Developmental Signaling Pathways Research Articles

BRAF/MAPK and GSK3 signaling converges to control MITF nuclear export

The close integration of the MAPK, PI3K, and WNT signaling pathways underpins much of development and is deregulated in cancer. In principle, combinatorial posttranslational modification of key lineage-specific transcription factors would be an effective means to integrate critical signaling events. Understanding how this might be achieved is central to deciphering the impact of microenvironmental cues in development and disease. The microphthalmia-associated transcription factor MITF plays a crucial role in the development of melanocytes, the retinal pigment epithelium, osteoclasts, and mast cells and acts as a lineage survival oncogene in melanoma. MITF coordinates survival, differentiation, cell-cycle progression, cell migration, metabolism, and lysosome biogenesis. However, how the activity of this key transcription factor is controlled remains poorly understood. Here, we show that GSK3, downstream from both the PI3K and Wnt pathways, and BRAF/MAPK signaling converges to control MITF nuclear export. Phosphorylation of the melanocyte MITF-M isoform in response to BRAF/MAPK signaling primes for phosphorylation by GSK3, a kinase inhibited by both PI3K and Wnt signaling. Dual phosphorylation, but not monophosphorylation, then promotes MITF nuclear export by activating a previously unrecognized hydrophobic export signal. Nonmelanocyte MITF isoforms exhibit poor regulation by MAPK signaling, but instead their export is controlled by mTOR. We uncover here an unanticipated mode of MITF regulation that integrates the output of key developmental and cancer-associated signaling pathways to gate MITF flux through the import-export cycle. The results have significant implications for our understanding of melanoma progression and stem cell renewal.

Solea senegalensis skeletal ossification and gene expression patterns during metamorphosis: New clues on the onset of skeletal deformities during larval to juvenile transition

Farmed Senegalese sole (Solea senegalensis) still show a high incidence of vertebral anomalies that limit its intensive production and hamper its economic profitability. A great effort towards understanding how fish species develops and grows in captivity has been undertaken in the last decade, with particular emphasis to how different biotic and abiotic factors affect its skeletal development. Although some work has been performed on its skeletal development and expression patterns of key genes in developmental signaling pathways, a detailed description of the above-mentioned processes is still lacking. Here, the progression of skeletal development of cranial, appendicular and axial skeleton has been described through the implementation of an acid free double staining protocol; while the gene expression pattern of vitamin A (VA) and thyroid hormones (THs) signaling pathways were assessed through quantitative PCR (qPCR) during larval fish development under a standard larval rearing protocol and in Senegalese sole larvae fed with increased dietary VA levels (8-fold increase) during the Artemia feeding phase (from 6 to 27 dph). The ontogenetic study allowed us to identify the onset and follow-up the development of most skeletal structures in this fish species, and to gather some insights on the potential onset of deformities originated at the larval to juvenile transition affecting Senegalese sole rearing. The pro-metamorphosis stage is when the caudal fin vertebrae are formed, where the most common skeletal deformities are found in Senegalese sole. A highly coordinated expression of VA- and TH-related genes has been revealed during larval to juvenile transition, while a comparative gene expression analysis in larvae fed control and high dietary VA content identified the specific timing of VA and THs signaling disruption through which VA and THs directly or indirectly might increase the incidence of skeletal deformities in this species. The present research work represents an important step forward towards the proper identification of the onset of some skeletal deformities and urge the investigation of nutritional and rearing conditions during the switch of larval behavior - from pelagic to benthonic - in order to overcome them in this important species for southwestern European aquaculture.

A Multidisciplinary Approach Reveals an Age-Dependent Expression of a Novel Bioactive Peptide, Already Involved in Neurodegeneration, in the Postnatal Rat Forebrain.

