Regulates Stem Cell Homeostasis Research Articles

Cytosolic isocitrate dehydrogenase regulates plant stem cell maintenance in response to nutrient deficiency.

WUSCHEL (WUS) and WUSCHEL-RELATED HOMEOBOX (WOX) proteins determine stem cell maintenance for continual plant growth and development under changing environmental conditions. Nutrient availability is an environmental factor that substantially controls plant growth and development. However, how plant stem cell homeostasis is regulated under nutrient deficiency remains to be elucidated. Here, we showed that cytosolic isocitrate dehydrogenase (ICDH) plays an important role in nutrient sensing of stem cells in Arabidopsis (Arabidopsis thaliana). Nutrient deficiency induced the cytoplasmic-to-nuclear translocation of cytosolic ICDH protein. ICDH can interact with WUS/WOX protein as a complex that further promotes WUS/WOX expression by binding to its promoter. WUS/WOX expression in the icdh-2 mutant was lower than that of wild-type plants under nutrient deficiency. Consistently, loss of ICDH function caused a more serious growth repression under nutrient deficiency that was independent of ICDH's catalytic activity. Therefore, cytosolic ICDH regulates stem cell homeostasis of plants in response to nutrient deficiency.

Mechanotransduction through adhesion molecules: Emerging roles in regulating the stem cell niche.

Stem cells have been shown to play an important role in regenerative medicine due to their proliferative and differentiation potential. The challenge, however, lies in regulating and controlling their potential for this purpose. Stem cells are regulated by growth factors as well as an array of biochemical and mechanical signals. While the role of biochemical signals and growth factors in regulating stem cell homeostasis is well explored, the role of mechanical signals has only just started to be investigated. Stem cells interact with their niche or to other stem cells via adhesion molecules that eventually transduce mechanical cues to maintain their homeostatic function. Here, we present a comprehensive review on our current understanding of the influence of the forces perceived by cell adhesion molecules on the regulation of stem cells. Additionally, we provide insights on how this deeper understanding of mechanobiology of stem cells has translated toward therapeutics.

Me31B regulates stem cell homeostasis by preventing excess dedifferentiation in the Drosophila male germline.

ABSTRACTTissue-specific stem cells maintain tissue homeostasis by providing a continuous supply of differentiated cells throughout the life of organisms. Differentiated/differentiating cells can revert back to a stem cell identity via dedifferentiation to help maintain the stem cell pool beyond the lifetime of individual stem cells. Although dedifferentiation is important for maintaining the stem cell population, it is speculated that it underlies tumorigenesis. Therefore, this process must be tightly controlled. Here, we show that a translational regulator, me31B, plays a critical role in preventing excess dedifferentiation in the Drosophila male germline: in the absence of me31B, spermatogonia dedifferentiate into germline stem cells (GSCs) at a dramatically elevated frequency. Our results show that the excess dedifferentiation is likely due to misregulation of nos, a key regulator of germ cell identity and GSC maintenance. Taken together, our data reveal negative regulation of dedifferentiation to balance stem cell maintenance with differentiation.

Gankyrin activates the hedgehog signalling to drive metastasis in osteosarcoma.

Gankyrin is a regulatory subunit of the 26‐kD proteasome complex and promotes the occurrence and progression of many malignancies. However, the role of gankyrin in osteosarcoma (OS) metastasis remains unclear. Hedgehog signalling has been shown to regulate stem cell homeostasis and cancer metastasis, but the mechanisms that activate this pathway in OS are still poorly understood. Here, a series of in vitro and in vivo assays were carried out to explore the function and mechanism of gankyrin regulating Hedgehog signalling in OS. We demonstrated that gankyrin promotes migration, invasion and regulates the expression of some stemness factors by up‐regulating Gli1 in OS. Importantly, our data showed an interaction between gankyrin and Gli1. Moreover, gankyrin suppresses the ubiquitin‐mediated degradation of Gli1 protein in OS. Gankyrin also significantly promotes the lung metastasis of OS in vivo. Our findings suggest that gankyrin drives metastasis and regulates the expression of some stemness factors in osteosarcoma by activating Hedgehog signalling, indicating that drug screening for compounds targeting gankyrin may contribute to the development of novel and effective therapies for OS.

A single-cell analysis of the Arabidopsis vegetative shoot apex.

