The Caenorhabditis elegans homolog of the Evi1 proto-oncogene, egl-43, coordinates G1 cell cycle arrest with pro-invasive gene expression during anchor cell invasion

Ting Deng; Przemyslaw Stempor; Alex Appert; Michael Daube; Julie Ahringer; Alex Hajnal; Evelyn Lattmann

doi:10.1371/journal.pgen.1008470

. 2020 Mar 23;16(3):e1008470. doi: 10.1371/journal.pgen.1008470

The Caenorhabditis elegans homolog of the Evi1 proto-oncogene, egl-43, coordinates G1 cell cycle arrest with pro-invasive gene expression during anchor cell invasion

Ting Deng ^1,², Przemyslaw Stempor ³, Alex Appert ³, Michael Daube ¹, Julie Ahringer ³, Alex Hajnal ^1,^*, Evelyn Lattmann ¹

Editor: David R Sherwood⁴

PMCID: PMC7117773 PMID: 32203506

Abstract

Cell invasion allows cells to migrate across compartment boundaries formed by basement membranes. Aberrant cell invasion is a first step during the formation of metastases by malignant cancer cells. Anchor cell (AC) invasion in C. elegans is an excellent in vivo model to study the regulation of cell invasion during development. Here, we have examined the function of egl-43, the homolog of the human Evi1 proto-oncogene (also called MECOM), in the invading AC. egl-43 plays a dual role in this process, firstly by imposing a G1 cell cycle arrest to prevent AC proliferation, and secondly, by activating pro-invasive gene expression. We have identified the AP-1 transcription factor fos-1 and the Notch homolog lin-12 as critical egl-43 targets. A positive feedback loop between fos-1 and egl-43 induces pro-invasive gene expression in the AC, while repression of lin-12 Notch expression by egl-43 ensures the G1 cell cycle arrest necessary for invasion. Reducing lin-12 levels in egl-43 depleted animals restored the G1 arrest, while hyperactivation of lin-12 signaling in the differentiated AC was sufficient to induce proliferation. Taken together, our data have identified egl-43 Evi1 as an important factor coordinating cell invasion with cell cycle arrest.

Author summary

Cells invasion is a fundamental biological process that allows cells to cross compartment boundaries and migrate to new locations. Aberrant cell invasion is a first step during the formation of metastases by malignant cancer cells. We have investigated how a specialized cell in the Nematode C. elegans, the so-called anchor cell, can invade into the adjacent epithelium during normal development. Our work has identified an oncogenic transcription factor that controls the expression of specific target genes necessary for cell invasion, and at the same time inhibits the proliferation of the invading anchor cell. These findings shed light on the mechanisms, by which cells decide whether to proliferate or invade.

Introduction

Cell invasion, which is initiated by the breaching of basement membranes (BMs), is a regulated physiological process allowing select cells to cross compartment boundaries during normal development. Cell invasion is also the first step that is activated during the formation of metastases by malignant cancer cells [1]. Anchor cell (AC) invasion in C. elegans is a genetically amenable and tractable model that has provided important insights into the molecular pathways regulating cell invasion and uncovered the molecular similarities between tumor cell and developmental cell invasion [2,3].

The AC is specified during the second larval stage (L2) of C. elegans development, when two equivalent precursor cells (Z1.ppp and Z4.aaa) adopt either the AC or the ventral uterine (VU) fate, depending on stochastic differences in LAG-2 Delta/ LIN-12 Notch signaling [4,5]. This initially small difference is amplified by two extra stochastic events, the division order of Z1 and Z4 and the expression onset of hlh-2 in Z1.pp and Z1.aa[6]. The cell that exhibits higher lag-2 expression levels adopts the “default” AC fate and activates LIN-12 Notch signaling in the adjacent cell to induce the VU fate [7]. Unlike the VU cells, which undergo three to four rounds of cell divisions, the AC never divides but remains arrested in G1 phase and adopts an invasive fate. NHR-67, a nuclear receptor of the tailless family, is required to maintain the G1 arrest of the AC by regulating the cyclin-dependent kinases (CDK) inhibitor CKI-1. In response to the G1 arrest established by NHR-67, the histone deacetylase HDA-1 promotes the invasive AC fate [8]. AC invasion occurs during the third larval stage (L3) of C. elegans development. During this process, the AC is guided ventrally by cues from the adjacent primary vulva precursor cells (VPCs) and the ventral nerve cord. The AC then breaches the two BM layers separating the uterus from the epidermis and establishes direct contact with the invaginating vulva epithelium [9].

Both AC/VU fate specification and AC invasion require the activity of the egl-43 gene, which encodes two isoforms of a Zinc finger transcription factor homologous to the mammalian Evi1 proto-oncogene in the MECOM (Myelodysplastic Syndrome 1(MDS1) and Ecotropic Viral Integration Site 1 (EVI1), MDS1-EVI1) locus [10–12]. Human Evi1 has been implicated in the development of different types of cancer, most notably in the hematopoietic system in acute myeloid leukemia (AML) [13,14]. Inhibition of C. elegans egl-43 during the mid-L2 stage leads to the formation of two ACs due to a defect in AC/VU cell specification [10]. However, egl-43 remains expressed in the AC after its specification, where EGL-43 is required together with the AP-1 transcription factor FOS-1 to induce the expression of genes that promote BM breaching (i.e. pro-invasive genes), such as zmp-1, cdh-3 and him-4 [10,11,15].

Despite the importance of egl-43 in regulating pro-invasive gene expression and BM breaching, its exact role during AC invasion remains poorly understood. Here, we report that egl-43 and fos-1 form a positive auto-regulatory feedback-loop. Moreover, egl-43 plays a previously unknown role in establishing the G1 arrest of the AC by repressing Notch-dependent AC proliferation. Thus, egl-43 acts as an important regulator of AC invasion that coordinates the G1 arrest with pro-invasive gene expression.

Results

Deletion of the long egl-43L isoform leads to the formation of multiple ACs that fail to invade

In order to study the role of the different egl-43 isoforms during AC invasion, we used CRISPR/Cas9 genome editing [16] to insert gfp tags at the 5’ or 3’ end of the egl-43 locus (Fig 1A). Since the transcriptional start site of the short isoform (egl-43S) is located within the 5th intron of the egl-43L locus, the insertion of the gfp cassette at the 5’ end of the first exon labels exclusively the protein encoded by the long isoform (gfp::egl-43L), whereas the insertion of the gfp tag at the 3’ end of the locus labels both, the short egl-43S and the long egl-43L isoforms (egl-43::gfp, Fig 1A). Furthermore, the gfp cassette inserted at the 5’ end contained two flippase recognition target (FRT) sites in the gfp introns, permitting the tissue-specific inactivation of the egl-43L isoform (S1 Fig). For the expression analysis, we focused on the mid-L3 stage (the Pn.pxx stage, after the VPCs had undergone two rounds of cell divisions), the time when AC invasion normally occurs [9]. Both reporters, gfp::egl-43L and egl-43::gfp, were expressed in the invading AC and in the surrounding ventral and dorsal uterine (VU and DU) cells (Fig 1B, for a quantification of the AC expression levels of the different reporters, see S2 Fig). We found no obvious difference in the expression pattern of the two egl-43 reporters, suggesting that the long egl-43L isoform accounts for most of the expression observed. In order to test if the uterine expression is indeed caused by egl-43L, we designed two RNAi clones, one specifically targeting egl-43L (exons 4 and 5 of egl-43L), and the other targeting the 5’ UTR of egl-43S, which is not included in the egl-43L transcript (Fig 1A). EGL-43::GFP expression was lost upon egl-43L RNAi, yet no difference in expression was observed after egl-43S RNAi (Fig 1C, S2C Fig), supporting the above conclusion that the uterine expression originates predominantly from the long egl-43L isoform.

Fig 1 — **(A)** Schematic overview of the *egl-43* locus and the CRISPR/Cas9 engineered alleles used in this study. Green triangles indicate *gfp* insertion sites and dashed red line indicate deleted regions. The red crosses indicate the sites of the 11 bp (TTACTCATCTT) deletion in the Fos-Responsive Element (FRE) and of the mutation in the initiation codon of the *egl-43S* isoform. Solid red lines refer to the regions targeted by RNAi. **(B)** Expression patterns of the different endogenous *egl-43* reporters depicted in **(A).** The basement membrane (BM) was simultaneously labelled with the LAM-1::GFP marker to score AC invasion. At least 25 animals were examined for each reporter. All animals contained one GFP expressing AC and exhibited BM breaching. A quantification of the AC expression levels is shown in **S2 Fig**. **(C)** RNAi of the different *egl-43* isoforms and FLP/FRT-mediated excision of *egl-43L*. Left panels show Nomarski (DIC) images and middle panels the fluorescence signals of endogenous EGL-43::GFP expression in the nuclei and the LAM-1::GFP BM marker. The right panels show the GFP signals merged with the ACs labelled by the *lin-3*^ACEL>mCherry reporter (rows 1–4) and *cdh-*3>mCherry::PH (row 5) in magenta. The black arrowheads point at the AC nuclei and the white arrows at the locations of the BM breaches. Control refers to animals exposed to the empty RNAi vector. The numbers to the right indicate the penetrance of the invasion defects. A quantification of the AC expression levels is shown in S2C Fig. The scale bars are 5 μm.