The basal forebrain has received much attention due to its involvement in multiple cognitive functions, but little is known about the basic neuronal mechanisms underlying its development, nor those mediating its primary role in Alzheimer’s disease. We have previously suggested that a novel 14-mer peptide, ‘T14’, could play a pivotal role in Alzheimer’s disease, via reactivation of a developmental signaling pathway. In this study, we have characterized T14 in the context of post-natal rat brain development, using a combination of different techniques. Ex-vivo rat brain slices containing the basal forebrain, at different stages of development, were used to investigate large-scale neuronal network activity in real time with voltage-sensitive dye imaging. Subsequent Western blot analysis revealed the expression profile of endogenous T14, its target alpha7 nicotinic receptor and the familiar markers of Alzheimer’s: amyloid beta and phosphorylated Tau. Results indicated maximal neuronal activity at the earliest ages during development, reflected in a concomitant profile of T14 peptide levels and related proteins. In conclusion, these findings show that the peptide, already implicated in neurodegenerative events, has an age-dependent expression, suggesting a possible contribution to the physiological mechanisms underlying brain maturation.

MagT1 is essential for Drosophila development through the shaping of Wingless and Decapentaplegic signaling pathways

Magnesium transporter subtype 1 (MagT1) is a magnesium membrane transporter with channel like properties. We have previously identified MagT1 (CG7830) in Drosophila genome and characterized its protein product by electrophysiological means. Here, we report the generation of fly MagT1 mutants and show that MagT1 is essential for early embryonic development. In wings and primordial wings, by clonal analysis and RNAi knock down of MagT1, we have found that loss of MagT1 results in enhanced/ectopic Wingless (Wg, a fly Wnt) signaling and disrupted Decapentaplegic (Dpp) signaling, indicating the crucial role of MagT1 for fly development at later stages. Finally, we demonstrate directly that magnesium transportations are proportional with the MagT1 expressional levels in Drosophila S2 cells. Taken together, these findings may suggest that MagT1 is a major magnesium transporter/channel profoundly involved in fly development by affecting developmental signaling pathways, such as Wg and Dpp signaling.

Abstract 2064: Differences in methylation patterns by pediatric germ cell tumor histologic subtype

Abstract Anomalous DNA methylation contributes to carcinogenesis by regulating gene expression and may be important in germ cell tumor (GCT) development as germ cells undergo tremendous epigenetic reprogramming during embryogenesis. Histologic subtypes of GCTs show differences in global methylation patterns with less differentiated tumors (e.g. embryonal carcinoma) experiencing lower levels of global methylation relative to more differentiated tumors (e.g. teratoma). Differences in promoter methylation have also been reported in GCTs. In particular, developmental signaling pathways and the BMP/TGF beta pathway have been consistently found to be differentially methylated. In this analysis, we conducted an epigenome wide association study to identify gene-specific methylation differences by GCT histology. Using the Illumina HumanMethylation450K array we determined methylation profiles for 154 pediatric GCTs (21 germinomas, 54 mixed, 9 teratomas, 70 yolk sac tumors [YST]) from fresh frozen and formalin fixed paraffin embedded tumor tissue samples. Fisher's exact tests were used to assess differences in the distributions of age group, sex, and tumor location by histologic subtype (alpha=0.05 for a two-sided test of significance [SAS v9.4]). Bioconductor package minfi was used to identify differential methylation between GCT histologic subtypes by comparing the methylation beta values. To identify differential methylation of genes predictive of tumor histology, a generalized linear model (glm) with LASSO was fit (R v3.4.1) to the quantile normalized beta values for all probes from all samples. There were significant differences in age, sex, and tumor location by histologic subtype (all p<0.01). We observed unique clusters for both germinoma and YST, but a single cluster for mixed tumors and teratoma. The methylation clusters did not differ by age, sex or FFPE status after accounting for histologic subtype. When stratifying by age group (0-10 vs. 11-19 years), we observed 2,043 differentially methylated regions (DMRs) (FWER <0.05); however, these differences did not persist once we adjusted for tumor histology. Differential methylation tests across histologic subtypes resulted in 8,049 DMRs (FWER < 0.05). Germinoma clustered most consistently and displayed a unique methylation pattern relative to other tumor types, which were more similar to one another. Fitting a glm with LASSO to the methylation data identified 21 probes that were predictive of histologic subtype with misclassification error < 20%: ANKRD24, ASL, ATG13, CCDC53, CKB, FAM224B, FZD10, KCNMB3, LINC00939, LOC101927248, MIR378C, MMP14, MUC1, OGFOD3, PLP2, PLPP3, PRR5, SERPINA6¸ SGSM2, VARS, and YWHAZ. In our analyses, we identified a large number of genes with differential methylation by tumor histology. These data may be informative in understanding GCT etiology and the timing of GCT initiation. Citation Format: Lindsay A. Williams, Lauren Mills, Anthony J. Hooten, Erica Langer, Michelle Roesler, A Lindsay Frazier, James Amatruda, Jenny N. Poynter, Jenny N. Poynter. Differences in methylation patterns by pediatric germ cell tumor histologic subtype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2064.

