The unfolded protein response is required for dendrite morphogenesis
The unfolded protein response is required for dendrite morphogenesis
Xing Wei 2 3
Audrey S Howell 2 3
Xintong Dong 2 3
Caitlin A Taylor 1 2 3
Roshni C Cooper 2 3
Jianqi Zhang 0 2
Wei Zou 2 4
David R Sherwood 2 4
Kang Shen 1 2 3
0 Division of Biostatistics, Department of Preventive Medicine, University of Southern California , Los Angeles , United States
1 Neuroscience Program, Stanford University School of Medicine , Stanford , United States
2 Reviewing editor: Graeme W Davis, University of California , San Francisco , United States
3 Department of Biology, Howard Hughes Medical Institute, Stanford University , Stanford , United States
4 Department of Biology, Duke University , Durham , United States
Precise patterning of dendritic fields is essential for the formation and function of neuronal circuits. During development, dendrites acquire their morphology by exuberant branching. How neurons cope with the increased load of protein production required for this rapid growth is poorly understood. Here we show that the physiological unfolded protein response (UPR) is induced in the highly branched Caenorhabditis elegans sensory neuron PVD during dendrite morphogenesis. Perturbation of the IRE1 arm of the UPR pathway causes loss of dendritic branches, a phenotype that can be rescued by overexpression of the ER chaperone HSP-4 (a homolog of mammalian BiP/ grp78). Surprisingly, a single transmembrane leucine-rich repeat protein, DMA-1, plays a major role in the induction of the UPR and the dendritic phenotype in the UPR mutants. These findings reveal a significant role for the physiological UPR in the maintenance of ER homeostasis during morphogenesis of large dendritic arbors. DOI: 10.7554/eLife.06963.001
The organization of dendritic arbors is fundamental to the shape and connectivity of the nervous
system (Ramo´ n y Cajal, 1911; Wassle et al., 1981). Complex and type specific dendritic arbors are
pivotal for many neurons to receive appropriate inputs from their receptive fields and to function
properly in a neural circuit (MacNeil and Masland, 1998). During development, dendrites acquire
their morphology by precisely regulated branch morphogenesis, which requires extracellular
interactions and intracellular signaling pathways (Jan and Jan, 2010). For example, several diffusive
or cell-surface molecules play instructive roles in guiding the growth and patterning of dendritic
arbors. The diffusible chemoattractant Semaphorin 3A instructs the dendritic extension of cortical
pyramidal neurons toward the pial surface (Polleux et al., 2000) while the graded expression of
transmembrane Semaphorin 1A regulates the precise targeting of the dendrites of projection neurons
in the Drosophila olfactory system (Komiyama et al., 2007). In the mammalian retina, a number of
neuronal homotypic adhesion molecules, including Sdk1, Sdk2 and Cntn2, restrict dendritic arbors of
amacrine cells and retinal ganglion cells in specific sublaminae in the inner plexiform layer (Yamagata
and Sanes, 2008, 2012; Sanes and Zipursky, 2010). Moreover, one common feature for dendrite
development is that the sister branches from the same neuron avoid each other, while coexist with the
branches of their neighboring neurons. This self-avoidance phenomenon has been elegantly
elucidated by the function of two classes of highly diversified, contact-mediated repulsive molecules:
Down syndrome cell adhesion molecules in Drosophila and protocadherins in vertebrates (Schmucker
et al., 2000; Wojtowicz et al., 2004; Matthews et al., 2007; Lefebvre et al., 2012).
eLife digest The brain consists of billions of cells called neurons that can rapidly send and
receive information. At one end of the neuron, branched structures called dendrites receive signals
from other cells. The number of dendrites and the amount of branching vary in different types of
neurons. These patterns are crucial for each neuron to receive the information it needs.
Abnormalities in dendrites affect brain activity and are associated with several diseases in humans.
To make dendrites, the neuron needs to increase the amount of protein and other cell materials it
produces. New proteins are made in a compartment called the endoplasmic reticulum and are folded
into particular three-dimensional shapes with the help of chaperone proteins. These chaperones may
be overwhelmed if protein production increases, leading to some proteins being folded incorrectly.
This can activate a system called the unfolded protein response, which increases the number of
chaperone proteins so that the proteins can be refolded correctly. However, it was not clear if
neurons rely on the unfolded protein response, or another system, to cope with the increased levels
of protein production needed to form complicated dendrite structures.
Wei et al. studied a type of neuron called PVD—which has an elaborate network of dendrites—in
nematode worms. The experiments show that the unfolded protein response is activated in these
neurons as the dendrites form. Mutant worms that were missing a protein called IRE1, which can
activate the unfolded protein response, had dendrites with fewer branches than normal worms.
The experiments also show that a protein called DMA-1—which is required for dendrites to
form—was not able to fold correctly in the mutant worms. As a result, this protein remained in the
endoplasmic reticulum instead of moving to the surface of the cell where it is usually found. Wei
et al.’s findings reveal that the unfolded protein response plays a major role in allowing cells to
increase protein production as the dendrites form. The next challenge is to understand how neurons
coordinate transcription and activation of the unfolded protein response.
These extrinsic cues must trigger intracellular signaling transduction that leads to cytoskeletal
rearrangement as well as membrane biogenesis and trafficking (Hanus and Ehlers, 2008). For
example, early endosome small G-protein RAB5 facilitates dendrite branching in Drosophila class IV
da neurons (Satoh et al., 2008). Large cells with highly branched dendrites such as Purkinje cells
accommodate the biosynthesis demand with a large soma containing extensive Golgi apparatus and
abundant mitochondria (Herndon, 1963). Molecularly, the secretory pathway components including
Sec23, Sar1, and Rab1 are particularly required for dendrite growth compared with axon
development in the highly branched mammalian and Drosophila neurons (Ye et al., 2007). As part
of the biosynthetic pathway, the production of membrane proteins requires protein folding in the
endoplasmic reticulum (ER). It is currently unclear whether protein folding pathways play a role in the
increased protein production required for dendrite development.