As reported previously [11], egl-43L RNAi led to an invasion defect with a penetrance comparable to that of total egl-43 RNAi (92% for egl-43L (n = 53) and 94% for total egl-43 RNAi (n = 49), combined data from two independent RNAi experiments). Consistent with a role of egl-43 during AC/VU specification [10,11], we detected early L3 larvae with two ACs upon egl-43L or total egl-43 RNAi (Fig 1C). However, in 36 out of 49 cases, total egl-43 RNAi led to the formation of more than two ACs, a phenotype that cannot be explained by an AC/VU specification defect (Fig 1C). Similar to the invasion defects, the occurrence of multiple ACs was also caused by selective inhibition of the long egl-43L isoform, with 42 out of 53 worms containing more than 2 ACs (Fig 1C). Moreover, the number of ACs increased progressively with the age of the larvae, indicating an ongoing proliferation of the AC after the L2 stage (Fig 2A and 2B). This pointed to an additional role of egl-43L in preventing the proliferation of the AC after its specification, independently of its function during VU fate specification.

Fig 2 — **(A)** Expression of the endogenous GFP::MCM-7 reporter after RNAi of the different *egl-43* isoforms. Elevated AC expression of GFP::MCM-7 was detected after *egl-43* RNAi and *egl-43L* RNAi (29/29 and 28/31 cases, respectively), but not after control or *egl-43S* RNAi (0/31 and 0/32 cases, respectively). The left panels depict Nomarski (DIC) images and the middle panels nuclear GFP::MCM-7 expression together with the LAM-1::GFP BM marker at the mid-L3 stage. The right panels show the GFP signals merged with the ACs labelled by the *cdh-3*>mCherry::PH reporter in magenta. (B) Quantification of the number of ACs formed after RNAi of the different *egl-43* isoforms from the early L3 (Pn.p) until the mid-L3 (Pn.pxx) stage. The error bars indicate the standard deviation. **(C)** Expression of the S-phase marker RNR-1::GFP together with LAM-1::GFP in control and *egl-43* RNAi-treated mid-L3 larvae. No RNR-1::GFP expression was detected in 48 control RNAi animals, while 30/39 *egl-43* RNAi- treated animals expressed the S-phase marker. **(D)** Elevated expression of the Cyclin D *cyd-1>gfp* reporter in the multiple ACs of *egl-43* RNAi-treated mid-L3 larvae was observed in 52/58 cases, while all 43 control larvae showed faint *cyd-1>gfp* expression in the single AC. **(E)** Expression of the CDK activity sensor in the AC of control and *egl-43* RNAi-treated mid-L3 larvae together with the LAM-1::GFP BM marker. The right panels show the CDK sensor signal merged with the ACs labelled by the *lin-3*^ACEL>mCherry reporter in magenta. **(F)** Quantification of CDK sensor activity in the AC. Scatter plots showing the cytoplasmic to nuclear intensity ratio as a measure of CDK activity. The error bars indicate the standard deviation and the vertical bars in **(B)** and horizontal bars in **(F)** the mean values. Statistical significance was determined with a Student’s t-test and is indicated with ** for p<0.01, *** for p<0.001, **** for p<0.0001 and n.s for p> 0.05. In each graph, the numbers of animals scored are indicated in brackets. The black arrowheads point at the AC nuclei and the white arrows at the locations of the BM breaches. The scale bars are 5 μm.

In order to specifically examine the role of the egl-43S isoform during AC invasion, we used CRISPR/Cas9 engineering to introduce an ATG→CTG mutation in the predicted egl-43S start codon (egl-43LΔS::gfp, Fig 1A and 1B). The egl-43LΔS::gfp strain was viable and showed a similar expression pattern as the egl-43::gfp and gfp::egl-43L strains (Fig 1B). Moreover, AC invasion occurred normally in all egl-43LΔS::gfp animals examined, and no AC proliferation was observed (n = 35), which confirms the predominant role of egl-43L in regulating AC invasion and proliferation.

Finally, we used the Flp/FRT system to generate an AC-specific knock-out of egl-43L [17]. Since two FRT sites had been inserted in introns of the gfp cassette at the 5’ end of the egl-43L locus, the expression of the FLPase under control of the AC-specific lin-3 enhancer element (lin-3^ACEL>flp) [18] specifically inactivated egl-43L in the AC (S1 Fig). Deletion of egl-43L in the AC led to invasion defects (9 out of 17 animals) and the formation of multiple ACs (4 out of 17 cases), similar to the phenotypes caused by egl-43 RNAi (Fig 1C). The relatively low penetrance observed after Flp/FRT-mediated excision compared to RNAi could be due to the perdurance of the EGL-43 protein in the AC, as faint GFP::EGL-43 expression in the AC of mid-L3 larvae could be observed in 2 out of 17 cases.

In summary, the long egl-43L isoform acts cell-autonomously to block AC proliferation and promote invasion.

Neither the N-terminal PR nor the ZF1 domains in EGL-43 are necessary for AC invasion

egl-43L encodes a transcription factor containing an N-terminal PRD1-BF1/RIZ-1 domain (PR) and two separate clusters of Zinc finger domain (ZF1 & ZF2) that could exhibit DNA binding activity [12]. PR domains are structurally similar to the SET (Su(var)3-9, Enhancer of zeste and Trithorax) domains, which contribute to histone lysine methyltransferase activity and have also been reported to mediate protein-protein interactions [19,20]. In order to dissect the requirement of the different EGL-43 domains during AC invasion, we generated in-frame deletions in the egl-43 coding region using the CRISPR/Cas9 system (Fig 1A). In the PR domain deletion allele (gfp::egl-43ΔPR, deleted amino acids 2–62), we detected an approximately 75% decrease in GFP::EGL-43L expression levels in the AC (Fig 1B, S2A Fig). Despite this strong reduction, neither the proliferation nor the invasion of the AC were affected as all 25 animals scored showed normal AC invasion and no proliferation. Thus, the PR domain in EGL-43 is not necessary for AC invasion. Though, the reduced expression levels suggested that the PR domain is either required for egl-43 autoregulation (see below), protein stability, or that there exist additional regulatory elements in the deleted first intron that promote egl-43 expression in the AC (Fig 1A).

Furthermore, no AC invasion defects and no change in expression levels were observed in egl-43ΔZF1::gfp mutants, which carry an in-frame deletion removing amino acids 161–235, which encode the N-terminal Zinc Finger domains (ZF1) (Fig 1B, S2B Fig). Note that this allele also deletes the promoter of the egl-43S isoform. By contrast, the egl-43 null mutant tm1802, which contains a 659 bp deletion removing the C-terminal Zinc Finger domains (ZF2), displays severe developmental defects and early embryonic lethality [11]. Thus, the regulation of AC invasion and proliferation most likely depends on the C-terminal ZF2 domains.

egl-43 is required for the G1 arrest of the AC

Previous studies have shown that the AC must remain arrested in the G1 phase in order to invade [8]. The occurrence of multiple (i.e. more than two) ACs upon inactivation of egl-43L indicated that the AC had bypassed the G1 arrest and begun to proliferate. In order to test this hypothesis, we performed RNAi knock down of the different egl-43 isoforms in a strain carrying an endogenous GFP::MCM-7 reporter. mcm-7 encodes a subunit of the pre-replication complex (pre-RC) that is highly expressed in cycling cells but down-regulated in non-proliferating cells [21,22]. No GFP::MCM-7 expression was detectable in the AC after control or egl-43S RNAi. However, egl-43 and egl-43L RNAi resulted in elevated GFP::MCM-7 levels in the multiple ACs that formed, indicating that these ACs had re-entered the cell cycle (Fig 2A). Consistent with this conclusion, the average number of ACs increased during the development from the Pn.p (late L2/early L3) stage to the Pn.pxx (mid to late L3) stage (Fig 2B).

To further characterize the cell cycle state of the AC, we analyzed RNR-1::GFP expression, which serves as an S-phase marker [23]. While RNR-1::GFP was absent in the AC of control RNAi animals, it was expressed in the multiple ACs formed after egl-43 RNAi (Fig 2C). Moreover, we detected elevated levels of a transcriptional cyd-1>gfp Cyclin D reporter in the multiple ACs produced after egl-43i, while the single AC in control animals showed only faint Cyclin D expression (Fig 2D). Finally, we expressed a CDK biosensor in the AC to directly quantify CDK kinase activity [24,25]. This sensor monitors CDK activity via the phosphorylation-induced nuclear export of a GFP-tagged kinase substrate. Thus, a high cytoplasmic-to-nuclear signal ratio indicates high CDK activity. The multiple ACs formed in egl-43 RNAi-treated animals showed a significantly increased CDK sensor activity compared to the single AC in control RNAi animals (Fig 2E and 2F).

Thus, loss of EGL-43 function increases CDK activity and triggers cell cycle entry of the AC.

egl-43 and nhr-67 play distinct roles in inducing the G1 arrest of the AC

As reported previously [8], RNAi of nhr-67 led to the appearance of multiple ACs that could not breach the BM (Fig 3A). Since loss of nhr-67 led to a similar phenotype as inhibition of egl-43, we tested whether the expression of nhr-67 depends on egl-43, or vice versa. We first measured egl-43 and nhr-67 GFP reporter expression in early L3 larvae at the Pn.p stage, shortly after AC specification had occurred and the G1 arrest had been established. Quantification of endogenous GFP::EGL-43L reporter levels after nhr-67 RNAi revealed no significant change (S3A and S3B Fig). Also, the expression of an nhr-67::gfp reporter at the early L3 stage was not significantly changed by egl-43 RNAi (Fig 3C and 3D). Moreover, the expression of the histone deacetylase hda-1, which acts downstream of nhr-67 to promote AC invasion [8], was not changed by egl-43 RNAi (S3E and S3F Fig). However, in mid-L3 larvae (Pn.pxx stage), around the time of AC invasion, nhr-67 RNAi caused an approximately 30% reduction in GFP::EGL-43 expression levels, and a similar reduction in NHR-67::GFP expression levels was observed after egl-43 RNAi (Fig 3A–3D). Thus, at later stages egl-43 and nhr-67 may positively regulate each other’s expression.

