacts IFT behavior and ciliogenesis in sensory neurons remains an open question.

Fig 7 — (A) Fluorescence micrographs of amphid and phasmid cilia in the indicated worm lines expressing OSM-6::GFP. (B) Phasmid cilia length quantification in the indicated worm lines. (C) Fluorescence micrographs of phasmid cilia in the indicated genotypes. (D) Percentage of severely collapsed dendrites in the phasmid in the indicated worm lines. Scale bars: 5 μm. Data are presented as the mean ± SEM (n ≥ 60 for each genotype). ***P < 0.001 (Mann-Whitney test for Fig 6B, Fisher’s Exact test for Fig 6D).

If the ciliary defects in dyf-4 or daf-6 mutants are non-cell autonomous consequences caused by dysfunctional glial cells, glial suppressors of dyf-4 and daf-6 might also rescue ciliogenesis defects. Consistent with this hypothesis, deletion of wsp-1, the suppressor of glial compartment formation of daf-6 mutants, indeed restored cilia length in dyf-4 or daf-6 mutants (Fig 7C and 7D).

Discussion

Previous studies demonstrated that the patched-related transmembrane protein DAF-6 functions in glia cells to negatively control the sensory compartment pore size [11]. Here, we identified a glial secretory protein DYF-4 as an apparent regulator of DAF-6. We showed that dyf-4 mutants completely recapitulate the phenotype of daf-6 mutants, and the suppressors of daf-6 mutants can also rescue defects of dyf-4 mutants. We demonstrated that DYF-4 interacts with DAF-6 and is required for the localization of DAF-6. Our results strongly indicate that DYF-4 functions in a same pathway as DAF-6 to regulate the sensory compartment formation (Fig 8).

Fig 8 — The glial channel morphogenesis coordinates with the dendrite tip anchoring and ciliogenesis. DAF-6 restricts glial channel growth, while suppressors of *daf-6* promote it [12]. DYF-4 regulates the function of DAF-6, which in turn restricts the expansion of glia channel.

It was originally proposed that DAF-6 is required for sensory pore lumen opening [11]. Later, it was discovered that DAF-6 may not be required for pore lumen opening, but restricts the pore lumen size [12]. EM studies of amphids in daf-6 mutants indicated that the initial stages of sheath and socket channel development are unperturbed, but sheath lumen starts bloating from embryo about 420 min post-fertilization (before cilia have formed) and gradually becomes more and more serious as it grows up [12]. Starting from L2, the sheath and socket channels are no longer continuous, and dendrite endings become disorganized [12,27]. As dendrite tips are anchored in the extracellular matrix of pore lumen, disorganized pore lumen will certainly affect the position of dendrite tips. Therefore, it is plausible to speculate that dendrite defects in daf-6 mutants are a secondary phenotype as the amphid channel structures become increasing disorganized. This hypothesis was supported by our observation that reported glial suppressors of daf-6 could suppress the dendrite defects of dyf-4 and daf-6 mutants. Since all the phenotypes of dyf-4 mutants observed so far are similar to daf-6 mutants, the short dendrite phenotype in dyf-4 mutants should also be a secondary phenotype. In future, we need to use TEM and fluorescence visualization techniques to characterize the sheath and socket channel defects of dyf-4 mutants at different stages of growth in more detail, and compared with daf-6 mutants to test our hypothesis and understand the underlying mechanism.

The molecular mechanism of DAF-6 regulating glia morphology is still not clear yet. According to the literature, it is generally believed that DAF-6 acts as a receptor like Patched. It is thought that DAF-6 regulates the size of glia channel by controlling the amount of membrane deposited on the glia channel surface through regulating exocytosis and/or endocytosis [9,11,12]. If DAF-6 is indeed a receptor, DYF-4 may be a ligand/ regulator/chaperone of DAF-6, and it may cooperate with DAF-6 to control glia compartment formation by regulating exocytosis and/or endocytosis related vesicular dynamics. In addition to function as receptors, several SSD-proteins have been shown to act as transporters in intracellular vesicles. For example, NPC1 (Niemann-Pick disease, type C1), a protein containing SSD, cooperates with a secreted protein NPC2 to transport cholesterol in lysosomes [31]. Interestingly, DYF-4 is a secreted protein containing a signal peptide and is localized to punctate vesicular structures, and DAF-6 has previously been observed in intracellular vesicles within the cytoplasm in vulva and excretory canal cells [11]. Therefore, an attractive possibility is that, like the relationship between NPC1 and NPC2, DAF-6 and DYF-4 cooperatively function in intracellular vesicular compartments to regulate the sorting and recycling of certain cargos critical for vesicular dynamics and/or membrane formation. So far, there is no evidence to support that DAF-6 is a receptor yet. In addition, our result that the neuronal expressed DYF-4 could not rescue the defective phenotype of dyf-4 mutants (Fig 3C) argues for a glial cell-autonomous requirement for DYF-4, which is inconsistent with the ligand-receptor relationship. In order to determine the molecular function of DAF-6 and DYF-4 in regulating the morphogenesis of glia channels, it will be necessary to conduct further detailed studies on the biochemical characteristics of DAF-6 and DYF-4, the characteristics and behaviors of intracellular vesicles of DYF-4 and their relationship with DAF-6 in future.

