mechanosensory abnormal) and unc-86 (unc for uncoor- dinated) are two genes that encode transcription factors (MEC-3 and UNC-86, respectively), which form heterodi- mers (UNC-86::MEC-3, a complex with increased DNA- binding speci!city) that regulate TRN development in C. elegans (Goodman, 2008). mec-3 encodes an LIM-type homeodomain protein (where LIM stands for the proteins Lin-11, Isl-1 and Mec-3) with an evolutionarily conserved role in neuronal di"erentiation, migration, and morpho- genesis (Hobert and Westphal, 2000). LIM-type protein expression in the TRNs, FLPs, and PVDs activates the downstream transcription of genes vital for TRN, FLP, or PVD function, respectively, when its promoters bind to the UNC-86::MEC-3 heterodimer (Figure 2 displays the com- binatorial action of these genes). MRN di"erentiation is dependent on the combinato- rial action of several genes: unc-86, mec-3, egl-44, egl-46 and lin-14. unc-86 encodes a POU-type (derived from the names of three transcription factors: the pituitary-speci!c Pit-1, the octamer-binding proteins Oct-1 and Oct-2, and the neural Unc-86 from C. elegans) homeodomain protein (from a highly conserved family of eukaryotic transcription factors) expressed in 59 cells, which is a key regulator in the development of cell lineages into TRNs, and activates expression of the mec-3 gene (Finney et al., 1988). #e development of TRNs requires unc-86 to produce appro- priate TRN lineages, and mec-3 for TRN di"erentiation (Way and Chal!e, 1989). In unc-86 mutants, the correct lineage of TRN precursors is disrupted, which prevents the six TRNs from being made. mec-3 mutants contain cells with the potential to become TRNs, but lack adequate dif- ferentiation and the typical features of TRNs. lin-14 is a gene that encodes a protein vital for regulating postem- bryonic cell division timing and acts as a switch between TRN and PVD di"erentiation: in the presence of lin-14, AVM and PVM di"erentiation occur, while the absence of lin-14 gives rise to PVDs. #e combined action of these genes ultimately restricts the expression of TRN fate to the six neurons observable in wild-type animals (Mitani et al., 1993). TRNs detect gentle touch to the body in a man- ner dependent on the expression of mec-3. In TRNs, combined action of mec-3 and unc-86 activates var- ious other mec genes (mec-1, 2, 4, 7, 8, 9, 10, 12, 14, 15, 17, 18) (Chal!e and Au, 1989) and the alr-1 gene (Topalidou et al., 2011), which de!ne TRN fate. #ese genes comprise the mechanoreceptor channel complex, encoding parts of the extracellular matrix, tubulins for the TRN speci!c 15-proto!lament micro- tubules, and other proteins of unidenti!ed function. Transduction of the touch stimulus itself is accomplished via proteins encoded by several mec genes (mec-2, 4, 6, 10) and unc-24 (Chal!e and Bounoutas, 2007). FLP neurons express the gene sto-5, which encodes a stomatin-like protein that regulates ion permeability (Stewart et al., 1993). In the absence of mec-3, sto-5 is no longer expressed in FLPs, indicating that sto-5 expression in FLPs depends on mec-3 (Topalidou and Chal!e, 2011). Although all FLPs and TRNs are regulated by mec-3, they have di"erent functions, which may be linked to how MRNs express their speci!c traits and acquire distinct cell fates (Way and Chal!e, 1988) (Figure 3 demonstrates the central role of MEC-3 in TRN and FLP di"erentiation). Since both mec-3 and unc-86 are also expressed in FLP neurons and sto-5 is only expressed in FLPs, there must be other co-factors that promote or block TRN fate. Although mec-3 is needed for sto-5 gene expression in FLPs (characteristic of FLP cell fate), FLPs do not express the alr-1 and other mec genes characteristic of TRNs. It is known that mec-3 activates the sto-5 gene directly (Topali- Mechanosensation, or converting mechanical