Combined Proteomic and In Silico Target Identification Reveal a Role for 5-Lipoxygenase in Developmental Signaling Pathways

Identification and validation of the targets of bioactive small molecules identified in cell-based screening is challenging and often meets with failure, calling for the development of new methodology. We demonstrate that a combination of chemical proteomics with in silico target prediction employing the SPiDER method may provide efficient guidance for target candidate selection and prioritization for experimental in-depth evaluation. We identify 5-lipoxygenase (5-LO) as the target of the Wnt pathway inhibitor Lipoxygenin. Lipoxygenin is a non-redox 5-LO inhibitor, modulates the β-catenin-5-LO complex and induces reduction of both β-catenin and 5-LO levels in the nucleus. Lipoxygenin and the structurally unrelated 5-LO inhibitor CJ-13,610 promote cardiac differentiation of human induced pluripotent stem cells and inhibit Hedgehog, TGF-β, BMP, and Activin A signaling, suggesting an unexpected and yet unknown role of 5-LO in these developmental pathways.

BRCA1 and BRCA2 gene variants and nonsyndromic cleft lip/palate.

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a debilitating condition that not only affects the individual, but the entire family. The purpose of this study was to investigate the association of BRCA1 and BRCA2 genes with NSCL/P. Twelve polymorphisms in/nearby BRCA1 and BRCA2 were genotyped using Taqman chemistry. Our data set consisted of 3,473 individuals including 2,191 nonHispanic white (NHW) individuals (from 151 multiplex and 348 simplex families) and 1,282 Hispanic individuals (from 92 multiplex and 216 simplex families). Data analysis was performed using Family-Based Association Test (FBAT), stratified by ethnicity and family history of NSCL/P. Nominal associations were found between NSCL/P and BRCA1 in Hispanics and BRCA2 in NHW and Hispanics (p < .05). Significant haplotype associations were found between NSCL/P and both BRCA1 and BRCA2 (p ≤ .004). Our results suggest a modest association between BRCA1 and BRCA2 and NSCL/P. Further studies in additional populations and functional studies are needed to elucidate the role of these genes in developmental processes and signaling pathways contributing to NSCL/P.

The 6xABRE Synthetic Promoter Enables the Spatiotemporal Analysis of ABA-Mediated Transcriptional Regulation.

The water stress-associated hormone abscisic acid (ABA) acts through a well-defined signal transduction cascade to mediate downstream transcriptional events important for acclimation to stress. Although ABA signaling is known to function in specific tissues to regulate root growth, little is understood regarding the spatial pattern of ABA-mediated transcriptional regulation. Here, we describe the construction and evaluation of an ABSCISIC ACID RESPONSIVE ELEMENT (ABRE)-based synthetic promoter reporter that reveals the transcriptional response of tissues to different levels of exogenous ABA and stresses. Genome-scale yeast one-hybrid screens complemented these approaches and revealed how promoter sequence and architecture affect the recruitment of diverse transcription factors (TFs) to the ABRE. Our analysis also revealed ABA-independent activity of the ABRE-reporter under nonstress conditions, with expression being enriched at the quiescent center and stem cell niche. We show that the WUSCHEL RELATED HOMEOBOX5 and NAC DOMAIN PROTEIN13 TFs regulate QC/SCN expression of the ABRE reporter, which highlights the convergence of developmental and DNA-damage signaling pathways onto this cis-element in the absence of water stress. This work establishes a tool to study the spatial pattern of ABA-mediated transcriptional regulation and a repertoire of TF-ABRE interactions that contribute to the developmental and environmental control of gene expression in roots.