Materials and methods

C. elegans strains

The C. elegans strains used in this study are listed in S1 Table. The worms were cultured and maintained on NGM agar seeded with OP50 bacteria at 20°C under standard conditions. The dyf-4(jhu431) and daf-6(jhu500) mutants were identified from an EMS screening for mutants showing dye filling defects. Standard genetic crossing was used to introduce reporter transgenes into worms of various genetic backgrounds and generate double or triple mutants. Genotyping was performed using PCR and DNA sequencing.

Dye-filling assay

Worms were rinsed into a 1.5 ml EP tube with M9 buffer, followed by centrifugation in a desktop centrifuge at low speed. Then, the supernatant was removed, and the worms were washed twice with M9 buffer. After washing, the worms were incubated with 10 μg/ml DiI in M9 buffer at room temperature for 2 h and were then washed again with M9 buffer three times. Dye filling in the amphid was observed with a Nikon SMZ18 stereomicroscope, and dye filling in the phasmid was observed with a Nikon Eclipse Ti microscope with a Plan Apochromat 100× objective.

Plasmids and transgene generation

To generate the Pdyf-4::DYF-4::GFP plasmid, a DNA fragment including the 1511 bp promoter and the whole genomic DNA sequence of dyf-4 was cloned into the SmaI and KpnI restriction sites of the GFP vector pPD95.75. For the construction of Pdyf-4::DYF-4^C366Y::GFP, the guanine (G) nucleotide at site 1097 of the dyf-4 CDS was replaced by adenosine (A). For the construction of Pdyf-4::GFP, 1511 bp of the promoter of dyf-4 was amplified by PCR and ligated into the SmaI and KpnI restriction sites of pPD95.75. To generate Pdaf-6::DAF-6::GFP, 3096 bp of the promoter of daf-6 was ligated into the BamHI and SmaI restriction sites of pPD95.75, and the CDS of daf-6 was ligated into the SmaI and KpnI restriction sites of pPD95.75. To construct Pdaf-6::DYF-4::GFP and Pdyf-7::DYF-4::GFP, the dyf-4 CDS was ligated into the SmaI and PstI restriction sites of pPD95.75, and 3096 bp of the promoter of daf-6 or 1758 bp of the promoter of dyf-7 was ligated into the PstI and SmaI restriction sites of pPD95.75. To obtain transgenic worms, plasmids were injected into wile-type animals at 30 ng/μl with the dominant roller marker pRF4 [rol-6(su1006)] at 30 ng/μl.

Microscopy and imaging

Worms were anaesthetized using 20 mM levamisole, mounted on 4% agar pads and then imaged by using a Nikon Eclipse Ti microscope or Olympus FV1000a confocal microscope. L4 worms were used to record phasmid dendrite length statistics. The fluorescence intensity was measured using NIS-Elements software. The relative fluorescence intensity of cilia was calculated by subtracting the average fluorescence intensity outside the cilia from the average fluorescence intensity inside the cilia.

Immunofluorescence of DYF-4::GFP

Worms harboring the DYF-4::GFP construct were placed on a glass slide and squashed with a coverslip. Then, the slide was frozen in liquid nitrogen, and the coverslip was removed. Then, the sample was fixed in −20°C methanol for 20 min, followed by fixation in −20°C acetone for 10 min, washing in PBS, blocking in 3% BSA, and sequential incubation with an anti-GFP primary antibody (mouse anti-GFP, 1:200, 11814460001, Roche) and a corresponding secondary antibody (goat anti-mouse Alexa Fluor 488). A Nikon Eclipse Ti-E microscope was used for observation and imaging.

GST pull-down assay

Plasmids for the expression of His-tagged DAF-6 fragments or GST-tagged DYF-4 were constructed using pET28a or pGEX-4T-1, respectively. All plasmids were transfected into Escherichia coli strain BL21 (DE3) to express the proteins. Protein expression was induced with 0.5 mM IPTG at 18°C overnight, and the proteins were then purified by using Ni-Sepharose beads (GE Healthcare) or GST Sepharose beads (GE Healthcare). GST pull-down was performed as described previously [32]. Briefly, the purified His-DAF-6 ECL1 or His-DAF-6 ECL4 protein was incubated with GST-DYF-4 or GST immobilized on glutathione Sepharose beads in binding buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM dithiothreitol, 10% glycerol, protease inhibitors) for 4 h at 4°C. After incubation, beads were washed 5 times with the binding buffer, after which the loading buffer was added, and the sample was boiled for 10 min. The samples were then subjected to SDS-PAGE and analyzed by western blotting with a monoclonal antibody against His.

Transmission electron microscopy

Your adult worms were immersed in 2.5% glutaraldehyde in cacodylate buffer for 24 h at 4°C, and then postfixed in 1% osmium tetroxide in cacodylate buffer for 4 h at 4°C. Then samples were dehydrated in a graded series of ethanol, and washed three times with pure acetone and infiltrated with a mixture of acetone and EPON 812 resin in a graded series, and finally embedded in EPON 812 resin. Ultrathin sections (~70 nm) were cut from worm heads and collected on mesh copper grids. Then samples were stained with uranyl acetate and lead citrate and imaged with a transmission electron microscope (Hitachi H-7650; Hitachi).

