Expression of the Caenorhabditis elegans Hox gene lin-39 begins in the embryo and continues in multiple larval cells, including the P cell lineages that generate ventral cord neurons (VCNs) and vulval precursor cells (VPCs). lin-39 is regulated by several factors and by Wnt and Ras signaling pathways; however, no cis-acting sites mediating lin-39 regulation have been identified. Here, we describe three elements controlling lin-39 expression: a 338-bp upstream fragment that directs embryonic expression in P5-P8 and their descendants in the larva, a 247-bp intronic region sufficient for VCN expression, and a 1.3-kb upstream cis-regulatory module that drives expression in the VPC P6.p in a Ras-dependent manner. Three trans-acting factors regulate expression via the 1.3-kb element. A single binding site for the ETS factor LIN-1 mediates repression in VPCs other than P6.p; however, loss of LIN-1 decreases expression in P6.p. Therefore, LIN-1 acts both negatively and positively on lin-39 in different VPCs. The Forkhead domain protein LIN-31 also acts positively on lin-39 in P6.p via this module. Finally, LIN-39 itself binds to this element, suggesting that LIN-39 autoregulates its expression in P6.p. Therefore, we have begun to unravel the cis-acting sites regulating lin-39 Hox gene expression and have shown that lin-39 is a direct target of the Ras pathway acting via LIN-1 and LIN-31.

Overexpression of SPARC, a collagen-binding glycoprotein, is strongly associated with tumor invasion through extracellular matrix in many aggressive cancers. SPARC regulates numerous cellular processes including integrin-mediated cell adhesion, cell signaling pathways, and extracellular matrix assembly; however, the mechanism by which SPARC promotes cell invasion in vivo remains unclear. A main obstacle in understanding SPARC function has been the difficulty of visualizing and experimentally examining the dynamic interactions between invasive cells, extracellular matrix and SPARC in native tissue environments. Using the model of anchor cell invasion through the basement membrane (BM) extracellular matrix in Caenorhabditis elegans, we find that SPARC overexpression is highly pro-invasive and rescues BM transmigration in mutants with defects in diverse aspects of invasion, including cell polarity, invadopodia formation, and matrix metalloproteinase expression. By examining BM assembly, we find that overexpression of SPARC specifically decreases levels of BM type IV collagen, a crucial structural BM component. Reduction of type IV collagen mimicked SPARC overexpression and was sufficient to promote invasion. Tissue-specific overexpression and photobleaching experiments revealed that SPARC acts extracellularly to inhibit collagen incorporation into BM. By reducing endogenous SPARC, we also found that SPARC functions normally to traffic collagen from its site of synthesis to tissues that do not express collagen. We propose that a surplus of SPARC disrupts extracellular collagen trafficking and reduces BM collagen incorporation, thus weakening the BM barrier and dramatically enhancing its ability to be breached by invasive cells.

