Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2018 Apr 1.
Published in final edited form as: Dev Biol. 2017 Feb 24;424(1):18–27. doi: 10.1016/j.ydbio.2017.02.016

The Caenorhabditis elegans matrix non-peptidase MNP-1 is required for neuronal cell migration and interacts with the Ror receptor tyrosine kinase CAM-1

Teresa R Craft 1, Wayne C Forrester 1
PMCID: PMC5399540  NIHMSID: NIHMS856164  PMID: 28238735

Abstract

Directed cell migration is critical for metazoan development. During Caenorhabditis elegans development many neuronal, muscle and other cell types migrate. Multiple classes of proteins have been implicated in cell migration including secreted guidance cues, receptors for guidance cues and intracellular proteins that respond to cues to polarize cells and produce the forces that move them. In addition, cell surface and secreted proteases have been identified that may clear the migratory route and process guidance cues. We report here that mnp-1 is required for neuronal cell and growth cone migrations. MNP-1 is expressed by migrating cells and functions cell autonomously for cell migrations. We also find a genetic interaction between mnp-1 and cam-1, which encodes a Ror receptor tyrosine kinase required for some of the same cell migrations.

Keywords: Caenorhabditis elegans, MNP-1, cell migration, secreted peptidase

Background

Cell migration is critical for metazoan development. Many cells migrate long distances during animal development. Cell migration is of particular importance in the developing nervous system, where many neuronal precursors move from their sites of birth to the positions they occupy in mature animals. In a related process the migrations of growth cones, specialized structures at the leading ends of axons and dendrites, establish the connections of the nervous system. Biochemical, genetic and molecular studies have identified many genes involved in cell migration but our understanding of the process remains incomplete.

We have utilized genetic screens to identify genes required for cell migration in the small nematode Caenorhabditis elegans. During C. elegans development, many cell types migrate extensively. For example, embryonic muscle cells migrate from lateral positions to flank the dorsal and ventral midlines (Hresko et al., 1994; Moerman et al., 1996; Sulston et al., 1983). Cell migration is also important during C. elegans nervous system development where several neuronal precursors migrate long distances during embryonic development (Fig. 1, Sulston et al., 1983) and migrating growth cones establish the connectivity of the nervous system (Durbin, 1987; White et al., 1986).

Figure 1.

Figure 1

Open in a new tab

Cell migrations. A. Schematic lateral view of a 450-minute old embryo showing the final positions of CAN, HSN and ALM (circles) and their migratory routes (arrows). B. Schematic lateral view of the right side of a late first stage larva. QR divides to produce AQR, AVM and SDQ and two cells that die (not shown). Their final positions and approximate migratory routes are shown (arrows).

Different classes of proteins have been implicated in the process of cell migration. For example, putative guidance cues and their receptors direct migrating cells along their proper pathways. Intracellular proteins coordinate signals from guidance cues with the proteins that regulate the polymerization of actin at the leading edges to direct migrating cells. Also implicated in cell migration are secreted proteases. Originally these proteins were thought to function by clearing the pathway of obstacles for migrating cells (Brooks et al., 1996; Sato et al., 1994) but more recent results suggest that the roles of these proteins is more complex (reviewed in McCawley and Matrisian, 2001; Seiki, 2002).

The mnp-1 gene encodes a membrane-associated member of the M1 aminopeptidase family that is required for embryonic muscle cell migration (Tucker and Han, 2008). Aminopeptidases remove one or a few amino acids from the N-termini of target proteins or peptides (Hooper, 1994). They are implicated in tumorigenesis, regulation of blood pressure, angiogenesis, cell migration, and immune response. M1 aminopeptidases include a conserved GXMEN catalytic domain and an HENNH+E sequence motif that functions in the coordination of zinc, an essential cofactor for their proteolytic activity (Hooper, 1994; Iturrioz et al., 2001; Laustsen et al., 2001). Interestingly, C. elegans MNP-1 lacks a recognizable GXMEN motif and replaces three of the four conserved amino acids of HENNH+E motif and is therefore presumed to be catalytically inactive (Tucker and Han, 2008).

To identify genes required for cell migration, we performed a genetic screen for mutations that disrupted canal-associated neuron (CAN) cell migration (Forrester and Garriga, 1997; Forrester et al., 1998). One such mutation we called fam-1(gm85), for fasciculation and cell migration defective. fam-1(gm85) mutants are defective in the migrations of multiple cell types and axon fasciculation, where axons fail to remain tightly bundled together (Forrester and Garriga, 1997). We cloned fam-1 and found that fam-1(gm85) is a mutation in the mnp-1 gene. We find that MNP-1 functions cell autonomously for CAN cell migration. Interestingly we find that mnp-1 interacts genetically with another gene, cam-1, that also is required for the migrations of multiple cell types including CAN (Forrester et al., 1999; Forrester and Garriga, 1997).

Materials and Methods

Strains

Nematodes were grown at 20°C as described (Brenner, 1974). Strains used in this study include: the N2 wild-type strain and cam-1(cw82), cam-1(gm105), cam-1(gm122) (Forrester et al., 1999; Forrester and Garriga, 1997; Forrester et al., 1998), mIn1[mIs14 dpy-10(e128)] (Edgley and Riddle, 2001), mnp-1(gm85) (Forrester and Garriga, 1997; Forrester et al., 1998), mnp-1(ok2434), otIs33[kal-1::gfp] (Bulow et al., 2002), gmEx129[ceh-10::gfp], gmIs18[ceh-23::gfp, rol-6(su1006)] (Lai and Garriga, 2004), juIs76[unc-25::gfp, lin-15] (Jin et al., 1999), jcIs1[ajm-1::gfp, unc-29, rol-6(su1006)] (Koppen et al., 2001), cwEx488[Pmnp-1::mnp-1::gfp], cwEx486[Pmnp-1::mnp-1::gfp], cwEx487[Pmnp-1::mnp-1::gfp], ; cwEx526[Punc-119::mnp-1::gfp], cwEx526[Punc-119::mnp-1::gfp], cwEx527[Punc-119::mnp-1::gfp], cwEx495[Pceh-10::mnp-1::gfp], cwEx498[Pceh-10::mnp-1::gfp], cwEx502[Pceh-23::mnp-1::gfp], cwEx510[Pceh-23::mnp-1::gfp], cwEx490[Phlh-1::mnp-1::gfp], cwEx489[Phlh-1::mnp-1::gfp], cwEx491[Pajm-1::mnp-1::gfp], cwEx491[Pajm-1::mnp-1::gfp], cwEx518[Pajm-1::mnp-1::gfp], cwEx519[Pajm-1::mnp-1::gfp + Phlh-1::mnp-1::gfp], and cwEx520[Pajm-1::mnp-1::gfp + Phlh-1::mnp-1::gfp]. Germline transformants were produced by microinjection as described (Mello et al., 1991).

mnp-1 cloning

To determine the chromosomal location of fam-1 we generated unc-93(e1500sd) fam-1(gm85) dpy-17(e164) animals and crossed them with CB4856, a polymorphic strain (Koch et al., 2000; Wicks et al., 2001). We identified Unc non Dpy and Dpy non Unc recombinant animals, determined whether they were also fam-1(gm85) homozygotes and asked whether they had the N2 or CB4856 polymorphism for SNPs in the interval to which fam-1 mapped. We found seven recombinants that showed that gm85 was to the right of a polymorphism within the C30D11 cosmid and four that showed it was to the left of F25F2. Of these, a single recombinant showed that gm85 lay to the right of a polymorphism within R10E4 and a second recombinant showed that it was to the left of C28A5.