The shoot apical meristem allows for reiterative formation of new aerial structures throughout the life cycle of a plant. We use single-cell RNA sequencing to define the cellular taxonomy of the Arabidopsis vegetative shoot apex at the transcriptome level. We find that the shoot apex is composed of highly heterogeneous cells, which can be partitioned into 7 broad populations with 23 transcriptionally distinct cell clusters. We delineate cell-cycle continuums and developmental trajectories of epidermal cells, vascular tissue, and leaf mesophyll cells and infer transcription factors and gene expression signatures associated with cell fate decisions. Integrative analysis of shoot and root apical cell populations further reveals common and distinct features of epidermal and vascular tissues. Our results, thus, offer a valuable resource for investigating the basic principles underlying cell division and differentiation in plants at single-cell resolution.

Loss of the Epigenetic Mark 5-hmC in Psoriasis: Implications for Epidermal Stem Cell Dysregulation

Epigenetic regulation has a profound influence on stem cell fate during normal development in maintenance of physiologic tissue homeostasis. Here we report diminished ten-eleven translocation (TET) methylcytosine dioxygenase expression and loss of the DNA hydroxymethylation mark 5-hydroxymethylcytosine (5-hmC) in keratinocyte stem cells and transit amplifying cells in human psoriasis and in imiquimod-induced murine psoriasis. Loss of 5-hmC was associated with dysregulated keratinocyte stem cell kinetics, resulting in accumulation of nestin and FABP5-expressing transit amplifying cells to produce classic psoriatic epidermal architecture. Moreover, 5-hmC loss was accompanied by diminished TET1 and TET2 mRNA expression. Genome-wide mapping of epidermal 5-hmC in murine psoriasis revealed loci-specific loss of 5-hmC in genes regulating stem cell homeostasis, including MBD1, RTN1, STRN4, PRKD2, AKT1, and MAPKAP2, as well as those associated with RAR and Wnt/β-catenin signaling pathways. Invitro restoration of TET expression by ascorbic acid was accomplished in cultured human keratinocyte stem cells to show similar Ca++-induced differentiation, resulting in increased 5-hmC levels and reduced nestin expression. To our knowledge, an epigenetic deficiency in psoriasis with relevance to stem cell dysregulation has not been previously reported. This observation raises the possibility that epigenetic modifiers that impact on the TET-5-hmC pathway may be a relevant approach of heretofore unappreciated therapeutic utility.

Interplay between Metabolites and the Epigenome in Regulating Embryonic and Adult Stem Cell Potency and Maintenance.

The environment surrounding stem cells has the ability to elicit profound, heritable epigenetic changes orchestrated by multiple epigenetic mechanisms, which can be modulated by the level of specific metabolites. In this review, we highlight the significance of metabolism in regulating stem cell homeostasis, cell state, and differentiation capacity, using metabolic regulation of embryonic and adult muscle stem cells as examples, and cast light on the interaction between cellular metabolism and epigenetics. These new regulatory networks, based on the dynamic interplay between metabolism and epigenetics in stem cell biology, are important, not only for understanding tissue homeostasis, but to determine in vitro culture conditions which accurately support normal cell physiology.

Global Analysis of WOX Transcription Factor Gene Family in Brassica napus Reveals Their Stress- and Hormone-Responsive Patterns.

The plant-specific WUSCHEL-related homeobox (WOX) transcription factor gene family is important for plant growth and development but little studied in oil crops. We identified and characterized 58 putative WOX genes in Brassica napus (BnWOXs), which were divided into three major clades and nine subclades based on the gene structure and conserved motifs. Collinearity analysis revealed that most BnWOXs were the products of allopolyploidization and segmental duplication events. Gene structure analysis indicated that introns/exons and protein motifs were conserved in each subclade and RNA sequencing revealed that BnWOXs had narrow expression profiles in major tissues and/or organs across different developmental stages. The expression pattern of each clade was highly conserved and similar to that of the sister and orthologous pairs from Brassica rapa and Brassica oleracea. Quantitative real-time polymerase chain reaction showed that members of the WOX4 subclade were induced in seedling roots by abiotic and hormone stresses, indicating their contribution to root development and abiotic stress responses. 463 proteins were predicted to interact with BnWOXs, including peptides regulating stem cell homeostasis in meristems. This study provides insights into the evolution and expression of the WOX gene family in B. napus and will be useful in future gene function research.

