The natural compound cordycepin (3’-deoxyadenosine) causes a reduction in breast cancer cell viability. Microarray analysis showed that growth related genes are down-regulated by cordycepin. Indeed, mTOR, ERK and AMPK signalling was shown to be altered by cordycepin, but the effect was too fast to be mediated by transcriptional changes. It was hypothesised that cordycepin affected signal transduction through translation. However, polysome profiling did not identify clear candidates for the effects of cordycepin on signal transduction but unveiled that cordycepin leads to translation repression on 5’ terminal oligopyrimidine (TOP) mRNAs. As TOP mRNAs are known to be regulated by mTOR signaling, this result consistently suggests mTOR signaling is inhibited by cordycepin treatment. To test if it is possible that cordycepin affects gene expression via signal transduction, we compared its effects to various signal transduction inhibitors and an activator. So far, Pictilisib, a pan-PI3K inhibitor, is the only inhibitor that mimics both the gene expression and signal transduction effects of cordycepin, indicating the PI3K-PDK1-AKT axis is affected by cordycepin. The RNAs upregulated by cordycepin were highly enriched in a group of non-coding RNAs, which are also appeared to induce during serum withdrawal. Knockdown of poly(A) polymerases induced these RNAs, indicating that they probably are degraded by the PABPN1 and poly(A) polymerase dependent nuclear RNA decay pathway. Thus the data suggest that cordycepin affects gene regulation by two distinct pathways, one affecting signal transduction and growth related mRNA expression and another affecting polyadenylation mediated decay of non-coding mRNAs.

Endogenous nitric oxide (NO) produced by the enzyme NO synthase (NOS) has an important role in a variety of physiological processes. However, NO becomes noxious to cells if produced in excess. Therefore, the production of NO is tightly regulated. A particularly exciting and novel aspect of the regulation of NO signalling is the possibility that the expression of NOS genes is controlled by unconventional mechanisms that depend on the presence of natural antisense transcripts (NATs). In this thesis I investigate the important properties of two distinct NOS-related NATs: Lym-antiNOS2 and Mm-antiNOS1. I show that Lym-antiNOS2 RNA is widely expressed in the CNS of the pond snail Lymnaea stagnalis. Furthermore, I demonstrate that the expression of Lym-antiNOS2 is differentially regulated by training leading to long-term memory formation. Moreover, my results indicate that Lym-antiNOS2 RNA is subjected to peripheral trafficking in neurons. As for Mm-antiNOS1, I find that its expression is restricted to embryonic brain tissue and is almost undetectable in the adult brain of Mus musculus.

In response to DNA double stranded breaks (DSBs), mammalian cells have evolved two major repair pathways, DNA Non Homologous End Joining (NHEJ) and Homologous Recombination (HR). The majority of DSB repair in G1 and G2 phase is repaired with fast kinetics by NHEJ in a pathway that involves the core NHEJ factors: Ku, DNA-PKcs, XLF, DNA Ligase IV and XRCC4. A subset of slow repairing DSBs also requires ATM and Artemis (Riballo et al, 2004). This slow component of repair represents DSBs that reside within highly compacted regions of the genome known as heterochromatin (HC) (Goodarzi et al, 2008). ATM functions at HC to mediate relaxation by phosphorylating the HC building factor KAP-1 (Goodarzi et al, 2008). Here I provide evidence that DSBs dependent upon Artemis for their repair also reside within regions of HC. However, unlike ATM, Artemis functions downstream of the HC relaxation process. In response to DSBs, ATM phosphorylates the histone variant H2AX (γH2AX). γH2AX acts as a docking site for the localized recruitment and activation of DNA Damage Response (DDR) proteins. The expansion of γH2AX can spread over megabases of DNA. Here I have shown that highly compacted KAP-1, MeCP2 and DNMT3B enriched chromatin acts as a barrier to IR induced γH2AX expansion. In patient cells deficient for MeCP2 or DNMT3B proteins, such as Rett syndrome (MeCP2 deficient) and Immunodeficiency centromeric-instability facial-anomalies syndrome (DNMT3B deficient), ATM and Chk2 signalling is heightened, which is reflected in a hypersensitive and prolonged G2/M checkpoint arrest. These findings suggest that higher order chromatin complexity is a barrier to ATM signalling to the checkpoint machinery. In the final section of my thesis, I addressed what affect DNA damage complexity exerts on checkpoint arrest. Using exposure to heavy ion irradiation, which induces complex DSBs, I observed larger γH2AX foci and prolonged G2/M checkpoint arrest.