We next looked for polymorphisms within the interval to which gm85 mapped by whole genome Illumina DNA sequencing (performed by the Indiana University Center for Genomics and Bioinformatics). This analysis identified four polymorphisms of which a single one altered the DNA sequence within a predicted gene. The mutation within mnp-1(gm85) was confirmed by amplifying the mnp-1 gene from gm85 mutants by polymerase chain reaction and subjecting the product to Sanger DNA sequencing.

Transgenes

We generated a 2431 bp PCR product derived from the Gateway pDONR221 vector that included the ccdB and chloramphenicol resistance genes flanked by attP1 and attP2 recombination sites and was flanked by HindIII sites that we inserted into the HindIII site of pPD95.75 (A. Fire). A 7908 bp full-length mnp-1 genomic PCR product (lacking its upstream regulatory regions) produced using N2 genomic DNA as template was inserted into the SbfI and AgeI restriction sites upstream of the gfp gene (Chalfie et al., 1994). This plasmid was used to insert promoters from ajm-1, ceh-10, ceh-23, hlh-1, mnp-1, and unc-119 upstream of mnp-1. In general, each promoter was amplified by PCR using N2 genomic DNA as template and then introduced into the plasmid by Gateway cloning following manufacturer’s protocols (ThermoFisher) using attB sites introduced into the PCR product. PCR primers used are listed in the Supplemental Table. Resulting plasmids were transformed into competent DH5-alpha cells (NEB). All constructs were verified by DNA sequencing. To produce ceh-10::gfp, we fused a 4.2 kb XhoI fragment from pS308 (kindly provided by James McGhee) into XhoI cut pPD95.69. The DNA sequence of the resulting plasmid was confirmed and transgenic animals were produced by microinjection (Mello et al., 1991).

Microscopy

The extent of cell migration was determined by comparing the positions of nuclei relative to non-migratory hypodermal nuclei using a Nikon E600 microscope equipped with Nomarski optics. For ALM, CAN, and HSN cells that migrate embryonically (Sulston et al., 1983), we scored the positions of the nuclei of these cells relative to non-migratory hypodermal V and P nuclei in newly-hatched first larval stage (L1) otIs33 and mnp-1; otIs33 hermaphrodites (Table 1). For the Q neuroblasts and their descendants, which migrate during the L1 stage (Sulston and Horvitz, 1977), we scored the final positions of the Q-cell-descendant nuclei relative to the non-migratory hypodermal nuclei Vn.a and Vn.p in mid-L1 stage hermaphrodites (Table 1). For mnp-1 transgenic animals (Tables 2 and 3), the positions of CAN and HSN were assessed in second and third larval stage Rol animals that carried a kal-1::gfp transgene (Bulow et al., 2002) that expressed GFP in CAN and HSN using a Nikon SMZ1500 stereomicroscope equipped with fluorescence. Images shown in figures 3 and 5 – 7 were taken using Leica SP5 and SP8 confocal microscopes using Leica Application Suite X. Images of embryos were taken using a Nikon Eclipse 90i and a CoolSnap HQ charge-coupled device camera (Photometrics, Tucson, AZ) controlled by MetaMorph (Molecular Devices, Sunnyvale, CA). Images were captured at 0.5 μm steps.

Table 1.

mnp-1 is required for multiple cell migrations*

Strain
CAN
HSN
ALM
QR
QL
posterior anterior
wild type 0 (38) 2.7 (38) 0 0 (38) 2.6 (38) 0 (42)
mnp-1(gm85) 46.7 (60) 18.2 (56) 9.1 20.7 (59) 59.3 (27) 7.1 (28)
mnp-1(ok2434) 36.3 (81) 27.0 (75) 2.7 15.8 (77) 39.3 (28) 0 (34)
Open in a new tab
*

Data presents percentage of cells misplaced.

The number in parentheses is the number of cells scored. CAN was scored as misplaced if it was anterior to V3. HSN was scored as posterior if it was posterior to V4 and anterior if it was anterior to P5/6. ALM was scored as misplaced if it was anterior to V2. QR was scored as misplaced if it was posterior to V2.a. QL was scored as misplaced if it was anterior to V4.p.

Table 2.

Tissue specific rescue of cell migration defects

Strain
CAN*
HSN
Lumpy
N
wild type 0 1.0 0 104
mnp-1(gm85) 38.7 7.5 37.0 93
mnp-1(ok2434) 35.6 14.1 27.7 101
mnp-1; cwEx522[rol-6] 28.2 13.8 34.5 110
MNP-1 promoter**
mnp-1; cwEx488[Pmnp-1::mnp-1] 0.9 13.9 0 109
mnp-1; cwEx486[Pmnp-1::mnp-1] 1.8 13.2 14.0 114
mnp-1; cwEx487[Pmnp-1::mnp-1] 3.0 17.2 13.5 101
Pan-neuronal
mnp-1; cwEx526[Punc-119::mnp-1] 7.5 3.8 20.8 106
mnp-1; cwEx527[Punc-119::mnp-1] 9.3 7.5 29.6 108
Expression in CAN
mnp-1; cwEx495[Pceh-10::mnp-1] 6.5 8.9 13.1 123
mnp-1; cwEx498[Pceh-10::mnp-1] 4.7 24.6 35.2 106
CAN + amphid neurons
mnp-1; cwEx502[Pceh-23::mnp-1] 15.1 13.2 41.5 108
mnp-1; cwEx510[Pceh-23::mnp-1] 24.5 16.7 25.9 110
Muscle
mnp-1; cwEx490[Phlh-1::mnp-1] 15.0 19.3 30.2 113
mnp-1; cwEx489[Phlh-1::mnp-1] 10.3 10.5 23.5 107
Hypodermis
mnp-1; cwEx491[Pajm-1::mnp-1] 24.1 13.4 17.9 112
mnp-1; cwEx518[Pajm-1::mnp-1] 35.3 19.8 22.4 116
Muscle + Hypodermis
mnp-1; cwEx519[Pajm-1&hlh-1::mnp-1] 24.0 13.9 10.0 100
mnp-1; cwEx520[Pajm-1&hlh-1::mnp-1] 20.8 9.4 12.5 96
Open in a new tab
*

CAN cell migration defects were rescued by Pmnp-1::mnp-1, Punc-119::mnp-1 and Pceh-10::mnp-1 (p<0.0001) and partially rescued by Phlh-1::mnp-1 (p<0.01). It was not rescued by Pajm-1&hlh-1::mnp-1, Pceh-23::mnp-1, and Pajm-1::mnp-1. p values were calculated by t-test comparing each transgenic line to mnp-1(ok2434)[rol-6].

**

Subheadings generalize major tissue types that express the relevant promoters.