PO-186 Generation of the GLI1, GLI2 and GLI3 knock-out ovarian cancer cell lines using the CRISPR/Cas9

Introduction Hedgehog signalling pathway is a developmental signalling pathway which is dormant in most adult differentiated tissues, but aberrantly activated in various tumours. In ovarian tumours it can be activated in a canonical way, by the SHH ligand, or the non-canonical way, by upregulation of the GLI transcription factors. Expression of GLI1 is usually associated with tumour progression in a clinical setting, but GLI2 and GLI3 also play a role by modifying the activity of GLI1 and transcription of their common transcriptional targets. In ovarian cancer, GLI3 protein is expressed in the full-length activator form, and not the shortened repressor form which is the predominant form for GLI3 protein. Material and methods To study the roles of the three GLI proteins in detail, we used the CRISPR/Cas9 guided gene editing to generate knock-out lines for each of the three GLI proteins. The sgRNA were designed using the online tool at crispr.mit.edu web site, and cloned into the pX330 vector expressing the sgRNA and the Cas9 protein. The sgRNA were designed to target the region close to the ATG site of each of the three GLI proteins, and after Cas9 nuclease activity the double stranded DNA break should be repaired by non-homologous end joining, which generates indels and leads to frameshift. Frameshift close to the ATG often leads to early termination of the protein, generating a knock-out. The SKOV3 ovarian cancer cell line was transfected with each of the vectors, the cells were split to the density of a single cell/well in a 96-well plate, and cell lines were expanded from single cells to obtain homogenous lines. Results and discussions SKOV3 ovarian cancer cell lines with knock outs in each of the three GLI proteins, GLI1, GLI2 and GLI3 were generated. The knock-out was confirmed by qRT-PCR and Western blot, and DNA was sequenced to show the exact genetic modification of the lines leading to the knock-out. The modified cell lines showed differences in cell morphology in relation to the original SKOV3 cell line immediately after expansion from the single cell. Conclusion The Hedgehog signalling pathway plays an important role in ovarian cancer. Cells harbouring knock out for the effector proteins of the Hedgehog signalling pathway, GLI1, GLI2 and GLI3 show distinct changes in cell morphology and viability. The Hedgehog signalling pathway, and more specifically GLI proteins, may be a good potential therapeutic target in management of ovarian cancer.

Ectopic expression of Hoxb1 induces cardiac and craniofacial malformations.

Members of the large family of Hox transcription factors are encoded by genes whose tightly regulated expression in development and in space within different embryonic tissues confer positional identity from the neck to the tips of the limbs. Many structures of the face, head, and heart develop from cell populations expressing few or no Hox genes. Hoxb1 is the member of its chromosomal cluster expressed in the most rostral domain during vertebrate development, but never by the multipotent neural crest cell population anterior to the cerebellum. We have developed a novel floxed transgenic mouse line, CAG-Hoxb1,-EGFP (CAG-Hoxb1), which upon recombination by Cre recombinase conditionally induces robust Hoxb1 and eGFP overexpression. When induced within the neural crest lineage, pups die at birth. A variable phenotype develops from E11.5 on, associating frontonasal hypoplasia/aplasia, micrognathia/agnathia, major ocular and forebrain anomalies, and cardiovascular malformations. Neural crest derivatives in the body appear unaffected. Transcription of effectors of developmental signaling pathways (Bmp, Shh, Vegfa) and transcription factors (Pax3, Sox9) is altered in mutants. These outcomes emphasize that repression of Hoxb1, along with other paralog group 1 and 2 Hox genes, is strictly necessary in anterior cephalic NC for craniofacial, visual, auditory, and cardiovascular development.

From intra- to extracellular vesicles: extracellular vesicles in developmental signalling

Signalling from cell-to-cell is fundamental for determining differentiation and patterning. This communication can occur between adjacent and distant cells. Extracellular vesicles (EVs) are membrane-based structures thought to facilitate the long-distance movement of signalling molecules. EVs have recently been found to allow the transport of two major developmental signalling pathways: Hedgehog and Wnt. These signalling molecules undergo crucial post-translational lipid modifications, which anchor them to membranes and impede their free release into the extracellular space. Preparation of these ligands in EVs involves intracellular vesicle sorting in an endocytosis-dependent recycling process before secretion. In the present review, we discuss the most recent advances with regard to EV involvement in developmental signalling at a distance. We focus on the role of the protein complexes involved in EV genesis, and provide a comprehensive perspective of the contribution of these complexes to intracellular vesicle sorting of developmental signals for their extracellular secretion, reception and transduction.