forces of external touch into electrical signals, is easily studied in Caenorhabditis elegans (C. elegans), a transparent nematode with a well-understood nervous system and a fully sequenced genome. The mechano- VHQVRU\�V\VWHP�PHGLDWHV�JURZWK�DQG�GHYHORSPHQW�LQ�PRVW�RUJDQLVPV��D�ODFN�RI�RU�GHIHFW�LQ�PHFKDQRVHQVRU\�VWLPXODWLRQ�FDQ�OHDG�WR� GHYHORSPHQWDO�GHOD\�LQ�FKLOGUHQ��UHVXOWLQJ�LQ�GHYHORSPHQWDO�GLVRUGHUV�VXFK�DV�DXWLVP�VSHFWUXP�GLVRUGHUV��$6'V��DQG�$WWHQWLRQ�'H¿FLW� Disorder (ADD). Studying touch in the animal model C. elegans can elucidate the underlying neuronal mechanisms of touch and their developmental effects. In C. elegans, mechanoreceptor neurons (MRNs) detect touch, with three types that require the gene mec-3 (one of the LIM-homeodomain transcription factors) for their proper differentiation: touch receptor neurons (TRNs) (gentle touch), FLP QHXURQV��WLS�RI�WKH�KHDG�WRXFK��DQG�39'�QHXURQV��KDUVK�WRXFK���:KLOH�WKHVH�WKUHH�051V�GLIIHUHQWLDWH�E\�WKH�VDPH�WUDQVFULSWLRQ�IDFWRU�� each cell type’s differentiation produces distinct morphology and express different genes. FLPs exclusively express the sto-5 gene, while TRNs express the mec-17 gene. The genes that determine and distinguish FLP vs. TRN differentiation are currently limited to egl-44, egl-46 and alr-1. To identify additional genes needed for the differential expression of TRNs and FLPs, the wild-type strain (TU3813), with the FLP neurons labeled with sto-5p::gfp and the TRNs labeled with mec-17p::rfp, was mutated and animals ZLWK�DOWHUHG�H[SUHVVLRQ�RU�QHXURQDO�PRUSKRORJ\�ZHUH�LVRODWHG��:KLOH�GHIHFWV�LQ�ERWK�H[SUHVVLRQ�DQG�PRUSKRORJ\�RI�WKH�)/3V�DQG� TRNs were observed in six animals, only one animal’s mutation affecting TRN appearance was true-breeding. This morphological mutant exhibits abnormal TRN axon morphology, due to an autosomal recessive mutation. The mutant strain will be sequenced to identify the mutated gene, and the role of this gene in TRN and FLP differentiation. Columbia Undergraduate Science Journal Open-Access Publication | http://cusj.columbia.edu 9 cusjVolume 6Spring 2012 Columbia Undergraduate Sci J http://cusj.columbia.edu % ,2 /2 * < cusjcolumbia undergraduate science journal Research Articles 10cusj Volume 6Spring 2012 Columbia Undergraduate Sci J http://cusj.columbia.edu % ,2 /2 * < Touch sensitivity via converting detected mechani- cal forces into electrical signals is a vital survival mechanism in most organisms, a behavior known as mechanosensation. To understand normal function of the mechanosensory sys- tem and its complex neuronal circuitry, defective genes can be studied in animals that are de!cient in mechanosensa- tion (touch insensitive). Touch insensitive mutants were !rst developed in the nematode Caenorhabditis elegans (C. elegans), which led to the discovery of a mechanosensory complex with the necessary components to allow certain cells to detect touch to the body. Mechanosensation is well studied as a basic behavioral component in C. elegans since the organism’s entire genome has been mapped. Investigat- ing mechanosensation is much more challenging in com- plex eukaryotes because the mechanosensing cells and their scarce transducing molecules are more di$cult to isolate and observe (Chal!e and Bounoutas, 2007). Development of touch sensitivity can be studied in C. elegans by analyzing genes expressed in a specialized set of 30 neurons that detect mechanosensation: mechano- receptor neurons (MRNs). #is experiment investigates expression mutants (with altered neuronal touch recep- tor-speci!c gene expression) and morphological mutants (with defects on genes that a"ect neuronal outgrowth, number, or branching patterns) of two types of MRNs (touch receptor neurons (TRNs) and FLP neurons) in order to better characterize MRN di"erentiation. TRNs are an important type of