The AtMC4 regulates the stem cell homeostasis in Arabidopsis by catalyzing the cleavage of AtLa1 protein in response to environmental hazards

The AtLa1 protein is an RNA binding factor that initiates the translation of WUSCHEL (WUS) mRNA in Arabidopsis. The AtLa1 protein can regulate the stem cell homeostasis via the nuclear-to-cytoplasmic translocation in response to environmental hazards. However, the translocation mechanism of AtLa1 protein remains to be elucidated. In the present study, we have explored the role of AtMC4 protein in the subcellular translocation of AtLa1 protein. Our results showed that the AtLa1 protein is a substrate of AtMC4 protein. The AtMC4 protein can interact with AtLa1 protein and catalyze the cleavage of the C-terminal nuclear localization signal peptide of AtLa1 protein. The AtMC4 protein is mainly distributed in the cytoplasm. In the presence of environmental stresses, the cytoplasmic-to-nuclear translocation of AtMC4 protein is enhanced with the result that more AtLa1 protein can be cleaved and transported from nucleus to cytoplasm, where AtLa1 protein further initiates the translation of WUS mRNA. By contrast, knockdown of AtMC4 expression inhibits the nuclear-to-cytoplasmic translocation of AtLa1 protein and the WUS protein translation. Based on these results, we conclude that the AtMC4 protein regulates stem cell homeostasis by catalyzing the cleavage of AtLa1 protein in response to environmental hazards.

Dual CLAVATA3 Peptides in Arabidopsis Shoot Stem Cell Signaling.

Plant shoot stem cell pool is constantly maintained by a negative feedback loop through peptide-receptor mediated signaling pathway. CLAVATA3 (CLV3) encode a 96 amino-acid protein which is processed to 12-amino-acid or arabinosylated 13-amino-acid peptides, acting as a ligand signal to regulate stem cell homeostasis in the shoot apical meristem (SAM). Although arabinosylated 13-amino-acid CLV3 peptide (CLV3p) shows more significant binding affinity to its receptors and biological activities in the SAM, the physiological function of two mature forms of CLV3p remained an unresolved puzzle in the past decade due to the technical difficulties of arabinosylation modification in the peptide synthesis. Here, we analyzed the role of two mature CLV3 peptides with newly synthesized arabinosylated peptide. Beside shoot meristem phenotypes, arabinosylated CLV3p showed the conventional trait of CLV2-dependent root growth inhibition. Moreover, both 12-amino-acid and arabinosylated 13-amino-acid CLV3 peptides have analogous activities in shoot stem cell signaling. Notably, we demonstrated that non-arabinosylated 12-amino acid CLV3p can affect shoot stem cell signaling at the physiological level unlike previously suggested (Ohyama et al., 2009; Shinohara and Matsubayashi, 2013; Shinohara and Matsubayashi, 2015). Therefore, these results support the physiological role of the 12-amino-acid CLV3p in shoot stem cell signaling in the deficient condition of arabinosylated 13-amino-acid CLV3p in Arabidopsis thaliana.

Abstract 5539: The phenotypic characterization of Lrig null mouse embryonic fibroblasts

Abstract The LRIG gene family consists of LRIG1, LRIG2 and LRIG3. The most studied of these is LRIG1, which is a tumor suppressor gene. The LRIG1 protein is downregulated in various types of cancer and it negatively regulates receptor tyrosine kinases including the EGF- and PDGF-receptors. Lrig1 also regulates stem cell homeostasis in mouse skin and intestine. The functions of LRIG2 and LRIG3 are less known, but some findings imply that they might antagonize LRIG1. Intriguingly, the sole LRIG homolog in C. elegans, Sma-10, positively regulates BMP signaling. Thus, it is not known to what degree the LRIG proteins have the same, overlapping, or opposing functions, and the molecular mechanisms involved are poorly characterized. To elucidate the functions of the three LRIG proteins we generated a series of wildtype and LRIG-null (Lrig1-/-;Lrig2-/-;Lrig3-/-) mouse embryonic fibroblast (MEF) lines. These cell lines were created from homozygous Lrig1flox/flox;Lrig2flox/flox;Lrig3flox/flox mouse embryos. The FLOXed Lrig genes were then deleted with CRE-recombinase, thereby creating Lrig-null MEFs. ddPCR analysis showed that the Lrig-null MEF lines were contaminated with less than 1% of cells carrying wildtype Lrig3 alleles. Lrig-null MEFs showed a higher proliferation rate and migrated more in trans-well chambers than the wildtype cells. These cell lines will also be useful to investigate the importance of Lrig proteins for other cellular functions such as, signal transduction, lipidomics, transcriptomics, secretomics, metabolomics, and oxidative stress sensitivity. We also transduced the Lrig-null cells with individual inducible Lrig-genes thus creating a powerful system for the examination of the biology of the specific Lrig proteins. Citation Format: Carl Herdenberg, Håkan Hedman. The phenotypic characterization of Lrig null mouse embryonic fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5539. doi:10.1158/1538-7445.AM2017-5539