Table 3.

mnp-1 and cam-1 act redundantly in cell migration*

Strain
CAN
HSN
n
posterior
anterior
kal-1::gfp 0 1.0 0 104
mnp-1(gm85); kal-1::gfp 38.7 7.5 6.5 93
mnp-1(ok2434); kal-1::gfp 35.6 14.1 2.0 101
cam-1(gm105); kal-1::gfp 23.2 1.8 12.6 112
cam-1(cw82); kal-1::gfp 11.4 0 2.9 105
cam-1(gm105); mnp-1(ok2434); kal-1::gfp 70.3 3.0 3.0 101
cam-1(gm105); mnp-1(gm85); kal-1::gfp 76.4 2.8 4.7 106
cam-1(cw82); mnp-1(ok2434); kal-1::gfp 79.6 7.8 4.9 103
cam-1(cw82); mnp-1(gm85); kal-1::gfp 78.4 6.0 8.0 100
Open in a new tab
*

Data presents percentage of cells misplaced.

n = number of cells scored.

Cells were scored as misplaced as described for Table 1.

Figure 3.

Figure 3

Open in a new tab

Mutations in mnp-1 disrupt migrations of neurons and their growth cones. In all figures, anterior is to the left and ventral is down. An asterisk (A, C, E, G) indicates the approximate center of the gonad primordium. Panels are presented in pairs where the first of each pair (A, C, E, G, I, K) shows an animals viewed by fluorescence microscopy and the second (B, D, F, H, J, L) shows the same animal viewed by DIC microscopy. Wild-type animals have normally positioned CANs (arrows, A and E) and HSN (arrowhead, A). mnp-1(ok2434) mutants have misplaced CANs (arrows, C and G) and HSN (arrowhead, C). In wild type, D-type motor neurons form a continuous line of UNC-25::GFP expression (I). In mnp-1(ok2434) mutants, D-type motor neuron axon migration is often defective producing gaps in UNC-25::GFP expression (I, arrows). Wild type animals have a smooth body surface (B, F, J) whereas mnp-1(ok2434) mutants often have misshapen bodies, especially anteriorly (D, H, L). E. Scale bar in panel A indicates 20 μm.

Figure 5.

Figure 5

Open in a new tab

An mnp-1 transgene rescues mnp-1 mutants. A. In mnp-1(ok2434) mutants CAN (arrow) and HSN (arrowhead) sometimes fail to migrate to their normal destinations. B. Same animal as A viewed using DIC microscopy showing misshapen body. C. A Pmnp-1::mnp-1::gfp transgene rescues the CAN (arrow) and HSN (arrowhead) cell migration defects. D. A Pmnp-1::mnp-1::gfp transgene rescues the body morphology defect of mnp-1(ok2434) mutants. Scale bar in panel A represents 20 μm.

Figure 7.

Figure 7

Open in a new tab

Mutations in mnp-1 and cam-1 mutually enhance cell migration and morphological defects. In panels A, C, E, and G, an arrow indicates CAN, an arrowhead indicates HSN and an asterisk marks the center of the gonad primordium. Panels B, D, F and H show the same animals as A, C, E and G, respectively, viewed under DIC microscopy. A. In wild type, CAN and HSN are positioned normally. C. In mnp-1(ok2434) mutant CAN and HSN are misplaced anteriorly. E. In weak cam-1 mutants, CAN and HSN usually migrate to their normal positions. G. In cam-1(cw82); mnp-1(ok2434) double mutants CAN cell migration defects are enhanced. Scale bar in panel A represents 20 μm. All panels show the same magnification.

Motorneuron defects were assessed using a Nikon E600 fluorescence microscope in wild type or mnp-1 mutants harboring the juIs76[unc-25::gfp, lin-15] transgene (Jin et al., 1999). The dorsal nerve cord appears as a continuous line of fluorescence in juIs76 animals. In wild-type animals we sometimes saw (24.4% of animals) a small gap in the fluorescence but rarely saw a large gap (7.7%). In mnp-1(ok2434); juIs76 animals we rarely saw a small gap (2.8%) but often saw a large gap in the dorsal nerve cord (29.2%). This defect is similar to that reported by Forrester and Garriga, 1997 for mnp-1(gm85) mutants.

Results

fam-1(gm85) affects neuronal cell migrations

We examined several embryonic cell migrations in fam-1(gm85) mutants (Fig. 1A). The canal-associated neurons (CANs), a pair of bilaterally symmetrical cells that are born in the anterior, migrate posteriorly in embryos to final positions near the middle of the animal (Sulston et al., 1983). The hermaphrodite-specific neurons (HSNs), a pair of bilaterally symmetrical cells that are born in the posterior, migrate anteriorly in embryos to occupy final positions slightly posterior and ventral to the CANs (Sulston et al., 1983). The anterior lateral microtubule cells (ALMs), a pair of bilaterally symmetrical neurons, migrate a short distance posteriorly during embryogenesis (Sulston et al., 1983). We also examined the postembryonic migrations of the Q neuroblasts, a pair of cells located on opposite sides of the animal that each divide to ultimately produce three neurons and two cells that die (Sulston and Horvitz, 1977). The left Q neuroblast (QL) and its descendants migrate posteriorly in first stage larvae whereas the right Q neuroblast (QR) and its descendants migrate anteriorly (Fig. 1B).

The migrations of multiple cell types were defective in fam-1(gm85) mutants (Figs. 2 and 3, Table 1, Forrester and Garriga, 1997). For example, in fam-1(gm85) mutants, CAN, HSN, ALM and QR descendants often failed to migrate to their normal positions. We previously found that CAN provides a signal that prevents HSN from migrating too far; when CAN was absent 15% of HSNs migrated beyond their normal positions (Forrester and Garriga, 1997). Accordingly, HSN migrated anterior to its normal position 9% of the time in fam-1(gm85) animals.

Figure 2.

Figure 2

Open in a new tab

The positions of CANs, HSNs and QR descendants are altered by mutations in mnp-1. Drawings at the top of the first two columns represent newly hatched first-stage larvae. Long vertical lines indicate the positions of V cell and Q cell nuclei and short vertical lines indicate the positions of P cell nuclei. The shaded oval shows the approximate location of the gonad primordium. The percent of CAN and HSN cells (first and second columns, respectively) relative to V, P, and Q cell nuclei are indicated. Drawing at the top of the third column represents a mid-first-stage larva. Shaded oval indicates the position of the gonad primordium. The percent of QR descendants SDQ and AVM relative to anterior (circles) and posterior (ovals) V cell daughter nuclei are indicated below. n indicates the number of cells scored.

