Sherwood DR et al. (2000) West Coast Worm Meeting "Mechanisms regulating the timing and specificity of anchor cell attachment to the vulval epithelium"

Sherwood DR, Sternberg PW

[West Coast Worm Meeting, 2000]

We are interested in understanding the basic cellular and molecular mechanisms that guide morphogenetic processes during development. Towards this goal we are examining the initial steps involved in the connection of the uterus and vulva during C.elegans development. The initial contact between the developing uterus and the vulva is established by the anchor cell (AC), which crosses the basement membranes separating both tissues and specifically attaches to the progeny of the P6.p cell during the mid- to late L3 stage. Following attachment, the AC invades between the inner descendants of the P6.p. cell and becomes positioned at the apex of the developing vulva. Using mutants lacking vulval induction, we have found that the descendants of the underlying induced vulval precursor cell, P6.p, send a signal to trigger the attachment behavior in the AC. We have also discovered that the competence of the AC to respond to this signal is regulated: AC attachment is either absent or delayed in the heterochronic mutant lin-28, which causes precocious vulval development. To understand the molecular mechanisms that regulate AC attachment, we have examined many known mutants, as well as initiated a genetic screen to search for new mutants with defective attachment behavior. Through these studies we have found that the evl-5 mutant, originally isolated in a screen for sterile p-vul's (Seydoux et al., (1993) Dev. Biol. 157(2): 423-36), has a defect in AC attachment to the vulval epithelium. In evl-5 animals vulval induction and AC positioning over the P6.p cell is normal; however, AC attachment either does not occur or is severely delayed. Visualization of AC behaviour in this mutant revealed the extension of cellular processes from the AC toward the vulva, indicating that the AC is still attracted to the developing vulva. However, in many cases the AC processes appeared to flatten or broaden out at the basement membrane of the developing gonad, suggesting that the AC may not be able to cross this basement membrane. We are currently molecularly cloning evl-5.

Spiri S et al. (2022) Development "Reciprocal EGFR signaling in the anchor cell ensures precise inter-organ connection during Caenorhabditis elegans vulval morphogenesis."

Mereu L, DeMello A, Berger S, Spiri S, Hajnal A

[Development, 2022]

During Caenorhabditis elegans vulval development, the uterine anchor cell (AC) first secretes an epidermal growth factor (EGF) to specify the vulval cell fates and then invades the underlying vulval epithelium. By doing so, the AC establishes direct contact with the invaginating primary vulF cells and attaches the developing uterus to the vulva. The signals involved and the exact sequence of events joining these two organs are not fully understood. Using a conditional let-23 EGF receptor (EGFR) allele along with novel microfluidic short- and long-term imaging methods, we discovered a specific function of the EGFR in the AC during vulval lumen morphogenesis. Tissue-specific inactivation of let-23 in the AC resulted in imprecise alignment of the AC with the primary vulval cells, delayed AC invasion and disorganized adherens junctions at the contact site forming between the AC and the dorsal vulF toroid. We propose that EGFR signaling, activated by a reciprocal EGF cue from the primary vulval cells, positions the AC at the vulval midline, guides it during invasion and assembles a cytoskeletal scaffold organizing the adherens junctions that connect the developing uterus to the dorsal vulF toroid. Thus, EGFR signaling in the AC ensures the precise alignment of the two developing organs.

David R Sherwood et al. (2001) International C. elegans Meeting "Cellular mechanisms governing the specificity and timing of anchor cell invasion into the vulval epithelium"

David R Sherwood, Paul W Sternberg

[International C. elegans Meeting, 2001]