Abstract 1167: Inhibition of WNT/ß-catenin signaling-related glutamine metabolism but not the Warburg effect in denervation-induced suppression of gastric tumorigenesis

Abstract Background/aim: Cholinergic nerves have been shown to regulate gastric WNT/ß-catenin signaling in leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5)-positive stem cells and have recently been implicated in gastric tumorigenesis. Denervation by local vagotomy or botulinum neurotoxin type A injection or the muscarinic acetylcholine receptor 3 antagonist limit gastric cancer growth. The aim of this study was to further unravel the metabolic pathways underlying the vagotomy-induced suppression of gastric tumorigenesis. Methods: Unilateral subdiaphragmatic truncal vagotomy (VT) was performed in wild-type (WT) and the INS-GAS mice, a genetic mouse model of spontaneous gastric cancer. Metabolic profiling and gene expression profiling in gastric tissues at 6 months post-surgery were performed using liquid chromatography/mass spectrometry, gas chromatography/mass spectrometry, high resolution magic angle spinning NMR spectroscopy, and microarray gene expression (Illumina). Results: VT-induced suppression of tumorigenesis was manifested by reduced proliferation rate and increased apoptotic and autophagic signaling pathways, leading to reduced tumor size and prolonged survival. Principal component analysis showed four distinct clusters among 343 metabolic compounds: WT without VT, INS-GAS without VT, WT with VT and INS-GAS with VT. In the gastric tumor, the metabolic pathways that regulate stem cell homeostasis were downregulated after VT. Glutaminolytic pathway, including glutamine, glutamate, glycine and glutathione-S-S-glutathione, was down-regulated. The tricarboxylic acid cycle (TCA), including citrate, cis-aconitate, acetyle-CoA, threonine and glycine, was also down-regulated. However, glycolytic pathway, including glucose, glucose 6-phosphate, fructose 6-phosphate and lactate, was not significantly down-regulated. Signaling pathways that regulate glutamine metabolism, such as WNT/ß-catenin signaling, WNT target genes Cyclin D1, Axin2, Myc, Lgr5 and Cd44, p53 signaling, and mTOR signaling were down-regulated. The central carbon metabolism in cancer (“The Warburg effect” signaling) and lactate production were unchanged after VT. In addition, the choline metabolism, the lipid-derived eicosanoids and prostaglandins were reduced after VT. Conclusions: The denervation-induced suppression of gastric tumorigenesis was associated with the inhibition of WNT/ß-catenin signaling-related glutamine metabolism but not the Warburg effect. We suggest that glutamine and choline phospholipid metabolisms can be used for metabolism-based tumor detection with MRS and/or positron emission tomography (PET) for gastric cancer diagnosis and that blocking these metabolic functions can be a therapeutic approach for gastric cancer treatment. Citation Format: Gøran Andersen, Riyas Vettukattil, Yoku Hayakawa, Jon Erik Grønbech, Timothy C. Wang, Duan Chen, Chun Mei Zhao. Inhibition of WNT/ß-catenin signaling-related glutamine metabolism but not the Warburg effect in denervation-induced suppression of gastric tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1167. doi:10.1158/1538-7445.AM2015-1167