MRNs because they exhibit the greatest electrophysiological response to mechanical stimuli (Kamkin and Kiseleva, 2007). Two other types of MRNs in C. elegans are PVD neurons, a pair of posterior interneu- rons that mediate harsh touch, and FLP neurons, a pair of ciliated mechanosensory neurons that detect touch at the very tip of the head. Collectively, TRNs (six cells), FLPs (2 cells) and PVDs (2 cells) comprise one-third of all MRNs (Goodman, 2008) (Figure 1). Several genes have been identi!ed as necessary for dif- ferentiation of the six touch receptor neurons (ALML, ALMR, PLML, PLMR, AVM and PVM). mec-3 (mec for Identifying Factors that Control Mechanoreceptor Neuron Development in C. elegans Alexis Tchaconas 1, ��0DUWLQ�&KDO¿H1 1Department of Biological Sciences, Columbia University, New York, NY 10027 Copyright: © 2012 The Trustees of Columbia University, Columbia University Libraries, some rights reserved, Tchaconas, et al. Received 12/15/2012. Accepted 1/31/2012. Published 4/1/2012 *To whom correspondence should be addressed: 1018 Fair- child Center, Columbia University, New York, NY 10027, e-mail: aet2129@columbia.edu Abstract Introduction Figure 1 Mechanosensory neurons in C. elegans Figure 2 Combinatorial MRN Differentiation dou and Chal!e, 2011), but it is not known why sto-5 is not expressed in the TRNs; this experiment aims to !nd genes that are involved in this process. Past studies have isolated egl-44 and egl-46 as two genes that restrict TRN fate in the FLP neurons: mutations of either gene cause the FLP neurons to transform into cells that resemble TRNs (Mitani et al., 1993) (Wu, Duggan and Chal!e, 2001). However, the genes that restrict FLP fate in other cells remain unknown. We hypothesize that another gene or network of genes is restricting sto-5 expression to FLP neurons and is not allowing mec gene expression in the FLPs (which inhibits TRN fate). One morphological mutant was identi!ed through this study, exhibiting ir- regularly wavy TRN axonal processes. General methods and strains To identify genes needed for the di"erential expres- sion of TRNs and FLPs, wild-type animals (TU3813 strain) were mutated. #ese C. elegans strains were main- tained on OP50 seeded agar plates at 20oC as outlined by Brenner (1974). #e expression of sto-5 and mec-17 was observed in the wild-type strain using $uorescent protein tags: sto-5 was tagged with green $uorescent protein (sto- 5p::gfp) and mec-17 was tagged with red $uorescent protein (mec-17p::rfp). sto-5 expression was used for FLP analysis since it is characteristic of FLP di"erentiation, and mec- 17 was used for TRN observations since it is needed for sustained TRN di"erentiation. Since C. elegans are trans- parent, these $uorescently tagged genes are visible under a dissecting microscope. Gene expression was monitored under a dissecting microscope, looking for any changes in expression upon mutagenesis (Figure 4 & Figure 5 are images from the dissecting microscope of the $uorescent protein tagged FLP and TRN cells in a wild-type animal). #e wild-type animals were synchronized to the same stage (L4) prior to mutagenesis using adecontaminating solution (20% bleach). Fluorescence Imaging #e appearance of FLPs and TRNs in the wild-type strains were observed and characterized via a stereo-$uo- rescence dissecting microscope (Leica MZ12) powered by a UV light source (Kramer Scienti!c Corporation) coupled with the X-Cite 120 Fluorescence Imaging System. Axiovi- sion 4.8.2 software was used to further characterize FLPs and TRNs. EMS Mutagenesis Mutagenesis was performed on the wild-type TU3813 strain with a standard protocol involving ethyl methane- sulfonate (EMS) (Brenner, 1974): two to three plates of healthy TU3813 L4 animals (approximately 100-300 ani- mals) were mutagenized, and 30 of the progeny animals (P0) were