Byrd, Dana et al. (2021) International Worm Meeting "A C. elegans model for human PACS1 syndrome"

Jin, Yishi, Byrd, Dana

[International Worm Meeting, 2021]

PACS1 syndrome (also known as Schuurs-Hoeijmakers syndrome) is a human neurogenetic disorder characterized by distinctive dysmorphic facial features, intellectual disability, and developmental delays. All known PACS1 patients have de novo mutations causing an R203W amino acid substitution in a highly conserved position of the PACS1 protein. While PACS1 (Phosphofurin Acidic Cluster Sorting Protein 1) was first identified by its role in sorting the proprotein convertase furin to the trans-Golgi network, most understanding of PACS1 function has been attained through studies of cultured cells. To better understand how the R203W disease variant changes PACS1 function in vivo, we have modeled PACS1 syndrome in C. elegans. The single C. elegans pacs-1 gene encodes a protein that shares all conserved domains, and is 27% identical/46% similar to human PACS1. The furin-binding region is especially well conserved between C. elegans and humans (45% identical/70% similar) with the disease variant site, R203, absolutely conserved and denoted as R116 in C. elegans. We edited the endogenous C. elegans pacs-1 gene to generate a PACS1 syndrome model expressing PACS-1(R116W). Using drugs to probe neuronal function, we found that pacs-1(R116W) animals are resistant to the paralyzing effect of the acetylcholine esterase inhibitor aldicarb. Consistent with its neuronal function, we also found that expression of PACS1 is enriched in the nervous system. Our C. elegans pacs-1(R116W) provides the first germline-expressed model of PACS1 syndrome that can be screened for potential chemical therapeutics and further characterized to understand molecular mechanisms of the PACS1 syndrome variant.

Byrd, Dana T. et al. (2013) International Worm Meeting "Distal tip cell processes provide extensive contact between the germline stem cell pool and its cellular niche."

Byrd, Dana T., Schmitt, Katharyn, Kimble, Judith, Knobel, Karla, Crittenden, Sarah L.

[International Worm Meeting, 2013]

The germline stem cell niche in C. elegans includes the distal tip cell (DTC). The DTC has a complex cellular architecture; there is a cap that covers germ cells with a sheet of membrane, and long external processes that extend along the outside of the germline syncytium, contacting a subset of germ cells. The role of this architecture in germline stem cell biology, stem cell maintenance and the pattern of mitosis and differentiation in the germ line remains an open question. We have identified an additional region of DTC to germline contact: a plexus of membranous processes that surround and intercalate between adjacent germ cells in the distal germ line. We find that the end of the plexus region corresponds with the start of expression of a meiosis-promoting protein marker, GLD-1::GFP. Thus, germ cells within the cap and plexus regions have a high level of niche contact and low levels of a meiosis-promoting factor. One model is that increased niche contact leads to increased Notch signaling in this region and is important for maintaining the "stem cell-like" state of germ cells in the distal germ line. In addition, we find that formation and maintenance of the DTC processes depends on continued germ cell divisions. Thus signals from mitotic germ cells appear to influence niche architecture. This work lays a foundation for studying the complex cellular architecture of a niche, its role in regulating the self-renewal versus differentiation decision of stem cells and two-way communication between niche and stem cells.

Dana Byrd et al. (2008) Development & Evolution Meeting "Subcellular Architecture of the Germline Stem Cell Niche"

Karla Knobel, Judith Kimble, Katharyn Schmitt, Dana Byrd, Audra Amasino

[Development & Evolution Meeting, 2008]

A stem cell's decision between self-renewal and differentiation is governed by extrinsic stimuli from the surrounding microenvironment, or niche, as well as by intrinsic cues. In C. elegans, the distal tip cell (DTC) provides the niche for germline stem cells and maintains a "mitotic region" by Notch signaling (e.g. Kimble and Crittenden 2007). DTC number and dosage of the LAG-2 DSL ligand, which is expressed by the DTC, can affect the number of germ cells in the mitotic region (Dyan Vogel and Myon-Hee Lee). To investigate DTC niche function, we made a panel of fluorescent protein markers to visualize its subcellular structure in vivo. These include markers for the nuclear envelope, endoplasmic reticulum, Golgi, endosomes, and plasma membrane. Using membrane markers, we found that contact between the DTC niche and germ cells in the mitotic region is extensive. The DTC extends membrane processes that surround adjacent germ cells. The intimate contact between the niche and germ cells may provide a mechanism to physically anchor the distal-most germ cells within the niche and/or provide more localized signaling to maintain the germline stem cells. With a combination of DTC, sheath cell, and germ cell markers, we are examining the relationship between the extent of niche contact and the region of cells with stem cell potential. We are also using tissue-specific RNAi to test which genes are required in the DTC for regulation of germline stem cells.