In the ER, a highly conserved protein quality control pathway, the unfolded protein response (UPR),
maintains the ER homeostasis by adjusting the ER folding capacity upon detection of unfolded proteins
(Schroder and Kaufman, 2005; Ron and Walter, 2007; Walter and Ron, 2011; Worby and Dixon,
2014). In higher eukaryotes, three proteins sense the ER stress and activate the UPR: the protein kinase
(PKR)-like ER kinase (PERK), the activating transcription factor 6 (ATF6) and the inositol-requiring
enzyme 1 (IRE1). Conserved in all eukaryotes, IRE1 contains an ER luminal domain, which is involved in
the recognition of misfolded proteins in the ER, and cytoplasmic kinase and endoribonuclease
domains, which can activate downstream pathways (Credle et al., 2005; Gardner and Walter, 2011)
(Figure 1—figure supplement 1A). Activated IRE1 mediates the non-conventional splicing of an intron
from the X box binding protein 1 (XBP1/HAC1) mRNA (Cox and Walter, 1996), and the IRE1-spliced
XBP1 acts as a transcription factor to up-regulate the expression of ER chaperones such as BiP and
other target genes to relieve the ER stress (Travers et al., 2000; Lee et al., 2003).
In the nematode Caenorhabditis elegans, the multidendritic polymodal nociceptive neuron PVD
has an elaborate and organized dendritic arbor (Figure 1A,B). PVD’s largely orthogonally arranged
secondary (2˚), tertiary (3˚) and quaternary (4˚) branches form repeated structural units resembling
menorahs (Smith et al., 2012). During development, PVD grows its entire dendrite arbor that spans
800 μm along the body of the animal in just 24 hr (Smith et al., 2010), suggestive of a high level of
Figure 1. ire-1 is required for PVD dendritic morphogenesis. (A) Cartoon showing the PVD dendritic arbor. The dash-boxed region is magnified to show
the PVD soma (asterisk), axon, primary dendrite (1˚), secondary dendrite (2˚), tertiary dendrite (3˚) and quaternary dendrite (4˚). (B) Representative wild type
(WT) dendritic morphology of PVD neuron expressing membrane associated mCherry (wyIs581). Starting from the cell body, the anterior and the posterior
sections of the primary dendrite are divided into 8 and 4 equal segments, respectively, indicated by dashed lines. Anterior, left; dorsal, top. Asterisk,
cell body; arrowhead, quaternary (4˚) dendrite. Scale bars, 50 μm. (C) Quantification of the number of quaternary (4˚) branches in each segment in WT.
The position of cell body is indicated by the black line. Error bars show mean ± s.e.m., n = 10. (D to G) Defective PVD dendritic morphogenesis in ire-1
(ok799) mutants (D and E) is rescued by expressing ire-1 cell-autonomously (F and G). (H and I) Representative dendritic morphology of FLP neurons
Figure 1. continued on next page
Figure supplement 1. Schematic diagrams of IRE-1 dependent UPR pathway and of C. elegans IRE-1 protein showing three mutations.
biosynthesis during the growth phase of this cell. The formation of PVD dendrites requires a single
transmembrane leucine rich repeat (LRR) protein DMA-1, which acts cell autonomously in PVD to
promote dendrite branching and stabilization (Liu and Shen, 2012). The elaborate dendritic branch
pattern is instructed by hypodermal derived ligands SAX-7/L1CAM and MNR-1. Subcellularly localized
stripes of SAX-7/L1CAM, together with MNR-1 form a tripartite receptor–ligand complex and guide
the growth and branching of the PVD dendritic arbor (Dong et al., 2013; Salzberg et al., 2013).
Using two ire-1 mutant alleles that we isolated from a dendrite morphology screen, we reveal that
the physiological UPR is induced and required in the PVD neuron during dendrite morphogenesis. The
IRE-1/XBP-1/BiP molecular cascade of the UPR pathway governs dendritic branching by regulating the
folding and processing of DMA-1. Surprisingly, our evidence indicates that among many cell surface
molecules required for dendrite formation, DMA-1 is largely responsible for the induction of the UPR.
Loss of ire-1 cause dendrite morphogenesis defects in highly branched
We visualized the PVD neurons using a membrane associated mCherry or GFP marker expressed
under the control of a cell-specific promoter (ser2prom3::myr-mCherry or ser2prom3::myr-GFP). From
a forward genetic screen for mutations that alter the PVD dendritic morphology, we identified two
loss-of-function mutations, wy762 and wy782. Both alleles cause dramatic loss of dendritic branches,
especially in the distal dendrites of PVD (Figure 1—figure supplement 2). Mapping and cloning of
these two alleles showed that each allele contains a single point mutation in the ire-1
(inositolrequiring 1 protein kinase) gene. In addition, a known null deletion allele of ire-1, ok799
(HenisKorenblit et al., 2010) showed indistinguishable phenotype in PVD compared with that of wy762 and
wy782 (Figure 1D). The complexity of the menorahs nearest to the cell body appeared unaffected in
these mutants (Figure 1E), as did the morphology of PVD axon (data not shown).
Interestingly, in the entire nervous system of C. elegans, the only other pair of highly branched
neurons in the head region, FLP also showed severe dendritic arbor defects in ire-1 mutants
(Figure 1H,I). Other neurons with fewer dendritic or axonal branches such as IL2, VC and ADL did not
show branching defects in ire-1 mutants (data not shown). Together, these results suggest that ire-1 is
required for establishing highly branched dendrites.
To investigate where IRE-1 functions to regulate dendritic development, we generated transgenic
mosaic animals. In the ire-1 mutant background, expression of IRE-1 with a PVD-specific promoter
(ser2prom3) fully restored the distal branch number and complexity of the whole dendritic arbor
(Figure 1F,G) indicating that IRE-1 functions cell-autonomously in PVD to regulate dendrite
morphogenesis. Expressing ire-1 cDNA did not cause overbranching in wild-type animals
(Figure 1—figure supplement 3B).