Neuronal axon migrations were often defective in fam-1 mutants (Forrester and Garriga, 1997). CAN normally produces two axons, one that runs anteriorly to the head and one that runs posteriorly to the tail (White et al., 1986). HSN sends an axon ventrally where it joins the nerve cord and turns anteriorly to extend to the head after skirting the vulva, where it can form one or two branches that innervate muscles involved in egg laying (Desai et al., 1988; White et al., 1986). CAN and HSN axons sometimes followed abnormal trajectories or stopped short along their normal pathways (Forrester and Garriga, 1997, not shown). Ventrally located D type motor neurons send processes longitudinally along the ventral nerve cord where they branch, with one branch extending along the lateral body wall to the dorsal nerve cord where it bifurcates to extend anteriorly and posteriorly within the dorsal nerve cord (White et al., 1978; White et al., 1986). In fam-1(gm85) mutants, dorsal projections often occurred along the left body wall instead of the right and gaps could be seen in the dorsal nerve cord, indicating that axons have failed to enter or extend along the dorsal nerve cord (Fig. 3, Forrester and Garriga, 1997).

gm85 is a mutation in mnp-1

To gain further insight into the role of fam-1 in cell migration, we cloned the gene. First, we mapped the position of the fam-1(gm85) mutation relative to polymorphisms located on the third chromosome using standard single nucleotide polymorphism (SNP) mapping (Wicks et al., 2001). We found that the mutation mapped to the third chromosome, between SNPs found within the cosmids R10E4 and C28A5, an interval encompassing less than 250,000 base pairs of DNA.

We next used whole genome Illumina DNA sequencing to identify mutations within the interval to which fam-1 mapped (Bentley, 2006; Sarin et al., 2008). DNA sequence of fam-1(gm85) genomic DNA identified four DNA sequence differences from wild type, only one of which was within a predicted gene. This gene was previously named mnp-1 (Tucker and Han, 2008). This mutation changed a C to a G at position +191 (where the A of the ATG is +1), thereby changing a serine to a stop codon (Fig. 4). Because this mutation was predicted to terminate translation after the 64th amino acid out of 781, it may be a null mutation. mnp-1 encodes a predicted metalloprotease family member but because of changes within the conserved region required for protease activity, MNP-1 is predicted to lack protease activity (Tucker and Han, 2008). Because of these and other experiments described below, we conclude that gm85 mutates mnp-1 and refer to the mutation as mnp-1(gm85) hereafter.

Figure 4.

Figure 4

Open in a new tab

Sequence of the MNP-1 protein. The mnp-1(gm85) mutation changes the serine at position 64 (asterisk and underlined) to a stop codon, which is predicted to truncate the protein after the first 63 amino acids. mnp-1(ok2434) deletes 630 nucleotides, which is predicted to produce a protein that lacks the final 213 amino acids (italicized). The M1 family protease consensus sequence is underlined.

The C. elegans gene knock-out consortium identified a mutation, mnp-1(ok2434), that deleted a portion of the mnp-1 gene (Fig. 4). We next asked whether mnp1(ok2434) and gm85 displayed similar phenotypes as expected if they mutate the same gene. mnp-1(ok2434) affected the migrations of the same cells and in a similar manner to mnp-1(gm85) (Fig. 2, Table 1). mnp-1 mutants exhibited visible defects in addition to or as a consequence of cell migration defects. Animals depleted for mnp-1 by RNA-mediated interference developed a distinctive notched head appearance (Tucker and Han, 2008). Both mnp-1(gm85) and mnp-1(ok2434) mutants often developed this notched-head defect (not shown, Tucker and Han, 2008). Both mnp-1 mutants often had a general lumpy appearance, where the cuticle did not form a smoothly tapering cylinder and produced similar uncoordinated locomotion (Fig. 3, Forrester et al., 1998). That both mnp-1(gm85) and mnp-1(ok2434) mutants shared all of these defects is consistent with both being defective in the same gene. Furthermore, mnp-1(gm85) and mnp-1(ok2434) failed to complement one another; mnp-1(gm85)/mnp-1(ok2434) transheterozygotes were indistinguishable from either homozygote both for cell migration and visible phenotypes (not shown).

MNP-1 is expressed in migrating cells

We next asked where MNP-1 is expressed. To do this we fused the mnp-1 upstream plus coding sequences to the gfp gene. First, we determined that the Pmnp-1::mnp-1::gfp reporter transgene rescued cell migration defects of mnp-1 mutants. In mnp-1(ok2434) mutants with the Pmnp-1::mnp-1::gfp transgene, only 0.9%, 1.8% or 3.0% (three independent transgenic lines) of CAN cells were misplaced, showing that the transgene rescued the CAN cell migration defect (Fig. 5, Table 2). The transgene also rescued the visible phenotypes of mnp-1 mutants but failed to rescue HSN cell migration defects (Table 2). That Pmnp-1::mnp-1::gfp rescued most defects of mnp-1 mutants further confirmed that gm85 was an mnp-1 mutant and suggested that it was expressed in cells that normally express the gene.

In embryos with the Pmnp-1::mnp-1::gfp transgene, we first detected GFP at about 250 minutes after first cell division. Expression continued throughout the rest of embryonic and post embryonic development. By 350 minutes after first division GFP was detected in many cells whose positions suggested that they were muscle cell precursors (Fig. 6A). In 1.5-fold stage and older embryos, expression continued in putative muscle precursors (Fig. 6B, C and D). In addition, we detected GFP expression in cells whose positions were consistent with their being neuronal precursors. In young larvae through adult, Pmnp-1::mnp-1::gfp was expressed in body wall muscle cells (Fig. 6A) and sometimes within neurons.

Figure 6.

Figure 6

Open in a new tab

mnp-1 is expressed throughout body wall muscle and in some neurons. All panels show wild type animals expressing Pmnp-1::mnp-1::gfp. A. Image shows GFP-expressing cells in an approximately 350-minute old embryo. Scale bar in panel A represents 10 μm. Panels A – D show the same magnification. B. Image shows GFP-expressing cells in an approximately 450-minute old embryo. C. Same as panel B but more medial focal plane. D. Image shows GFP-expressing cells in an approximately 500-minute old embryo. E. Confocal microscope Z-series of one side of a young adult, flattened to show Pmnp-1::mnp-1::gfp expression in two rows of muscle cells along the dorsal and ventral quadrant. Arrows indicate GFP associated with the membrane along one side of a single trapezoid-shaped muscle cell. The animal is twisted because of the presence of the dominant rol-6 transgene. Scale bar in panel E represents 25 μm.

Consistent with its predicted subcellular localization, GFP appeared around the periphery of muscle cells in animals after hatching (Fig. 6E). Interestingly, in embryos protein appeared to be largely cytoplasmic. We cannot say whether this protein is within the endoplasmic reticulum (ER) prior to its transport to the cell membrane or is within cytoplasm. However, we sometimes saw a punctate pattern similar to that seen for proteins within the ER.

MNP-1 acts autonomously in migrating cells

To determine whether MNP-1 acted autonomously in migrating or in surrounding cells, we utilized tissue-specific promoters to drive MNP-1 expression in different cell types (Table 2). In each case we looked for rescue from two independent transgenic lines. We first asked whether MNP-1 acted autonomously in CAN cells. unc-119 is expressed throughout much of the nervous system as well as some non-neuronal cells (Knobel et al., 2001; Maduro and Pilgrim, 1995). Expression of MNP-1 from the unc-119 promoter rescued CAN cell migration defects. ceh-10 is expressed in several neurons including the CANs (Svendsen and McGhee, 1995). A Pceh-10::mnp-1 transgene rescued CAN cell migration defects. ceh-23 is expressed in the CAN cells and several chemosensory neurons (Wang et al., 1993; Zallen et al., 1999). Only one Pceh-23::mnp-1 transgenic line may have provided weak rescue CAN cell migration defects. The lack of or weak rescue by Pceh-23::mnp-1 may reflect later timing of ceh-23 expression (Altun-Gultekin et al., 2001; Forrester et al., 1998). Together our results suggest that mnp-1 functions autonomously in CANs for CAN cell migration.