Dana Byrd et al. (2006) Development & Evolution Meeting "Understanding the Molecular Properties of a Stem Cell Niche"

Judith Kimble, Dana Byrd, Daniel Hesselson

[Development & Evolution Meeting, 2006]

Distal tip cells (DTCs) serve at least two critical roles during development of the C. elegans hermaphrodite gonad. They function as migratory leader cells to regulate morphogenesis of the U-shaped gonad arms and as niche cells to regulate germline proliferation. We have taken a microarray approach to determine the gene expression profiles of the DTC during development to further explore the molecular basis of its leader and niche cell functions. We are enriching for DTC transcripts by immunoprecipitation of Flag-tagged PAB-1 (poly-A binding protein) with bound RNAs (Roy et al. 2002). DTC expression will be validated with promoter-GFP constructs, and function will be assessed by cell-specific RNAi. Our progress will be described.

Dana Byrd et al. (2004) Mid-west Worm Meeting "Regulation of germline stem cells by the DTC niche"

Sarah L Crittenden, Dana Byrd, Judith Kimble, Kim Leonhard

[Mid-west Worm Meeting, 2004]

Stem cells are defined by their capacity for self-renewal and ability to produce at least one differentiated cell type. Their decision between self-renewal and differentiation is governed by both intrinsic signals and extrinsic stimuli from the surrounding microenvironment, or niche. The mitotic region in the C. elegans germline includes stem cells* since it is self-renewing and continuously generates gametes. In young wild-type adults, the mitotic region is composed of about 225 germ cells that extend 18-20 cell diameters along the distal-proximal axis. The somatic distal tip cell (DTC), which lies adjacent to the distal-most germ cells, promotes proliferation by GLP-1/Notch signaling and provides the stem cell niche. Since stem cells defined in other systems divide slowly and/or asymmetrically, we are characterizing cell cycles and orientation of mitotic spindles within the germline mitotic region. Preliminary results using BrdU, Cy3-dUTP, and anti-PH3 reveal no differences along the distal-proximal axis with respect to frequency of mitosis or time from BrdU incorporation (S phase) to M phase. In addition, the orientation of mitotic spindles appears random throughout the region. We suggest that stem cells in the C. elegans germ line may be controlled at a population level rather than by asymmetric divisions. We are currently developing methods to track the lineage of individual cells within the mitotic region to examine this further. The DTCs provide the LAG-2 Notch ligand required for maintenance of the germline stem cells. Whether the LAG-2 signal must reach all of the cells within the mitotic region is still unknown. To ask whether direct contact between the DTC and the germ cells defines the extent of the mitotic region, we examined the DTC and its processes using lag-2 ::GFP and lag2 ::myrGFP reporters. In animals of different ages and in mutants with longer and shorter mitotic regions, we found that DTC process length does not correlate with the length of the mitotic region. These findings extend the work of Hall et al. (1999) and confirm the idea that the length of DTC processes does not define the extent of the mitotic region. To identify additional genes that are important for DTC niche function, we are planning to use a DTC-specific RNAi screen. ------------------------------------ Hall et al. (1999) Developmental Biology 212, 101-123. * Note: While nuclei in the germline are syncytial, we call them (capital O, grave accent)cells(capital O, acute accent) for simplicity as they are partially enclosed by membranes and appear to function individually with respect to cell division regulation.

Dana Byrd et al. (2001) International C. elegans Meeting "UNC-16, A JNK signaling scaffold, regulates vesicle transport in C. elegans"

Yishi Jin, Masato Kawasaki, Naoki Hisamoto, Kunihiro Matsumoto, Mercy Walcoff, Dana Byrd

[International C. elegans Meeting, 2001]