Lack of folding capacity in the ER contributes to dendritic defect of PVD
in ire-1 mutants
IRE1 is conserved in all eukaryotes and contains an ER luminal domain for recognizing misfolded
proteins in the ER, and a cytoplasmic kinase and an endoribonuclease domain, which lead to the
non-conventional cytoplasmic splicing of xbp-1 (Figure 1—figure supplement 1A). One missense
mutation (wy782) of ire-1 is a substitution of a conserved residue in the kinase domain while another
missense mutation (wy762) is a substitution of a conserved residue in the endoribonuclease domain
(Figure 1—figure supplement 1), indicating both domains might be required for dendrite
morphogenesis. Since these two domains are required for splicing of the xbp-1 mRNA, we reasoned
that the neurons should be able to bypass the requirement of IRE-1 if a spliced form xbp-1 was
provided in PVD. Consistent with this hypothesis, PVD-specific expression of spliced xbp-1 cDNA in
ire-1 mutants rescued the loss of distal dendrite branches phenotype. In contrast, expression of
unspliced xbp-1 genomic DNA at the same concentration did not rescue branching defect
(Figure 2A–D). Expressing spliced xbp-1 cDNA did not cause overbranching in wild type animals
(Figure 1—figure supplement 3C). These data offer compelling evidence that XBP-1 functions
downstream of IRE-1 to establish complex dendritic arbor in PVD. Hence, the IRE-1 arm of the UPR
pathway is likely involved in dendrite morphogenesis.
Because of the well-established role of the IRE-1/XBP-1 pathway in enhancing protein folding
capacity in the ER, we hypothesized that IRE-1/XBP-1 upregulates specific ER chaperones to promote
dendrite morphogenesis. We searched the PVD-specific gene profiling data (Smith et al., 2010) and
found that two abundant ER chaperones of the Hsp70 family (homologous to mammalian grp78/BiP),
HSP-3 and HSP-4, are enriched in PVD and therefore might be the targets of XBP-1 in PVD neurons
(Urano et al., 2002). Consistent with this idea, overexpression of hsp-4 in PVD restored normal
dendritic branches in ire-1 mutants (Figure 2E,F). However, overexpression of hsp-3 or daf-21
(a cytoplasmic chaperone of the Hsp90 family) did not rescue the phenotype (Figure 2—figure
supplement 1B,C). Furthermore, hsp-4 single mutant did not show the dendritic arbor defects
(Figure 2—figure supplement 1D), indicating other ER chaperones or co-chaperones functioning in
parallel with HSP-4. These results indicate that the dendritic defect in the ire-1 mutants is likely due to
lack of specific chaperones in the ER.
Importantly, xbp-1 mutant (Figure 2—figure supplement 2B) did not show the dendritic arbor
defects. This suggests that other pathways downstream of IRE-1 but independent of XBP-1 can play
redundant roles in dendrite morphogenesis. During ER stress, Ire1 can promote the degradation of
mRNAs encoding some ER proteins to maintain homeostasis through regulated Ire1-dependent
decay (RIDD) (Hollien and Weissman, 2006; Hollien et al., 2009). The RIDD pathway has been shown
to affect cell fate in various organisms, such as photoreceptor development in Drosophila (Coelho
et al., 2013; Maurel et al., 2014). We next investigated whether the RIDD pathway functions in
parallel with XBP-1 to regulate dendrite morphogenesis. mRNA degradation is initiated by internal
cleavage mediated by RIDD, and the resulting RNA fragments would be subject to degradation by
cytoplasmic 5′-3′ mRNA degradation machinery. However, all null mutants of the RIDD pathway
components are lethal and difficult to examine dendrite phenotypes. Therefore, we used somatic
clustered regularly interspaced short palindromic repeat (CRISPR) to create mosaic viable and
conditional knock out of various genes (Jinek et al., 2012; Cong et al., 2013; Shen et al., 2014).
Using this method, we found that in the xbp-1 mutant background, somatic knockout of xrn-1, which
encodes a 5′-3′ exoribonuclease and is a key component of the 5′-3′ mRNA degradation pathway
(Newbury and Woollard, 2004), phenocopied the ire-1 dendritic phenotype in PVD neurons in about
10% of the animals (Figure 2—figure supplement 2C). Somatic CRISPR is intrinsically mosaic and
often generates low-penetrance phenotypes compared with viable null alleles. These results indicate
that the RIDD pathway functions in parallel to the XBP-1 to regulate dendrite branching of PVD.
We also examined mutations in the other two arms of the UPR pathway, ATF-6 and PERK/PEK-1,
and found that they did not show any dendrite morphogenesis phenotype in PVD (Figure 2—figure
supplement 3A,B). However, xbp-1 pek-1 double mutant showed a low-penetrance (about 25%)
ire1-like phenotype (Figure 2—figure supplement 3C). This suggests that the ER homeostasis mediated
by other UPR pathways also contribute to dendrite morphogenesis.
DMA-1 is a key target of the IRE-1 UPR pathway in PVD dendrite
We next asked which protein(s) are potential targets of the IRE-1 UPR pathway in PVD executing
dendrite morphogenesis. The severe decrease of distal dendritic branches of PVD in ire-1 mutants is
reminiscent of dma-1 mutants. DMA-1 is a single transmembrane leucine-rich repeat (LRR) protein
prominently expressed in PVD, and mutations in dma-1 result in severely reduced dendritic branching
and complexity (Liu and Shen, 2012) (Figure 3—figure supplement 1A). DMA-1 acts in PVD as
a receptor to recognize the SAX-7/L1CAM and MNR-1 ligand complex in the surrounding skin cell to
promote branching and precisely pattern the dendritic arbor (Dong et al., 2013; Salzberg et al.,
2013). We reasoned that the folding of DMA-1 might require IRE-1. Consistent with this hypothesis,
ire-1 dma-1 double mutants showed a phenotype that is indistinguishable from the dma-1 single
mutant phenotype, suggesting that these two molecules function in the same genetic pathway in
dendrite morphogenesis (Figure 3—figure supplement 1B). Furthermore, hsp-4 overexpression in
ire-1 dma-1 double mutants was not able to rescue the dendritic arbor defect (Figure 3—figure
supplement 1C), suggesting that DMA-1 might be a target of HSP-4.
As a single transmembrane protein, DMA-1 is synthesized in the ER and delivered to the plasma
membrane through the secretory pathway. In wild type animals, GFP-tagged DMA-1 was detected on
all the PVD dendritic processes and at the cortex of the cell body as diffusive fluorescence. In addition,
numerous discrete intracellular puncta were found in the cell body and along the dendrites, which
presumably represent the membrane trafficking organelles that carry DMA-1 (Figure 3B) (Liu and
Shen, 2012). In ire-1 mutants, the punctate DMA-1::GFP in the cell body was lost (Figure 3E). Instead,
the somatic DMA-1::GFP in the ire-1 mutants co-localized with an general ER marker, cytochrome b5
(cb5) (Rolls et al., 2002) (Figure 3G). Moreover, the diffuse DMA-1::GFP signal on the distal dendrites
was dramatically reduced in the ire-1 mutant while the signal on the proximal dendrites in ire-1
mutants was the same as in wild type (Figure 3H,J,K). These observations suggest that DMA-1 is
trapped in the ER and is not delivered to the distal dendrite plasma membrane, leading to the distal
dendritic phenotype. Consistent with this hypothesis, overexpression of the ER chaperone HSP-4,
restored the DMA-1::GFP subcellular localization in ire-1 mutants to the normal distribution
(Figure 3J,K). Taken together, these data suggest that ER chaperones such as HSP-4 help to fold
DMA-1, which is required for the plasma membrane localization of DMA-1 and dendrite branching.