picked and plated individually, and stored at 25oC for three days. After three days, four animals from each P0 plate were plated individually (F1 animals) onto new plates and then stored at 25oC for three days (produc- ing F2 animals). #ese F2 plates were manually screened three days later for any apparent abnormalities in FLP or TRN expression. F2 animals with noticeable abnormali- ties in either set of cells were picked individually onto new plates and their progeny were observed from subsequent generations to determine if the mutation is heritable (true- breeding) (Figure 6 schematically outlines the mutageniz- ing process). Mutant screens via !uorescence imaging #e sto-5 promoter was expressed with gfp (sto-5p::gfp), and the mec-17 promoter with rfp (mec-17p::rfp) to ob- serve and compare FLPs and TRNs among the wild-type and mutant strains. #e $uorescent constructs are fu- Columbia Undergraduate Science Journal Open-Access Publication | http://cusj.columbia.edu 11 cusjVolume 6Spring 2012 Columbia Undergraduate Sci J http://cusj.columbia.edu % ,2 /2 * < Figure 3 unc-86 and mec-3 are both required for TRN and FLP differenti- ation, although they express different genes upon differentiation. sions of promoters to gfp and rfp injected and integrated into the wild-type TU3813 animals. F2s were !rst screened abnormal expression of the gfp and rfp markers, indicating abnormal gene expression. #en, the morphology of FLPs and TRNs was characterized in identi!ed mutant strains, noting the shape (axonal branching patterns), position, and changes in the number of FLPs or TRNs. Any animals with abnormal traits in these cells were isolated and screened for the abnormality over several generations. If the isolated mutant consistently produced progeny with the same ab- normality, the mutation was further investigated through genetic crosses. Characterizing mutants via genetic crosses #e isolated mutants were crossed with the N2 wild-type strain and then with the dpy-5 mutant strain to determine the nature of the mutation: whether it was dominant or re- cessive, and X-linked or autosomal. Two successive crosses were set up: the !rst was a cross between three hermaphro- dite mutants and seven N2 (wild-type) males, and the sec- ond was a cross between seven of the male mutant progeny and three dpy-5 hermaphrodites. dpy-5 mutants are char- acterized by a short, “dumpy” body (Dpy phenotype) due to the disruption of the dpy-5 gene, which encodes a cuticle procollagen (#acker, Sheps and Rose, 2006). #e dpy-5 animals were used to distinguish hermaphroditic self-prog- eny (dpy/dpy genotype with Dpy phenotype) from her- maphroditic cross progeny (m/dpy genotype with normal phenotype, since dpy-5 is a recessive mutation). All animals crossed were in the L4 stage, a standard procedure used in order to enable mating between males and hermaphrodites and to minimize hermaphroditic self-progeny (Brenner, 1974). Six mutant strains exhibit abnormal FLP and TRN fea- tures Seven mutageneses were performed on the TU3813 strain, yielding 710 observable F1 mutant animals. 32 can- didate mutants were isolated from the mutant screens, six of which produced heritable FLP and/or TRN defects. One mutant is true-breeding for abnormal TRN morphology Among the six viable mutants, two mutants exhibited abnormalities in FLP neuron quantity and appearance. Observed morphological mutations included multiple and vertically moving FLP neurons, and abnormal TRN pro- cess morphology (wavy, crossed, or shortened). Expression mutations involved simultaneous FLP and TRN expression when observed in the GFP channel on the dissecting mi- croscope. Over successive generations, only one mutant (4A1) retained its mutant phenotype (‘true-breed- ing’). #e mutation is particularly observable along the processes of ALM and PLM