Localization of synaptic components must be both precise and dynamic to allow proper information flow between neurons and their targets. In first larval stage (L1) animals, the GABAergic DD motor neurons form synapses with ventral body wall muscle, and the localization of SNB-1::GFP, a synaptic vesicle marker, is restricted to the ventral processes. Loss-of-function mutations in the unc-16 gene result in the mislocalization of SNB-1::GFP to dorsal dendritic processes of L1 stage DD neurons without a detectable change in a postsynaptic GABA receptor marker in the muscle. The localization of presynaptic markers in many neurons is similarly altered in unc-16 mutants. SNB-1::GFP is mislocalized to the dorsal dendritic processes of the VD motor neurons and out along the dendritic endings of several sensory neurons in the head. Intriguingly, GLR-1::GFP, a postsynaptic marker, is also mislocalized along the ventral cord and in the anterior endings of some interneurons. unc-16 encodes a protein homologous to mouse JSAP1/JIP3 and Drosophila Sunday Driver (dSYD). Like its mammalian homologs, UNC-16 physically interacts with JNK signaling components. Deletion mutants of KNK signaling genes result in slight mislocalization of synaptic vesicle markers in L1 DD motor neurons and exacerbate the phenotype of weak unc-16 alleles. UNC-16 and JNK signaling molecules are expressed in neurons and function cell autonomously in DD neurons. Our results suggest that UNC-16 may regulate synaptic vesicle transport through its combined interactions with both JNK signaling molecules and motor proteins. In support of a role for UNC-16 in regulating vesicle transport, unc-16 mutations partially suppress mutations in unc-104 , a synaptic vesicle specific kinesin. The suppression is not allele specific, suggesting that the interaction of unc-16 and unc-104 may not be direct, but that both UNC-104 and UNC-16 may contribute to a similar process. Mutations in unc-16 do not suppress unc-116 kinesin heavy chain mutations. Instead, a weak unc-116 mutation results in SNB-1::GFP mislocalization in L1 DD motor neurons similar to that seen in unc-16 mutants. We propose that UNC-16 functions by both forming a linkage between vesicular cargo and motor proteins and serving as a scaffold for signaling molecules that direct cargo selection, motor activity, or motor direction.

Dana Byrd et al. (2002) West Coast Worm Meeting "UNC-16, the C. elegans JIP3, plays kinesin-1-dependent and independent roles in directing vesicular traffic"

Masato Kawasaki, Dana Byrd, Rie Sakamoto, Naoki Hisamoto, Yishi Jin, Kunihiro Matsumoto

[West Coast Worm Meeting, 2002]

The proper localization of synaptic components is a regulated process that requires the pairing of multiple cargo vesicles with different motor proteins. Despite the identification of many motors, the specific interactions of cargoes and motors are poorly understood. We have previously shown that loss-of-function mutations in unc-16, the C. elegans homolog of JSAP1/JIP3/dSYD, cause mislocalization of synaptic vesicle and glutamate receptor markers (Neuron 32: 787-800). Moreover, mutations in unc-16partially suppress the synaptic vesicle retention defect in unc-104 KIF1A mutants. UNC-16 physically interacts with the KLC-2/UNC-116 kinesin-1 complex via KLC-2. Mutations in unc-116 KHC/KIF5 and klc-2 share a similar synaptic vesicle mislocalization phenotype with unc-16 mutants, and mislocalize a functional UNC-16::GFP. Conversely, UNC-116::GFP is mislocalized in unc-16 and klc-2 mutants. These results are consistent with a direct interaction of UNC-16 with the kinesin-1 motor complex and support an evolutionarily conserved function of UNC-16/JIP3 in mediating kinesin-1 transport. However, the function of UNC-16 may be more complex than simply serving to regulate kinesin-1-cargo interactions because UNC-16 and kinesin-1 have separable functions. For example, mutations in unc-116do not suppress the unc-104synaptic vesicle retention defect; unc-116 mutants, but not unc-16, have axon outgrowth defects; and GLR-1::GFP is mislocalized in unc-16 mutants, but not in unc-116, jnk-1, or unc-104 mutants. Therefore, we propose that UNC-16 likely has kinesin-1-independent functions.

Dana T. Byrd et al. (2007) International Worm Meeting "Interactions between the germline stem cells and their niche."

Dana T. Byrd, Audra Amasino, Katharyn Schmitt, Judith Kimble

[International Worm Meeting, 2007]

Stem cells are defined by their unlimited or prolonged capacity for self-renewal and ability to produce at least one differentiated cell type. Their decision between self-renewal and differentiation is governed by both intrinsic signals and extrinsic stimuli from the surrounding microenvironment, or niche. The niche for C. elegans germline stem cells is provided by the distal tip cell (DTC) since it is both necessary and sufficient for maintenance of cells in the mitotic region. The DTC controls germline stem cells by Notch signaling (Crittenden et al. 2003). Specifically, germ cells in the mitotic region express the GLP-1 Notch receptor and respond to the LAG-2 DSL ligand expressed by the DTC. However, there may be additional factors required in the DTC for LAG-2 signaling and maintenance of germline stem cells. We have taken several approaches to further dissect the molecular and cellular properties of the DTC that enable it to function as a niche for the germline stem cells. Using a candidate gene approach, we are screening for genes required in the DTC for fertility by tissue-specific RNAi. To both narrow down the list of candidate genes and to determine the gene expression profile of the DTC, we have used a microarray approach, enriching for DTC transcripts by immunoprecipitation of Flag-tagged PAB-1 (poly-A binding protein) with associated RNAs (Roy et al. 2002). To investigate the cellular characteristics of the DTC that may contribute its role as a stem cell niche, we made a panel of fluorescent protein markers to examine the subcellular structure of the DTC in vivo. These include markers for the nuclear envelope, endoplasmic reticulum, Golgi, endosomes, and plasma membrane. Using a membrane marker, we found that contact between the DTC niche and germ cells in the mitotic region is extensive and dynamic. The DTC extends membrane processes that surround adjacent germ cells. The intimate contact between the niche and germ cells may provide a mechanism to physically anchor the distal-most germ cells within the niche and/or provide more localized signaling to maintain the germline stem cells.