To further understand why the dendrite loss in the ire-1 mutants was restricted to the distal
dendrites, we investigated where the synthesis and folding of membrane proteins took place in PVD.
This is an important question because the existence of local translation in dendrites might provide
a source of DMA-1 production (Holt and Schuman, 2013; Tom Dieck et al., 2014). Since HSP-4 is
capable of folding DMA-1, we first examined the subcellular localization of HSP-4 and found that
HSP4::GFP was exclusively localized in the PVD soma (Figure 3—figure supplement 2A,D), co-localizing
with a rough ER marker TRAM (Figure 3—figure supplement 2G–I), HSP-4’s ER localization pattern is
consistent with the observation that its mammalian homolog BiP is localized in rough ER (Bole et al.,
1989; Lai et al., 2010).These data suggests that the main protein synthesis and folding capacity for
DMA-1 is likely in the cell body. In ire-1 mutants, lack of the upregulation of hsp-4 by spliced XBP-1
results in less DMA-1 in the secretory pathway and insufficient diffusion of DMA-1 to the distal region
might be responsible for the specific loss of distal dendrites.
If the ire-1 phenotype was the result of diminished DMA-1 levels in the distal dendrites, we
reasoned two potential outcomes of DMA-1 overexpression in ire-1 mutants. The increased
expression of DMA-1 might reach the plasma membrane and rescue the ire-1 phenotype.
Alternatively, the DMA-1 overexpression might increase the protein-folding load and exacerbate
the already strained protein folding machinery and lead to a more severe dendrite defect.
Interestingly, we observed both effects: about 70% of animals showed efficient rescue of the dendritic
arbor (Figure 4B,D), while about 25% of animals showed a more severe phenotype, with the loss of
proximal branches in addition to the distal ones (Figure 4C,D). We hypothesized that in the absence
of IRE-1, the remaining protein folding capacity is at a critical level where overexpression of DMA-1
can produce functional or misfolded proteins, possibly depending on the slightly variable levels of
endogenous chaperones in individual animals (Burga et al., 2011). Consistent with the hypothesis,
high level of chaperon HSP-4 expression together with DMA-1 decreased the percentage of
dma-1like phenotype, in a dose-dependent manner (Figure 4D). To further test this hypothesis, we
separated the transgenic animals into the phenotypically rescued animals and the severely defective
animals based on their dendrite morphology, we found that there was much less accumulation or
aggregation of DMA-1::GFP in the PVD cell bodies with the rescued morphology compared to more
severely defective animals (Figure 4—figure supplement 1A–G). Together, these rescuing results
argue that the insufficient level of functional DMA-1 due to decreased protein folding capacity
accounts for a large part of PVD dendritic defect in the ire-1 mutants.
The UPR activity in the PVD neuron is correlated with dendritic
branching during development
We have shown that the UPR machinery is required for dendrite morphogenesis in PVD. However, it is
not clear whether the dendritic branching activates the UPR in PVD during development. To answer
this question, we designed a UPR activity reporter which contains the genomic fragment of xbp-1
fused with a GFP in frame followed by an SL2::mCherry cassette (Figure 5A). Upon UPR activation, the
intron in genomic xbp-1 DNA will be spliced out by IRE-1, leading to the production of XBP-1::GFP
(Iwawaki et al., 2004). The SL2::mCherry cassette permits the bicistronic expression of XBP-1::GFP
Figure supplement 2. Subcellular localization of HSP-4::GFP, general ER marker cb5::mCherry and rough ER marker BFP::TRAM in PVD cell body (CB)
region (A to C and G to I) and in distal (D) dendritic region (D to F) in wild type animals.
and mCherry (Spieth et al., 1993), and its function is similar to the viral IRES sequence in mammalian
system. The whole reporter is driven by a PVD specific promoter (Pdes-2). The XBP-1::GFP intensity
indicates the endogenous UPR activity in PVD while the intensity of mCherry is used to normalize to
transgene expression levels among different animals. Consistent with the requirement of IRE-1 to
activate XBP-1, the XBP-1::GFP intensity in PVD neurons was diminished in ire-1 mutants compared
with wild-type animals (Figure 6—figure supplement 1A,D).
PVD neurons are derived postembryonically during the mid-L2 larval stage (Sulston and Horvitz,
1977), and starting from the late L2/early L3, 2˚ branches begin to form followed by extension of 3˚
branches in the L3 stage. Dendrite morphogenesis is completed in the early L4 stage after 4˚ branches
have sprouted from the 3˚ branches to form a network of menorah-shaped processes (Smith et al.,
2010). Using this PVD specific UPR activity reporter, we observed XBP-1::GFP in the nucleus of PVD
starting at the L3 stage. The normalized XBP-1::GFP intensity increased between L3 and late L4
animals, coincidental with the stage of rapid dendrite branch addition. The XBP-1::GFP intensity
subsequently decreased in mid-adult animals (Figure 5B–K). We verified this result by using another
UPR activity reporter (Phsp-4::HIS-24::GFP). As an ER chaperone, HSP-4 is a transcriptional target of
activated XBP-1 (Calfon et al., 2002; Urano et al., 2002), and its transcriptional level shows tight
correlation with activation of the UPR with high sensitivity (Iwawaki et al., 2004). We used the hsp-4
promoter to drive the expression of the C. elegans H1 histone, HIS-24 fused with GFP to detect the
UPR activity in PVD neurons labeled with cytoplasmic mCherry. We found that the HIS-24::GFP
signal became clearly detectable in L3 and further increased in L4 animals during which the menorahs
form. The GFP fluorescence is dramatically downregulated in adult animals, (Figure 5—figure
supplemental 1). Taken together, these results suggest that the UPR activity occurs most strongly
during the time of PVD dendritic branching.