LIN-1 is an ETS domain protein. A receptor tyrosine kinase/Ras/mitogen-activated protein kinase signaling pathway regulates LIN-1 in the P6.p cell to induce the primary vulval cell fate during Caenorhabditis elegans development. We identified 23 lin-1 loss-of-function mutations by conducting several genetic screens. We characterized the molecular lesions in these lin-1 alleles and in several previously identified lin-1 alleles. Nine missense mutations and 10 nonsense mutations were identified. All of these lin-1 missense mutations affect highly conserved residues in the ETS domain. These missense mutations can be arranged in an allelic series; the strongest mutations eliminate most or all lin-1 functions, and the weakest mutation partially reduces lin-1 function. An electrophoretic mobility shift assay was used to demonstrate that purified LIN-1 protein has sequence-specific DNA-binding activity that required the core sequence GGAA. LIN-1 mutant proteins containing the missense substitutions had dramatically reduced DNA binding. These experiments identify eight highly conserved residues of the ETS domain that are necessary for DNA binding. The identification of multiple mutations that reduce the function of lin-1 as an inhibitor of the primary vulval cell fate and also reduce DNA binding suggest that DNA binding is essential for LIN-1 function in an animal. INTRACELLULAR signaling specifies many cell fates guanine nucleotide exchange factor. LET-341 is likely to cause LET-60 Ras to release GDP, resulting in GTP during development. The Caenorhabditis elegans vulva is a useful model system for understanding how signal binding and LET-60 activation. Activated LET-60 Ras can bind and activate the serine/threonine kinase LINtransduction cascades regulate cell fates. The vulva is a specialized epidermal structure used for egg laying and 45 Raf. Activated LIN-45 phosphorylates and thereby activates the MEK-2 mitogen-activated protein (MAP) sperm entry that is formed by the descendants of three kinase kinase. MEK-2 phosphorylates and thereby actiectodermal blast cells, P5.p, P6.p, and P7.p (Horvitz vates the MPK-1 extracellular signal-regulated kinase and Sternberg 1991). In wild-type hermaphrodites, the (ERK) MAP kinase. MPK-1 appears to phosphorylate anchor cell of the somatic gonad signals to P6.p using multiple target proteins, including the LIN-1 ETS tranthe LIN-3 epidermal growth factor-like ligand (reviewed scription factor, and these modifications cause P6.p to by Greenwald 1997; Kornfeld 1997; Sternberg and adopt the 1Њ vulval cell fate (eight descendants). When Han 1998). LIN-3 presumably binds to the LET-23 re-P6.p is induced to adopt the 1Њ vulval cell fate, it signals ceptor tyrosine kinase (RTK). This is likely to stimulate to P5.p and P7.p through the LIN-12 Notch receptor, receptor autophosphorylation and create docking sites causing these cells to adopt the 2Њ vulval cell fate (seven for the SEM-5 adaptor protein and the LET-341 Ras descendants

Overexpression of SPARC, a collagen-binding glycoprotein, is strongly associated with tumor invasion through extracellular matrix in many aggressive cancers. SPARC regulates numerous cellular processes including integrin-mediated cell adhesion, cell signaling pathways, and extracellular matrix assembly; however, the mechanism by which SPARC promotes cell invasion in vivo remains unclear. A main obstacle in understanding SPARC function has been the difficulty of visualizing and experimentally examining the dynamic interactions between invasive cells, extracellular matrix and SPARC in native tissue environments. Using the model of anchor cell invasion through the basement membrane (BM) extracellular matrix in Caenorhabditis elegans, we find that SPARC overexpression is highly pro-invasive and rescues BM transmigration in mutants with defects in diverse aspects of invasion, including cell polarity, invadopodia formation, and matrix metalloproteinase expression. By examining BM assembly, we find that overexpression of SPARC specifically decreases levels of BM type IV collagen, a crucial structural BM component. Reduction of type IV collagen mimicked SPARC overexpression and was sufficient to promote invasion. Tissue-specific overexpression and photobleaching experiments revealed that SPARC acts extracellularly to inhibit collagen incorporation into BM. By reducing endogenous SPARC, we also found that SPARC functions normally to traffic collagen from its site of synthesis to tissues that do not express collagen. We propose that a surplus of SPARC disrupts extracellular collagen trafficking and reduces BM collagen incorporation, thus weakening the BM barrier and dramatically enhancing its ability to be breached by invasive cells. Author SummarySPARC is an extracellular matrix protein that is present at high levels in many metastatic cancers where it promotes tumor invasion into neighboring tissues. The mechanism linking a surplus of SPARC to cell invasion, however, is not clear due to the challenge of examining SPARCs function in complex tumor environments. We have used anchor cell Data Availability Statement: All relevant data are within the paper and its Supporting Information files.Funding: This study was funded by the Pew Scholars Program in the Biomedical Sciences (DRS), a National Science Foundation Graduate Research Fellowship (MAM), and the National Institute of General Medicine grant GM079320 (DRS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.invasion in C. elegans development to understand how an excess of SPARC promotes invasion in a native tissue setting. Anchor cell invasion allows experimental examination and visualization of the interactions between an invasive cell, neighboring tissues, and the basement membrane, a sheet-like extracellular matrix that surrounds tissues. We find that increased SPARC expression potently enhances the ability of weakly invasive anchor cells to breach the basement membrane. Our data ind...