Ceramide Synthase 4 Regulates Stem Cell Homeostasis and Hair Follicle Cycling

Ceramides are crucial for skin barrier function, but little is known about the regulation of epidermal appendages and whether stem cell populations that control their regeneration depend on specific ceramide species. Here we demonstrate that ceramide synthase 4 (CerS4) is highly expressed in the epidermis of adult mice where it is localized in the interfollicular epidermis and defined populations within the pilosebaceous unit. Inactivation of CerS4 in mice resulted in precocious activation of hair follicle bulge stem cells while expanding the Lrig1(+) junctional zone and sebaceous glands. This was preceded first by a decrease in bone morphogenetic protein (BMP) and a subsequent increase in Wnt signaling. This imbalance in quiescent versus activating signals likely promoted a prolonged anagen-like hair follicle state after the second catagen, which exhausted stem cells over time ultimately resulting in hair loss in aged mice. K14-Cre-mediated deletion of CerS4 revealed a similar phenotype, thus suggesting an epidermis intrinsic function of CerS4 in regulating the regeneration of the pilosebaceous unit. The data indicate that CerS4-directed epidermal ceramide composition is essential to control hair follicle stem and progenitor cell behavior potentially through its regulation of BMP and Wnt signaling.

Competitive balance of intrabulge BMP/Wnt signaling reveals a robust gene network ruling stem cell homeostasis and cyclic activation

Hair follicles facilitate the study of stem cell behavior because stem cells in progressive activation stages, ordered within the follicle architecture, are capable of cyclic regeneration. To study the gene network governing the homeostasis of hair bulge stem cells, we developed a Keratin 15-driven genetic model to directly perturb molecular signaling in the stem cells. We visualize the behavior of these modified stem cells, evaluating their hair-regenerating ability and profile their molecular expression. Bone morphogenetic protein (BMP)-inactivated stem cells exhibit molecular profiles resembling those of hair germs, yet still possess multipotentiality in vivo. These cells also exhibit up-regulation of Wnt7a, Wnt7b, and Wnt16 ligands and Frizzled (Fzd) 10 receptor. We demonstrate direct transcriptional modulation of the Wnt7a promoter. These results highlight a previously unknown intra-stem cell antagonistic competition, between BMP and Wnt signaling, to balance stem cell activity. Reduced BMP signaling and increased Wnt signaling tilts each stem cell toward a hair germ fate and, vice versa, based on a continuous scale dependent on the ratio of BMP/Wnt activity. This work reveals one more hierarchical layer regulating stem cell homeostasis beneath the stem cell-dermal papilla-based epithelial-mesenchymal interaction layer and the hair follicle-intradermal adipocyte-based tissue interaction layer. Although hierarchical layers are all based on BMP/Wnt signaling, the multilayered control ensures that all information is taken into consideration and allows hair stem cells to sum up the total activators/inhibitors involved in making the decision of activation.

Plant stem cell maintenance involves direct transcriptional repression of differentiation program

In animal systems, master regulatory transcription factors (TFs) mediate stem cell maintenance through a direct transcriptional repression of differentiation promoting TFs. Whether similar mechanisms operate in plants is not known. In plants, shoot apical meristems serve as reservoirs of stem cells that provide cells for all above ground organs. WUSCHEL, a homeodomain TF produced in cells of the niche, migrates into adjacent cells where it specifies stem cells. Through high-resolution genomic analysis, we show that WUSCHEL represses a large number of genes that are expressed in differentiating cells including a group of differentiation promoting TFs involved in leaf development. We show that WUS directly binds to the regulatory regions of differentiation promoting TFs; KANADI1, KANADI2, ASYMMETRICLEAVES2 and YABBY3 to repress their expression. Predictions from a computational model, supported by live imaging, reveal that WUS-mediated repression prevents premature differentiation of stem cell progenitors, being part of a minimal regulatory network for meristem maintenance. Our work shows that direct transcriptional repression of differentiation promoting TFs is an evolutionarily conserved logic for stem cell regulation.