Statistical analysis

Statistical differences between two samples were analyzed by Fisher’s Exact test (for categorical data) or Mann-Whitney test (for continuous data). P values >0.05 were considered to indicate a nonsignificant difference. P values < 0.001 (marked as ***) were considered to indicate a significant difference.

Supporting information

S1 Fig. Protein structure prediction for DYF-4.

(A) Signal peptide prediction for DYF-4 by SignalP4.1. (B) Sequence alignment of the PLAC domain. *, the conserved cysteines in the PLAC domain.

(TIF)

Click here for additional data file.^{(2.4MB, tif)}

S2 Fig. DYF-4 is expressed in glial cells.

(A) The subcellular localization pattern of DYF-4^C366Y::GFP stained by anti-GFP. Scale bar: 5 μm. (B) The subcellular localization pattern of DYF-4 ^Δ(1–16)::GFP. Scale bar: 5 μm. (C) The localization of DYF-4::OPT-GFP in a 3 fold embryo. White arrow indicates the localization of DYF-4 in amphid glial cells. White asterisk indicates the localization of DYF-4 between the embryo and the eggshell. (D) Subcellular localization of DAF-6::GFP in young adults of WT and dyf-4 worms.

(TIF)

Click here for additional data file.^{(4.1MB, tif)}

S3 Fig. The glial compartment is compromised in dyf-4 mutants.

(A) Fluorescence micrographs of curved cilia in dyf-4 and daf-6 mutant worms. Curved cilia are indicated by red arrowheads. Scale bar = 5 μm. (B) Phasmids of WT and dyf-4 worms expressing F16F9.3pro::mCherry (Red, sheath glia) and OSM-6::GFP (Green, neurons). Sheath glia cells in dyf-4 mutants could not extend as in WT. Scale bar = 10 μm. (C) WSP-1A localization at the head and tail in WT, dyf-4(m158) and daf-6(e1377) worms. Scale bars: 2 μm. (D) Quantification of GFP::WSP-1A signal length in the amphids of WT, dyf-4(m158) and daf-6(e1377) worms. (E) Quantification of GFP::WSP-1A signal length in the phasmids of WT, dyf-4(m158) and daf-6(e1377) worms. (F) ARX-2 localization at the head and tail in WT, dyf-4 and daf-6(e1377) worms. Scale bars: 2 μm. (G) Quantification of ARX-2::GFP signal length in the amphids of WT, dyf-4(m158) and daf-6(e1377) worms. (H) Quantification of ARX-2::GFP signal length in the phasmids of WT, dyf-4(m158) and daf-6(e1377) worms. All data are presented as the mean ± SEM (n ≥ 50 for each genotype). ***P < 0.001 (Mann-Whitney test).

(TIF)

Click here for additional data file.^{(3.1MB, tif)}

S4 Fig. Genetic interaction between glia compartment formation regulators, DYF-7 and the transition zone in dendrite extension.

(A) Quantification of phasmid dendrite length in the indicated worm lines. The defective dendrite phenotype in dyf-7(ns117) mutants and nphp-1(ok500); mks-6(gk674) double mutants was rescued by wsp-1a(gm324). Each data point represents a single measurement. n represents number of dendrites analyzed. ***P≤0.001 (Mann-Whitney test).

(TIF)

Click here for additional data file.^{(598.4KB, tif)}

S5 Fig. The localization of IFT components in WT, dyf-4 and daf-6 worms.

(A) Fluorescent micrographs and quantification of the relative fluorescence intensities in the phasmid cilia of WT, dyf-4(m158) and daf-6(e1377) worms expressing various IFT markers: IFT-B components OSM-6::GFP and OSM-5::GFP, IFT-A component CHE-11::GFP and BBsome component BBS-7::GFP. Data are presented as the mean ± SEM (n ≥ 60 for each genotype). ***P≤0.001 (Mann-Whitney test). Scale bars: 5 μm.

(TIF)

Click here for additional data file.^{(3.2MB, tif)}

S6 Fig. Full scans of western blots for Fig 5A and 5B.

(TIF)

Click here for additional data file.^{(6.8MB, tif)}

S1 Table. Worm strains used in this study.

(DOCX)

Click here for additional data file.^{(23KB, docx)}

S2 Table. Peptide sequence information of extracellular loops in DAF-6 and CHE-14.

(DOCX)

Click here for additional data file.^{(15.3KB, docx)}

Acknowledgments

We thank the Caenorhabditis Genetics Center, the Japanese National Bioresource Project, and Dr. Guangshuo Ou for worm strains.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by National Natural Science Foundation Youth Project of China (Grant 31702019) to H.H., and National Natural Science Foundation of China (Grant 31671549) to Q.W. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Genet. doi: 10.1371/journal.pgen.1009618.r001

Decision Letter 0

Gregory S Barsh, Andrew D Chisholm

22 Dec 2020

Dear Dr Wei,

Thank you very much for submitting your Research Article entitled 'DYF-4 regulates patched-related/DAF-6-mediated sensory compartment formation in C. elegans' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a much-revised version. We cannot, of course, promise publication at that time.