Phosphatase and tensin homologue deleted on chromosome ten (PTEN) is one of the most commonly altered tumor suppressors in human cancer. It is a dual-specificity phosphatase that by converting the lipid second messenger PIP3 to PIP2 antagonizes the PI3K/AKT signaling pathway. PTEN also has numerous, albeit controversial nuclear functions, which thus far have been shown to be independent of its phosphatase activity. Although a number of studies have described that loss or gain of PTEN protein expression alters gene expression patterns, relatively little is known about the exact mechanism. In this research study, we investigated PTEN’s influence on gene expression and its role in transcription regulation. First, we established mouse embryonic fibroblasts (MEFs) as a suitable model system to study the effects of PTEN loss on gene expression. Using an Adeno-virus containing Cre-recombinase, Pten expression could be ablated efficiently in MEFs carrying loxP sites flanking exon 5 of the endogenous Pten locus. Genome-wide mRNA microarray analysis revealed that Pten deletion decreased the transcript levels of a subset of genes and increased the transcript levels of a different subset of genes. Moreover, by uncoupling these effects from PTEN’s role in the PI3K/AKT pathway we discovered that Pten loss can alter gene expression in a PI3K/AKT-dependent as well as a PI3K/AKT-independent manner. The upregulated genes were enriched for genes involved in DNA binding, replication, and repair, but also for regulation of gene expression. Gene expression can be influenced by histone modifications. However, loss of PTEN did not affect histone modifications globally as evidenced by western blotting. Using native ChIP-Seq experiments we showed that loss of PTEN altered the levels of H3K36me3 and H3K27me3 on a subset of genes and markedly decreased levels of H3K27ac at most enhancers as well as super-enhancers. However, RNAPII occupancy on enhancer-associated genes did not decrease, suggesting that the modulation of enhancer strength did not affect RNAPII recruitment to TSS. In Chapter 3 we identify a nuclear pool of Pten that could associate with chromatin. Furthermore, we are the first to report that nuclear PTEN can directly interact with components of the transcription machinery including CDK7, CDK9, Cyclin T1, AFF4, and RNAPII. Loss of PTEN increased phosphorylation of Ser2 and Ser5 of the RNAPII CTD as well as RNAPII occupancy on promoters of expressed genes indicating an increase in transcriptional activity in PTEN-/- cells. Furthermore, PTEN deletion resulted in the upregulation of genes which are part of the important “Achilles cluster”, previously shown to confer sensitivity to transcription inhibition. We believe that it is over-expression of those genes that render PTEN deficient cells especially sensitive to transcription inhibitors such as THZ1, Triptolide, Flavopiridol and LDC000067. Over-expression of wild type PTEN but not a phosphatase-dead mutant of PTEN could decrease cells’ sensitivity to treatment with THZ1 or Flavopiridol. It also decreased protein levels of p-AKT Ser473 as well as RNAPII Ser2P and Ser5P suggesting that the phosphatase activity of PTEN is important for its role in transcription regulation. In sum, we propose a model in which PTEN binds to CDK7, CDK9, Cyclin T1, RNAPPII and/or AFF4 thereby exerting a negative regulatory effect on the activity of transcription complexes. Upon loss of PTEN the negative regulatory effect is eliminated and transcription of a subset of genes increases. It is most likely these genes that confer sensitivity to transcription inhibition on PTEN-/- cells. The better understanding of this oncogenic mechanism may reveal novel therapeutic opportunities, and ultimately we propose that the sensitivity of PTEN deficient cells to inhibitors of transcription could provide an effective clinical strategy to target PTEN deficient cancers.

The following dissertation herein discusses the role of axonal protein synthesis in Aβ1-42-dependent neurodegeneration, which has important implications in AD pathogenesis. In Part 1, I provide a brief introduction to relevant topics including neurodegeneration and axonal protein synthesis. In Part 2, I discuss findings that we published in 2014 describing a mechanism by which axonal exposure to Aβ1-42 induces cell death via axonal synthesis and retrograde transport of a transcription factor, ATF4. In Part 3, I discuss a follow-up project that I conducted independently, which is not yet published but is in preparation for submission describing the immediate effect of Aβ1-42 on axonal protein synthesis, which mediates the downstream axonal ATF4 signaling events described in Part 2. In Part 4, I discuss the key findings from these two projects including their significance and potential future directions. In the Appendix, I provide details regarding experimental methods and statistical analyses performed in Part 3.