Abstract IA13: Imaging stem cell signals in cancer heterogeneity and therapy resistance

Abstract Our research focuses on the signals that control stem cell self-renewal and how these signals are hijacked in cancer. Using a series of genetic models, we have studied how classic developmental signaling pathways such as Wnt, Hedgehog, and Notch play key roles in hematopoietic stem cell growth and regeneration and are dysregulated during leukemia development. In addition, using real-time imaging strategies we have found that hematopoietic stem cells have the capacity to undergo both symmetric and asymmetric division, and that shifts in the balance between these modes of division are subverted by oncogenes. Further, regulators of this process, including the cell fate determinant Musashi, are critical players in driving progression of solid and liquid cancers and could serve as targets for diagnostics and therapy. Ongoing work is focused on understanding the mechanisms that drive therapy resistance after drug delivery, as well as developing high-resolution in vivo imaging approaches to map normal stem cell behavior and interactions within living animals, and to define how these change during cancer formation. Citation Format: Tannishtha Reya. Imaging stem cell signals in cancer heterogeneity and therapy resistance [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr IA13.

Regulation of seizure-induced MeCP2 Ser421 phosphorylation in the developing brain

Neonatal seizures disrupt normal synaptic maturation and often lead to later-life epilepsy and cognitive deficits. During early life, the brain exhibits heightened synaptic plasticity, in part due to a developmental overabundance of CaV1.2 L-type voltage gated calcium (Ca2+) channels (LT-VGCCs) and Ca2+-permeable AMPARs (CP-AMPARs) lacking GluA2 subunits. We hypothesized that early-life seizures overactivate these channels, in turn dysregulating Ca2+-dependent signaling pathways including that of methyl CPG binding protein 2 (MeCP2), a transcription factor implicated in the autism spectrum disorder (ASD) Rett Syndrome. Here, we show that in vivo hypoxia-induced seizures (HS) in postnatal day (P)10 rats acutely induced phosphorylation of the neuronal-specific target of activity-dependent MeCP2 phosphorylation, S421, as well as its upstream activator CaMKII T286. We next identified mechanisms by which activity-dependent Ca2+ influx induced MeCP2 phosphorylation using in vitro cortical and hippocampal neuronal cultures at embryonic day (E)18 + 10 days in vitro (DIV). In contrast to the prevalent role of NMDARs in the adult brain, we found that both CP-AMPARs and LT-VGCCs mediated MeCP2 S421 and CaMKII T286 phosphorylation induced by kainic acid (KA) or high potassium chloride (KCl) stimulation. Furthermore, in vivo post-seizure treatment with the broad-spectrum AMPAR antagonist NBQX, the CP-AMPAR blocker IEM-1460, or the LT-VGCC antagonist nimodipine blocked seizure-induced MeCP2 phosphorylation. Collectively, these results demonstrate that early-life seizures dysregulate critical activity-dependent developmental signaling pathways, in part via CP-AMPAR and LT-VGCC activation, providing novel age-specific therapeutic targets for convergent pathways underlying epilepsy and ASDs.

Stimulation-dependent remodeling of the corticospinal tract requires reactivation of growth-promoting developmental signaling pathways

The corticospinal tract (CST) can become damaged after spinal cord injury or stroke, resulting in weakness or paralysis. Repair of the damaged CST is limited because mature CST axons fail to regenerate, which is partly because the intrinsic axon growth capacity is downregulated in maturity. Whereas CST axons sprout after injury, this is insufficient to recover lost functions. Chronic motor cortex (MCX) electrical stimulation is a neuromodulatory strategy to promote CST axon sprouting, leading to functional recovery after CST lesion. Here we examine the molecular mechanisms of stimulation-dependent CST axonal sprouting and synapse formation. MCX stimulation rapidly upregulates mTOR and Jak/Stat signaling in the corticospinal system. Chronic stimulation, which leads to CST sprouting and increased CST presynaptic sites, further enhances mTOR and Jak/Stat activity. Importantly, chronic stimulation shifts the equilibrium of the mTOR repressor PTEN to the inactive phosphorylated form suggesting a molecular transition to an axon growth state. We blocked each signaling pathway selectively to determine potential differential contributions to axonal outgrowth and synapse formation. mTOR blockade prevented stimulation-dependent axon sprouting. Surprisingly, Jak/Stat blockade did not abrogate sprouting, but instead prevented the increase in CST presynaptic sites produced by chronic MCX stimulation. Chronic stimulation increased the number of spinal neurons expressing the neural activity marker cFos. Jak/Stat blockade prevented the increase in cFos-expressing neurons after chronic stimulation, confirming an important role for Jak/Stat signaling in activity-dependent CST synapse formation. MCX stimulation is a neuromodulatory repair strategy that reactivates distinct developmentally-regulated signaling pathways for axonal outgrowth and synapse formation.