We are examining the initial steps that regulate the connection of the uterus and vulva during C. elegans development. The initial contact between the developing uterus and vulva is established by the anchor cell (AC), which crosses the basement membranes separating both tissues and specifically attaches to the inner descendants of the 1 o fated vulval precursor cell, P6.p, during the mid- to late L3 stage. Following attachment, the AC extends cellular processes from its basolateral surface and invades between the inner descendants of the P6.p. cell, positioning itself at the apex of the developing vulva. Using mutants lacking vulval induction, we show that underlying vulval cells trigger AC attachment. We have further discovered that AC attachment is not stimulated by 2 o fated vulval cells (the vulval cells that flank the centrally located 1 o cells), but rather appears to be specific for the 1 o vulval cells. Using a basement membrane marker, we show that isolated 1 o cells do not break down the basement membrane in the absence of the AC, suggesting that 1 o vulval cells do not stimulate attachment by removing the basement membrane. Instead we provide evidence indicating that the 1 o vulval cells secrete a signal that triggers AC invasive behavior: when the AC is placed at a distance from the 1 o vulval cells, it sends out cellular extensions toward the 1 o vulval cells that are similar to those seen during normal invasion. We further show that the competence of the AC to respond to this signal is regulated: AC attachment is either absent or delayed in the heterochronic mutant lin-28 , which causes precocious vulval development. To understand the molecular mechanisms that regulate AC attachment and invasion, we are examining many known mutants. Through these studies we have found that the evl-5 mutant, originally isolated in a screen for sterile mutants with everted vulvae (Seydoux et al., (1993) Dev. Biol. 157(2): 423-36), has a defect in AC attachment to the vulval epithelium. In evl-5 animals vulval induction and AC positioning over the P6.p cell is normal; however, AC attachment either does not occur or is severely delayed. Visualization of AC behavior in this mutant revealed the extension of cellular processes from the AC toward the vulva, indicating that the AC is still attracted to the developing vulva. However, in many cases the AC processes appeared to flatten or broaden out at the basement membrane of the developing gonad, suggesting that the AC may not be able to cross this basement membrane. We are currently molecularly cloning evl-5 .

Grimbert S et al. (2016) Dev Biol "Anchor cell signaling and vulval precursor cell positioning establish a reproducible spatial context during C. elegans vulval induction."

Grimbert S, Braendle C, Sternberg PW, Tietze K, Felix MA, Barkoulas M

[Dev Biol, 2016]

How cells coordinate their spatial positioning to successfully communicate is poorly understood. Here we address this topic using C. elegans vulval patterning during which hypodermal vulval precursor cells (VPCs) adopt distinct cell fates determined by their relative positions to the gonadal anchor cell (AC). LIN-3/EGF signaling by the AC induces the central VPC, P6.p, to adopt a 1 vulval fate. Exact alignment of AC and VPCs is thus critical for correct fate patterning, yet, as we show here, the initial AC-VPC positioning is both highly variable and asymmetric among individuals, with AC and P6.p only becoming aligned at the early L3 stage. Cell ablations and mutant analysis indicate that VPCs, most prominently 1 cells, move towards the AC. We identify AC-released LIN-3/EGF as a major attractive signal, which therefore plays a dual role in vulval patterning (cell alignment and fate induction). Additionally, compromising Wnt pathway components also induces AC-VPC alignment errors, with loss of posterior Wnt signaling increasing stochastic vulval centering on P5.p. Our results illustrate how intercellular signaling reduces initial spatial variability in cell positioning to generate reproducible interactions across tissues.

Costa DS et al. (2023) Development "The C. elegans anchor cell transcriptome: ribosome biogenesis drives cell invasion through basement membrane."

Park K, Kenny-Ganzert IW, Kelley LC, Pani AM, Park J, Matus DQ, Goldstein B, Yogev S, Chi Q, Garde A, Sherwood DR, Gibney TV, Costa DS

[Development, 2023]

Cell invasion through basement membrane (BM) barriers is important in development, immune function, and cancer progression. As invasion through BM is often stochastic, capturing gene expression profiles of actively invading cells in vivo remains elusive. Using the stereotyped timing of C. elegans anchor cell (AC) invasion, we generated an AC transcriptome during BM breaching. Through a focused RNAi screen of transcriptionally enriched genes, we identified new invasion regulators, including TCTP (translationally controlled tumor protein). We also discovered gene enrichment of ribosomal proteins. AC-specific RNAi, endogenous ribosome labeling, and ribosome biogenesis analysis revealed a burst of ribosome production occurs shortly after AC specification, which drives the translation of proteins mediating BM removal. Ribosomes also enrich near the AC endoplasmic reticulum (ER) Sec61 translocon and the endomembrane system expands prior to invasion. We show that AC invasion is sensitive to ER stress, indicating a heightened requirement for translation of ER trafficked proteins. These studies reveal key roles for ribosome biogenesis and endomembrane expansion in cell invasion through BM and establish the AC transcriptome as a resource to identify mechanisms underlying BM transmigration.