Due to the high penetrance of the AC proliferation phenotype caused by single nhr-67 or egl-43 RNAi, we were unable to test if the simultaneous knock-down of both transcription factors had an additive effect. Consistent with a previous report [8], the AC-specific expression of the CDK inhibitor cki-1 together with an mNeonGreen (mNG) marker on a bi-cistronic mRNA under control of a cdh-3 enhancer/promoter fragment (cdh-3>cki-1::SL2::mNG) partially rescued the AC proliferation and invasion defects caused by nhr-67 RNAi (Fig 3E–3G). By contrast, CKI-1 overexpression did not rescue the invasion defects caused by egl-43 RNAi (Fig 3E and 3F), and it only slightly inhibited AC proliferation (Fig 3G). Notably, around 10% of egl-43 RNAi treated and CKI-1 overexpressing animals contained one AC, yet their BMs were not breached (Fig 3F).

These data suggested that that egl-43 acts in parallel with nhr-67 and hda-1 to maintain the G1 arrest and promote invasion of the AC.

egl-43 inhibits AC proliferation by repressing lin-12 Notch expression

LIN-12 Notch signaling is not only critical during the AC/VU decision, but it also links differentiation to cell cycle progression in different tissues [4,26,27]. We therefore tested whether egl-43 regulates lin-12 Notch expression in the AC by examining a translational LIN-12::GFP reporter [28]. In control animals at the mid-L3 stage, LIN-12::GFP expression had disappeared in the AC, while expression persisted in the adjacent VU cells (Fig 4A). By contrast, the multiple ACs that formed after egl-43 RNAi continued to express LIN-12::GFP (Fig 4A). The ACs in egl-43 RNAi-treated larvae still expressed a lin-3::mNG reporter, which serves as a marker to distinguish the AC from the VU fate [29], as well as a reporter for the LIN-12 ligand LAG-2 (S4A and S4B Fig) [30]. Thus, the inhibition of egl-43 did not cause a transformation of the AC into a VU fate, but rather resulted in the ectopic expression of LIN-12 in the proliferating ACs.

Fig 4 — **(A)** Expression of LIN-12::GFP after control, *lin-12*, *egl-43* or *nhr-67* single and *egl-43; lin-12* or *nhr-67; lin-12* double RNAi. The left panels shows Nomarski (DIC) images, middle panels LIN-12::GFP expression in green and the right panels the GFP signal merged with the AC labelled by the *cdh-3>mCherry*::PH reporter in magenta. Elevated LIN-12::GFP expression was observed in the ACs of 93/128 *egl-43* and 119/130 *nhr-67* RNAi treated animals, while none of 111 control RNAi animals showed LIN-12::GFP expression in the AC. **(B)** Quantification of the AC numbers in mid-L3 larvae treated with the different RNAi combinations shown in **(A)**. **(C)** Quantification of the AC invasion defects caused by the different RNAi treatments shown in **(A)**. **(D)** GFP::MCM-7 expression together with LAM-1::GFP after control, *egl-43* or *lin-12* single and *egl-43; lin-12* double RNAi, and in larvae expressing NICDΔCT::mCherry. The left panels show Nomarski (DIC) images and the right panels the GFP::MCM-7 signal in green merged with the AC labelled by the *cdh-3>mCherry*::PH reporter in magenta. 32/35 *egl-43* single RNAi and 13/40 *egl-43; lin-12* double RNAi treated animals showed GFP::MCM-7 expression in the AC. None of the 32 control and of the 40 *lin-12* single RNAi treated animals exhibited GFP::MCM-7 expression in the AC. Expression of NICDΔCT::mCherry induced GFP::MCM-7 expression in 32/33 cases. **(E)** AC-specific expression of *nicdΔct* from the *cdh-3* enhancer/promoter leads to the formation of multiple ACs. Left panels shows Nomarski (DIC) images and middle panels the BM marker LAM-1::GFP. The right panels show the mCherry expression from the *cdh-3* promoter as a control (row 1) or co-expressed with *nicdΔct* from a bi-cistronic transcript (row 2). (F) Quantification of the AC numbers in three independent control lines expressing mCherry alone and in three lines expressing NICDΔCT together with mCherry in the AC. **(G)** Quantification of the AC invasion defects in control lines and in lines expressing NICDΔCT together with mCherry in the AC. The pooled results of the three indepdnet lines are shown. The error bars indicate the standard deviation and the horizontal bars in **(B)** and **(F)** the mean values. Statistical significance was determined with a Student’s t-test and is indicated with ** for p<0.01 and **** for p<0.0001. The black arrowheads point at the AC nuclei. The numbers in brackets in the graphs refer to the numbers of animals analyzed. The scale bars are 5 μm.

To test whether an over-activation of lin-12 Notch signaling in the AC is responsible for the AC proliferation phenotype, we performed double RNAi of egl-43 and lin-12 and scored the number of ACs, as well as their ability to invade. While 63% of egl-43 single RNAi-treated animals formed multiple (i.e. more than one) ACs, only 16% of egl-43; lin-12 double RNAi-treated animals contained multiple ACs. The average number of ACs per animals decreased from 2.6 in egl-43 single to 1.2 in egl-43; lin-12 double RNAi-treated animals (Fig 4B), and the penetrance of the AC invasion defect decreased from 72% to 14% (Fig 4C). Eight out of the 19 animals that had been treated with egl-43; lin-12 double RNAi and exhibited an invasion defect contained a single AC. This suggested that the egl-43 invasion phenotype is not exclusively caused by the over-proliferation of the AC.

To test if reducing lin-12 activity restored the cell cycle arrest of the AC, we examined the GFP::MCM-7 reporter, which is expressed exclusively in proliferating cells. Control or single lin-12 RNAi did not induce GFP::MCM-7 expression in the AC of mid-L3 larvae, while egl-43 RNAi resulted in the formation of multiple GFP::MCM-7 positive ACs in 91% of the cases (Figs 2A & 4D). By contrast, the single ACs formed in egl-43; lin-12 double RNAi-treated animals did not express GFP::MCM-7 in 68% of the cases (Fig 4D).

LIN-12::GFP expression was also up-regulated in the AC of nhr-67 RNAi treated animals (Fig 4A). However, inhibition of lin-12 only partially suppressed the AC over-proliferation caused by nhr-67 RNAi from 3.8 to 2.6 ACs per animal (Fig 4B) and only slightly reduced the nhr-67 invasion defects from 94% to 81% (Fig 4C).

Thus, reducing lin-12 activity suppressed the AC proliferation and invasion defects caused by egl-43 RNAi. By contrast, the nhr-67 phenotype was less sensitive to a reduction in lin-12 levels, suggesting that NHR-67 inhibits AC proliferation predominantly through another pathway.

Activation of LIN-12 Notch signaling in the differentiated AC triggers proliferation

These results led to the hypothesis that the ectopic activation of LIN-12 Notch signaling caused by loss of egl-43 function may trigger the re-entry of the AC into the cell cycle. In order to test if LIN-12 signaling is sufficient to induce AC proliferation, we expressed a fragment of the intracellular LIN-12 Notch domain, in which the C-terminal PEST degradation motif had been deleted (NICDΔCT) [26], under the control of the AC-specific cdh-3 enhancer/promoter fragment. Expression of NICDΔCT in the VPCs was shown to hyper-activate the Notch signaling pathway [26], and cdh-3-driven expression occurs only after the AC fate has been specified [31]. Three independent transgenic lines carrying the cdh-3>nicdΔct::SL2::mCherry transgene (co-expressing an mCherry marker on a bi-cistronic mRNA) exhibited an AC proliferation phenotype with an average of 3.4 ACs per animal, which is comparable to the phenotype observed after egl-43 RNAi (Fig 4E and 4F). In addition, the GFP::MCM-7 reporter was up-regulated (Fig 4D), even though GFP::EGL-43L continued to be expressed in the multiple ACs of cdh-3>nicdΔct::SL2::mCherry animals (S4C Fig). In addition, NICDΔCT expression caused a penetrant AC invasion defect (Fig 4G).

Thus, the ectopic activation of the LIN-12 Notch pathway in the differentiated AC was sufficient to trigger cell cycle entry. Taken together, we propose that EGL-43 inhibits LIN-12 Notch expression to maintain the G1 arrest of the invading AC.

A positive regulatory feedback loop between egl-43 and fos-1 activates pro-invasive gene expression

It has previously been reported that fos-1 positively regulates egl-43 expression in the AC and that egl-43 is required for the expression of zmp-1, cdh-3 and him-4 [10,11]. To further characterize the role of egl-43 in regulating pro-invasive gene expression, we investigated a possible mutual regulation of fos-1 and egl-43. The expression of the endogenous GFP::EGL-43L reporter in the AC was reduced approximately two-fold in homozygous fos-1(ar105) mutants compared to heterozygous fos-1(ar105)/+ control siblings at the mid-L3 stage (Fig 5A and 5B). Using CRISPR/Cas9 genome editing, we deleted the 11 bp (TTACTCATCTT) FOS-Responsive Element (FRE) [11] in the promoter region of the endogenous gfp::egl-43L reporter strain (ΔFRE>gfp::egl-43L, Fig 1A). In a heterozygous fos-1(ar105)/+ background, the expression of the mutant ΔFRE>gfp::egl-43L reporter was reduced to a similar extent as the wild-type gfp::egl-43L reporter was reduced in a homozygous fos-1(ar105) background. Since the levels of the FRE mutant reporter did not further decrease in homozygous fos-1(ar105) larvae, FOS-1 appears to control egl-43 expression mainly through this FRE (Fig 5A and 5B). While this experiment confirmed that FOS-1 up-regulates endogenous egl-43 expression, it also pointed at the existence of additional factors that activate egl-43 expression in the AC. In particular, the deletion of the FRE in egl-43 did not cause an obvious defect in AC invasion (all 23 animals scored showed normal AC invasion), suggesting that the reduction in egl-43 expression after the deletion of the FRE can be compensated by the AC.