Should you decide to revise the manuscript for further consideration here, your revisions should address the specific points made by each reviewer. We will also require a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please pay particular attention to the comments from Reviewer 3, as some of these will require further experimental work to address, e.g. analysis of the glial channel, analysis of earlier developmental stages, and additional controls for the GST pulldown studies.

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Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: From a forward genetics dye filling screen, this study identifies dyf-4 as a likely extracellular regulator of sensory neuron dendrite extension and cilium formation. Although dyf-4 had been previously described in an earlier screen, it had not been cloned. The current study’s screen also identified alleles in daf-6 that phenocopy the dendrite/cilium defects of dyf-4 worms. daf-6 (Patched homologue) is a known positive regulator of dye filling and negative regulator of amphid sensory pore size. The present study reports that DYF-4 is expressed in the sensory pore glial cells, colocalising via an interdependent manner with DAF-6 at the sensory pore part of those cells; they also report that both proteins biochemically interact. The paper also shows that dyf-4 and dyf-6 interact in a suppressive manner to control dendrite extension and cilium length/IFT with wsp-1 and lit-1, which are known suppressors of the daf-6 dye filling and amphid sensory pore size defects. From these data, the authors propose that DYF-4 is an extracellular ligand for DAF-6 at the glial cell membrane, serving to regulate the glial sensory pore channel, and control dendrite extension and cilium formation.

DYF-4 is therefore a new glial regulator of C. elegans sensory pores, joining a group that includes DAF-6, WSP-1 and LIT-1. Overall, we do not know very much about how sensory pores form, and how cell-cell communication between the glial cells and ciliated sensory neurons is coordinated during this process. Thus, the topic is certainly of importance and broad interest.

Major comments:

1. The evidence for DYF-4 being an extracellular DAF-6 ligand is not very strong. Whilst the authors show that DYF-4 can bind two of the DAF-6 ECLs and demonstrate interdependent localisations, clearly more needs to be done to demonstrate a ligand-receptor relationship. Indeed, it is not clear from the data if DYF-4 is an extracellular protein, within the sensory compartment lumen, as the images do not provide enough resolution to demonstrate this. The findings of glial cell mislocalisation for DYF-4(delta 1-16) and DYF-4(C366Y) do not prove the extracellular properties of DYF-4. Also, it would be nice to show that the DAF-6/DYF-4 interaction from worm lysates. In fairness to the authors, they state in their discussion that more work needs to be performed to prove the extracellular ligand-receptor hypothesis. Therefore, they need to be more cautious about over interpretation. For example, in lines 218-219 of the results, and line 38 of abstract. I don’t expect the authors to do any more experiments for this comment, but just tone down the language in places and make it very clear what still needs to be done to prove the ligand-receptor model.

2. The paper’s major conclusion is that DYF-4 regulates sensory compartment formation, and this in turn leads to dendrite anchoring problems. Whilst the authors show mispositioning of amphid/phasmid cilia in dyf-4 L4-aged mutant, which by implication means the corresponding pores are abnormal, there is no actual data that shows dyf-4 pore lumen morphogenesis defects, as was presented previously for dyf-6 embryos/larvae by the Shaham group. Ideally, this new work would perform some TEM analysis of the dyf-4 amphid pore lumen at early ages to help prove the cause and effect relationship that is proposed. At the very least, they should include a statement in the discussion saying that TEM should be performed and results compared to dyf-6 worms.

Minor comments:

1. Some quantification of the data in S3H should be provided, as was done in Fig. 1D.

2. Fig. S4A. What exactly does ‘severely collapsed’ mean. How was this determined for the analysis shown?

3. Line 361: I think the word ‘act’ is missing.

Reviewer #2: Sensory neurons in C. elegans extend ciliated endings into the environment by protruding through tube-like glia called the sheath and socket cells. These glia contain a sensory matrix that shapes the dimensions of the glial channel while also anchoring neuronal dendrites at the glial socket. Prior studies implicated the Patched-related transmembrane protein DAF-6 in secretion or endocytosis of sensory matrix components (or some related aspect of matrix organization). Here, Hong et al identify a secreted protein, DYF-4, as an apparent partner of DAF-6.

I found this study to be very exciting! Patched-related proteins are an intriguing group of putative transporters, probably of lipids or other hydrophobic cargo. Their regulation is not well understood, though there is precedence for them acting in tandem with a soluble partner (e.g. NPC1 acts with NPC2). The relationship between DYF-4 and DAF-6 proposed here is strongly supported by shared mutant phenotypes and shared genetic interactions with suppressors, double mutant analysis, co-localization experiments, and physical binding interactions. It is a beautiful example of the power of forward genetics to find genes that act together in a biological process.

Most of my comments below have to do with improving clarity, addressing “rigor and reproducibility” issues, and adding some better context to the discussion.

Specific comments

1. Figure 1: Rather than a defect in “dendrite extension” (as the title states), this appears to be a defect in dendrite anchoring. Does the vertical line at left represent the nose tip position? If so, it appears amphid cell bodies have migrated to the appropriate location, but dendrite tips are not anchored at the nose. Similar comment applies to the phasmid defect. Actual dendrite length or the distance from nose tip to dendrite should be quantified here to capture this major aspect of the phenotype. To better understand the mutant phenotype, it would be helpful to visualize the socket and sheath glia positions and morphology (e.g. as in Low et al 2019).