Sara M Vassalli et al. (2005) International Worm Meeting "Finding genes that control the attachment of the anchor cell to P6.p during vulval development"

Sara M Vassalli, ALEX HAJNAL, Amit Dutt

[International Worm Meeting, 2005]

During hermaphrodite vulval development three out of six equivalent vulval precursor cells (VPCs, P3.p to P8.p) are induced by a signal from the anchor cell (AC) in the somatic gonad to adopt the vulval cell fates. The LIN-3 EGF growth factor produced by the AC activates in the VPCs the RTK/RAS/MAPK pathway to specify the primary cell fate. Prior to vulval induction, the gonad is separated from the VPCs by two basal laminas covering each tissue such that the position of the AC relative to the VPCs is variable. However, at the time of induction in early L3 larvae the AC cell attaches to the basal side of P6.p, the future primary cell, assumes a hat-like shape and remains fixed just above the nucleus of P6.p. While the mechanism of vulval induction is well studied, it is not known how P6.p attracts the AC. To elucidate the mechanism of AC attachment we are examining the morphology changes and position of the AC relative to the VPCs in different mutant backgrounds using a membrane-tethered GFP marker to label the plasma membrane of the AC. Mutations that inactivate the EGFR/RAS/MAPK pathway prevent AC attachment to P6.p, suggesting that P6.p produces an attractive signal in response to the inductive signal.

Hanako Yashiro et al. (2007) International Worm Meeting "b (PAT-3) Integrin Plays Multiple Roles during Anchor Cell Invasion in C. elegans."

Hanako Yashiro, David R Sherwood

[International Worm Meeting, 2007]

Cell invasion through basement membranes (BM) is required in various developmental processes and is misregulated in diseases such as cancer. We investigate the underlying mechanisms regulating cell-invasive behavior by studying anchor cell (AC) invasion in C. elegans. During development, the gonadal AC invades through the BM to contact the underlying vulval precursor cells, which send a diffusible cue that guides AC invasion. In a whole-genome RNAi screen, we found that RNAi against both the <font face=symbol>b</font> integrin ina-1 and <font face=symbol>b</font>pat-3 caused defects in AC invasion. We also found that a loss-of-function allele in ina-1(gm39) showed a moderately penetrant invasion defect. Furthermore, in wild-type transgenic animals, PAT-3::GFP localization is increased at the basolateral invasive surface of the AC, suggesting a direct role in promoting invasion To further investigate the role of integrin, we have driven a dominant-negative construct of pat-3 (pAC&#62;HA-<font face=symbol>b</font>tail, a gift from Dr. Jean Schwarzbauer) specifically in the AC. In integrated lines of pAC&#62;HA-<font face=symbol>b</font>tail, AC invasion is severely disrupted. Examination of laminin::GFP, a BM marker, showed that the AC fails to breach the BM in pAC&#62;HA-<font face=symbol>b</font>tail worms and instead detaches from the membrane at the time of invasion. Notably, the AC fails to send cellular protrusions toward the vulval cells after detachment, suggesting an inability to respond to the vulval cue. Recent work in our lab has shown that the Rac protein MIG-2, F-actin, and PtdIns(4,5)P₂ localize to the basolateral membrane of the AC where they promote the formation of filapodia in response to the vulval cue. We have found that GFP::MIG-2 and PH::mCherry (a PtdIns(4,5)P₂-specific promoter) polarity at the invasive AC membrane is retained in pAC&#62;HA-<font face=symbol>b</font>tail animals as long as there is contact between the AC and the BM. Once the AC is detached, however, the polarization is significantly reduced, suggesting that integrin-mediated attachment of the AC to the BM is required to maintain polarity along the invasive membrane. Finally, we studied the loss of integrin in the AC on BM dynamics at the site of invasion by examining the localization of hemicentin::GFP. Hemicentin is a conserved extracellular matrix protein that is deposited under the AC at the site of invasion, where it promotes the movement of the AC through the BM. Strikingly, in pAC&#62;HA-<font face=symbol>b</font>tail worms, hemicentin is still made in the AC but is not deposited. Together, these results indicate that integrin function in the AC is crucial for establishing a properly organized invasive cell membrane and regulates the composition of the BM at the site of invasion.

Martinez MAQ et al. (2022) Biol Open "Reevaluating the relationship between EGL-43 (EVI1) and LIN-12 (Notch) during C. elegans anchor cell invasion."