Single nucleotides in the first intron of FTO convey effects on adiposity by mechanisms that remain unclear, but appear to include modulation of expression of FTO itself, as well as other genes (e.g. RPGRIP1L, IRX3) in the vicinity of FTO. This locus affects food intake, the browning of white adipose tissue and risk of type 2 diabetes (independent of its effects on body weight). FTO and RPGRIP1L expression are decreased in fibroblasts and iPSC-derived human neurons of individuals segregating for obesity risk alleles of FTO at rs8050136 and rs1421085. These alleles exhibit decreased binding of isoform p110 of the CUX1 transcription factor. This isoform activates transcription of both FTO and RPGRIP1L. The FTO locus conveys effects on adiposity via hyperphagia, in part, by regulating FTO and RPGRIP1L expression in the hypothalamus. We examined whether FTO and RPGRIP1L also modify adipogenesis and adipose tissue lipid storage. Such effects would influence systemic consequences of the hyperphagia driven by the actions of the genes in the hypothalamus. Given the role in energy homeostasis of genes encoding elements of the primary cilium, we hypothesized that mice hypomorphic for Rpgrip1l would display increased adiposity. In confirmation, we find that Rpgrip1l+/− mice are hyperphagic and obese, and display diminished suppression of food intake in response to leptin administration. These findings suggest that RPGRIP1L may be partly or exclusively responsible for the obesity susceptibility signal at the FTO intronic locus. We describe effects of Rpgrip1l in adipocytes which may contribute to the adiposity phenotype observed in these animals, and possibly humans. Loss of Rpgrip1l in 3T3-L1 preadipocytes increased the number of cells capable of differentiating into mature adipocytes. Knockout of Rpgrip1l in mature adipocytes (using Adipoq-Cre) did not increase adiposity in mice fed chow or high fat diet. Neither did we observe any effects of Rpgrip1l knockdown in mature 3T3-L1 adipocytes in vitro. Thus, to the extent that Rpgrip1l affects cell-autonomous adipose tissue function, it appears to do so by effects conveyed in preadipocytes, a cell type in which the primary cilium – as a mediator of developmental signals – may have functional importance. We propose that decreased RPGRIP1L expression in preadipocytes in humans segregating for FTO-associated obesity risk alleles increases the potential storage capacity of adipose tissue. Such capacity would influence the metabolic consequences of positive energy balance due to the action of these alleles within the brain. Fto expression is upregulated during adipogenesis in murine and human cells in vitro, and is more highly expressed in isolated mouse adipocytes than in preadipocytes. Here we demonstrate that FTO is required for the maintenance of adipocyte lipid filling and endocrine function in murine 3T3-L1 cells and human adipose tissue-derived stromal cells. RNAseq analysis indicates that this effect on adipocyte programming is conveyed in part by modulation of C/ebpβ- and C/ebpδ-regulated transcription, consistent with reports that Fto acts a transcriptional coactivator. Fto-/- mice have normal fat mass in early life, but spontaneously lose adipose tissue as they age. We propose that Fto is required to maintain adipocyte viability, a function critical to the prevention of ectopic lipid accumulation in obese states. Such accumulation – both total and in specific anatomic regions – has adverse metabolic consequences. In addition to the developmental effects on adiposity mediated by RPGRIP1L, and the effects conveyed on adipocyte function related to FTO, the FTO locus could also impact systemic energy homeostasis by modifying production of humoral signals that are integrated centrally to regulate energy balance. We explored molecular modifiers of adipocyte production of leptin identified by GWAS that may modify obesity risk. The FTO locus was associated with circulating leptin concentration, but this association was abrogated when corrected for BMI, indicating that this locus does not contribute to adiposity by dysregulating leptin production. Our in vitro findings are consistent in this regard, as knockdown of Rpgrip1l and Fto in 3T3-L1 cells did not affect leptin production per adipocyte. These results, however, are not inconsistent with a role for FTO in maintenance of adipocyte viability.