neurons, producing irregularly wavy axons in these animals that are no- ticeably distinct from the straight axons in wild-type animals (Figure7-12 display side-by-side comparisons of TU3813 wild-type TRN morphology and 4A1 mutant TRN morphology from the anterior, mid- body, and posterior sections of the body). cusjcolumbia undergraduate science journal Research Articles 12cusj Volume 6Spring 2012 Columbia Undergraduate Sci J http://cusj.columbia.edu % ,2 /2 * < Materials and Methods Figure 4 sto-5p::gfp expression in wild-type TU3813 animals Figure 5 PHF���S��UIS�H[SUHVVLRQ�LQ�ZLOG�W\SH�78�����DQLPDOV Figure 6 Growth timeline and observation scheme for a standard EMS mutagenesis Results Figure 7 Anterior view of TU3813 wild-type (WT) animal (left to right: ALM process, ALML). The ALM process is straight as it extends from the cell body. to-5p::gfp expression in wild-type TU3813 animals 4A1 Mutation Characterization via Genetic Crosses !e "rst cross between hermaphrodite mutants and N2 (wild-type) males yielded male o#spring and hermaphro- dite o#spring without the mutant phenotype (wild-type). !e second cross between the male mutant progeny and dpy-5 hermaphrodites also produced wild-type male and hermaphrodite o#spring (Figure 13 summarizes the cross). Columbia Undergraduate Science Journal Open-Access Publication | http://cusj.columbia.edu 13 cusjVolume 6Spring 2012 Columbia Undergraduate Sci J http://cusj.columbia.edu Figure 8 Anterior view of 4A1 mutant (AVM), with irregularly wavy ALM process (left to right: ALM process, AVM, ALMR). The ALM process is wavy as it extends from the ALMR cell body !is study isolated genes that disrupt the function or de- velopment of gene expression, or morphology in the TRNs and FLP neurons (by isolating an observed phenotype and then identifying the responsible gene). Based on the speci"c abnormality observed in a mutant, the normal function of the mutated gene can be inferred from characteristics in its absence, and its role in MRN di#erentiation can then be established. !e seven mutageneses performed enabled screening of 710 F1 animals, yielding 32 candidate mutants, only one of which bred true. Typically, there is a 1/2000 probability of "nding a mutant via mutagenesis, so an appropriate sample size would be screening 10,000 F1 animals, and their F2 progeny (Brenner, 1974). !e F1 population size in our study was relatively small, and thus should be increased in the future by mutagenizing and screening more animals. As mentioned above, this work has isolated one viable mutant via EMS mutagenesis (4A1). !is mutant has dis- played a consistent, morphological mutant phenotype in every successive generation, indicating that it is a true- breeding mutant. !e 4A1 mutant phenotype, which in- volves irregularly wavy TRN morphology (along the ALM and PLM processes), suggests that the induced mutation may have disrupted the axonal development of the ALM and PLM neurons. Although none of the putative mutants with FLP abnormalities appeared to be true-breeding mu- tants, the only true-breeding mutant identi"ed exhibits in- teresting TRN abnormalities. While "nding a TRN or FLP morphological mutant was a secondary goal of the project (the primary goal was to "nd expression mutants), the mu- tant nonetheless appears to have a signi"cant mutation that may reveal more about MRN functionality. !us, the 4A1 mutant is worthy of further exploration and genetic char- acterization. !e results of the cross between 4A1 mutant hermaphro- dites and N2 males, followed by the cross of the male o#- spring and dpy-5 hermaphrodites indicate that the mutation is autosomal recessive. !e male o#spring only inherited one X chromosome, from the mutant hermaphrodite. Since the cross with 4A1 mutant hermaphrodites and N2 males produced male o#spring that did not exhibit the