We next asked whether MNP-1 also functioned in surrounding cell types (Table 2). hlh-1 is expressed in muscle cells (Krause et al., 1994). Phlh-1::mnp-1 provided partial rescue of CAN cell migration suggesting a possible role in neuronal migration for mnp-1 in muscle. ajm-1 is expressed throughout epithelia including hypodermis, pharynx, intestine and others (Koppen et al., 2001). Pajm-1::mnp-1 failed to rescue CAN cell migration. To determine whether MNP-1 might be required simultaneously in both muscle and epithelial cells, we generated animals that had both the Pajm-1::mnp-1 + Phlh-1::mnp-1 transgenes. CAN cell migration was not rescued in these lines.

Expression of MNP-1 in any of the cell types tested did not provide clear rescue of HSN cell migration defects (Table 2). Expression throughout much of the nervous system with the unc-119 promoter may produce partial rescue (Table 2) in one line but the observation that expression of MNP-1 from its own promoter failed to rescue HSN migration makes interpretation of this result difficult.

Expression of MNP-1 individually in neuron, muscle and epithelial cells failed to rescue the lumpy phenotype of mutants but simultaneous expression in both muscle and hypodermis provided partial rescue (Table 2). These results suggest that MNP-1 may be required in muscle and epithelia as well as other cell types to produce normal morphology.

Mutations in mnp-1 appear not to substantially alter cell fates

A possible explanation for the cell migration defects is that cell fates are altered in mnp-1 mutants. To address this possibility, we examined the expression of reporter transgenes in mnp-1 mutants. kal-1 is expressed in several neurons in the anterior, CAN, HSN, and PVM in the middle, and a few neurons in the tail (Bulow et al., 2002). ceh-10 is expressed in the CAN cells as well as several other neurons in the head (Svendsen and McGhee, 1995; Altun-Gultekin et al., 2001). ceh-23 is expressed in several neurons and the CAN cells (Zallen et al., 1999). unc-25 is expressed by γ-aminobutyric acid (GABA)-ergic neurons including the DD and VD motorneurons (Jin et al., 1999). ajm-1 is expressed at the apical junctions of many cells, including the laterally located seam cells (Koppen et al., 2001). In all cases, GFP expression appears indistinguishable from wild type in mnp-1(ok2434) mutants with kal-1, ceh-10, ceh-23, unc-25 and ajm-1 promoters driving gfp expression (Fig. 2, Supp. Fig. 1). These results suggest that mnp-1 mutations do not dramatically alter cell fates. Tucker and Han reached a similar conclusion with respect to MNP-1 function during muscle cell migration (Tucker and Han, 2008).

mnp-1 and cam-1 mutations are synthetic lethal

cam-1 encodes a Ror-family receptor tyrosine kinase (RTK) that functions to direct the migrations of several cell types including CAN and HSN (Forrester et al., 1999; Forrester and Garriga, 1997; Koga et al., 1999). In cam-1 strong loss-of-function mutants, CANs usually remain in the head and HSNs migrate too far 65% of the time (Forrester and Garriga, 1997). Since mutation in mnp-1 affects many of the same cell migrations, we constructed mnp-1 and cam-1 double mutants.

To produce mnp-1 and cam-1 double mutants, we first generated mnp-1(gm85 or ok2434); cam-1(gm122)/mIn1 animals. mIn1 is a gfp-marked chromosome balancer (Edgley and Riddle, 2001) and cam-1(gm122) is a putative null mutation of cam-1 (Forrester et al., 1999; Forrester et al., 1998). To identify mnp-1; cam-1 double homozygotes, we looked for animals that lacked GFP expression, indicating that they no longer retained the balancer chromosome and therefore were homozygous for cam-1(gm122). We found few of these animals. When transferred to individual culture dishes, the presumptive mnp-1; cam-1 double mutants invariably either died or produced few progeny, which in turn died. Occasionally animals would survive a few generations but we were unable to establish homozygous double mutant lines. Double mutants died at all stages although we noted that they most frequently died either as early stage embryos, by about 350 minutes of embryonic development, or as first stage larvae. This shows that mutations in mnp-1 and cam-1 are synthetically lethal, suggesting that the genes act redundantly for some essential function.

To look further at the genetic interaction, we made mnp-1; cam-1 double mutants using the hypomorphic gm105 and cw82 alleles of cam-1 (Chien et al., 2015; Forrester et al., 1999; Forrester and Garriga, 1997). In these weak cam-1 mutants CAN and HSN cell migrations were usually normal (Forrester and Garriga, 1997; Forrester et al., 1998 and Table 3). mnp-1; cam-1(hypomorph) double mutants were viable, although they grew more slowly and produced fewer offspring than either single mutant. We looked at CAN and HSN position in the double mutants and found that CAN cell migrations were substantially more defective in double mutants than in either single mutant (Fig. 7, Table 3). In addition, uncoordinated locomotion, withered tail and the overall lumpy, misshapen appearance all were enhanced in the double mutants relative to single mutants (not shown), consistent with redundant roles for mnp-1 and cam-1.

We wondered whether cam-1 expression might be altered by mutation in mnp-1. A reporter transgene that fused cam-1 along with its regulatory sequences to gfp was expressed in many cells, particularly in the anterior region of the animals (Forrester et al., 1999). We compared cam-1::gfp expression in wild-type animals to mnp-1 mutants and, as expected if cam-1 and mnp-1 act independently, found no detectable difference (Supp. Fig. 2).

Discussion

We used DNA sequencing in conjunction with single nucleotide polymorphism genetic mapping to identify mnp-1 as the gene mutated in gm85 mutants. The C. elegans Gene Knockout Consortium identified mnp-1(ok2434). mnp-1(ok2434) and mnp-1(gm85) mutants appear similar for all phenotypes examined. A transgene that included the mnp-1 upstream and coding regions rescued the mutant phenotypes of both. Collectively these results demonstrate convincingly that gm85 is a mutation in mnp-1. Tucker et al. showed that mnp-1 is required for normal muscle cell migration (Tucker and Han, 2008). Our results extend the role of MNP-1 to include directed neuronal cell and growth cone migration, demonstrating that MNP-1 plays a general role in the migrations of multiple cell types.

Both mnp-1 mutations are likely to be strong loss-of function mutations. mnp-1(gm85) changes serine 63 to a stop codon. This is predicted to remove more than 90% of the MNP-1 protein, which would be expected to reduce if not eliminate protein function. mnp-1(ok2434) is predicted to delete the final 213 amino acids. Because both mutations are predicted to delete large portions of the protein, both are likely to reduce or eliminate gene function.