Efficient Derivation of Basal Progenitor Cells from Human Pluripotent Stem Cells for Modeling Esophageal Development

Many esophageal diseases including malignancy are associated with stem/progenitor cell abnormalities involving reactivation of developmental signaling pathways. However, anatomical and structural differences between species render mouse a suboptimal organism for studying esophageal development and disease mechanism. To address this issue we established an efficient protocol to generate esophageal epithelial progenitors (EPCs) from human pluripotent stem cells (hPSCs) including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Inhibition of TGFs and BMP signaling is required for the sequential differentiation of hPSCs into EPCs which can be further purified with the cell surface markers EPCAM and Integrin s4. The hPSC-derived EPCs recapitulate the normal development of the stratified squamous epithelium in the human esophagus. More importantly, a combination of hPSC differentiation and mouse genetics allows us to elucidate the critical role for NOTCH signaling in the formation of the stratified squamous epithelium. Our studies therefore not only provide an efficient approach to generate human EPCs, but also offer a new model to study the regulatory mechanisms underlying the development of the human esophagus.

Differentiation of hepatocyte-like cells from human pluripotent stem cells using small molecules

A variety of approaches have been developed for the derivation of hepatocyte-like cells from pluripotent stem cells. Currently, most of these strategies employ step-wise differentiation approaches with recombinant growth-factors or small-molecule analogs to recapitulate developmental signaling pathways. Here, we tested the efficacy of a small-molecule based differentiation protocol for the generation of hepatocyte-like cells from human pluripotent stem cells. Quantitative gene-expression, immunohistochemical, and western blot analyses for SOX17, FOXA2, CXCR4, HNF4A, AFP, indicated the stage-specific differentiation into definitive endoderm, hepatoblast and hepatocyte-like derivatives. Furthermore, hepatocyte-like cells displayed morphological and functional features characteristic of primary hepatocytes, as indicated by the production of ALB (albumin) and α-1-antitrypsin (A1AT), as well as glycogen storage capacity by periodic acid-Schiff staining. Together, these data support that the small-molecule based hepatic differentiation protocol is a simple, reproducible, and inexpensive method to efficiently drive the differentiation of human pluripotent stem cells towards a hepatocyte-like phenotype, for downstream pharmacogenomic and regenerative medicine applications.

In silico identification of microRNAs predicted to regulate N-myristoyltransferase and Methionine Aminopeptidase 2 functions in cancer and infectious diseases.

Protein myristoylation is a key protein modification carried out by N-Myristoyltransferase (NMT) after Methionine aminopeptidase 2 (MetAP2) removes methionine from the amino-terminus of the target protein. Protein myristoylation by NMT augments several signaling pathways involved in a myriad of cellular processes, including developmental pathways and pathways that when dysregulated lead to cancer or immune dysfunction. The emerging evidence pointing to NMT-mediated myristoylation as a major cellular regulator underscores the importance of understanding the framework of this type of signaling event. Various studies have investigated the role that myristoylation plays in signaling dysfunction by examining differential gene or protein expression between normal and diseased states, such as cancers or following HIV-1 infection, however no study exists that addresses the role of microRNAs (miRNAs) in the regulation of myristoylation. By performing a large scale bioinformatics and functional analysis of the miRNAs that target key genes involved in myristoylation (NMT1, NMT2, MetAP2), we have narrowed down a list of promising candidates for further analysis. Our condensed panel of miRNAs identifies 35 miRNAs linked to cancer, 21 miRNAs linked to developmental and immune signaling pathways, and 14 miRNAs linked to infectious disease (primarily HIV). The miRNAs panel that was analyzed revealed several NMT-targeting mRNAs (messenger RNA) that are implicated in diseases associated with NMT signaling alteration, providing a link between the realms of miRNA and myristoylation signaling. These findings verify miRNA as an additional facet of myristoylation signaling that must be considered to gain a full perspective. This study provides the groundwork for future studies concerning NMT-transcript-binding miRNAs, and will potentially lead to the development of new diagnostic/prognostic biomarkers and therapeutic targets for several important diseases.