ERECTA-Family Receptor Kinases Regulate Stem Cell Homeostasis via Buffering its Cytokinin Responsiveness in the Shoot Apical Meristem

Shoot apical meristems (SAMs), which are maintained at the tips of stems, are indeterminate structures and sources of stem cells from which all aerial organs are ultimately derived. Although mechanisms that regulate the homeostasis of the stem cells have been extensively investigated, identification of further unknown regulators should provide better understanding of the regulation. Here, we report that members of the Arabidopsis ERECTA (ER) receptor kinase family redundantly play a significant role in the regulation of stem cell homeostasis. In wild-type seedlings, the expression of WUSCHEL (WUS), a central regulator of the stem cell population, is stimulated by cytokinin. Interestingly, however, the SAM morphology and the expression of CLAVATA3 (CLV3), which is expressed in stem cells and therefore serves as a stem cell marker, are relatively stable against cytokinin treatment regardless of increased WUS expression. These findings indicate the presence of a mechanism to buffer stem cell homeostasis against an increase in cytokinin. Mutant seedlings lacking all ER-family members, which are expressed in the SAM, show an increase in the stem cell population and also the up-regulation of a cytokinin-responsive gene in the SAM. In this mutant, WUS expression is stimulated by cytokinin treatment as efficiently as in wild-type plants. However, in contrast to wild-type plants, SAM morphology and CLV3 expression respond drastically to cytokinin treatment, suggesting that the buffering mechanism to maintain stem cell homeostasis against an increase in cytokinin is severely impaired in this mutant. We suggest that the ER family regulates stem cell homeostasis via buffering its cytokinin responsiveness in the SAM.

Hymyc1 Downregulation Promotes Stem Cell Proliferation in Hydra vulgaris

Hydra is a unique model for studying the mechanisms underlying stem cell biology. The activity of the three stem cell lineages structuring its body constantly replenishes mature cells lost due to normal tissue turnover. By a poorly understood mechanism, stem cells are maintained through self-renewal while concomitantly producing differentiated progeny. In vertebrates, one of many genes that participate in regulating stem cell homeostasis is the protooncogene c-myc, which has been recently identified also in Hydra, and found expressed in the interstitial stem cell lineage. In the present paper, by developing a novel strategy of RNA interference-mediated gene silencing (RNAi) based on an enhanced uptake of small interfering RNAi (siRNA), we provide molecular and biological evidence for an unexpected function of the Hydra myc gene (Hymyc1) in the homeostasis of the interstitial stem cell lineage. We found that Hymyc1 inhibition impairs the balance between stem cell self renewal/differentiation, as shown by the accumulation of stem cell intermediate and terminal differentiation products in genetically interfered animals. The identical phenotype induced by the 10058-F4 inhibitor, a disruptor of c-Myc/Max dimerization, demonstrates the specificity of the RNAi approach. We show the kinetic and the reversible feature of Hymyc1 RNAi, together with the effects displayed on regenerating animals. Our results show the involvement of Hymyc1 in the control of interstitial stem cell dynamics, provide new clues to decipher the molecular control of the cell and tissue plasticity in Hydra, and also provide further insights into the complex myc network in higher organisms. The ability of Hydra cells to uptake double stranded RNA and to trigger a RNAi response lays the foundations of a comprehensive analysis of the RNAi response in Hydra allowing us to track back in the evolution and the origin of this process.

Membrane distributions of two ligand-binding receptor complexes in the CLAVATA pathway

Genetic studies have suggested that transmembrane proteins CLAVATA1 (CLV1), CLV2, CORYNE (CRN), BAM1 and BAM2 all play a role in relaying the CLV3 signal and thus regulating stem cell homeostasis at the shoot meristem (SM). The extracellular domain of CLV1 was previously shown to bind the CLE peptide derived from CLV3, providing direct evidence that CLV3-CLV1 function as a ligand-receptor pair. How the other putative receptors function in the CLV pathway, however, remained unclear. We demonstrated in a recent Plant Journal article that the receptor-like protein CLV2 and the receptor-kinases BAM1 and BAM2 also bind to the CLV3 CLE peptide ligand with an affinity similar to that of CLV1. Critically, these ligand binding receptors form two distinct complexes in both transient expression in tobacco and in Arabidopsis meristem cells: a CLV2/CRN multimer and a CLV1/BAM multimer. Here we examine in detail the subcellular membrane partitioning for the receptor proteins in transient expression by two-phase partitioning and co-expression with known subcellular markers. All tested proteins measurably accumulate at the plasma membrane. While CLV1 primarily co-localizes with a plasma membrane marker, CLV2 shows greater co-localization with an endoplasmic reticulum (ER) marker.

Membrane distributions of two ligand-binding receptor complexes in the CLAVATA pathway.