Matus DQ, Shen K, Yee C, Martinez MAQ, Zhang W, Zhao CZ, Mullarkey AA

[Biol Open, 2022]

Development of the C. elegans reproductive tract is orchestrated by the anchor cell (AC). This occurs in part through a cell invasion event that connects the uterine and vulval tissue. Several key transcription factors regulate AC invasion, such as EGL-43, HLH-2, and NHR-67. Specifically, these transcription factors function together to maintain the post-mitotic state of the AC, a requirement for AC invasion. Recently, a mechanistic connection has been made between loss of EGL-43 and AC cell-cycle entry. The current model states that EGL-43 represses LIN-12 (Notch) expression to prevent AC proliferation, suggesting that Notch signaling has mitogenic effects in the invasive AC. To reexamine the relationship between EGL-43 and LIN-12, we first designed and implemented a heterologous co-expression system called AIDHB that combines the auxin-inducible degron (AID) system of plants with a live cell-cycle sensor based on human DNA helicase B (DHB). After validating AIDHB using AID-tagged GFP, we sought to test it by using AID-tagged alleles of egl-43 and lin-12. Auxin-induced degradation of either EGL-43 or LIN-12 resulted in the expected AC phenotypes. Lastly, we seized the opportunity to pair AIDHB with RNAi to co-deplete LIN-12 and EGL-43, respectively, which revealed that LIN-12 is not required for AC proliferation following loss of EGL-43.

Hwang BJ et al. (2007) Development "C. elegans EVI1 proto-oncogene, EGL-43, is necessary for Notch-mediated cell fate specification and regulates cell invasion."

Hwang BJ, Meruelo AD, Sternberg PW

[Development, 2007]

During C. elegans development, LIN-12 (Notch) signaling specifies the anchor cell (AC) and ventral uterine precursor cell (VU) fates from two equivalent pre-AC/pre-VU cells in the hermaphrodite gonad. Once specified, the AC induces patterned proliferation of vulva via expression of LIN-3 (EGF) and then invades into the vulval epithelium. Although these cellular processes are essential for the proper organogenesis of vulva and appear to be temporally regulated, the mechanisms that coordinate the processes are not well understood. We computationally identified egl-43 as a gene likely to be expressed in the pre-AC/pre-VU cells and the AC, based on the presence of an enhancer element similar to the one that transcribes lin-3 in the same cells. Genetic epistasis analyses reveal that egl-43 acts downstream of or parallel to lin-12 in AC/VU cell fate specification at an early developmental stage, and functions downstream of fos-1 as well as upstream of zmp-1 and him-4 to regulate AC invasion at a later developmental stage. Characterization of the egl-43 regulatory region suggests that EGL-43 is a direct target of LIN-12 and HLH-2 (E12/47), which is required for the specification of the VU fate during AC/VU specification. EGL-43 also regulates basement membrane breakdown during AC invasion through a FOS-1-responsive regulatory element that drives EGL-43 expression in the AC and VU cells at the later stage. Thus, egl-43 integrates temporally distinct upstream regulatory events and helps program cell fate specification and cell invasion.

Ivo Rimann et al. (2006) European Worm Meeting "The Transcription Factor EGL-43 is Necessary for Anchor Cell Invasion during Vulval Development"

ALEX HAJNAL, Ivo Rimann

[European Worm Meeting, 2006]

Ivo Rimann and Alex Hajnal. For proper egg-laying by the hermaphrodite, the uterus has to become firmly connected to the vulva during late larval development. The anchor cell (AC), a specialized cell in the somatic gonad, coordinates vulval and uterus development and initiates the formation of the connection between these two tissues.. During the L3 larval stage, the AC induces six neighboring ventral uterine (VU) cells to adopt the Pi cell fate. Around the same time of development, the basal laminas separating the uterus from the vulva begin to dissolve and the AC invades the vulval tissue by extending a process between the primary cells (Sherwood et al., 2003). In this way, the AC connects the vulval and uterine tissues.. In an RNAi-based screen for genes regulating vulval morphogenesis, we have found that knock-down of egl-43 causes a penetrant protruding vulva (Pvl) phenotype because the connection between the uterus and vulva is not formed correctly. egl-43 encodes a zinc finger transcription factor homologous to the human EVI1 proto-oncogene. Closer examination showed that in egl-43 RNAi animals the basal lamina under the AC is not removed, which prevents the AC from invading the vulval tissue. In addition, egl-43 RNAi animals show defects in the specification of the Pi cell fate. Similar defects in AC invasion have been reported for fos-1 mutants (Sherwood et al., 2005), and like fos-1, a transcriptional egl-43p::gfp reporter is expressed at high levels in the AC and at lower levels in the adjacent VU cells. Moreover, egl-43p::gfp expression in the AC is 5-fold reduced in a fos-1(0) background, indicating that FOS-1 promotes AC invasion at least in part by up-regulating egl-43 expression in the AC.