This dissertation reviews the development and implementation of two systems biology meth- ods: ADVOCATE and hpARACNE. ADVOCATE was designed to deconvolve epithelium and stroma compartments fractions and virtual expression profiles from bulk gene expression profiles from human patients. We used laser capture microdissection and RNA sequencing to disentangle the transcriptional programs active in the malignant epithelium and stroma of pancreatic ductal adenocarcinoma (PDA), an aggressive malignancy with a prominent stromal component. We learned that distinct molecular subtypes are present in both the epithelium and the stroma of pancreatic cancer, and that the subtype identity of these two compartments are independent of one another. Critically, we discovered that specific com- binations of epithelial and stromal subtypes are strongly associated with patient survival across multiple external datasets, exhibiting both an effect-size and a level of reproducibility that was absent from previous efforts. These analyses were made possible by a new proba- bilistic algorithm (Adaptive DeconVolution Of CAncer Tissue Expression - ADVOCATE) that can extract compartment-specific gene expression profiles from bulk gene expression data. ADVOCATE accurately predicted the compartment fractions of bulk tumor samples and improved the performance of molecular classifiers by controlling for the diverse cellular compositions of independent datasets. This approach provides a much-needed framework to handle solid tumor tissue heterogeneity, allowing integrated analysis of both epithelial and stromal transcriptional programs from individual bulk samples. Reverse engineering approaches have been used to systematically dissect regulatory in- teractions based on gene expression profiles in different context and data types, thus im- proving our mechanistic understanding of molecular programs under perturbations. Pro- teomics data, on the other hand, provides direct evidence of cell functions. Particularly, signaling molecules are best candidates for drug targets. Previous efforts have shown that targeting signaling proteins could potentially lead to cancer remission. In this work, I introduce hybrid proteomics Algorithm for the Reconstruction of Accurate Cellular Network (hpARACNE), a re-design of gene expression based ARACNE algorithm. Us- ing Clinical Proteomics Tumor Analysis Consortium (CPTAC) breast cancer proteomics data, hpARACNE reconstructs a network that significantly outperforms ARACNE when compared with curated Kinase/Phosphatase-substrates interactions from public databases. Compared with Stable Isotope Labeling with Amino acid in Cell Culture (SILAC) ex- perimentally identified substrates for EGFR, hpARACNE predicts substrates with high accuracy. Integrative network analysis of breast cancer transcriptome and phosphopro- teome reveals potential drug targets for Triple Negative Breast Cancer (TNBC) treat- ment. hpARACNE has three innovations that adapt it to proteomics data and signaling process: 1) Refinement of the kinase/phosphatase peptides by integrating matched whole proteomic and whole phosphoproteomic profiles; 2) Establishment of association based on newly designed Mutual Information (MI) estimator for missing data; 3) Network pruning using directional Data Processing Inequality (dDPI) for signalling process.

Cordycepin (3′-deoxyadenosine) is a medicinal bioactive component of the caterpillar fungi (Cordyceps and Ophicordyceps). It is reported to have nephroprotective, antiapoptotic, anti-metastatic, hepatoprotective (Yue et al. 2013), inflammatory effects, antioxidant, anti-tumor, immunomodulatory and vasorelaxation activities. Cordycepin is well known to terminate and inhibit polyadenylation, both in vitro and in vivo. Other proposed mechanisms of action of cordycepin include activation of adenosine receptors, activation of AMP dependent kinase (AMPK) and inhibition of PARP1. The purpose of this study is to elucidate the biological and pharmacological effects of cordycepin on cancer cell lines such as MCF-7 cells. In this study I found that cordycepin reduces the cell proliferation in all examined cell lines without always exerting an effect on 4EBP phosphorylation and protein synthesis rates. Therefore, the effects on protein synthesis via inhibition of mTOR, which were previously reported, are not only the sole reason for the effect of cordycepin on cell proliferation. Knockdown of poly (A) polymerases reduces cell proliferation and survival, indicating that poly (A) polymerases are potential targets of cordycepin. I studied different adenosine analogues and found that 8 aminoadenosine, the only one that also consistently inhibits polyadenylation, also reduces levels of P-4EBP. It also inhibits the expression of specific genes indicating that the effects on polyadenylation, mTOR signalling and gene expression are linked. Also consistent with polyadenylation inhibition as the major mode of action is the fact that the effects of cordycepin on gene expression are predominantly post-transcriptional. However, knockdown of poly (A) polymerases did not have the same effects on gene expression or on polyadenylation, indicating that cordycepin may act as a dominant negative rather than as a null mutant. This is consistent with the fact that cordycepin is known to arrest a normally transient polyadenylation complex. We performed microarray analysis of cordycepin treated MCF-7 cells and found that the downregulated mRNAs were predominantly involved in transcriptional regulation, cell proliferation, cell cycle and cell migration. These data show that cordycepin is a promising new drug for cancer and indicates that the mode of action it is likely to be through the inhibition of polyadenylation.