Fig 5 — **(A)** Expression of endogenous GFP::EGL-43L and of the mutant *ΔFRE*>GFP::EGL-43L reporter carrying a deletion of the Fos-responsive element TTACTCATCTT (ΔFRE), each in a *fos-1(ar105)/+* heterozygous and *fos-1(ar105)* homozygous background at the mid-L3 stage. **(B)** Quantification of GFP::EGL-43L expression levels in the ACs of the indicated mutant backgrounds. (C) Expression of a FOS-1a::YFP reporter after control and *egl-43* RNAi. **(D)** Quantification of FOS-1a::YFP levels in the ACs after control RNAi. **(E)** Expression of a transcriptional *egl-43>gfp* reporter in the ACs after control and *egl-43* RNAi. **(F)** Quantification of the transcriptional *egl-43>gfp* reporter expression shown in **(E)**. For each reporter, the left panels show Nomarski (DIC) images, the middle panels the GFP signals of the indicated reporters in green (in **(A)** and **(C)** together with the LAM-1::GFP BM marker) and the right panels the GFP reporter signals merged with the ACs labelled with the *cdh-3>mCherry*::PH reporter in magenta. The black arrowheads point at the AC nuclei and the white arrows at the locations of the BM breaches. The error bars indicate the standard deviation and the horizontal bars the mean values. Statistical significance was determined with a Student’s t-test and is indicated with n.s. for p>0.05, * for p<0.05 and **** for p<0.0001. The numbers in brackets refer to the numbers of animals analyzed. The scale bars are 5 μm.

Since egl-43 and fos-1 regulate some of the same target genes [11,15], we tested if egl-43 regulates fos-1a expression. The expression of a fos-1a::yfp reporter in the AC was reduced approximately three-fold by egl-43 RNAi (Fig 5C and 5D), indicating that egl-43 and fos-1 positively regulate each other’s expression. Moreover, the expression of a transcriptional egl-43L reporter (egl-43L>gfp is a strain containing an insertion of the self-excising cassette [16] after the gfp coding sequences to terminate transcription 5’ of the egl-43 coding sequences) was reduced by egl-43 RNAi (Fig 5E and 5F). Thus, egl-43 positively regulates its own expression in the AC.

Unlike nhr-67 or egl-43, fos-1 was not required to maintain the G1 arrest of the AC, as neither the S-phase maker RNR-1::GFP nor the proliferation marker GFP::MCM-7 were up-regulated after fos-1 RNAi (S5A and S5B Fig). We thus speculated that the regulation of fos-1 by EGL-43 and the cell cycle inhibition via lin-12 repression represent two independent functions of EGL-43. Supporting this hypothesis, LIN-12::GFP expression was not up-regulated by fos-1 RNAi, while expression of NICDΔCT did not affect fos-1 expression levels in the AC (S5C–S5E Fig).

Taken together, the expression analysis indicated that egl-43 and fos-1 form a positive feedback loop that maintains high expression of both transcription factors in the AC to induce the expression of target genes required for invasion. The auto-regulation of egl-43 likely adds further robustness to this network.

EGL-43 binding is enriched at the fos-1, egl-43 and lin-12 loci

To test if the changes in fos-1, lin-12 and egl-43 reporter expression caused by egl-43 RNAi could be due to a direct regulation by EGL-43, we performed chromatin immuno-precipitation and sequencing (ChIP-seq) analysis of the endogenous EGL-43::GFP reporter at the L3 stage using anti-GFP antibodies, in two biological replicates (see extended methods). At this stage, most of the EGL-43::GFP expression was confined to cells in the somatic gonad and to approximately 30 head and 6 tail neurons [11] (and this study). The ChIP-seq analysis identified 6276 peaks of significant enrichment, 5257 of which we could associate with 3977 genes (S5 Table).

EGL-43 binding was found at the egl-43, fos-1 and lin-12 loci (Fig 6). Notably, EGL-43 was also enriched at the jun-1 locus, which encodes the homolog of the human c-JUN proto-oncogene that forms together with c-FOS the AP-1 transcription factor (S5 Table). Even though no function of JUN-1 in AC invasion has been reported, this observation might indicate that EGL-43 regulates AP-1 activity in other processes, such as ovulation or lifespan [32]. Moreover, EGL-43::GFP was enriched at other previously reported targets, including mig-10 [33], hlh-2 [34] and zmp-1 [11] (S5 Table). On the other hand, no specific binding to the nhr-67 [34] or cdh-3 [11] genes was observed, suggesting that these two genes may be indirectly regulated by EGL-43.

Taken together, the ChIP-seq analysis suggested that fos-1, lin-12 and egl-43 are direct EGL-43 targets.

Discussion

An uncontrolled activation of cell invasion is one of the hallmarks of malignant cancer cells that form metastases [1]. Genetic studies in model organisms have indicated that invasive tumor cells re-activate the same molecular pathways that control cell invasion during normal animal development. AC invasion in C. elegans has served as an excellent model to dissect the genetic pathways regulating the invasive phenotype of a single cell [3].

Here, we report that the egl-43 gene, the C. elegans ortholog of the human Evi1 proto-oncogene, functions in a regulatory network together with the transcription factors NHR-67 and FOS-1 to control AC invasion (Fig 7). Our results are consistent with a recent report by Medwig-Kinney et al. [34] demonstrating similar interactions between egl-43, nhr-67 and fos-1 during AC invasion. The inclusion of the LIN-12 NOTCH pathway in our model further expands the network and differentiates between the functions of EGL-43 and the nuclear receptor NHR-67. Even though egl-43 and nhr-67 positively regulate each other’s expression at a later stage, our data indicate that EGL-43 and NHR-67 inhibit AC proliferation through distinct mechanisms.

Fig 7 — EGL-43L plays two distinct functions during AC invasion. Left side: EGL-43L represses LIN-12 Notch expression in the differentiated AC to prevent proliferation. In addition, EGL-43 and NHR-67 positively regulate each other, and NHR-67 controls CKI-1 expression to maintain the G1 arrest of the AC. Right side: EGL-43L activates in a positive feedback loop together with FOS-1 the expression of pro-invasive genes in the AC.

Firstly, EGL-43 maintains the AC arrested in the G1 phase of the cell cycle by repressing the expression of the LIN-12 Notch receptor. The ectopic activation of Notch signaling in the differentiated AC was sufficient to induce proliferation in the presence of EGL-43, while reducing lin-12 expression efficiently suppressed the AC proliferation and invasion defects caused by inhibition of egl-43. Thus, LIN-12 is an essential downstream target of EGL-43 that can reactivate the cell cycle in the AC. A recent study in C. elegans has highlighted the importance of the LIN-12 Notch pathway in keeping an equilibrium between the proliferation and differentiation of somatic cells [27]. Furthermore, the regulation of different cell cycle genes by the Notch pathway has been reported in several cases. For example, Notch signaling induces cyclin D1 expression in mammalian kidney and breast epithelial cells [35,36] and activates dE2F1 and cyclin A expression in the photoreceptor precursors of the Drosophila eye to promote S-phase entry [37].

It was previously shown that NHR-67 maintains the G1 arrest of the AC by inducing the expression of the CDK inhibitor CKI-1 [8]. While overexpression of CKI-1 efficiently rescued the AC proliferation and invasion phenotype caused by nhr-67 RNAi, CKI-1 only slightly suppressed the AC proliferation phenotype and had no effect on the invasion defects caused by egl-43 RNAi. However, it should be noted that the transgene we used to overexpress CKI-1 did not fully suppress the nhr-67 AC proliferation and invasion phenotypes, while a different cki-1 overexpression transgene used by Medwig-Kinney et al. [34] caused a complete rescue of nhr-67 RNAi, probably due to higher levels of CKI-1 expression. It is therefore possible that a further increase in CKI-1 levels beyond the concentration we reached may also suppress the egl-43 phenotype. Taken together, we suggest that the AC proliferation phenotype caused by inhibition of egl-43 is less sensitive to an increase in the CKI-1 dosage than the nhr-67 phenotype.

On the other hand, the nhr-67 AC proliferation and invasion phenotypes were less sensitive to lin-12 inhibition when compared to the egl-43 phenotype, even though NHR-67 RNAi also increased lin-12 expression in the AC. We thus propose that EGL-43 inhibits AC proliferation predominately by repressing LIN-12 NOTCH signaling, while NHR-67 acts primarily by enhancing CKI-1 expression (Fig 7). One possible explanation for the different sensitivities could be that the hyper-activation of LIN-12 signaling caused by loss of egl-43 results in elevated CDK/Cyclin activity, which overcomes a threshold set by CKI-1-mediated cell cycle inhibition. The inhibition of nhr-67, on the other hand, may reduce the threshold by decreasing CKI-1 expression. Since nhr-67 and egl-43 positively regulate each other’s expression in the proliferating ACs, EGL-43 may promote cki-1 expression indirectly via nhr-67.