2. Figure1F: what is the “phasmid dendrite defect ratio”? It is not defined in the text or legend. Is it something different from the % of severely collapsed dendrites?

3. Figure 2 and 2F: similar comments apply here as for Figure 1. Legend: “daf-6(e1377) was previously reported”. Please cite reference for this statement.

4. Figure 3: Photos are representative of how many animals imaged for these expts? What stage is being imaged? Is DYF-4 present in the developing glia of embryos (as implied in Figure 7)? Unfortunately, it is impossible to judge if DYF-4 is extracellular or in a vesicular compartment close to the apical membrane.

5. Figure 4A,B: Photos are representative of how many animals imaged for these expts? What stage is being imaged? Given the eventual loss of the channel in daf-6 mutants, it may be more informative to look at an earlier stage of channel development, when it is still intact. The word “extracellular” appears within panel A erroneously.

6. Figure 4C,D. What specific sequences are included in the ECL1 and ECL4 constructs? I do not see this information in the Methods section.

7. Figure 5: What igbd-2 allele was used here? It should be listed in legend.

8. Figures 5, S4: How were “severely collapsed dendrites” defined?

9. Figure 7 (model): The model should more clearly show what is being newly proposed based on the current work. Currently, this model is mostly summarizing data from prior Shaham or Heiman lab papers about daf-6 or dyf-7 (which should therefore be cited in the legend). There are no data shown in this paper to address whether the glial channel is initially open or whether socket and sheath glia detach in dyf-4 mutants. The placement of DYF-4 inside the sheath cell is particularly confusing, and it should be more clearly explained – are you proposing that DYF-4 is in a vesicular compartment (e.g. like NPC2) or in the extracellular matrix? Perhaps both models should be shown.

10. It would be informative to mention NPC1/NPC2 precedent in the Discussion.

11. Table S1: What is IsOSM-6? This is non-standard nomenclature. Also, Ex transgene names should have lab and numerical identifiers.

12. Statistical methods should be re-evaluated in consultation with a statistician. The authors use a student’s t-test for both categorical and continuous data. Categorical data (e.g. dyf or not dyf; severely collapsed or not) should be analyzed with a Fisher’s Exact test instead. A t-test is appropriate for continuous data (e.g. distance measurements), but only if the data have a normal distribution and similar variance between groups, which doesn’t always seem to be the case here. Consider a non-parametric test such as Mann-Whitney instead.

13. There are many typos or grammatical errors, some of which are listed below. A copy editor will be needed.

line 50: “how DAF-6 functions and be regulated”

lines 99-100: glial channel formation requires the dendrite tip anchoring protein DYF-7 (Low et al., 2019), sheath cells fail to undergo extension in dyf-7 mutants (Heiman and Shaham, 2009).

Line 150: “defects described here is a new phenotype”

Line 180: “DYF-4 is at least partially colocalizes with DAF-6”

Line 188: “DYF-4 function in a same pathway as DAF-6”

Line 203: “is the results of”

Lines 248-249: “results in severely dendrite elongation defects …”

Reviewer #3: This manuscript presents the identification of dyf-4, a gene involved in sensory dendrite/cilia development. It encodes a secreted protein that the authors show is expressed in glia and localizes to a glial channel around the dendrites/cilia. The authors identify an interesting relationship with DAF-6, a glial multipass transmembrane protein that has been previously shown to shape the glial channel. Specifically, each protein fails to localize normally in the absence of the other, and the authors use a GST pull-down assay to show a physical interaction between DYF-4 and the extracellular loops of DAF-6. The identity of DYF-4 and its potential interaction with DAF-6 are interesting, however the data seem preliminary and do not support the main conclusions of the paper.

1. The authors interpret their results in terms of morphogenesis of the glial channel. However, the data mostly relate to dendrite length and the glial channel is not examined directly at all. This is a major shortcoming. The authors seem to use dendrite length as a proxy for glial channel morphogenesis but it is not clear what the glial channel defects are, when they appear, or how (if) they relate to dendrite/cilia development.

2. The authors interpret their results in terms of developmental defects. However, the data relate to L4 stage phenotypes. Embryos (or even L1s) are not examined. This is another major shortcoming, especially in light of the relationship to DAF-6, which has progressively more severe defects, becoming extremely disorgnized by L4/adult. daf-6 dendrites are short only in adults, not in L2 larvae (Albert et al. 1981). It is not clear if dyf-4 affects dendrite/cilia development directly or if these are secondary defects that develop later as the structure becomes increasingly disorganized, as is the case for daf-6.

3. Many of the results are inconsistent with previously published work. In principle this is not an issue but there should be some attempt to acknowledge/reconcile the differences. The defining phenotype of daf-6 mutants is the accumulation of vacuoles/enlarged secretory structures at the distal glial channel which does not seem to be seen here. Conversely the authors report shortened cilia which is not consistent with previous studies. Can the authors account for these differences?

4. The localization defects are interesting but are more consistent with a chaperone role in which DYF-4 and DAF-6 mutually require each other for trafficking to the cell surface. However the authors' interpretation is that DYF-4 is an extracellular ligand for DAF-6, acting in the glial channel, despite the fact that DYF-4 cannot rescue when expressed from the neurons. This is very confusing and seems to contradict their results.