Liu, Yang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 99-104). / Abstracts in English and Chinese. / Table of Contents / Thesis/Assessment Committee --- p.ii / Statement --- p.iii / Abstract --- p.iv / 摘要 --- p.vi / Acknowledgements --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xi / List of Figures --- p.xii / List of Abbreviations --- p.xiv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1. --- The plant secretory pathway --- p.2 / Chapter 2. --- Vesicular pathways in plant cells --- p.3 / Chapter 3. --- Prevacuolar Compartments --- p.6 / Chapter 4. --- Vacuolar sorting receptors (VSRs) --- p.7 / Chapter 5. --- BP-80 & Arabidopsis VSR Proteins --- p.8 / Chapter 6. --- Research Objectives --- p.9 / Chapter Chapter 2 --- Development and Expression of GFP-OsVSRs Fusion Reporters in Tobacco BY-2 and Rice Suspension Cultured Cells --- p.11 / Chapter 1. --- Introduction --- p.12 / Chapter 2. --- Materials and methods --- p.14 / Chapter 2.1 --- Construction of GFP-OsVSR chimeric reporters --- p.14 / Chapter 2.2 --- Construction of Golgi Marker and PVC marker --- p.18 / Chapter 2.3 --- Agrobacterium electroporation --- p.27 / Chapter 2.4 --- Transformation of tobacco BY-2 cells --- p.27 / Chapter 2.5 --- Transient expression of GFP-OsVSRs in protoplasts of tobacco BY-2 cells and rice suspension cultured cells --- p.28 / Chapter 2.6 --- Screening of transgenic BY-2 cells expressing GFP-OsVSR reporters --- p.30 / Chapter 2.7 --- Chemicals --- p.33 / Chapter 3. --- Results --- p.34 / Chapter 3.1 --- Study subcellular localization of OsVSR proteins with chimeric GFP-OsVSR reporters --- p.34 / Chapter 3.2 --- Generation of transgenic tobacco BY-2 cell lines expressing GFP-OsVSR reporter constructs --- p.38 / Chapter 3.3 --- Transient expression of GFP-OsVSR reporters in tobacco BY-2 and rice cell protoplasts --- p.40 / Chapter 4. --- Conclusion --- p.42 / Chapter Chapter 3 --- Subcellular Localization of GFP-OsVSR Fusion Reporters in Tobacco BY-2 and Rice Suspension Cultured Cells --- p.43 / Chapter 1. --- Introduction --- p.44 / Chapter 2. --- Materials and methods --- p.45 / Chapter 2.1 --- Confocal immunofluorescence studies --- p.45 / Chapter 2.2 --- Antibodies --- p.46 / Chapter 2.3 --- Wortmannin and BFA drug treatment --- p.46 / Chapter 2.4 --- Electron microscopy of resin-embedded cells --- p.47 / Chapter 2.5 --- Two-dimensional (2-D) gel analysis --- p.47 / Chapter 3 --- Results --- p.49 / Chapter 3.1 --- "Distinct subcellular localizations of GFP-OsVSRl, GFP-OsVSR2 and GFP-OsVSR4 reporters in transgenic BY-2 cell lines" --- p.49 / Chapter 3.2 --- "Subcellular localizations of GFP-OsVSRl, GFP-OsVSR2 and GFP-OsVSR4 in protoplasts of rice suspension cultured cells" --- p.58 / Chapter 3.3 --- Distinct localizations of GFP-OsVSR3 and GFP-OsVSR5 --- p.62 / Chapter 3.4 --- Immunogold EM localization of VSR proteins in rice suspension cultured cells --- p.65 / Chapter 3.5 --- 2-D western blot detection of VSR proteins in various plants --- p.68 / Chapter 4. --- Conclusions --- p.70 / Chapter Chapter 4 --- Summary and Discussion --- p.71 / Chapter 1. --- The significance of this study --- p.72 / Chapter 2. --- The hypothesis in this study --- p.73 / Chapter 3. --- A reporter system to study subcellular localization of OsVSR proteins in both tobacco BY-2 cells and rice suspension cultured cells --- p.75 / Chapter 4. --- Transiently expression of GFP-OsVSR reporters in BY-2 and rice protoplasts ..… --- p.76 / Chapter 5. --- Distinct PVC and Golgi localizations of GFP-OsVSR fusions --- p.77 / Chapter 6. --- Summary and future perspective --- p.78 / Appendix --- p.79 / Characterization of A Novel Rice Protein --- p.79 / Chapter 1. --- Introduction --- p.80 / Chapter 2. --- Materials and methods --- p.83 / Chapter 2.1 --- Antibodies --- p.83 / Chapter 2.2 --- Sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) and western blot analysis of proteins from different plant species --- p.84 / Chapter 2.3 --- Sucrose gradient fractionation with protein F antibody --- p.84 / Chapter 2.4 --- Confocal immunofluorescence studies --- p.85 / Chapter 2.5 --- Affinity purification of Protein F by its antibody --- p.85 / Chapter 3. --- Results --- p.87 / Chapter 3.1 --- Protein F is presented in different plant species --- p.87 / Chapter 3.2 --- Protein F is an integral membrane protein --- p.89 / Chapter 3.3 --- Subcelluar localization of Protein F --- p.91 / Chapter 3.4 --- Affinity purification of Protein F for identification --- p.95 / Chapter 4. --- Summary and future perspectives --- p.97 / Chapter 4.1 --- Summary --- p.97 / Chapter 4.2 --- Future Perspectives --- p.97 / References --- p.99