mutant phenotype, the mutation is not X-linked, which is further veri"ed by the next cross. Furthermore, from both crosses the o#spring do not show a mutant phenotype, which in- dicates that the mutation is recessive. An additional cross will be performed to recon"rm the mutant allele’s mode of inheritance, by crossing dpy-5 (dpy/dpy) hermaphro- dites with N2 (+/+) males. !e heterozygous male o#spring (dpy/+) will then be crossed with 4A1 mutant hermaphro- dites (m/m), producing two types of heterozygous progeny (m/dpy and m/+). Since a recessive hermaphroditic allele was crossed with a male to produce heterozygote mutant o#spring in the second generation, the cross further clari- "es whether the mutation is dominant or recessive (based on the second cross male progeny’s phenotype – if all males are mutant, the mutation is dominant; if the males are wild- type, the mutation is recessive). Future studies will continue to follow the 4A1 mutant strain, speci"cally to determine on which chromosome the mutation is located. First, additional two-step crosses will be performed with mutants that have a known chromosomal location (i.e. dpy-5, with an autosomal recessive mutation on chromosome I). Such crosses will indicate which chro- mosome the 4A1 mutation is on, based on the progeny phe- notypes. !en, complementation tests will be performed to determine whether the mutation is on a previously isolated gene, or an entirely unexplored gene. In addition, if it is a novel mutation for MRN di#erentiation (based on the chromosome and gene on which it is located), then the mu- tant strain will have its genome fully sequenced. Genomic sequencing of a 4A1 mutant animal can be compared with the wild-type genome in order to identify the gene that contains the mutation. Additional mutageneses will also be performed (increasing F1 sample size) to "nd more genes po- tentially involved in the combinatorial regulation of MRN di#erentiation. Once these genes have been identi"ed via genetic sequencing, they can be fused with GFP to further characterize their normal expres- sion. With an understanding of the normal and ab- normal function of these combinatorial genes, each gene’s role in FLP and TRN di#erentiation can be determined. Other experiments can be performed to determine how these genes mechanistically function in the de"nition of MRN development or function. !e irregularly wavy TRN processes observed in the 4A1 mutant could be due to a defect in axonal attachment. An- other researcher in our lab has found a mutant with a cuti- cle attachment defect (from a mutation on the gene mec-5), so the 4A1 mutant will be compared to this other mutant’s phenotype. Such comparisons can determine phenotypic similarities between mutants, and potentially identify genes that have a combinatorial interaction with one another in MRN di#erentiation. Once the combinatorial mechanisms regulating di#erentiation of the touch neurons is better understood, more insight into mechanosensory systems in higher organisms can be gained, due to the cross-organis- mal homology of touch. We thank the Chal"e lab (Dr. Irini Topalidou, Dr. Emalick Njie, Dr. Charles Keller, Xiaoyin Chen, Chaogu Zheng, Yushu Chen, and Ana Pozo). AET was funded by the Columbia Department of Biological Sciences from the cusjcolumbia undergraduate science journal Research Articles 14cusj Volume 6Spring 2012 Columbia Undergraduate Sci J http://cusj.columbia.edu Discussion Figure 9 Mid-body view of a TU3813 WT animal’s PLM process displays its normally straight morphology. Figure 10 Mid-body view of a 4A1 mutant’s PLM process displays an ir- regularly wavy morphology throughout its midsection Figure 11 3RVWHULRU�YLHZ�RI�D�78�����:7�DQLPDO¶V�3/05�/�DQG�QRUPDOO\� VWUDLJKW�3/0�SURFHVV��OHIW�WR�ULJKW��3/0�SURFHVV��3/05�/�� % ,2 /2 * < Figure 12 4A1 mutant