DMA-1 is largely responsible for the activation of the UPR in PVD
The next question we wanted to address was how the UPR in PVD is induced during dendrite
morphogenesis. The rapid dendritic growth of PVD requires high level of biosynthesis of plasma
membrane proteins and efficient folding of them in the ER. PVDs are one of the only two pairs of
highly branched neurons in C. elegans. Several transcription factors have been implicated in the cell
fate determination of PVD. We considered two possibilities for the induction of the UPR activity in
PVD neuron. In a ‘top down’ cell fate model, the enhanced UPR might be part of the cell fate decision
controlled by transcription factors. Alternatively, the UPR might be induced because of the protein
folding demand, in particular, maybe due to the translation of DMA-1, an essential membrane
molecule for PVD dendrite branching (Figure 6A).
To distinguish these models, we first tested this PVD specific UPR activity reporter in dma-1
knockout mutants and we found that the normalized XBP-1::GFP fluorescence level was significantly
lower compared with wild type (Figure 6B,E,K), suggesting that a functional dma-1 gene is required
to turn on the UPR activity in PVD. Conversely, overexpression of dma-1 cDNA in PVD leads to
an increase of UPR activity (Figure 6H,K). Consistently, in dma-1 mutants, another UPR activity
reporter, Phsp4::HIS-24::GFP also showed dramatic decrease in PVD neurons (Figure 6—figure
These surprising results argue that DMA-1 is
necessary for UPR induction in PVD, despite the
fact that there must be many membrane proteins
necessary to build dendrites. To test whether
DMA-1’s role in UPR induction is specific, we
asked if mutations in other membrane proteins
required for PVD dendrite morphogenesis also
result in a decrease in UPR activation. Deletion
mutations in kpc-1 (a Kex2/subtilisin-like
proprotein convertase and a Furin homolog) (Schroeder
et al., 2013) and hpo-30 (a claudin homolog)
(Smith et al., 2013) cause severe reduction of
PVD dendrites. While both of these gene
products are processed in ER, neither mutation
causes reduced UPR reporter activity in PVD
(Figure 6K and Figure 6—figure supplement
1G–L). In addition, this reporter also showed
activity in the unbranched neuron PVC, indicating
that there might be UPR activity that is unrelated
to branched dendrite morphogenesis.
Nevertheless, the dma-1 mutation did not change the UPR
activity in PVC (Figure 6—figure supplement 3).
These results demonstrate that the activation of
UPR in PVD specifically depends on DMA-1
Coexpression of HSP-4 and DMA-1
induces ectopic branches more
Surveying morphological phenotypes of other
types of neurons, we found that the dendritic
arbor defects in ire-1 mutants were restricted to
PVD and FLP the only two pairs of highly branched
neurons in the C. elegans nervous system
(Figure 1I). Coincidentally, only PVD and FLP
showed sustained expression of DMA-1(Liu and
Shen, 2012). These observations suggest that the
establishment of a complex dendritic arbor not
only requires instructive cell surface molecules but
also physiological UPR to increase the protein
folding capacity and maintain cellular
homeostaFigure supplement 1. The UPR activity is correlated sis. Since PVD and FLP are also the largest neurons
with dendritic branching during development in the in worms with complicated dendritic arbor, we
PVD neuron with another UPR reporter. wondered if the UPR is particularly activated in
DOI: 10.7554/eLife.06963.017 these large cells. To directly test this idea, we
examine the PVD morphology in dpy-5 ire-1
double mutants. dpy-5 mutants have reduced
body length (about two third of that of wild type) due to bearing a deletion in the cuticle procollagen
DPY-5 gene (Thacker et al., 2006) and correspondingly reduced PVD size (Figure 7—figure
supplement 1B). Interestingly, the defective PVD phenotype of ire-1 was dramatically rescued with
some animals showing wild type morphology (Figure 7—figure supplement 1C–E), indicating the UPR
is particularly required for neurons with large and complicated dendritic arbors.
To further test this hypothesis, we determined the sufficiency of UPR activation to induce ectopic
branches in neurons that normally do not branch extensively. The sensory neuron PDE shares the same
lineage with PVD and does not express detectable levels of dma-1. The cell body of PDE is positioned
close to the PVD’s and has a single processes running adjacent to the PVD dendrites. Consequently,
the extracellular environment for PDE including the molecular ligands for DMA-1 is similar to that of
PVD (Figure 7A,B).
Overexpression of dma-1 in PDE resulted in ectopic orthogonal branches that were similar to the
PVD tertiary level branches (Figure 7C) (Liu and Shen, 2012). However, the low efficiency of ectopic
branch induction (21% of animals bearing transgene) suggests there might be other intrinsic
mechanisms that are necessary to establish exuberant branches. Notably, increasing the ER folding
capacity by expressing HSP-4 together with DMA-1 in PDE induced ectopic branches more efficiently
(47% of animals) while expressing other PVD-branching cell surface molecules such as HPO-30 with
HSP-4 did not induce any ectopic branches (Figure 7D–G). Further, these ectopic branches were
more branched and significantly longer than overexpressing DMA-1 alone (Figure 7H), and the
Phsp4::HIS-24::gfp reporter also showed increased UPR activity in PVD (Figure 7—figure supplement 2).
These data support our model that the UPR is required for highly branched dendrites.
The unfolded protein response is an intrinsic requirement for highly
Conserved in all eukaryotes, the UPR pathway plays significant roles in dealing with cellular stress and
balancing homeostasis and apoptosis (Walter and Ron, 2011). Failure to mitigate the ER stress and
reestablish homeostasis correlates with cell death, playing a central role in numerous human diseases
such as pancreatic β-cell loss in diabetes (Fonseca et al., 2011), retinal degeneration triggered by
misfolded proteins in retinal dystrophies (Lin and Lavail, 2010) and dopaminergic neuron
degeneration in Parkinson disease models (Valdes et al., 2014). In addition, under other biological
conditions involving intense ER functions such as viral infection (Dimcheff et al., 2003) or pathogen
defense (Richardson et al., 2010), the UPR is activated to relieve the ER stress. In the nervous system,
several reports indicate normal regulatory roles of the IRE-1. For example, IRE-1 was shown to be
involved in trafficking cell surface molecules such as AMPA receptor in cultured cells (Vandenberghe
et al., 2005) or glutamate receptor GLR-1 in C. elegans interneurons (Shim et al., 2004), and
rhodopsins in Drosophila photoreceptors (Coelho et al., 2013).