Dana T Byrd et al. (2003) International Worm Meeting "Intracellular Transport and Localization Mechanisms of Synaptic Components"

Dana T Byrd, Yishi Jin

[International Worm Meeting, 2003]

Localization of synaptic components must be both precise and dynamic to allow proper information flow between neurons and their targets. We have shown that loss-of-function mutations in unc-16, the C. elegans homolog of JSAP1/JIP3/dSYD, cause mislocalization of synaptic vesicle (SNB-1::GFP) and glutamate receptor (GLR-1::GFP) markers (Byrd et al. 2001). UNC-16 functions cell autonomously in presynaptic neurons to regulate synaptic vesicle marker localization. To test whether the mislocalization of a postsynaptic glutamate receptor marker in unc-16 mutants is a secondary consequence of, or homeostatic response to, reduced presynaptic input, we asked where unc-16 is required to restore GLR-1::GFP localization in unc-16 mutants. We found that while expression of unc-16 in neurons presynaptic to GLR-1::GFP expressing cells with Posm-10-UNC-16 failed to rescue GLR-1::GFP localization, expression of unc-16 in the postsynaptic GLR-1::GFP expressing neurons with Pglr-1-UNC-16 rescued GLR-1::GFP mislocalization in unc-16 mutants. Therefore, UNC-16 most likely functions cell autonomously in the GLR-1-expressing neurons to regulate the transport and/or localization of the GLR-1 glutamate receptor. Similar to what is observed in unc-16 mutants, a SNB-1::GFP synaptic vesicle marker is mislocalized in let-981 mutant L1 larvae. We previously reported that alleles of let-981 could be allelic with unc-16 based on non-complementation tests. However, we were unable to find lesions in the unc-16 ORF of let-981 mutants. Repeating the non-complementation tests revealed that the non-complementing L1 progeny are most likely aneuploids that are hemizygous for unc-16 and that unc-16 and let-981 are not allelic. Additional characterization of the let-981 alleles suggests that the early larval lethality of s453, s529, and s222 and sterility of s1072 may be due to post-embryonic mitotic defects. Since let-981 function is required for viability and proper synaptic vesicle precursor localization, the molecular identity of let-981 may yield some insight on the possible ties between growth or cell cycle control and vesicle transport.

Gilbert, Jennifer et al. (2013) International Worm Meeting "SPCH-1/2/3 localize to mature sperm chromatin and may play a role in fertility and genome stability."

Chu, Diana, Berry, Jordan, Gilbert, Jennifer, Byrd, Dana

[International Worm Meeting, 2013]

During spermatogenesis, chromatin becomes highly compacted to ensure efficient delivery of DNA to the oocyte. Compaction of sperm chromatin in most animals is facilitated by deposition of small nuclear basic proteins called protamines. While protamines share molecular features, their sequence variability makes identification across species challenging. From a proteomic screen, we identified three nearly identical C. elegans proteins, SPCH-1/2/3, that share molecular features of protamines and are highly enriched in sperm chromatin. Shared molecular features include low molecular weight, high isoelectric point, and a high percent of arginine and serine residues. Using proteomic analysis of acid solubilized sperm chromatin, we also found that SPCH-1/2/3 are highly phosphorylated. Based on the feature similarity to protamines, we hypothesize that SPCH-1/2/3 may play a role in fertility. We first examined the localization of SPCH-1/2/3 by immunofluorescence to assess where SPCH-1/2/3 may function. Consistent with a role in male fertility, SPCH-1/2/3 localizes to the compact chromatin of mature sperm. Immediately after fertilization, SPCH-1/2/3 mark the paternal pronuclei and then are removed as the sperm pronucleus decondenses. In order to examine localization of SPCH-1, 2 and 3 independently in live animals and in real time during fertilization, we are currently generating transgenic animals expressing SPCH-1/2/3::GFP. To test whether loss of SPCH-1/2/3 function results in declined fertility, we counted viable progeny from spch deletion mutant animals. Surprisingly, disruption in a single spch gene, spch-2, results in a statistically significant reduction in the number of viable progeny compared to wild type. We have also found that spch-2 mutants have a higher percentage of male progeny than wild type, suggesting that SPCH proteins, and perhaps protamines more generally, may play a role in both fertility and genome stability.