with irregularly wavy posterior process (PLM) (left to right: PVM, PLM process), exhibiting an apparent deviation from the WT process morphology (in Figure 11). Figure 13 4A1 crosses for mutational characterization Symbol Key Acknowledgments % ,2 /2 * < Summer Undergraduate Research Fellowship. Bounoutas, A., and Chal!e, M. 2007. Touch sensitivity in Cae- norhabditis elegans. P"ugers Arch. 454, 691–702. Brenner, S. 1974. #e genetics of Caenorhabditis elegans. Genet- ics 77, 71–94. Chal!e, M., & Au, M. 1989. Genetic control of di$erentiation of the Caenorhabditis elegans touch receptor neurons. Science 243, 1027 – 1033. Finney, M., Ruvkun, G., & Horvitz, H.R. 1988. #e C. elegans cell lineage and di$erentiation gene unc-86 encodes a protein with a homeodomain and extended similarity to transcription factors. Cell 55, 757 – 769. Goodman, Miriam B. “Mechanosensation.” WormBook (May 2008): 9 June 2011. Hobert, Oliver, and Heiner Westphal. “Functions of LIM- homeobox genes.” Trends in Genetics 16.2 (2000): 75-82. Trends in Genetics. 21 Jan. 2012. Kamkin, Andre Glebovich, and Irina S. Kiseleva, eds. Mechanosensitive ion channels. New York: Springer, 2007. #e Tavernarakis Lab. 3 Aug. 2011. Mitani, S. 1995. Genetic regulation of mec-3 expression implicated in the speci!cation of the mechanosensory neuron cell types in Caenorhabditis elegans. Dev. Growth Di$er., 37, 551 - 557. Mitani, S., Du, H., Hall, D. H., Driscoll, M., & Chal!e, M. 1993. Combinatorial control of touch receptor neuron expression in Caenorhabditis elegans. Development 119, 773 – 783. Stewart GW, Argent AC, Dash BC, 1993. Stomatin: a putative cation transport regulator in the red cell membrane. Biochim Bio- phys Acta 1225,15–25 #acker C, Rose AM, and Sheps JA. “Caenorhabditis elegans dpy-5 is a cuticle procollagen processed by a proprotein convertase.” Cellular and Molecular Life Sciences 63.10 (2006): 1193-204. Topalidou, Irini, Alexander Van Oudenaarden, and Martin Chal- !e.” Caenorhabditis elegans aristaless/Arx gene alr-1 restricts vari- able gene expression.” Proceedings of the National Academy of Sci- ences 108.10 (2011): 4063-4068. Topalidou, Irini, and Martin Chal!e. “Shared gene expression in distinct neurons expressing common selector genes.” Proceedings of the National Academy of Sciences 108.48 (2011): 1-6. PNAS. 25 Feb. 2012. Way, J.C. and Chal!e, M. 1988. mec-3, a homeobox-containing gene that speci!es di$erentiation of the touch receptor neurons in C. elegans. Cell 54, 5–16. Way, J. C., & Chal!e, M. 1989. #e mec-3 gene of Caenorhab- ditis elegans requires its own product for maintained expression and is expressed in three neuronal cell types. Genes Dev. 3, 1823 – 1833. Wu J, Duggan A, Chal!e M. Inhibition of touch cell fate by egl- 44 and egl-46 in C.elegans. Genes Dev 2001; 15, 789-802. Zhang S, Arnadottir J, Keller C, Caldwell GA, Yao CA, Chal!e M (2004) MEC-2 is recruited to the putative mechanosensory com- plex in C. elegans touch receptor neurons through its stomatin-like domain. Curr Biol 14, 1888 –1896. Zhang, Y., Ma, C., Delohery, T., Nasipak, B., Foat, B.C., Bounoutas, A., Bussemaker, H.J., Kim, S.K., Chal!e, M., 2002. Identi!cation of genes expressed in C. elegans touch receptor neu- rons. Nature 418, 331–335. Columbia Undergraduate Science Journal Open-Access Publication | http://cusj.columbia.edu 15 cusjVolume 6Spring 2012 Columbia Undergraduate Sci J http://cusj.columbia.edu % ,2 /2 * < References Breast cancer affects one in eight women per year, and 70% of patients with stage IV breast cancer develop metastases in bone, FDXVLQJ�OLIH�WKUHDWHQLQJ�VLGH�HIIHFWV��:H�H[DPLQH�WKH�XVH�RI�DQ�H[SODQW�V\VWHP�WR�PLPLF�EUHDVW�FDQFHU�PHWDVWDVLV�WR�ERQH�ZLWKRXW� confounding cell types and to determine the role that osteocytes, the bone mechanosensing cells, may have in the development of metastasis. Using