During neural development, axons rely on extracellular cues to reach their target regions. Although extracellular signaling is one of the principal determinants for the growth of developing axons, only a small handful of known signaling cues has been identified. The existence of some 86 billion neurons of different subtypes, which ultimately form numerous functional circuits in the human nervous system, means an enormous number of extracellular cues would be required during development. Current views hold that even if more extracellular cues were to be discovered, they would never number large enough to account for the complexity of the human nervous system. Rather, intracellular signaling pathways and other cell-intrinsic mechanisms expand the ways in which a neuron can respond to extracellular cues by tuning the degree of responsiveness to them. Cell-intrinsic signaling pathways also give axons the ability to actively control their own development. These pathways can operate independently of the extracellular environment or even independently of the cell body, where the majority of protein synthesis takes place. For example, the local translation of proteins in the axon gives it autonomous control to immediately respond to changing demands in the environment. Local translation also occurs in other cell types, but the compartmentalized control over growth is especially important for neurons since the axon can extend up to a meter away from the cell body. In addition to local translation, axonally derived transcription factors, which can be locally synthesized in or localized to the axon, provide another means to control axon development. Axonally derived transcription factors act as physiological sensors and relay information about events happening in the periphery back to the cell body in order to effectuate a global response. It has recently been shown that transcription factors belonging to the OASIS family are activated by proteolysis in axons. Following their activation by proteolytic cleavage, the transcriptionally active N-terminus of these factors is transported to the cell body to activate global transcriptional pathways. For at least one OASIS family member, CREB3L2, this cleavage event simultaneously produces the C-terminus, which is capable of undergoing secretion. The secreted C-terminus of CREB3L2 acts as an accessory ligand for the activation of Hh pathways in chondrocytes. The generation of two bioactive proteins from one transcription factor, a transcriptionally active portion and a secreted portion, raised the question of whether there was a local function for OASIS transcription factors in axons. Through my research, I identified a mechanism in which DRG axons secrete the C-terminus of CREB3L2, which promotes axon growth in a paracrine manner. CREB3L2 is a transcription factor whose translation is induced by physiological ER stress. For CREB3L2 to be active, it must be cleaved by S2P, which I found is expressed in developing axons. Following proteolysis of CREB3L2 by S2P, the secreted C-terminus of CREB3L2 promotes the formation of Shh and Ptch1 complexes along axons. I found that upon depletion of the secreted CREB3L2 C-terminus, binding of Shh to the Ptch1 receptor is diminished. Returning the CREB3L2 C-terminus to the cultures exogenously was sufficient to rescue the formation of these complexes. These results highlight an intrinsic role for Shh signaling in developing DRG axons. Moreover, these results demonstrate how ER stress machinery is recruited to axons and promotes axon outgrowth. Finally, these results illustrate a novel, neuron-intrinsic mechanism by which developing axons actively regulate their own growth.