However, to the best of our knowledge, the role of the UPR pathway in the development of the
nervous system is poorly understood except in a few isolated cases. For example, a different arm of
the UPR, PERK1, was recently shown to be required for the olfactory receptor choice in mammalian
olfactory sensory neurons through a feedback loop (Dalton et al., 2013). Our results on the IRE-1/
XBP-1/BiP/DMA-1 molecular cascade regulating dendrite morphogenesis in PVD neurons provide
another clear example that UPR is directly involved in the development of neuronal cell morphology.
More importantly, these results provide a link between dendrite morphogenesis and cellular
homeostasis. First, only highly branched neurons such as PVD and FLP require the UPR to establish
their dendritic arbors; second, the UPR activity in PVD is correlated with the development of
dendrites; finally the induction of UPR is dependent on the expression of a pivotal molecule for
dendritic branching DMA-1 in a homeostatic manner. Thus, in addition to the instructive extracellular
cues required for complex branch formation and guidance, intrinsic mechanisms are also required.
The UPR and secretory pathways are critical for dendrite morphogenesis
The secretory and endocytic pathways constitute the main membrane trafficking pathway in cell
(Sallese et al., 2006; Brandizzi and Barlowe, 2013). In order to form dendrites, membrane and
transmembrane proteins need to be synthesized and delivered to the growing dendritic arbor. It is
therefore not surprising that molecular components, in these pathways such as the early endosome
protein RAB-5 are involved in dendrite morphogenesis (Satoh et al., 2008). Interestingly, one
previous study showed that secretory pathway mutants preferentially alter dendrite morphology and
not axon extension (Ye et al., 2007). Similarly, we found that the dendrite but not axon
morphogenesis is specifically compromised in the ire-1 mutants, suggesting that specific molecular
program and membrane trafficking pathways are required for dendrite development. Thus, our
studies add weight to the idea that different molecular and trafficking pathways are utilized during
dendrite morphogenesis and axon extension.
The UPR for dendrite morphogenesis is largely triggered by A single
The physiological functions of the UPR have been best demonstrated in highly differentiated cells that
produce specific types of proteins in large amounts. One best-characterized example is the
requirement of IRE1 and XBP1 for differentiation of B cells into plasma cells, where the UPR is
activated to accommodate the secretion of large amounts of immunoglobulins (Reimold et al., 2001).
In these cells, the ER and secretory system are highly specialized for antibody biosynthesis, which
accounts for half of the total protein production in these cells (Askonas, 1975). In Ig heavy chain
knockout mice, the UPR activity was diminished in B cells, indicating that the production of
immunoglobulins in B cells is required for induction of the UPR (Iwakoshi et al., 2003). Another
example is in the mammalian olfactory sensory neurons. Olfactory receptor (OR) genes are among the
most highly transcribed G-protein-coupled receptors (GPCRs) in these neurons and trigger the UPR
feedback loop required for specific OR choice (Dalton et al., 2013).
In contrast to these specialized cell types, a large number of diverse proteins and lipids are
required in a developing neuron to establish its dendritic arbor. Many of these proteins are folded and
processed in the ER. Surprisingly, our results suggest that among these proteins, a single
transmembrane protein, DMA-1, appears to be largely responsible for the activation of the UPR
pathway in PVD during dendritic development. The characteristics of the DMA-1-like LRR proteins
including their non-globular flexible solenoid like structure, repetitive amino acid sequences and high
content of hydrophobic leucines, might make them particularly challenging to fold and assemble
properly (Freiberg et al., 2004). These characteristics may trigger the UPR in these cells and thus
make this system required for dendrite morphogenesis. Other evidence also supports the notion that
specific proteins have higher folding demands. For example, although IRE-1 has been shown to
function in the normal secretory pathway (Safra et al., 2013), the protein trafficking defect in ire-1
mutants is not general. It has been shown that for several different membrane-spanning proteins
including Golgi-resident mannosidase, a TWK-type potassium channel, a single transmembrane
synaptic vesicle protein synaptobrevin and a vesicular monoamine transporter CAT-1, their subcellular
localizations in ire-1 mutants were unaffected in interneurons (Shim et al., 2004). These results
suggest that DMA-1 is specifically regulated by the UPR pathway in the PVD neuron.
Together, these findings indicate that certain proteins are intrinsically more challenging for folding
and specific cell types have to employ the UPR pathway to accommodate the influx of these proteins
and maintain homeostasis in the ER.
Materials and methods
Strains and genetics
Strains were grown at 20˚C on NGM agar plates seeded with Escherichia coli OP50 except the UPR
mutants and UPR reporter strains growing at 16˚C. The wild-type strain was C. elegans N2 Bristol. The
following mutant alleles and transgenes were used in this study:
LGI: dma-1(wy686), kpc-1(gk8) dpy-5 (e907); LGII: ire-1(ok799), hsp-4(gk514); LGIII: xbp-1(tm2482)
wyIs592 [ser2prom3::myrGFP, odr-1::dsRed]; LGIV: wyIs581[ser2prom3::myr-mCherry, odr-1::dsRed];
LGV: hpo-30(ok2047); LGX: atf-6(ok551), pek-1(ok275), qyIs369[ser2prom3::dma-1::GFP,unc-119+]),
wyIs378[ser2prom3::myrGFP, Prab3::mCherry, odr-1::dsRed].
Isolation and mapping of mutants
The wy762 and wy782 alleles were isolated from an F2 semiclonal screen of 3000 haploid genomes in
the strain containing wyIs378 (Dong et al., 2013). Based on SNIP-SNP mapping and whole genome
sequencing (Sarin et al., 2008), we got the missense mutation information on ire-1 locus and verified
by Sanger sequencing and complementation test with the null allele.