5. The genetic interaction experiments are also very confusing. dyf-4 and daf-6 do not interact genetically with dyf-7, but suppressors of dyf-4 and daf-6 suppress dyf-7. What is the significance of this? The authors conclude "there are genetic interaction between glia compartment morphogenesis, dendrite anchoring complex and the TZ in dendrite extension". But, it is not clear that any of these mutants are affecting the same steps – for example, dyf-7 dendrite extension defects occur in embryos, and daf-6 dendrite length defects do not occur until late larval development. These experiments also use a hypomorphic allele of dyf-7 which complicates the analysis. All of these suppressor/enhancer experiments seem quite preliminary and inconclusive.

6. The GST pull-down experiments are potentially promising but also seem preliminary and to lack appropriate controls. It is curious that both the extracellular loops of DAF-6 independently bind DYF-4 equally well. Perhaps DYF-4 is just "sticky"? It would help to include some negative controls, for example extracellular loops of related proteins like CHE-14 that are not expected to bind DYF-4, or point mutations in the DAF-6 loops that have been shown to disrupt its function. S253P in loop 1 and F568L in loop 4, which disrupt localization of DAF-6 similarly to loss of dyf-4 (Perens et al. 2005), would be especially good candidates to investigate.

7. The model figure seems to place WSP-1 and LIT-1 upstream of DAF-6 and DYF-4. This is not consistent with the genetic data, in which loss of these WSP-1/LIT-1 suppresses loss of DAF-6/DYF-4. The model figure itself adds very little to the paper other than re-stating the phenotypes, while omitting the key features of the daf-6 mutant (over-expansion of the sheath pore in the embryo).

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: No: numerical data underlying graphs are not provided

Reviewer #3: Yes

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

PLoS Genet. 2021 Jun 11;17(6):e1009618. doi: 10.1371/journal.pgen.1009618.r002

Author response to Decision Letter 0

24 Mar 2021

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Submitted filename: Response to reviewers 0320.docx

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PLoS Genet. doi: 10.1371/journal.pgen.1009618.r003

Decision Letter 1

Gregory S Barsh, Andrew D Chisholm

15 Apr 2021

Dear Dr Wei,

Thank you very much for submitting your Research Article entitled 'DYF-4 regulates patched-related/DAF-6-mediated sensory compartment formation in C. elegans' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some concerns that we ask you address in a revised manuscript

We therefore ask you to modify the manuscript according to the review recommendations. Your revisions should address the specific points made by each reviewer. We agree with the reviewers' comments that data shown in the rebuttal letter needs to be incorporated into the final manuscript, most likely as additional supplemental data.

The C. elegans transgenic nomenclature needs to conform with standards in this field, specifically all transgenes should have unique identifying Ex or Is numbers. The Methods or Supplementary Table should also state the concentrations of experimental and coinjection marker DNAs used in transgene generation.

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1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

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Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: Authors have done a good job addressing my comments.

Reviewer #2: Hong et al provide compelling evidence that the secreted protein DYF-4 functions with the Patched-related protein DAF-6 to affect the morphology of C. elegans glial sensory channels and their cognate sensory neurons. The discovery of a DAF-6 partner is very significant given the widespread importance of Patched, Dispatched, NPC1, and other Patched-related proteins; therefore this paper should be of broad interest.

The revision now provides more direct evidence for glial channel defects by visualizing the sheath and socket glia with fluorescent markers and by TEM; this confirms that the dyf-4 and daf-6 phenotypes are really similar.

The authors have also removed claims for a ligand-receptor relationship between DYF-4 and DAF-6, and instead more appropriately consider several possible models for their relationship.

There are remaining issues:

There are many places where the authors provided clarifying information in the "response to reviewers", but then failed to include that information in the actual manuscript. This information is needed for readers to understand (and believe) results shown in the paper.

1. Figure 1B, 2B legends should explain that the vertical line at left represents the nose tip position.

2. Figure1F, 5C, 6D: The “phasmid dendrite defect ratio” and/or "severely collapsed dendrites" should be defined in the Methods or legends.

3. Regarding the timing of the phenotype, L1 and L2 data shown in the "response to reviewers" (Rev 3 Question 2) should be shown and discussed in the manuscript (could be supplemental).

4. The DYF-4::OPT-GFP embryo image included in the "response to reviewers" should be shown in Figure 3 or S2, as it clearly shows that the protein is present during embryonic development and is secreted, accumulating between the embryo and the eggshell.

5. Figures 3 and 4: The legends should list the stage and say that these are "representative of more than 100 worms" (or whatever is the case).

6. Figure 3E. The specific sequences included in the ECL1 and ECL4 constructs should be listed in the Methods section or in a supplemental Figure/Table.

7. Figure 3E. The control pull-down experiments done with CHE-14 (shown in response to reviewer 3) should also be shown in the manuscript (could be supplemental).

8. Table S1: The OSM-6::GFP nomenclature was fixed, but a large number of QW strains still use non-standard nomenclature. All Ex transgenes should be named with lab and numerical identifiers.