MNP-1 acts autonomously for CAN cell migration. Neuronal MNP-1 expression rescued CAN cell migration defects. Notably, the observation that a Pceh-10::mnp-1::gfp transgene rescued CAN cell migration strongly supports this model because ceh-10 is expression in only a few neurons in the head plus the CANs. Expression in muscle also provided partial rescue, suggesting that MNP-1 in muscle many contribute to neuronal migration. Consistent with our results, Tucker et al. obtained results suggesting that MNP-1 functioned cell autonomously in migrating muscle cells (Tucker and Han, 2008).

MNP-1 is a member of the M1 family of aminopeptidases, proteins that remove N-terminal amino acids from target proteins. Several extracellular proteases have been implicated in cell migration where they are thought to “clear a path” for migrating cells, to degrade cell attachments to permit cells to migrate, or to process regulators of cell migration (reviewed in McCawley and Matrisian, 2001; Seeds et al., 1997; Stefansson and Lawrence, 2003). Because of the replacement of several amino acids within the HENNH + E motif that is conserved amongst M1 family proteases, MNP-1 is unlikely to be an active peptidase and therefore unlikely to function in any of these ways.

Interestingly of 17 M1 aminopeptidases within the C. elegans genome, only 9 are predicted to be catalytically active (Althoff et al., 2014). Inactive proteases are common in other species as well (reviewed in Reynolds and Fischer, 2015). Some inactive peptidases have been found to act as regulators of active proteases. For example, the catalytically inactive caspase CASPS18 stimulates the activity of the catalytically active caspase CASPS19 (Bryant et al., 2010). Others have been postulated to act as inhibitors of active proteases. These observations suggest possible roles for MNP-1 as a positive or negative regulator of another protease that in turn might degrade extracellular matrix components or process guidance molecules. Alternatively, MNP-1 may function independent of any other proteases. Todd et al. (Todd et al., 2002) have found that proteins inactivated by mutation of catalytically active sites often perform functions unrelated to their ancestral roles. For example, the inactive serine protease heparin-binding protein (HBP) can act as a chemoattractant (Flodgaard et al., 1991). Still other inactive proteases, such as UNC-71, have been postulated to function in cell adhesion (Huang et al., 2003). Further studies will be required to determine the biochemical activity of MNP-1.

At least two independent pathways direct CAN cell migrations. cam-1 encodes a receptor tyrosine kinase of the Ror family (Forrester et al., 1999; Koga et al., 1999) that is required for cell migration (Forrester et al., 1999; Forrester and Garriga, 1997), neuronal development (Forrester et al., 1999; Forrester and Garriga, 1997; Kennerdell et al., 2009; Koga et al., 1999; Song et al., 2010), synaptic development (Francis et al., 2005; Jensen et al., 2012), neuronal pruning (Hayashi et al., 2009), and vulval development (Green et al., 2007, 2008). Some CAM-1 functions require its kinase activity whereas others do not. mnp-1 and cam-1 function redundantly for CAN migration. In weak cam-1 single mutants CAN cell migration defects are minimal, consistent with the mutations weakly compromising CAM-1’s cell migration function. In weak cam-1; mnp-1 double mutants CAN cell migration defects are substantially enhanced relative to either single mutant, showing that cam-1 and mnp-1 act independently in cell migration. Furthermore, the synthetic lethality seen in strong cam-1; mnp-1 doubles demonstrates that the two genes together provide an essential function for animals.

Supplementary Material

1

Supplemental Figure 1. mnp-1 does not alter transgene expression. A and B. Wild type and mnp-1(ok2434) mutants expressing kal-1::gfp, respectively. C and D. Wild type and mnp-1(ok2434) mutants expressing Pceh-23::gfp, respectively. E and F. Wild type and mnp-1(ok2434) mutants expressing Punc-25::gfp, respectively. G and H. Wild type and mnp-1(ok2434) mutants expressing Punc-25::gfp, respectively. I and J. Wild type and mnp-1(ok2434) mutants expressing Pkrp-95::gfp, respectively. Scale bar represents 20 μm.

2

Supplemental Figure 2. mnp-1 mutations do not alter cam-1::gfp expression. A. Wild type expressing cam-1::gfp. Scale bar represents 20 μm. B. The same animal viewed by DIC microscopy. C. mnp-1(ok2434) expressing cam-1::gfp. D. The same animal viewed by DIC microscopy.

3

Highlights.