Defining Adult Stem Cells by Function, not by Phenotype.

Central to the classical hematopoietic stem cell (HSC) paradigm is the concept that the maintenance of blood cell numbers is exclusively executed by a discrete physical entity: the transplantable HSC. The HSC paradigm has served as a stereotypic template in stem cell biology, yet the search for rare, hardwired professional stem cells has remained futile in most other tissues. In a more open approach, the focus on the search for stem cells as a physical entity may need to be replaced by the search for stem cell function, operationally defined as the ability of an organ to replace lost cells. The nature of such a cell may be different under steady state conditions and during tissue repair. We discuss emerging examples including the renewal strategies of the skin, gut epithelium, liver, lung, and mammary gland in comparison with those of the hematopoietic system. While certain key housekeeping and developmental signaling pathways are shared between different stem cell systems, there may be no general, deeper principles underlying the renewal mechanisms of the various individual tissues.

Jasmonate Negatively Regulates Stomatal Development in Arabidopsis Cotyledons

Stomata are ports that facilitate gas and water vapor exchange between plants and their environment. Stomatal development is strictly regulated by endogenous signals and environmental cues. Jasmonate is an important signal that modulates multiple physiological processes in plants, yet the molecular mechanisms underlying its interactions with other developmental signaling pathways remain poorly understood. Here, we show that jasmonate negatively regulates stomatal development in Arabidopsis (Arabidopsis thaliana) cotyledons. Cotyledons of the wild type and stomata-overproliferating mutants (such as too many mouths-1 and stomatal density and distribution1-1) treated with methyl jasmonate exhibit a clear reduction in stomata number. By contrast, blocking endogenous jasmonate biosynthesis or perception enhanced stomatal development. Moreover, three MYC transcription factors involved in jasmonate signaling, MYC2, MYC3, and MYC4, were found to redundantly modulate jasmonate-inhibited stomatal development. A genetic analysis showed that these MYC proteins act upstream of the SPEECHLESS and FAMA transcription factors to mediate stomatal development. Furthermore, jasmonate repression of stomatal development is dependent on these three MYC transcription factors, as stomatal development of the myc2 myc3 myc4 triple mutant was insensitive to methyl jasmonate treatment. Collectively, our study demonstrates that jasmonate and MYC transcription factors negatively regulate stomatal development in Arabidopsis cotyledons.

Vascular Development.

The vascular system forms as a branching network of endothelial cells that acquire identity as arterial, venous, hemogenic, or lymphatic. Endothelial specification depends on gene targets transcribed by Ets domain-containing factors, including Ets variant gene 2 (Etv2), together with the activity of chromatin-remodeling complexes containing Brahma-related gene-1 (Brg1). Once specified and assembled into vessels, mechanisms regulating lumen diameter and axial growth ensure that the structure of the branching vascular network matches the need for perfusion of target tissues. In addition, blood vessels provide important morphogenic cues that guide or direct the development of organs forming around them. As the embryo grows and lumen diameters increase, smooth muscle cells wrap around the nascent vessel walls to provide mechanical strength and vasomotor control of the circulation. Increasing mechanical stretch and wall strain promote smooth muscle cell differentiation via coupling of actin cytoskeletal remodeling to myocardin and serum response factor-dependent transcription. Remodeling of artery walls by developmental signaling pathways reappears in postnatal blood vessels during physiological and pathological adaptation to vessel wall injury, inflammation, or chronic hypoxia. Recent reports providing insights into major steps in vascular development are reviewed here with a particular emphasis on studies that have been recently published in Arteriosclerosis, Thrombosis, and Vascular Biology.