The egl-43 AC invasion and proliferation phenotypes did not completely correlate, as a fraction of egl-43 depleted animals -especially in combination with lin-12 RNAi- contained a single AC that failed to invade. Hence, EGL-43 appears to have an additional function besides merely preventing AC proliferation. EGL-43 likely exerts the proliferation-independent function through its interaction with fos-1, which is not required for the G1 arrest of the AC but necessary to induce the expression of pro-invasive genes. Deleting the FOS-1 responsive element (FRE) in the endogenous egl-43 locus confirmed our earlier findings based on transgenic reporter analysis that egl-43L expression is positively regulated by FOS-1 [11]. FOS-1 is not absolutely required for the expression of EGL-43, because additional factors such as HLH-2 activate egl-43 expression in the AC [10]. Moreover, EGL-43 positively regulates fos-1 as well as its own expression in the AC. Thanks to the positive feedback loop between egl-43 and fos-1, low levels of either of the two transcription factors may be sufficient to induce a stable expression of both transcription factors and thereby irreversibly determine the invasive fate of the AC. Interestingly, a similar self-activating function has been described for mammalian Evi1 in hematopoietic stem cells [12].

In summary, EGL-43 coordinates the expression of pro-invasive genes with the cell cycle arrest of the AC by inducing fos-1 and inhibiting lin-12 Notch expression. Thus, EGL-43 is a central component in a regulatory network, which decides whether to divide or invade.

Material and methods

C. elegans culture and maintenance

C. elegans strains were maintained at 20°C on standard nematode growth plates as described [38]. The wild-type strain was C. elegans Bristol, variety N2. We refer to translational protein fusions with a :: symbol between the gene name and the tag, while transcriptional fusions are indicated with a > symbol between the promoter/enhancer and the tag. The genotypes of the strains used in this study are listed in S1 Table. The construction of the plasmids, oligonucleotides and sgRNAs used to generate the different reporters are described in the Extended Methods in S1 Text and in S2–S4 Tables.

Scoring the AC invasion phenotype

AC invasion was scored in mid-L3 larvae after the VPC had divided twice (Pn.pxx stage) as described [11]. We monitored the continuity of the BM by DIC or fluorescence microscopy using the qyIs10[lam-1>lam-1::gfp] transgene as a marker.

RNA interference

RNAi interference was done by feeding dsRNA-producing E. coli [39]. Larvae were synchronized at the L1 stage by hypochlorite treatment of gravid adults and plated on NGM plates containing 3mM IPTG. P0 animals were analyzed after 30–36 hours of treatment. For double RNAi experiments, bacteria of the indicated clones were mixed at a 1:1 ration. RNAi clones targeting genes of interest were obtained from the C. elegans genome-wide RNAi library or the C. elegans open reading frame (ORFeome) RNAi library (both from Source BioScience). RNAi vectors targeting egl-43L and egl-43S were subcloned into the L4440 vector by Gibson assembly (S3 Table). The empty L4440 vector (labelled “control” in the figures) was used as negative control in all experiments.

Microscopy and image analysis

Fluorescent and Nomarski images were acquired with a LEICA DM6000B microscope equipped with a Leica DFC360 FX camera and a 63x (N.A. 1.32) oil-immersion lens, or with an Olympus BX61 wide-field microscope equipped with a X-light spinning disc confocal system, a 100x Plan Apo (N.A. 1.4) lens, a lumencor light engine as light source and an iXon ultra888 EMCCD camera. Worms were imaged with 100 x magnification and z-stacks with a spacing of 0.1 to 0.8 μm were recorded. The Fiji software [40] was used for image analysis and fluorescent intensity quantifications using the built-in measurement tools as follows. To quantify expression of the different reporters, deconvolved optical z-sections across the AC were used to generate summed z- projections. The region of the AC nucleus was manually selected, and the integrated signal intensity was measured in the AC nucleus, from which a background value measured in an identically sized region outside of the animal was subtracted. To quantify the CDK sensor activity, images were processed with a Gaussian blur filter (sigma = 50), and a single mid-sagittal z-slice through the AC nucleus was selected for the measurements. The average of the integrated intensities in three equally sized and randomly selected areas, each in the cytoplasm (I_c) and the nucleus (I_n) of the AC were measured to calculate the cytoplasmic to nuclear (I_c/I_n) intensity ratios, which are plotted in Fig 2F.

Supporting information

S1 Fig. Structure of the FRT-tagged gfp::egl-43L allele zh144.

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S2 Fig. The PR domain deletion reduces egl-43 expression levels.

(A) Quantification of GFP::EGL-43L and GFP::EGL-43LΔPR and (B) EGL-43LΔS::GFP and EGL-43LΔZF1::GFP expression levels in the AC of mid-L3 larvae. N- and C-terminal GFP fusions were quantified separately because the site of insertion potentially affects GFP signal intensity. (C) Quantification of the AC expression levels of EGL-43::GFP, a reporter for both the EGL-43L and EGL-43S isoforms, upon control, egl-43, egl-43L, and egl-43S RNAi. The numbers of animals analyzed for each condition are shown in bracket. The error bars indicate standard deviations and the horizontal bars the mean values. Statistical significance was determined by Student’s t-tests and is indicated with n.s. for p>0.05 and **** for p<0.0001.

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S3 Fig. Regulation of egl-43, nhr-67 and hda-1 expression in early L3 larvae.

(A) NHR-67::GFP expression in the ACs of early-L3 larvae (Pn.p stage) after egl-43 RNAi. (B) Quantification of NHR-67::GFP expression levels after egl-43 RNAi. (C) GFP::EGL-43L expression in the ACs of early-L3 larvae after nhr-67 RNAi. (D) Quantification of GFP::EGL-43 expression levels after nhr-67 RNAi. (E) HDA-1::RFP expression in mid-L3 larvae after egl-43 RNAi. (F) Quantification of the HDA-1::RFP expression shown in (E). For all reporters, left panels show Nomarski (DIC) images, middle panels the respective reporter together with the LAM-1::GFP BM marker, and right panels merged images with the ACs labelled by cdh-3>mCherry::PH (A), cdh-3>mCherry::moeABD (C) or cdh-3>gfp (E). The error bars indicate standard deviations and the horizontal bars the mean values. Statistical significance was determined with a Student’s t-test and is indicated with ** for p<0.01 and n.s. for p>0.05. The numbers in brackets refer to the numbers of animals analyzed. The scale bars are 5 μm.

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S4 Fig. AC fate markers remain expressed after egl-43 RNAi, while Notch signaling does not affect EGL-43L expression.

(A) lin-3 reporter expression in the control and egl-43 RNAi ACs. Left panels show Nomarski (DIC) images and right panels the fluorescence image with LIN-3::mNG. (B) lag-2 expression in the ACs of control and egl-43 RNAi. Left panels shows Nomarski (DIC) images, middle panel the fluorescence image with lag-2>GFP reporter, and right panels the reporter merged with AC marker cdh-3>mCherry::PH. (C) GFP::EGL-43L expression in the ACs of control and NICDΔCT expressing ACs. Left panels show Nomarski (DIC) images, middle panels the GFP::EGL-43 signal with the LAM-1::GFP BM marker, and right panels merged with the ACs labelled with cdh-3>PH::mCherry (control, row 1) and cdh-3>NICDΔCT::SL2::mCherry (row 2) respectively. (D) Quantification of the GFP::EGL-43L expression shown in (C). The error bars indicate standard deviations and the horizontal bars the mean values. Statistical significance was determined with a Student’s t-test and is indicated with n.s. for p>0.05. The numbers in brackets refer to the numbers of animals analyzed. The scale bars are 5 μm.

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S5 Fig. FOS-1 neither regulates cell cycle markers nor LIN-12 expression, while LIN-12 does not regulate FOS-1 expression.

(A) Expression of the S-phase marker RNR-1::GFP after control and fos-1 RNAi. None of 19 control or 23 fos-1i animals showed RNR-1::GFP expression in the AC. (B) Expression of GFP::MCM-7 after control and fos-RNAi. None of 25 control or 25 fos-1i animals showed GFP::MCM-7 expression. (C) LIN-12::GFP expression is not up-regulated after control (0/20) or fos-1 (0/24) RNAi treatment. (D) Expression of FOS-1a::YFP in control and NICDΔCT-expressing ACs of mid-L3 larvae. (E) Quantification of the FOS-1a::YFP expression shown in (D). For each reporter, the left panels show Nomarski (DIC) images, the middle panels the GFP or YFP signals of the indicated reporters in green (in (B) and (D) together with the LAM-1::GFP BM marker) and the right panels the GFP reporter signals merged with the ACs labelled with the cdh-3>mCherry::moeABD (A, C), lin-3^ACEL>mCherry (B) or cdh-3>nicdΔct::sl2::mCherry (D) reporters in magenta. The black arrowheads point at the AC nuclei and the white arrows at the locations of the BM breaches. The error bars indicate standard deviations and the horizontal bars the mean values. Statistical significance was determined with a Student’s t-test and is indicated with n.s for p>0.05. The numbers in brackets refer to the numbers of animals analyzed. The scale bars are 5 μm.

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S1 Table. List of strains used.

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S2 Table. Design of plasmids used.

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S3 Table. Oligonucleotide primers used.

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S4 Table. Sequences of the guide RNAs used.

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S5 Table. List of EGL-43 binding sites identified by ChIP-seq analysis.

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S1 Text. Extended methods.