  • MNP-1 is required for neuronal cell migration

  • MNP-1 functions autonomously in migrating cells

  • mnp-1 functions redundantly with cam-1 for viability and in cell migration

Acknowledgments

We thank James McGhee for pS308 and Oliver Hobert for otIs33. We thank Brittany Flores for technical assistance. We thank the C. elegans gene knock out consortium for generating the mnp-1(ok2434) mutation and the C. elegans stock center for providing strains used in these studies. We thank the Indiana University Center for Genomics and Bioinformatics for DNA sequence analysis. This work was supported in part by NIH grant GM080745. We are grateful for project support from the Medical Sciences Program and the Department of Biology at Indiana University. We also thank Jim Powers and the Light Microscopy Imaging Center at Indiana University, which is supported in part by the Office of the Vice Provost for Research at IU.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Althoff MJ, Flick K, Trzepacz C. Collaboration within the M1 aminopeptidase family promotes reproductive success in Caenorhabditis elegans. Dev Genes Evol. 2014;224:137–146. doi: 10.1007/s00427-014-0470-3. [DOI] [PubMed] [Google Scholar]
  2. Altun-Gultekin Z, Andachi Y, Tsalik EL, Pilgrim D, Kohara Y, Hobert O. A regulatory cascade of three homeobox genes, ceh-10, ttx-3 and ceh-23, controls cell fate specification of a defined interneuron class in C. elegans. Development. 2001;128:1951–1969. doi: 10.1242/dev.128.11.1951. [DOI] [PubMed] [Google Scholar]
  3. Bentley DR. Whole-genome re-sequencing. Curr Opin Genet Dev. 2006;16:545–552. doi: 10.1016/j.gde.2006.10.009. [DOI] [PubMed] [Google Scholar]
  4. Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77:71–94. doi: 10.1093/genetics/77.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brooks PC, Stromblad S, Sanders LC, von Schalscha TL, Aimes RT, Stetler-Stevenson WG, Quigley JP, Cheresh DA. Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin alpha v beta 3. Cell. 1996;85:683–693. doi: 10.1016/s0092-8674(00)81235-0. [DOI] [PubMed] [Google Scholar]
  6. Bryant B, Ungerer MC, Liu Q, Waterhouse RM, Clem RJ. A caspase-like decoy molecule enhances the activity of a paralogous caspase in the yellow fever mosquito, Aedes aegypti. Insect Biochem Mol Biol. 2010;40:516–523. doi: 10.1016/j.ibmb.2010.04.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bulow HE, Berry KL, Topper LH, Peles E, Hobert O. Heparan sulfate proteoglycan-dependent induction of axon branching and axon misrouting by the Kallmann syndrome gene kal-1. Proc Natl Acad Sci USA. 2002;99:6346–6351. doi: 10.1073/pnas.092128099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. Green fluorescent protein as a marker for gene expression. Science. 1994;263:802–805. doi: 10.1126/science.8303295. [DOI] [PubMed] [Google Scholar]
  9. Chien SC, Gurling M, Kim C, Craft T, Forrester W, Garriga G. Autonomous and nonautonomous regulation of Wnt-mediated neuronal polarity by the C. elegans Ror kinase CAM-1. Dev Biol. 2015;404:55–65. doi: 10.1016/j.ydbio.2015.04.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Desai C, Garriga G, McIntire SL, Horvitz HR. A genetic pathway for the development of the Caenorhabditis elegans HSN motor neurons. Nature. 1988;336:638–646. doi: 10.1038/336638a0. [DOI] [PubMed] [Google Scholar]
  11. Durbin R. PhD Thesis. Cambridge University; UK: 1987. Studies on the development and organization of the nervous system of Caenorhabditis elegans. [Google Scholar]
  12. Edgley ML, Riddle DL. LG II balancer chromosomes in Caenorhabditis elegans: mT1(II;III) and the mIn1 set of dominantly and recessively marked inversions. Mol Genet Genomics. 2001;266:385–395. doi: 10.1007/s004380100523. [DOI] [PubMed] [Google Scholar]
  13. Flodgaard H, Ostergaard E, Bayne S, Svendsen A, Thomsen J, Engels M, Wollmer A. Covalent structure of two novel neutrophile leucocyte-derived proteins of porcine and human origin. Neutrophile elastase homologues with strong monocyte and fibroblast chemotactic activities. Eur J Biochem. 1991;197:535–547. doi: 10.1111/j.1432-1033.1991.tb15942.x. [DOI] [PubMed] [Google Scholar]
  14. Forrester WC, Dell M, Perens E, Garriga G. A C. elegans Ror receptor tyrosine kinase regulates cell motility and asymmetric cell division. Nature. 1999;400:881–885. doi: 10.1038/23722. [DOI] [PubMed] [Google Scholar]
  15. Forrester WC, Garriga G. Genes necessary for C. elegans cell and growth cone migrations. Development. 1997;124:1831–1843. doi: 10.1242/dev.124.9.1831. [DOI] [PubMed] [Google Scholar]
  16. Forrester WC, Perens E, Zallen JA, Garriga G. Identification of Caenorhabditis elegans genes required for neuronal differentiation and migration. Genetics. 1998;148:151–165. doi: 10.1093/genetics/148.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Francis MM, Evans SP, Jensen M, Madsen DM, Mancuso J, Norman KR, Maricq AV. The Ror receptor tyrosine kinase CAM-1 is required for ACR-16-mediated synaptic transmission at the C. elegans neuromuscular junction. Neuron. 2005;46:581–594. doi: 10.1016/j.neuron.2005.04.010. [DOI] [PubMed] [Google Scholar]
  18. Green JL, Inoue T, Sternberg PW. The C. elegans ROR receptor tyrosine kinase, CAM-1, non-autonomously inhibits the Wnt pathway. Development. 2007;134:4053–4062. doi: 10.1242/dev.005363. [DOI] [PubMed] [Google Scholar]
  19. Green JL, Inoue T, Sternberg PW. Opposing Wnt pathways orient cell polarity during organogenesis. Cell. 2008;134:646–656. doi: 10.1016/j.cell.2008.06.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hayashi Y, Hirotsu T, Iwata R, Kage-Nakadai E, Kunitomo H, Ishihara T, Iino Y, Kubo T. A trophic role for Wnt-Ror kinase signaling during developmental pruning in Caenorhabditis elegans. Nat Neurosci. 2009;12:981–987. doi: 10.1038/nn.2347. [DOI] [PubMed] [Google Scholar]
  21. Hooper NM. Families of zinc metalloproteases. FEBS Lett. 1994;354:1–6. doi: 10.1016/0014-5793(94)01079-x. [DOI] [PubMed] [Google Scholar]
  22. Hresko MC, Williams BD, Waterston RH. Assembly of body wall muscle and muscle cell attachment structures in Caenorhabditis elegans. J Cell Biol. 1994;124:491–506. doi: 10.1083/jcb.124.4.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Huang X, Huang P, Robinson MK, Stern MJ, Jin Y. UNC-71, a disintegrin and metalloprotease (ADAM) protein, regulates motor axon guidance and sex myoblast migration in C. elegans. Development. 2003;130:3147–3161. doi: 10.1242/dev.00518. [DOI] [PubMed] [Google Scholar]
  24. Iturrioz X, Rozenfeld R, Michaud A, Corvol P, Llorens-Cortes C. Study of asparagine 353 in aminopeptidase A: characterization of a novel motif (GXMEN) implicated in exopeptidase specificity of monozinc aminopeptidases. Biochemistry. 2001;40:14440–14448. doi: 10.1021/bi011409j. [DOI] [PubMed] [Google Scholar]
  25. Jensen M, Hoerndli FJ, Brockie PJ, Wang R, Johnson E, Maxfield D, Francis MM, Madsen DM, Maricq AV. Wnt signaling regulates acetylcholine receptor translocation and synaptic plasticity in the adult nervous system. Cell. 2012;149:173–187. doi: 10.1016/j.cell.2011.12.038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Jin Y, Jorgensen E, Hartwieg E, Horvitz HR. The Caenorhabditis elegans gene unc-25 encodes glutamic acid decarboxylase and is required for synaptic transmission but not synaptic development. J Neurosci. 1999;19:539–548. doi: 10.1523/JNEUROSCI.19-02-00539.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kennerdell JR, Fetter RD, Bargmann CI. Wnt-Ror signaling to SIA and SIB neurons directs anterior axon guidance and nerve ring placement in C. elegans. Development. 2009;136:3801–3810. doi: 10.1242/dev.038109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Knobel KM, Davis WS, Jorgensen EM, Bastiani MJ. UNC-119 suppresses axon branching in C. elegans. Development. 2001;128:4079–4092. doi: 10.1242/dev.128.20.4079. [DOI] [PubMed] [Google Scholar]