the explant system, a custom cell seeder and sterile cell culture techniques, we introduced metastatic MDA-MB-231/ *)3�FHOOV�WR�D�WKUHH�GLPHQVLRQDO�ERQH�PDWUL[�ZLWK�RVWHRF\WHV�RQO\��&RQIRFDO�LPDJLQJ�FRQ¿UPV�WKDW�EUHDVW�FDQFHU�FHOOV�ZHUH��LQ�IDFW�� successfully seeded onto bone cores, mimicking metastasis. Though additional experiments will be necessary to determine the im- portance of breast cancer-osteocyte interactions, this study shows that the explant system is a viable methodology for studying breast cancer in bone. Cancer is a devastating disease that is responsible for thir- teen percent of deaths worldwide and has a$ected count- less families and individuals throughout the world (Cancer, World Health Organization). It a$ects 1 in 8 women every year (U.S. breast cancer statistics). Breast cancer originates from the inner lining of the lobules that supply the milk ducts in the breast (Wolf et al., 2003). Stage IV breast can- cer is metastatic, meaning that it is violent and transcends the host organ (the breast) and spreads to a secondary site. #e cancer that metastasizes is still considered breast can- cer. It has been reported that up to 70% of stage IV breast cancer patients will experience some form of metastasis of breast cancer to bone (Roth et al., 2009). Patients often experience pathological fractures, intense pain, hypercal- cemia, and various nervous compression complications (Zhang et al., 2010). #ese devastating e$ects are caused by an imbalance of bone remodeling, which involved the interactions of the three main bone cell types. Bone is comprised of three types of cells: osteocytes (OCY), osteoblasts (OB) and osteoclasts (OCL). Osteo- cytes are the primary mechanosensing cells in bones (Burg- er et al., 1995). #ey regulate the activity of osteoblasts and osteoclasts. Osteocytes are “trapped” in the mineralized bone matrix, and are thus they are thought to have only sig- naling functions, both intercellular and intracellular. After osteocytes sense a mechanical load, that load is transduced into a chemical signal is sensed by the cells. #is stress is translated into a biochemical signal that is communicated to the osteoblasts and osteoclasts, the bone forming and bone resorbing cells (Burger et al., 1995). Osteoblasts syn- thesize the bone matrix, which is subsequently de- posited and calci!ed to become bone mineral. When osteoblasts secrete too much matrix, they become stuck in the bone, and as a result they completely di$erentiate into osteocytes (Saladin, 2007; Buck- walter et al. 1995). Osteoclasts, on the other hand, resorb bone. To accomplish this, they use their “ruf- "ed” membrane (as shown in Figure 1) to create a seal around bone and then pump enzymes and hy- drochloric acid to degrade the matrix (Saladin, 2007; Buckwalter et al. 1995). #ere are two types of metastasis: osteolytic and os- teoblastic. Osteolytic metastases break down bone and are the most common type of metastasis for breast cancer. Os- teoblastic lesions, characterized by excess bone formation, a$ect 15-20% of patients. Mixed types also exist, wherein the patient experiences unnecessary bone excess as well as dearth (Zhang et al., 2010). #e large majority of stage IV breast cancer cases end in metastasis to bone because bone has high levels of growth factors that breast cancer uses to survive. We can see that bone is a likely candidate for breast cancer metastasis due to the presence of “transforming growth factor B (TGFB), insulin-like growth factors I and II (IGF), !broblast growth factors (FGFs), platelet-derived !e Veri"cation of a Novel Explant System Used to De- termine the Role of Osteocytes in the Breast Cancer Vicious Cycle Sonia Bansal*, Genevieve N. Brown, X. Edward Guo %RQH�%LRHQJLQHHULQJ�/DERUDWRU\��'HSDUWPHQW�RI�%LRPHGLFDO�(QJLQHHULQJ��&ROXPELD�8QLYHUVLW\��1HZ