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Acknowledgments

We wish to thank members of the Hajnal laboratory for numerous discussions, the Caenorhabditis Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440), and the van der Heuvel laboratory for providing strains. We are also grateful to Andrew Fire for making gfp vectors available.

Data Availability

All relevant data are within the manuscript and its Supporting Information files. The ChIP-seq data generated in this study are available at the NCBI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE144292.

Funding Statement

This work was supported by the Swiss National Science Foundation grant no. 31003A-166580 and by the Kanton of Zürich. Julie Ahringer's laboratory was supported by Wellcome grant 101863 and by core funding from the Wellcome Trust (092096) and Cancer Research UK (C6946/A14492). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Genet. doi: 10.1371/journal.pgen.1008470.r001

Decision Letter 0

Gregory P Copenhaver, David R Sherwood

6 Nov 2019

Dear Dr Hajnal,

Thank you very much for submitting your Research Article entitled 'The C. elegans homolog of the Evi1 proto-oncogene, egl-43, coordinates G1 cell cycle arrest with pro-invasive gene expression during anchor cell invasion' to PLOS Genetics. Your manuscript was fully evaluated at the editorial level and by three independent peer reviewers. The reviewers appreciated the attention to an important problem, were excited about the potential of the work, but raised some substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a much-revised version. We cannot, of course, promise publication at that time.

Should you decide to revise the manuscript for further consideration here, your revisions should address the shared concerns of reviewer #2 and #3 on rigor--better quantification and outlining methodology more clearly. All the points raised by reviewer #1 and #2 should be addressed. For Reviewer #3, please address points 1 and 3 (it is not clear that 4 can be addressed and 5 and 6 are beyond the scope of the work). We will require a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

[GPC Note: two of the reviewers indicated that the data underlying the figures in the manuscript have not been provided. In particular they mentioned numerical data for the bar graphs, sequence of the egl-43Si 5'UTR used in the generation of the RNAi targeting clone to the short isoform of egl-43, and data on expression of the different GFP-tagged alleles. PLOS Genetics, as part of its Open Data policy, requires that all such data be provided in the manuscript (as supplemental material), or be deposited in a public database. Please address this issue in your revised manuscript, and describe the amendments you make in your response-to-review letter.]

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Gregory P. Copenhaver

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Reviewer #1: This article by Deng and coworkers is a study of EGL-43, the C. elegans ortholog of the human Evi1 proto-oncogene, and how it regulates the fate and function of the invading anchor cell (AC). The authors show that egl-43 inhibits AC proliferation by repressing lin-12 expression. (G1 arrest is known to be essential for the AC to realize its invasive potential.) Furthermore the authors show that a positive feed-back loop exists between egl-43 and fos-1 in the AC, maintaining high expression of both for the induction of genes required for AC invasion.

I have no major criticisms of this work. It appears solid and complete, and the text is well-written and clear. The only thing I felt was lacking was more discussion of the role of egl-43 in the fate decision of the AC as compared to other studies on this subject, in particular the recent work from the Greenwald lab (HLH-2/E2A Expression Links Stochastic and Deterministic Elements of a Cell Fate Decision during C. elegans Gonadogenesis, Current Biology, Sept 2019). How does egl-43 function fit in with the HLH-2 expression clock described in this paper?

Reviewer #2: Summary:

The research described in this manuscript examined the function of egl-43 in regulating C. elegans anchor cell (AC) invasion. Using CRISPR/Cas9 mediated genome editing, the authors have engineered several new egl-43 alleles that provide valuable insight into egl-43 function. Specifically, they identify the long isoform (egl-43L) as the predominant isoform functioning during AC invasion as well as regulation of egl-43 by fos-1 via a cis-regulatory FOS-Responsive Element. Using multiple cell cycle reporters, the authors also show that egl-43 is required for the AC to undergo G1 cell cycle arrest, and that egl-43 depletion results in proliferative ACs. The authors claim that this cell cycle dependent function of egl-43 is independent of the nhr-67 / CKI-1 pathway known to be involved in AC invasion, although I feel that this is not well-supported by the evidence currently provided in the manuscript. Opportunities to include more convincing controls and to quantify data were sometimes missed by the authors, though should be easily remedied by either better quantification of existing data or new experiments. Suggested revisions to the manuscript include validation of RNAi reagents, explicit indication of sample sizes, and additional experiments (outlined below). Despite the fact that some of this data (i.e., the relationship between egl-43 and fos-1; egl-43’s role in cell cycle arrest) has been recently shown in a bioRxviv preprint (Medwig-Kinney et al. (2019)), I feel that the mechanistic insights gained through the authors’ careful dissection of the egl-43 locus is complementary and I am enthusiastic about seeing this work published. Furthermore, the novel finding that ectopic expression of Notch intracellular domain is sufficient to induce proliferation in a normally post-mitotic differentiated cell is a very exciting finding and would be of interest to a broad readership.

Comments:

General comments:

Assuming PLoS Genetics allows for citations of preprints, given the nature of overlap between this work and that of a recently updated bioRxviv preprint (Medwig-Kinney et al. (2019)), I think it would be useful for the field if the authors discussed their results in the context of data showing that egl-43 regulates both hlh-2 and nhr-67 in a cell-cycle dependent manner as well as feedback between EGL-43 and FOS-1. The main discrepancy between this work and Medwig-Kinney et al. (2019) is whether or not egl-43 and nhr-67 function independently of each other in mediating G1/G0 arrest in the AC. See below for specific experimental suggestions that might clear this discrepancy up.

I would recommend for showing single channel fluorescence images to use grey scale, which the human eye can see subtle differences in easier than false colored images, and only use false colored images for overlays.

In the results and brief Discussion section the authors miss the chance to put their data using endogenously-tagged alleles in the context of what has been shown by their and other labs previously using transgenes - for example, autoregulation of egl-43 has been shown multiple times based on transgenes and the first potential explanation for this is that levels of egl-43 are extremely important - see Wang et al. 2014 (doi: 10.1016/j.bbrc.2014.08.049) - where they show that egl-43 functions through an incoherent feed forward circuit with negative feedback in regulating MIG-10 levels in the AC.

For the most part, the authors represent fluorescence quantification data through box plots, which depict median values. However, given the wide spread of some of this data (e.g., Fig. 4B), median may not be the best statistic to show. I would recommend using an alternative method of data visualization, such as violin plots including mean values and standard deviation.

Introduction:

Potential typos (minor):

“selected” → “select” (paragraph 1)

“trackable” → “tractable” (paragraph 1)

“EGl-43” → “EGL-43” (paragraph 3)

“the VU cell undergoes three rounds of cell divisions” - This is not 100% accurate, as the ρ cells undergo an extra round of division. See Newman, White, & Sternberg (1996).

Results:

“FRT” should be defined upon 1st use of the acronym. The FRT experiments are really elegant - I’m wondering, is the reduced penetrance in these lines as compared to RNAi due to produrance of the protein during the length of time it takes for the flipase to remove the genomic region flanked by FRT sites? I couldn’t tell from the images - it looks like there is no expression, but it would be useful to quantify this.

“We found no obvious difference in the expression pattern of the two egl-43 reporters, suggesting that the long egl-43L isoform accounts for most of the expression observed.” - This claim can be supported by evidence showing quantitative comparison of expression levels in both reporters (not directly provided).

How was the egl-43Si RNAi construct validated? The targeting sequence is presumably much smaller (although this information is missing from the supplement) than typical RNAi constructs, so the efficiency may be significantly lower. Also, how did the authors determine the 5’ UTR sequence, as I could not find it annotated on WormBase? The authors also may want to consider that there is evidence (Bosher et al., 1999) that RNAi can act on pre-mRNA, which would indicate that this construct may recognize the introns of pre-spliced egl-43L transcripts.

Figure 1B-C: It would be helpful to see quantification of this data presented as well.

How was the sample size for the egl-43L RNAi vs. egl-43 RNAi experiment (Results paragraph #2) determined? Typically a minimum sample size of 28 is required to perform a significance test at ɑ = 0.05.

Figure 2: The number of animals observed with the representative phenotype shown, with respect to the total number of animals observed, should be indicated in Figures 2A,C-D. The n indicated in the bar graph in panel B is difficult to read due to the small font size (and I expect the font size would need to be increased for publication per journal standards anyway).

The source of the RNR-1::GFP strain/construct (Park & Krause, 1999) should be cited in addition to the WormBook chapter.

Is the characterization of the endogenously-tagged MCM-7::GFP described elsewhere? I know that the transgene has been used as a reporter for actively cycling cells by the van den Heuvel lab (I would recommend citing the data paper, Korzelius et al. 2011, rather than the wormbook chapter here). If this is the first description of the endogenous MCM complex as a reporter for S-phase onset/cycling cells it would be worth characterizing it first and then using it as a reporter. I believe the data, I just think it would be nice to highlight that it’s a GFP-knock in - you could cross the allele into the MCM-4::mCherry transgene from the van den Heuvel lab and just demonstrate that they show the same exact pattern of localization in a cell cycle-dependent way as a supplemental figure?

The original CDK biosensor citation should be included as well from Spencer et al. 2011 when citing its use as it was co-opted from mammalian cell culture.

Image quantification: The Materials and Methods section is specifically lacking a description of how the CDK sensor was quantified, and in general more information is needed in reference to image quantification for all of the data in the manuscript - “built-in measurement tools” in Fiji/ImageJ could mean many different things - how did the authors correct for background/camera noise? Were measurements made from single confocal z-planes? Are the authors’ reported mean grey values or integrated density (either is fine, just more details are needed). Did the authors use thresholding and the wand tool to select the region of interest, or did they hand draw regions of interest?