Genetic studies have suggested that transmembrane proteins CLAVATA1 (CLV1), CLV2, CORYNE (CRN), BAM1 and BAM2 all play a role in relaying the CLV3 signal and thus regulating stem cell homeostasis at the shoot meristem (SM). The extracellular domain of CLV1 was previously shown to bind the CLE peptide derived from CLV3, providing direct evidence that CLV3-CLV1 function as a ligand-receptor pair. How the other putative receptors function in the CLV pathway, however, remained unclear. We demonstrated in a recent Plant Journal article that the receptor-like protein CLV2 and the receptor-kinases BAM1 and BAM2 also bind to the CLV3 CLE peptide ligand with an affinity similar to that of CLV1. Critically, these ligand binding receptors form two distinct complexes in both transient expression in tobacco and in Arabidopsis meristem cells: a CLV2/CRN multimer and a CLV1/BAM multimer. Here we examine in detail the subcellular membrane partitioning for the receptor proteins in transient expression by two-phase partitioning and co-expression with known subcellular markers. All tested proteins measurably accumulate at the plasma membrane. While CLV1 primarily co-localizes with a plasma membrane marker, CLV2 shows greater co-localization with an endoplasmic reticulum (ER) marker.

Notch Signaling Is Necessary and Sufficient for Runx1-Dependent Stem Cell Induction in the Embryonic Aorta-Gonad-Mesonephros (AGM) Region.

Vertebrate hematopoiesis can be divided into two embryonic phases: a short primitive wave predominantly generating erythrocytes and a definitive (fetal/adult) wave producing long-term hematopoietic stem cells (HSCs). The definitive wave occurs in the embryonic aorta-gonad-mesonephros (AGM) region through the asymmetric induction of HSCs from the ventral, but not dorsal, aortic endothelial wall. Since Notch signaling is critical for orchestrating a variety of developmental cell fate choices from invertebrates to humans and has been implicated in affecting the differentiation of some hematopoietic lineages, we analyzed whether the Notch pathway regulates definitive HSC induction in vivo. The zebrafish mutant mindbomb harbors a mutation in an essential E3 ligase that ubiquitylates Delta, which in turn allows the Notch intercellular domain to be released and activate downstream target gene transcription. Thus, in the absence of Mindbomb function Notch signaling does not occur. We found that although mindbomb mutants show normal primitive hematopoiesis, definitive c-myb and runx1 HSC expression is lacking. Since embryos injected with synthetic morpholinos designed to inhibit proper splicing of runx1 RNA ( runx morphants) show the same hematopoietic phenotype as mindbomb mutants, we next addressed the epistatic relationship between notch and runx1 using classic gain-of-function and loss-of-function analyses. In runx1 morphants expression of a notch receptor, notch3, and a delta ligand, deltaC, in the developing dorsal aorta was normal. Moreover, injection of runx1 RNA rescued HSCs in the AGM of mindbomb mutants. Together, these results suggest that Runx1 functions downstream of Notch in promoting HSC fate. We next analyzed whether a constitutively activated form of Notch (NICD) is sufficient for HSC specification in the AGM using an inducible binary transgenic system. Zebrafish carrying the heat-shock promoter driving the activator gal4 were mated to animals carrying 6 gal4 -responsive tandem upstream activating sequences (UAS) driving NICD. At the 10 somite-stage the embryos were heat-shocked at 37°C for 1 hour to activate NICD throughout the double transgenic animals. Surprisingly, expression of both HSC markers, c-myb and runx1, were expanded from their normal restricted domain in the ventral endothelium to the entire circumference of the dorsal aorta. Most interestingly, the presence of ectopic c-myb and runx1 transcripts were observed in the developing post-cardinal vein, a vessel that normally does not produce HSCs. These data imply that activation of the Notch pathway generates increased numbers of HSCs in vivo. When runx1 RNA is injected into wild-type embryos a similar expansion of c-myb transcripts is seen throughout the entire dorsal aorta and post-cardinal vein, further indicating that Runx1 functions downstream of Notch in HSC induction. In summary, discovery of the molecular programs essential and sufficient for fetal/adult hematopoietic ontogeny will lead to a further understanding of the physiologic and pathologic processes regulating stem cell homeostasis and translate into more effective therapies for blood disorders.