Expression clones were made in the pSM vector, a derivative of pPD49.26 (A Fire) with extra cloning
sites (a kind gift from S McCarroll and CI Bargmann). The ser2prom3 (PVD) and Pdat-1 (PDE)
promoters were used for cell-specific expression. cDNAs of ire-1, xbp-1(long isoform), hsp-3 and
his24 were amplified from cDNA library while genomic DNAs of xbp-1, hsp-4, cb5 (C31E10.7) and tram-1
were amplified from genomic templates. The XBP-1 UPR reporter construct was driven by Pdes-2
(PVD), containing xbp-1 genomic DNA fused with GFPnovo2 (Arakawa et al., 2008) followed by
gpd-2 SL2::mCherry (from pBALU12) (Tursun et al., 2009). For hsp-4 transcriptional activity reporter,
the 1.1 kb 5′ upstream of hsp-4 ATG was cloned, driving HIS-24 fused with GFPnovo2. For HSP-4::
GFP, GFPnovo2 was inserted right before the C-terminus HDEL sequence of genomic HSP-4.
For somatic CRISPR, two DNA templates of xrn-1 sgRNA were 5′- GATATCGCTCCGATGTCCAT-3′
and 5′- AACGTGACGTCATCGTCATT-3′, under the control of U6 promoter as in (Chen et al., 2013).
The transgenic extrachromosomal arrays were generated via injection using standard microinjection
techniques (Mello and Fire, 1995).
For rescue experiments, wyEx7329[ser2prom3::ire-1 (40 ng/μl), pBluescript (60 ng/μl), odr-1::dsRed
(90 ng/μl)]; wyEx7332[ser2prom3::xbp-1(cDNA) (20 ng/μl), pBluescript (60 ng/μl), odr-1::dsRed(90 ng/μl)];
wyEx6502[ser2prom3::xbp-1(genomic DNA) (40 ng/μl), pBluescript (60 ng/μl), odr-1::dsRed(90 ng/μl)];
wyEx6816[ser2prom3::hsp-4 (50 ng/μl), pBluescript (60 ng/μl), odr-1::dsRed(90 ng/μl)]; wyEx7333
[ser2prom3::hsp-3 (50 ng/μl), pBluescript (60 ng/μl), odr-1::dsRed(90 ng/μl)];
wyEx7335[ser2prom3::daf21 (50 ng/μl), pBluescript (60 ng/μl), odr-1::dsRed(90 ng/μl)].
For ER markers and chaperone co-labeling, wyEx8074[ser2prom3::cb5::mCherry PCR fusion
product (20 ng/μl), ser2prom3::hsp-4::GFPnovo2::HDEL (10 ng/μl), pBluescript (30 ng/μl), odr-1::
dsRed(90 ng/μl)]; wyEx8075[Pdes-2::tagBFP::TRAM (15 ng/μl), ser2prom3::hsp-4::GFPnovo2::HDEL
(10 ng/μl), pBluescript (30 ng/μl), odr-1::dsRed(90 ng/μl)].
For DMA-1 overexpression experiment, in wyIs581 background, wyEx7338[ser2prom3::dma-1::
GFP (50 ng/μl), pBluescript (60 ng/μl), Pmyo-2::mCherry(1.5 ng/μl)];
For HSP-4 dose-dependent rescue experiments, with wyEx7338 and wyIs581, wyEx7859
[ser2prom3::hsp-4 (30 ng/μl), pBluescript (60 ng/μl), odr-1::dsRed(60 ng/μl), pBluescript (60 ng/μl)];
wyEx7770[ser2prom3::hsp-4 (60 ng/μl), pBluescript (30 ng/μl), odr-1::dsRed(60 ng/μl)].
For the UPR activity reporter, wyEx6766[Pdes-2::xbp-1(genomic)::GFPnovo2::SL2-mCherry (80 ng),
Punc-122::dsRed(30 ng/μl), pBluescript (30 ng/μl)]; wyEx6812[ser2prom3::dma-1 (50 ng/μl), odr-1::
dsRed(60 ng/μl), pBluescript (60 ng/μl)] For UPR activation experiment with hsp-4 transcriptional
reporter, with wyIs581, wyEx7820[Phsp-4::HIS-24::GFPnovo2 (20 ng/μl), pBluescript (60 ng/μl), odr-1::
For somatic CRISPR, in xbp-1 (tm2482) background, wyEx7862[Phsp-16.2::Cas9 (50 ng/μl), PU6::
xrn-1-sgRNA1 temp (30 ng/μl), PU6::xrn-1-sgRNA2 temp (30 ng/μl),odr-1::GFP(40 ng/μl)].
For PDE ectopic branching experiments, wyEx7035 [Pdat-1::hsp-4 (40 ng/μl), Pdat-1::GFP (20 ng/μl),
odr-1::dsRed(60 ng/μl), pBluescript (30 ng/μl)] injected into wyEx4287 strain with overexpression of
dma-1 in PDE (Liu and Shen, 2012); wyEx8063[Pdat-1::GFP (20 ng/μl), Pdat-1::hsp-4 (40 ng/μl),
Pdat1::hpo-30 (30 ng/μl), odr-1::dsRed (90 ng/μl)];
For PDE UPR reporter experiments, wyEx8049[Phsp-4::HIS-24::GFPnovo2 (20 ng/μl), Pdat-1::
mCherry (2 ng/μl), pBluescript (60 ng/μl), odr-1::dsRed(70 ng/μl)]; then use this line to ectopic express
dma-1 in PDE, wyEx8065 [Pdat-1::dma-1::BFP (50 ng/μl), Pdat-1::mCherry (20 ng/μl), pBluescript
(30 ng/μl), odr-1::GFP(20 ng/μl)].
wyEx4280 was used for FLP labeling (Liu and Shen, 2012).
Somatic xrn-1 CRISPR
Following the protocol in (Shen et al., 2014) with some modifications, we first synchronized the
culture by allowing 100–150 adult worms containing transgenic arrays (raised at 20˚C) to lay eggs for
3 hr on seeded NGM plates. The eggs were heat-shocked at 33˚C for 2 hr and then shifted to 20˚C.
After 60 hr, the PVD morphology was checked at the young adult stage.