In the updated Medwig-Kinney et al. pre-print, it is shown that the regulatory relationships between egl-43 and nhr-67 do not exist until post-AC-specification. I recognize that this data was not available at the time of submission. However, this could explain why the authors do not see a significant change in nhr-67::GFP expression in the AC following egl-43(RNAi). More importantly, however, I would suggest that the authors examine mitotic ACs for regulation of gene expression rather than looking at earlier stages, as it is impossible to know whether or not an AC is out of cycle (beyond using a second set of reporters for cell cycle state) so you can not assess whether the single AC you are measuring is going to invade or not. As to the authors’ statement that the proliferation of the AC results in dilution of protein expression - this data exists. I would point the authors to data using the transgene containing the full (~5kb) cdh-3 promoter fused to GFP. In Matus et al. (2015), I found that this promoter was expressed at ~97% of wild-type levels in proliferating ACs (see Figure 4B from Matus et al. 2015), while other reporters are clearly down-regulated, suggesting that GFP is not simply diluted as ACs become mitotic and proliferate but that the actual transcriptional program is changing due to inappropriate cell cycle entry.

This also brings up an important point on the use of the cdh-3 promoter for driving constructs of interest in the AC. We have found that the smaller ACEL used to drive cdh-3 (~1.5kb) for many of the transgenes from the Sherwood lab, also used in this paper (qyIs23 and qyIs50), is regulated by nhr-67 and egl-43, which is why we used the full cdh-3 promoter (~5kb) to generate new AC reporters for studying nhr-67 loss of function in Matus et al. 2015. I bring this up because if anything, the use of the smaller cdh-3>constructs could under-report the number of ACs due to depletion of the promoter. It took a little digging for me to figure out that the new constructs were designed with the full cdh-3 promoter, so it would be helpful to distinguish this in the text/methods. We used cdh-31.5 vs cdh-3 in our original paper if that nomenclature is helpful.

Figure 3G,I: The overexpression of CKI-1 in a lineage should cause G1/G0 arrest. It would appear that the authors are making the claim that egl-43 mediates cell cycle arrest independent of CKI-1, but a more parsimonious explanation would be that depletion of egl-43 results in downregulation of the cdh-3 promoter driving CKI-1 expression, and in cases where you see multiple ACs, those ACs do not have a critical threshold of CKI-1 activity to prevent cell cycle entry. One suggestion would be to quantify levels of CKI-1 in all of the animals and see if there is a statistical correlation between CKI-1 levels and number of ACs observed. While, the 2 AC phenotype could be the result of perturbing AC/VU specification, 3+ ACs shouldn’t be observed if the cdh-3>CKI-1 is functioning 100% of the time.

Figure 3G-I: The key says “control siblings” - does this mean that these are the progeny resulting from a cross? I assume not, but this terminology may be misleading and whether animals of homozygous or heterozygous is an important distinction.

Figure 4A: This data should be quantified. Also how was expression of lin-12 determined? How were the boundaries of ACs versus VUs determined in adjacent cells? The endogenously- tagged lin-12::GFP reporter from Attner et al. (2019) has both membrane bound and nuclear localization, making it easier to distinguish which cell has active Notch signaling - this strain might be easier to use and would better make this really stunning point that active Notch signaling post-AC/VU decision can force the differentiated AC into the cell cycle and inhibit invasion.

Figures 4A-C: When was lin-12 RNAi treatment administered? Knockdown of lin-12 prior to AC/VU specification may confound the number of ACs observed. It would be worthwhile to try an L2 plating of lin-12(RNAi) and see if, at some penetrance, you can repeat your experimental results.

Figure 4D: What percentage of animals are the phenotypes shown indicative of?

Figure 5B: It would be helpful to show GFP::EGL-43L expression without fos-1(ar105) in the background here.

The authors postulate that egl-43 has cell cycle dependent (lin-12) and independent (fos-1) roles, but this is not supported by the data showing that lin-12(RNAi) rescues AC invasion in egl-43(RNAi) animals.

The authors should mention the potential effects that the endogenous transcriptional reporters (with pre-floxed SEC) have on protein function.

The introduction and discussion need elaboration, specifically with regard to links between Notch signaling and cell cycle regulation, in C. elegans and other model systems.

The authors argue that egl-43 and nhr-67 control cell cycle arrest in distinct pathways. To show this convincingly, they should perform the lin-12/Notch experiments with nhr-67 RNAi perturbation experiments, the expectation would be that nhr-67(RNAi) does not induce lin-12 expression if the two pathways are independent. Alternatively, as we believe that egl-43 does regulate nhr-67 activity, it would be interesting if this was still the case - that nhr-67(RNAi) does not regulate lin-12, as we have recently shown that endogenous lin-12::GFP is strongly down-regulated pre-AC/VU decision in our bioRxiv preprint. If you find that nhr-67(RNAi) doesn’t turn on lin-12::GFP, it could also suggest that egl-43 has nhr-67-dependent and nhr-67-independent roles in maintaining the AC in a post-mitotic state, and provide an explanation why nhr-67(RNAi) on an nhr-67(pf88) hypomorphic allele doesn’t significantly increase the AC invasion/proliferation defect (it makes it slightly worse, but there are still a small population of ACs that invade).

Supplementary tables:

Tables S2 and S3 is missing the plasmids and primers used to generate the egl-43Si and egl-43Li RNAi constructs. Information regarding the targeting sequences used would also be helpful to include.

Table S2 contains primers whose sequences are not provided in Table S3. Namely oTD140-143.

Table S3 contains sequences of primers that are not defined in Table S2 or elsewhere. Namely the OEL316-319.

Reviewer #3: The C. elegans AC-VU cell fate decision is a classic example of Notch-based lateral inhibition, and the two fate outcomes differ in proliferation and invasive (basement membrane breaching) activity. In this manuscript, the authors follow up on prior studies from >10 years ago that showed that EGL-43, an Evi1-related transcription factor, is required for both the VU cell fate and for AC invasion. They focus particularly on EGL-43 function during AC invasion.

Key findings:

-The long isoform of egl-43 is required for AC invasion despite unimportance of several specific protein domains unique to this isoform (based on RNAi, AC-specific deletion, and CRISPR-generated in-frame deletions).

-EGL-43 inhibits AC proliferation by promoting G1 arrest, and does so by (directly or indirectly) repressing LIN-12/Notch, which promotes proliferation and inhibits invasion (based on increasing AC numbers in egl-43i over time, with increased expression of LIN-12::GFP and a variety of S-phase reporters, and epistasis analysis with lin-12 rf and gf). This is the most impactful result in the paper.

-EGL-43 does not affect expression of two other factors involved in AC invasion (NHR-67 and HDA-1), or vice versa, but EGL-43 and FOS-1 mutually upregulate each others’ expression

Overview:

The paper is generally well written, though data presentation needs clarification in several places. The presented results seem solid and the paper definitely moves the field forward, but it stops short of a really impactful, mechanistic understanding. Therefore, my enthusiasm for the current version is modest.

Specific points:

1. There are some “rigor and reproducibility” issues in the data presentation that need to be addressed:

Fig 1B quantification: how many animals were examined, how many showed these expression patterns, and how many had multiple ACs and/or no invasion?

Fig 2B: statistics? Are the changes over time significant?

Fig 2C and 2D quantification: how many animals were examined, how many showed these expression patterns?

Figure 3H and 3I: What % of animals had multiple ACs? How does this relate to the % that had invasion defects?

Figure 4F and 4G: Are the %s in G based on pooling the 3 lines of each genotype, or are they based on one specific line? What % of the animals scored in G had multiple ACs and is there an absolute correlation between the two types of defects?

Addressing any of the below would add more meat and impact to this paper, but addressing points 2,3,4 would be especially relevant:

2. Circumstantial evidence suggests that the egl-43 long isoform might be required because it is the predominant one transcribed in the AC, but the authors can't exclude that there is something unique about the protein made by this isoform. Transgenic rescue experiments with each isoform could address this question more definitively.

3. The authors present more evidence for the previously observed correlation between cell cycle arrest and invasive activity, but the basis for this correlation remains unclear. On p. 10 the authors speculate that "EGL-43 might perform two functions in the AC; first to repress AC proliferation and second to activate pro-invasive gene expression". But data in Figure 4 suggest that EGL-43 might do one thing (inhibit lin-12) to accomplish both results. What is the explanation for why lin-12(gf) suppresses proliferation and invasion defects (Figure 4), but CKI expression mostly does not (Figure 3)? At what step of the cell cycle is lin-12 acting? Does lin-12 affect expression of the mentioned "pro-invasive" genes?

4. Is the cross-regulation of egl-43 and fos-1 functionally important? Can cdh-3>fos-1 rescue egl-43 defects or vice versa? Or are they both needed in parallel?

5. The paper does not show whether lin-12, fos-1, or any of the other downstream effectors of EGL-43 are direct targets, so it does not provide insights into EGL-43 binding properties or mechanisms of transcriptional regulation (e.g. is EGL-43 both a tx repressor and activator?).

6. Given that egl-43 is expressed in both AC and VU, and seems to regulate different target genes in each, what is the basis for target specificity?

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

Reviewer #1: Yes

Reviewer #2: No: We cannot find information pertaining to the sequence of the egl-43Si 5'UTR used in the generation of the RNAi targeting clone to the short isoform of egl-43. Also, I could not find the data supporting the claim that the different GFP-tagged alleles have the same expression levels in the AC - Fig. S2 should use the unmodified allele to normalize data to.