9. There are still typos or grammatical errors, some of which are listed below.

Fig. S6: The figure panel itself says it refers to Fig. 4C, whereas the legend refers to Fig. 3E.

Lines 146-148: "It is likely that the abnormal morphology of glial channels changes the anchoring position of the tips of dendrites, which indirectly leads to the shortening of dendrites". This sentence sounds like pure speculation - please add "see below" or give some indication that data about glial channel morphology will be shown later in the manuscript.

Line 190: "only partial of amphids"

Line 202: "Dendrite defects are much more severed" (severe)

10. Some of the data in the Supplemental Figures are quite important and really should be shown in a main Figure instead.

For example, rescue data in Fig. S2B (text lines 156-159) argue for a glia cell-autonomous requirement for DYF-4, which is more consistent with a role in DAF-6 trafficking or some other vesicular process, and less consistent with a ligand-receptor relationship on the external cell surface (as pointed out by Reviewer 3). The data could be highlighted and discussed in this context.

DYF-4::OPT-GFP imaging data in Fig. S2F shows the punctate localization that leads authors to propose DYF-4 might be in vesicles (text lines 177-178).

Double mutant data in Fig. S3I is critical for the conclusion that dyf-4 and daf-6 act in the same pathway.

Reviewer #3: The authors have clearly made a diligent attempt to respond to the comments. The manuscript has been improved by their revisions. By being somewhat more cautious in its conclusions and providing better support for the central claims, the revised manuscript really highlights the strengths of this exciting story.

Regarding my previous major comments:

1. The EM and other additional data related to visualizing defects in the glial channel have improved the story.

2. Regarding timing of the phenotypes, the data that are shared privately in the reviewer response are excellent. In my view this result, showing that dendrite defects only appear at the L2 stage, is essential to interpreting the results. **It needs to be included in the manuscript.**

3. These clarifications and additions are very helpful.

4. The revised discussion of the DYF-4-DAF-6 relationship is a major improvement.

5. The discussion of genetic interactions remains somewhat confusing, and possibly misleading. It is really important to clarify in the text that dendrite defects in dyf-7, TZ mutants, and daf-6/dyf-4 arise at different developmental stages, suggesting that any genetic interactions among them would be indirect.

The authors respond to my comments, "Although DYF-7 anchoring complex forms in the embryo, glia channel defects in later stage will definitely change the position or composition of extracellular matrix which will in turn affect the position of dendrites." My understanding is that DYF-7 controls dendrite length by preventing rupture of the developing sheath-socket-hyp epithelium in the embryo. It is not clear to me how the ECM would affect dendrite anchoring after the epithelium has already ruptured. It seems like these genes are acting at different developmental stages, and probably through different mechanisms.

6. The additional figure shared privately in the rebuttal letter is very helpful and strengthens an important conclusion of the paper. Because it addresses an important point that many readers will consider, I would **strongly encourage** the authors to add it to the manuscript.

I have a few other minor comments that might improve the manuscript, but these should be handled at the authors' discretion and are not an impediment to publication:

1. In the introduction, perhaps revise to "the distal portions of *most* cilia extend through a pore formed by the membrane of the socket cell" to clarify that this is not the case for some cilia (AWA, AWB, AWC, AFD).

2. Several minor typos – "compartmentation" for "compartmentalization", "severed" for "severe", "socked" for "socket", "Gila" for "Glia" in Fig 7.

3. It is surprising that DAF-6 ECL1 and ECL4 both bind DYF-4. It would help to note whether these sequences share any motifs (does the 12 TM structure of DAF-6 look like a duplication of a more ancestral 6 TM sequence, with ECL4 homologous to ECL1?). As noted, the additional CHE-14 control included in the rebuttal letter would really help to shore up the specificity of this result.

4. "the localization of DYF-4::OPT-GFP is basically normal, only partial of amphids had abnormal DYF-4 signals". I think this should read "only a fraction of amphids", and it would be very helpful to cite what proportion (even a ballpark estimate, e.g. "~5%", "<20%", etc.).

5. In Fig. 4B, a zoomed-out view would help – is DAF-6 mostly stuck in the cell body? I am confused why a 12-TM protein is giving a diffuse cytoplasmic signal. It looks like soluble GFP. Perhaps clarify in the text what we are looking at.

6. In Fig. 5, the difference between % Dye-filling and % severely collapsed is potentially confusing. For example, wsp-1a; dyf-4 appears to be almost fully rescued for dendrite collapse but nevertheless fails to dye-fill. I think this is because many dendrites are of intermediate length – it might help to clarify this in the text. On a related note, it would help to be consistent between Fig. 5A and 5C in whether you show the fraction that are normal or defective (so WT would be 100% or 0% in both plots).

7. The claim that "the actin cytoskeleton is likely the major downstream functional site of DYF-4-DAF-6-regulated glial function" seems like it goes far beyond what the data support. There does not seem to be any evidence yet to distinguish whether WSP-1 acts downstream of DYF-4/DAF-6 rather than in a parallel pathway. For example, if dyf-4/daf-6 defects arise by excess vesicle fusion to the forming channel lumen, then wsp-1 mutants might suppress this defect by reducing vesicle delivery to the apical surface. This seems quite possible, and dyf-4/daf-6 would not affect actin in this case at all. I would be very careful here.