Images of fluorescently tagged fusion proteins were captured in live C. elegans using Plan-Apochromat
40×/1.3NA objective for whole PVD morphology and 63×/1.4NA for subcellular localization of
fluorescent proteins on a Zeiss LSM710 confocal microscope (Carl Zeiss, Germany). Animals were
immobilized on 2% agarose pad using 10 mM levamisole (Sigma–Aldrich, St. Louis, MO) and oriented
anterior to the left and dorsal up. Z-stacks were collected and the maximum intensity projection was
used for additional analysis. For analyzing DMA-1::GFP intensity on tertiary dendrites (middle and
bottom panels in Figure 3H–J), XBP-1::GFP intensity during development (Figure 5B–J) and HIS-24::
GFP UPR activity reporter (Figure 5—figure supplement 1, Figure 6—figure supplement 2 and
Figure 7—figure supplement 2), images were acquired using a Zeiss Axio Observer Z1 microscope
equipped with a Plan-Apochromat 63×/1.4NA objective, Yokogawa spinning disk head (Japan), 488 nm
and 561 nm diode lasers (Coherent, Santa Clara, CA), and a Hamamatsu ImagEm EMCCD camera
(Japan) driven by MetaMorph (Molecular Devices, Sunnyvale, CA).
For 4˚ dendrite number counting, two PVD images (labeled by wyIs581) from late L4 or young adults
were stitched together in Adobe Photoshop (San Jose, CA). The general shape and location of the
primary dendrite (the ‘backbone’) was recognized by a model-based neurite fiber tracing method
(Peng et al., 2008). Then the length of primary dendrite was determined by tracing the backbone and
calculating the distance between adjacent identified pixels. Finally, the anterior part from cell body was
divided into 8 equal segments while the posterior part was divided into 4 equal segments (written in
custom Matlab scripts (Mathworks, Natick, MA)). It should be noted that the length of each anterior
segment is not equal to each posterior segment. The numbers of 4˚ dendrites whose secondary
dendrites grew in each segment were counted manually.
For 2˚ dendrite number counting, two PVD images (labeled by wyIs581 or wyIs592) from late L4 or
young adults were stitched together in Photoshop. Then the length of primary dendrite was
determined manually by tracing the backbone and calculating the distance between adjacent
identified pixels. The 2˚ dendrite number in each animal was counted manually, and this number was
divided by the length of PVD primary dendrite (per 100 μm).
For measuring DMA-1::GFP intensity on 3˚ dendrites, we chose menorahs around the vulva region
as ‘Proximal’ to avoid numerous puncta in dendrites close to cell body and chose menorahs around
the middle point of anterior primary dendrite as ‘Distal’ to make sure we could get T-like branches in
this region in ire-1 mutants. Two channel images were combined together by ImageJ (Wayne
Rasband), and a 2-pixel wide line was drawn along the tertiary branches (avoiding obvious puncta) and
then the mean intensity values of two separated channels along this line were measured. After
subtracting the background signal, the DMA-1::GFP signal was normalized to cytoplasmic mCherry.
3–5 tertiary branches were chosen for each spinning-disk image.
To quantify the UPR activity, for different genotypes, the XBP-1::GFP or HIS-24:GFP mean intensity in
the nucleus (after background subtraction) measured with ImageJ was normalized to the mean intensity
of cytoplasmic mCherry in the same region using custom written Python (Python Software Foundation,
Beaverton, OR) scripts. HIS-24::GFP intensity (Figure 5—figure supplement 1 and Figure 7—figure
supplement 2) was measured and quantified without normalization to cytoplasmic mCherry.
All custom matlab and Python codes are provided in the Source code 1.
In comparisons of measurements such as fluorescence intensity or length of ectopic branches, we first
tested for normality using a D’Agostino-Pearson test (alpha = 0.05). For data sets with normal
distribution, we applied a two-tailed Student’s t test for comparisons of two groups (Figure 6—figure
supplement 3G and Figure 4—figure supplement 1G). Comparisons involving multiple groups with
multiple factors used two-way ANOVA and post hoc Sidak’s multiple comparisons test (Figure 3K). For
data sets without normal distribution, we applied a two-tailed Mann–Whitney U-test for comparisons of
two groups (Figure 7H and Figure 7—figure supplement 2G). Comparisons involving multiple groups
used Kruskal–Wallis one-way test and post hoc Dunn’s test (Figures 5K, 6K, Figure 5—figure
supplement 1J and Figure 6—figure supplement 2J). To compare variables such as proportions we
used χ2 test with Sidak correction for multiple comparisons (Figure 4D and Figure 7F). All statistical
tests were performed in Graphpad Prism (San Diego, CA) or in R (R Development Core Team).
This work was supported by the Howard Hughes Medical Institute and by the NIH (1R01NS082208).
We thank Shohei Mitani, the C. elegans Gene Knockout Consortium, the Caenorhabditis Genetics
Center provided mutant strains. We thank R Kopito and J Frydman for discussion, C Richardson,
P Kurshan and members of Shen laboratory for thoughtful comments on the manuscript. We thank
Caroline Yu for contribution of the screen. XW was supported by a predoctoral fellowship from the
American Heart Association, Western States Affiliate (13PRE14000009).
Xing Wei, Audrey S Howell,
Xintong Dong, Caitlin A Taylor,
Roshni C Cooper, Kang Shen
Xing Wei, Audrey S Howell,
Xintong Dong, Caitlin A Taylor,
Roshni C Cooper, Kang Shen
The funders had no role in study design, data collection and interpretation, or the
decision to submit the work for publication.
XW, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or
revising the article; ASH, Mapped the mutants and performed the initial characterization of the
mutants, Drafting or revising the article; XD, Isolated the two mutants of ire-1; CAT, Performed the
initial characterization of the mutants; RCC, Wrote the Matlab codes for tracing and dividing primary
dendrite; JZ, Did the non-parametric statistical analysis in R; WZ, DRS, Generated the qyIs369
marker; KS, Conception and design, Drafting or revising the article
· Source code 1. Custom built software in Matlab and Python codes. Zip file contains: findPrimary.m to
find the PVD primary dendrite, and it calls BDB function (Peng, H, Long, F, Liu, X, Kim, SK, and Myers,
EW (2008). Straightening Caenorhabditis elegans images. Bioinformatics 24, 234–242. Cited paper in
the manuscript), which is available from http://penglab.janelia.org/proj/wormatlas/bdb_minus_demo_
download.html. SplitWorm.m to split the primary dendrite into equal length fragments. PrintSplits.m to
draw lines to indicate the segmentation of the primary dendrite. ImageProcessing.py: the python code
for image processing and data analysis.
5R01NS082208-02 Xing Wei, Audrey S Howell, Xintong Dong , Caitlin A Taylor, Roshni C Cooper, Kang Shen
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