Reviewer #3: No: numerical data for bar graphs has not been provided

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Reviewer #1: No

Reviewer #2: Yes: David Matus

Reviewer #3: No

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PLoS Genet. 2020 Mar 23;16(3):e1008470. doi: 10.1371/journal.pgen.1008470.r002

Author response to Decision Letter 0

24 Jan 2020

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PLoS Genet. doi: 10.1371/journal.pgen.1008470.r003

Decision Letter 1

Gregory P Copenhaver, David R Sherwood

17 Feb 2020

* Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. *

Dear Dr. Hajnal

After carefully reading through Reviewer#1's comments (self identified as David Matus), I think he brings up a reasonable editorial suggestion in improving the manuscript--more strongly suggesting an alternative model that EGL-43 might also function to repress the cell cycle in part by promoting cki-1 expression. I don't think you need to bring up the ChIP data, but I leave that up to you. If you can make this change and outline how you have done so in your letter, I will make sure this elegant work is accepted quickly--it will not be sent out for re-review.

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Reviewer #2: Summary:

I commend the authors on the extensive revisions they performed to address many of the comments provided by myself and other reviewers previously. Both the text and graphs are much clearer now. The only result that we’d like to bring attention to is still the discrepancy in data between this report and recent work from our laboratory. We are not asking for additional experiments at this time, but would just like to offer some caveats that might change the interpretation of the data, regarding the role of EGL-43 in maintaining the post-mitotic state of the AC independent of the activity of CKI-1.

In 2015 we reported that an integrated transgene (cdh-3>CKI-1::GFP) completely rescued the AC proliferation and invasion defects in the hypomorph, nhr-67(pf88). In our 2020 paper we demonstrated this same transgene also completely rescued invasion and proliferation in nhr-67(RNAi) using the enhanced RNAi vector in the T444T backbone. In Figure 3F and 3G it appears that your extrachromosomal cdh-3>CKI-1::SL2::mNG transgene only partially rescues nhr-67(RNAi) [>30% invasion defect, and >10% with 2+ ACs]. Both the use of extrachromosomal arrays, which can vary in expression levels and the construction of your transgene using an SL2::mNG may explain the difference between our results and your results presented in Figure 3. Specifically, Ahier and Jarriault (Genetics 2014, doi: 10.1534/genetics.113.160846) argue for using 2A viral peptides rather than SL2 sequences in generating fusion proteins where stoichiometry between the protein of interest and the fluorescent protein are important to maintain. Here’s the relevant text from their discussion section:

“This is in contrast to the SL2 approach as operon sequences may be under the influence of cis-regulating elements (enhancers or silencers) that are difficult to predict (Pfleger et al. 2006). Moreover, when using an SL2-based approach, several additional parameters can impact on the expression levels of protein products, which could vary widely. For example, after trans-splicing, each subsequent mRNA molecule may have its own specific stability, and each transcript is read individually by different ribosomes, permitting variable translation.”

In our integrated transgene, cdh-3>CKI-1::GFP, we avoid this by generating the fusion between CKI-1 and GFP.

*If* the extrachromosomal lines more robustly rescued nhr-67(RNAi), I would be inclined to believe that egl-43 could be functioning independently of CKI-1 through LIN-12, but the more parsimonious explanation is that you’re not generating enough CKI-1 to restore the AC to a G0 cell cycle arrested state. I think this only affects the summary model - that the repression of LIN-12 is required with NHR-67 to maintain G0 arrest is an amazing result. I think that we don’t know (yet) whether LIN-12 functions to either activate cyclins and CDK complexes or repress CKI-1. I think this is made even more complex because of the likely feedback between CKIs and CDK/Cyclin complexes. I would be fine with showing dotted arrows with a question mark between LIN-12 and the cell cycle machinery, to show that the current data can’t resolve this relationship.

Finally, I also noticed that cki-1 might actually be a direct target of egl-43, from your L3 ChIP data presented in the excel file. This is exciting, but obviously potentially counter to your proposed model. It suggests that nhr-67 and egl-43 might co-regulate CKI-1 activity in the AC to maintain it in a post-mitotic G0 arrested state, though we still don’t know if nhr-67 directly regulates CKI-1.

chrII:7810217-7810482 cki-1; cki-1 356.7129 15.22800064

chrII:7809138-7809378 cki-1 170.3209 6.874500275

Reviewer #3: The authors have responded satisfactorily to my prior comments, and the revisions have significantly improved the paper.

**********

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PLoS Genet. 2020 Mar 23;16(3):e1008470. doi: 10.1371/journal.pgen.1008470.r004

Author response to Decision Letter 1

21 Feb 2020

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PLoS Genet. doi: 10.1371/journal.pgen.1008470.r005

Decision Letter 2

Gregory P Copenhaver, David R Sherwood

27 Feb 2020

Dear Dr Hajnal

We are pleased to inform you that your manuscript entitled "The C. elegans homolog of the Evi1 proto-oncogene, egl-43, coordinates G1 cell cycle arrest with pro-invasive gene expression during anchor cell invasion" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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PLoS Genet. doi: 10.1371/journal.pgen.1008470.r006

Acceptance letter

Gregory P Copenhaver, David R Sherwood

12 Mar 2020

PGENETICS-D-19-01671R2

The Caenorhabditis elegans homolog of the Evi1 proto-oncogene, egl-43, coordinates G1 cell cycle arrest with pro-invasive gene expression during anchor cell invasion

Dear Dr Hajnal,

We are pleased to inform you that your manuscript entitled "The Caenorhabditis elegans homolog of the Evi1 proto-oncogene, egl-43, coordinates G1 cell cycle arrest with pro-invasive gene expression during anchor cell invasion" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

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Associated Data

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

Supplementary Materials

S1 Fig. Structure of the FRT-tagged gfp::egl-43L allele zh144.

(TIF)

Click here for additional data file.^{(218.7KB, tif)}

S2 Fig. The PR domain deletion reduces egl-43 expression levels.

(TIF)

Click here for additional data file.^{(270.1KB, tif)}

S3 Fig. Regulation of egl-43, nhr-67 and hda-1 expression in early L3 larvae.

(TIF)

Click here for additional data file.^{(6.6MB, tif)}

S4 Fig. AC fate markers remain expressed after egl-43 RNAi, while Notch signaling does not affect EGL-43L expression.

(TIF)

Click here for additional data file.^{(3.9MB, tif)}

S5 Fig. FOS-1 neither regulates cell cycle markers nor LIN-12 expression, while LIN-12 does not regulate FOS-1 expression.

(TIF)

Click here for additional data file.^{(6.5MB, tif)}

S1 Table. List of strains used.

(DOCX)

Click here for additional data file.^{(13.6KB, docx)}

S2 Table. Design of plasmids used.

(DOCX)

Click here for additional data file.^{(15.6KB, docx)}

S3 Table. Oligonucleotide primers used.

(DOCX)

Click here for additional data file.^{(13.9KB, docx)}

S4 Table. Sequences of the guide RNAs used.

(DOCX)

Click here for additional data file.^{(12.7KB, docx)}

S5 Table. List of EGL-43 binding sites identified by ChIP-seq analysis.

(XLSX)

Click here for additional data file.^{(328.1KB, xlsx)}

S1 Text. Extended methods.

(DOCX)

Click here for additional data file.^{(19.3KB, docx)}

Attachment

Submitted filename: Deng_etal_review.pdf

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Attachment

Submitted filename: Point-by-point response.pdf

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Attachment

Submitted filename: Response.pdf

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Data Availability Statement

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PERMALINK

The Caenorhabditis elegans homolog of the Evi1 proto-oncogene, egl-43, coordinates G1 cell cycle arrest with pro-invasive gene expression during anchor cell invasion

Ting Deng

Przemyslaw Stempor

Alex Appert

Michael Daube

Julie Ahringer

Alex Hajnal

Evelyn Lattmann

Roles

Abstract

Author summary

Introduction

Results

Deletion of the long egl-43L isoform leads to the formation of multiple ACs that fail to invade

Fig 1. Loss of egl-43L function leads to the formation of multiple ACs.

Fig 2. egl-43 is required for the G1 arrest of the AC.

Neither the N-terminal PR nor the ZF1 domains in EGL-43 are necessary for AC invasion

egl-43 is required for the G1 arrest of the AC

egl-43 and nhr-67 play distinct roles in inducing the G1 arrest of the AC

Fig 3. egl-43 functions in parallel with nhr-67 and cki-1.

egl-43 inhibits AC proliferation by repressing lin-12 Notch expression

Fig 4. egl-43 and nhr-67 repress lin-12 Notch expression to prevent AC proliferation.

Activation of LIN-12 Notch signaling in the differentiated AC triggers proliferation

A positive regulatory feedback loop between egl-43 and fos-1 activates pro-invasive gene expression

Fig 5. egl-43 and fos-1 regulate each other’s expression.

EGL-43 binding is enriched at the fos-1, egl-43 and lin-12 loci

Fig 6. EGL-43 binding is enriched at the egl-43, fos-1 and lin-12 loci.

Discussion

Fig 7. EGL-43L is part of a regulatory network controlling AC invasion.

Material and methods

C. elegans culture and maintenance

Scoring the AC invasion phenotype

RNA interference

Microscopy and image analysis

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Gregory P Copenhaver

David R Sherwood

Roles

Author response to Decision Letter 0

Decision Letter 1

Gregory P Copenhaver

David R Sherwood

Roles

Author response to Decision Letter 1

Decision Letter 2

Gregory P Copenhaver

David R Sherwood

Roles

Acceptance letter

Gregory P Copenhaver

David R Sherwood

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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