  29. Koch R, van Luenen HG, van der Horst M, Thijssen KL, Plasterk RH. Single nucleotide polymorphisms in wild isolates of Caenorhabditis elegans. Genome Res. 2000;10:1690–1696. doi: 10.1101/gr.gr-1471r. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Koga M, Take-uchi M, Tameishi T, Ohshima Y. Control of DAF-7 TGF-(alpha) expression and neuronal process development by a receptor tyrosine kinase KIN-8 in Caenorhabditis elegans. Development. 1999;126:5387–5398. doi: 10.1242/dev.126.23.5387. [DOI] [PubMed] [Google Scholar]
  31. Koppen M, Simske JS, Sims PA, Firestein BL, Hall DH, Radice AD, Rongo C, Hardin JD. Cooperative regulation of AJM-1 controls junctional integrity in Caenorhabditis elegans epithelia. Nat Cell Biol. 2001;3:983–991. doi: 10.1038/ncb1101-983. [DOI] [PubMed] [Google Scholar]
  32. Krause M, Harrison SW, Xu SQ, Chen L, Fire A. Elements regulating cell- and stage-specific expression of the C. elegans MyoD family homolog hlh-1. Dev Biol. 1994;166:133–148. doi: 10.1006/dbio.1994.1302. [DOI] [PubMed] [Google Scholar]
  33. Lai T, Garriga G. The conserved kinase UNC-51 acts with VAB-8 and UNC-14 to regulate axon outgrowth in C. elegans. Development. 2004;131:5991–6000. doi: 10.1242/dev.01457. [DOI] [PubMed] [Google Scholar]
  34. Laustsen PG, Vang S, Kristensen T. Mutational analysis of the active site of human insulin-regulated aminopeptidase. Eur J Biochem. 2001;268:98–104. doi: 10.1046/j.1432-1327.2001.01848.x. [DOI] [PubMed] [Google Scholar]
  35. Maduro M, Pilgrim D. Identification and cloning of unc-119, a gene expressed in the Caenorhabditis elegans nervous system. Genetics. 1995;141:977–988. doi: 10.1093/genetics/141.3.977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McCawley LJ, Matrisian LM. Matrix metalloproteinases: they’re not just for matrix anymore! Curr. Opin Cell Biol. 2001;13:534–540. doi: 10.1016/s0955-0674(00)00248-9. [DOI] [PubMed] [Google Scholar]
  37. Mello CC, Kramer JM, Stinchcomb D, Ambros V. Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 1991;10:3959–3970. doi: 10.1002/j.1460-2075.1991.tb04966.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Moerman DG, Hutter H, Mullen GP, Schnabel R. Cell autonomous expression of perlecan and plasticity of cell shape in embryonic muscle of Caenorhabditis elegans. Dev Biol. 1996;173:228–242. doi: 10.1006/dbio.1996.0019. [DOI] [PubMed] [Google Scholar]
  39. Reynolds SL, Fischer K. Pseudoproteases: mechanisms and function. Biochem J. 2015;468:17–24. doi: 10.1042/BJ20141506. [DOI] [PubMed] [Google Scholar]
  40. Sarin S, Prabhu S, O’Meara MM, Pe’er I, Hobert O. Caenorhabditis elegans mutant allele identification by whole-genome sequencing. Nat Methods. 2008;5:865–867. doi: 10.1038/nmeth.1249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sato H, Takino T, Okada Y, Cao J, Shinagawa A, Yamamoto E, Seiki M. A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature. 1994;370:61–65. doi: 10.1038/370061a0. [DOI] [PubMed] [Google Scholar]
  42. Seeds NW, Siconolfi LB, Haffke SP. Neuronal extracellular proteases facilitate cell migration, axonal growth, and pathfinding. Cell Tissue Res. 1997;290:367–370. doi: 10.1007/s004410050942. [DOI] [PubMed] [Google Scholar]
  43. Seiki M. The cell surface: the stage for matrix metalloproteinase regulation of migration. Curr Opin Cell Biol. 2002;14:624–632. doi: 10.1016/s0955-0674(02)00363-0. [DOI] [PubMed] [Google Scholar]
  44. Song S, Zhang B, Sun H, Li X, Xiang Y, Liu Z, Huang X, Ding M. A Wnt-Frz/Ror-Dsh pathway regulates neurite outgrowth in Caenorhabditis elegans. PLoS Genet. 2010:6. doi: 10.1371/journal.pgen.1001056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Stefansson S, Lawrence DA. Old dogs and new tricks: proteases, inhibitors, and cell migration. Sci STKE. 2003;2003:pe24. doi: 10.1126/stke.2003.189.pe24. [DOI] [PubMed] [Google Scholar]
  46. Sulston JE, Horvitz HR. Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Dev Biol. 1977;56:110–156. doi: 10.1016/0012-1606(77)90158-0. [DOI] [PubMed] [Google Scholar]
  47. Sulston JE, Schierenberg E, White JG, Thomson JN. The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev Biol. 1983;100:64–119. doi: 10.1016/0012-1606(83)90201-4. [DOI] [PubMed] [Google Scholar]
  48. Svendsen PC, McGhee JD. The C. elegans neuronally expressed homeobox gene ceh-10 is closely related to genes expressed in the vertebrate eye. Development. 1995;121:1253–1262. doi: 10.1242/dev.121.5.1253. [DOI] [PubMed] [Google Scholar]
  49. Todd AE, Orengo CA, Thornton JM. Sequence and structural differences between enzyme and nonenzyme homologs. Structure. 2002;10:1435–1451. doi: 10.1016/s0969-2126(02)00861-4. [DOI] [PubMed] [Google Scholar]
  50. Tucker M, Han M. Muscle cell migrations of C. elegans are mediated by the alpha-integrin INA-1, Eph receptor VAB-1, and a novel peptidase homologue MNP-1. Dev Biol. 2008;318:215–223. doi: 10.1016/j.ydbio.2008.02.062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wang BB, Muller-Immergluck MM, Austin J, Robinson NT, Chisholm A, Kenyon C. A homeotic gene cluster patterns the anteroposterior body axis of C. elegans. Cell. 1993;74:29–42. doi: 10.1016/0092-8674(93)90292-x. [DOI] [PubMed] [Google Scholar]
  52. White JG, Albertson DG, Anness MA. Connectivity changes in a class of motoneurone during the development of a nematode. Nature. 1978;271:764–766. doi: 10.1038/271764a0. [DOI] [PubMed] [Google Scholar]
  53. White JG, Southgate E, Thomson JN, Brenner S. The structure of the nervous system of Caenorhabditis elegans. Philos Trans R Soc Lond B Biol Sci. 1986;314:1–340. doi: 10.1098/rstb.1986.0056. [DOI] [PubMed] [Google Scholar]
  54. Wicks SR, Yeh RT, Gish WR, Waterston RH, Plasterk RH. Rapid gene mapping in Caenorhabditis elegans using a high density polymorphism map. Nat Genet. 2001;28:160–164. doi: 10.1038/88878. [DOI] [PubMed] [Google Scholar]
  55. Zallen JA, Kirch SA, Bargmann CI. Genes required for axon pathfinding and extension in the C. elegans nerve ring. Development. 1999;126:3679–3692. doi: 10.1242/dev.126.16.3679. [DOI] [PubMed] [Google Scholar]

Associated Data

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

Supplementary Materials

1

Supplemental Figure 1. mnp-1 does not alter transgene expression. A and B. Wild type and mnp-1(ok2434) mutants expressing kal-1::gfp, respectively. C and D. Wild type and mnp-1(ok2434) mutants expressing Pceh-23::gfp, respectively. E and F. Wild type and mnp-1(ok2434) mutants expressing Punc-25::gfp, respectively. G and H. Wild type and mnp-1(ok2434) mutants expressing Punc-25::gfp, respectively. I and J. Wild type and mnp-1(ok2434) mutants expressing Pkrp-95::gfp, respectively. Scale bar represents 20 μm.

NIHMS856164-supplement-1.tif (17.9MB, tif)
2

Supplemental Figure 2. mnp-1 mutations do not alter cam-1::gfp expression. A. Wild type expressing cam-1::gfp. Scale bar represents 20 μm. B. The same animal viewed by DIC microscopy. C. mnp-1(ok2434) expressing cam-1::gfp. D. The same animal viewed by DIC microscopy.

NIHMS856164-supplement-2.tif (12.9MB, tif)
3
NIHMS856164-supplement-3.docx (98KB, docx)

RESOURCES