8. In the model drawn in Fig. 7, showing DAF-6 as an inhibitor of glia channel morphogenesis suggests that loss of DAF-6 should improve channel morphogenesis – but in fact, loss of DAF-6 disrupts morphogenesis. I understand what you are trying to convey, but something like "glial channel growth" or "expansion" might be better.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Reviewer #1: Yes

Reviewer #2: No: numerical data underlying graphs has not been provided

Reviewer #3: Yes

**********

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Reviewer #1: Yes: Oliver Blacque

Reviewer #2: No

Reviewer #3: No

PLoS Genet. 2021 Jun 11;17(6):e1009618. doi: 10.1371/journal.pgen.1009618.r004

Author response to Decision Letter 1

20 May 2021

Attachment

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PLoS Genet. doi: 10.1371/journal.pgen.1009618.r005

Decision Letter 2

Gregory S Barsh, Andrew D Chisholm

24 May 2021

Dear Dr Wei,

We are pleased to inform you that your manuscript entitled "DYF-4 regulates patched-related/DAF-6-mediated sensory compartment formation in C. elegans" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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----------------------------------------------------

Comments from the reviewers (if applicable):

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PLoS Genet. doi: 10.1371/journal.pgen.1009618.r006

Acceptance letter

Gregory S Barsh, Andrew D Chisholm

8 Jun 2021

PGENETICS-D-20-01746R2

DYF-4 regulates patched-related/DAF-6-mediated sensory compartment formation in C. elegans

Dear Dr Wei,

We are pleased to inform you that your manuscript entitled "DYF-4 regulates patched-related/DAF-6-mediated sensory compartment formation in C. elegans" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Protein structure prediction for DYF-4.

(A) Signal peptide prediction for DYF-4 by SignalP4.1. (B) Sequence alignment of the PLAC domain. *, the conserved cysteines in the PLAC domain.

(TIF)

Click here for additional data file.^{(2.4MB, tif)}

S2 Fig. DYF-4 is expressed in glial cells.

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S3 Fig. The glial compartment is compromised in dyf-4 mutants.

(TIF)

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S4 Fig. Genetic interaction between glia compartment formation regulators, DYF-7 and the transition zone in dendrite extension.

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S5 Fig. The localization of IFT components in WT, dyf-4 and daf-6 worms.

(TIF)

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S6 Fig. Full scans of western blots for Fig 5A and 5B.

(TIF)

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S1 Table. Worm strains used in this study.

(DOCX)

Click here for additional data file.^{(23KB, docx)}

S2 Table. Peptide sequence information of extracellular loops in DAF-6 and CHE-14.

(DOCX)

Click here for additional data file.^{(15.3KB, docx)}

Attachment

Submitted filename: Response to reviewers 0320.docx

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Attachment

Submitted filename: Response to reviewers 2nd.docx

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

DYF-4 regulates patched-related/DAF-6-mediated sensory compartment formation in C. elegans

Hui Hong

Huicheng Chen

Yuxia Zhang

Zhimao Wu

Yingying Zhang

Yingyi Zhang

Zeng Hu

Jian V Zhang

Kun Ling

Jinghua Hu

Qing Wei

Roles

Abstract

Author summary

Introduction

Results

dyf-4 encodes C13C12.2 and is required for dendrite extension in C. elegans

Fig 1. dyf-4 encodes C13C12.2 and is required for dendrite extension in C. elegans.

Fig 2. The daf-6 mutant mimics the phenotype of the dyf-4 mutant.

daf-6 mimics the phenotype of dyf-4

DYF-4 is a glial secretory protein required for DAF-6 localization

Fig 3. DYF-4 is a glial secretory protein.

Fig 4. DYF-4 is required for DAF-6 localization and glia channel formation.

DYF-4 is required for sensory compartment formation

DYF-4 acts in a same pathway as DAF-6

Fig 5. DYF-4 interacts with DAF-6 and acts in a same pathway.

daf-6 suppressors effectively restore dyf-4 mutant phenotypes

Fig 6. Suppressors of daf-6 inhibit defects in dendrite extension in both dyf-4 and daf-6 mutants.

Genetic interaction between glia compartment formation regulators, DYF-7 and the transition zone (TZ) in dendrite elongation

The ciliogenesis defects of dyf-4 and daf-6 mutants can be rescued by suppressors

Fig 7. Ciliogenesis is compromised in dyf-4 and daf-6 mutants and can be rescued by wsp-1.

Discussion

Fig 8. A model for the role of DYF-4 and DAF-6 in sensory compartment morphogenesis.

Materials and methods

C. elegans strains

Dye-filling assay

Plasmids and transgene generation

Microscopy and imaging

Immunofluorescence of DYF-4::GFP

GST pull-down assay

Transmission electron microscopy

Statistical analysis

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Gregory S Barsh

Andrew D Chisholm

Roles

Author response to Decision Letter 0

Decision Letter 1

Gregory S Barsh

Andrew D Chisholm

Roles

Author response to Decision Letter 1

Decision Letter 2

Gregory S Barsh

Andrew D Chisholm

Roles

Acceptance letter

Gregory S Barsh

Andrew D Chisholm

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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