bHLH Factor Research Articles

Phosphorylation in intrinsically disordered regions regulates the activity of Neurogenin2.

BackgroundNeuronal differentiation is largely under the control of basic Helix-Loop-Helix (bHLH) proneural transcription factors that play key roles during development of the embryonic nervous system. In addition to well-characterised regulation of their expression, increasing evidence is emerging for additional post-translational regulation of proneural protein activity. Of particular interest is the bHLH proneural factor Neurogenin2 (Ngn2), which orchestrates progression from neural progenitor to differentiated neuron in several regions of the central nervous system. Previous studies have demonstrated a key role for cell cycle-dependent multi-site phosphorylation of Ngn2 protein at Serine-Proline (SP) sites for regulation of its neuronal differentiation activity, although the potential structural and functional consequences of phosphorylation at different regions of the protein are unclear.ResultsHere we characterise the role of phosphorylation of specific regions of Ngn2 on the stability of Ngn2 protein and on its neuronal differentiation activity in vivo in the developing embryo, demonstrating clearly that the location of SP sites is less important than the number of SP sites available for control of Ngn2 activity in vivo. We also provide structural evidence that Ngn2 contains large, intrinsically disordered regions that undergo phosphorylation by cyclin-dependent kinases (cdks).ConclusionsPhosphorylation of Ngn2 occurs in both the N- and C-terminal regions, either side of the conserved basic Helix-Loop-Helix domain. While these phosphorylation events do not change the intrinsic stability of Ngn2, phosphorylation on multiple sites acts to limit its ability to drive neuronal differentiation in vivo. Phosphorylated regions of Ngn2 are predicted to be intrinsically disordered and cdk-dependent phosphorylation of these intrinsically disordered regions contributes to Ngn2 regulation.

Transcriptional regulatory events initiated by Ascl1 and Neurog2 during neuronal differentiation of P19 embryonic carcinoma cells.

As members of the proneural basic-helix-loop-helix (bHLH) family of transcription factors, Ascl1 and Neurog2 direct the differentiation of specific populations of neurons at various times and locations within the developing nervous system. In order to characterize the mechanisms employed by these two bHLH factors, we generated stable, doxycycline-inducible lines of P19 embryonic carcinoma cells that express comparable levels of Ascl1 and Neurog2. Upon induction, both Ascl1 and Neurog2 directed morphological and immunocytochemical changes consistent with initiation of neuronal differentiation. Comparison of Ascl1- and Neurog2-regulated genes by microarray analyses showed both shared and distinct transcriptional changes for each bHLH protein. In both Ascl1- and Neurog2-differentiating cells, repression of Oct4 mRNA levels was accompanied by increased Oct4 promoter methylation. However, DNA demethylation was not detected for genes induced by either bHLH protein. Neurog2-induced genes included glutamatergic marker genes while Ascl1-induced genes included GABAergic marker genes. The Neurog2-specific induction of a gene encoding a protein phosphatase inhibitor, Ppp1r14a, was dependent on distinct, canonical E-box sequences within the Ppp1r14a promoter and the nucleotide sequences within these E-boxes were partially responsible for Neurog2-specific regulation. Our results illustrate multiple novel mechanisms by which Ascl1 and Neurog2 regulate gene repression during neuronal differentiation in P19 cells.

Notch signaling differentially regulates Atoh7 and Neurog2 in the distal mouse retina.

Notch signaling regulates basic helix-loop-helix (bHLH) factors as an evolutionarily conserved module, but the tissue-specific mechanisms are incompletely elucidated. In the mouse retina, bHLH genes Atoh7 and Neurog2 have distinct functions, with Atoh7 regulating retinal competence and Neurog2 required for progression of neurogenesis. These transcription factors are extensively co-expressed, suggesting similar regulation. We directly compared Atoh7 and Neurog2 regulation at the earliest stages of retinal neurogenesis in a broad spectrum of Notch pathway mutants. Notch1 and Rbpj normally block Atoh7 and Neurog2 expression. However, the combined activities of Notch1, Notch3 and Rbpj regulate Neurog2 patterning in the distal retina. Downstream of the Notch complex, we found the Hes1 repressor mediates Atoh7 suppression, but Hes1, Hes3 and Hes5 do not regulate Neurog2 expression. We also tested Notch-mediated regulation of Jag1 and Pax6 in the distal retina, to establish the appropriate context for Neurog2 patterning. We found that Notch1;Notch3 and Rbpj block co-expression of Jag1 and Neurog2, while specifically stimulating Pax6 within an adjacent domain. Our data suggest that Notch signaling controls the overall tempo of retinogenesis, by integrating cell fate specification, the wave of neurogenesis and the developmental status of cells ahead of this wave.

Sources of CNS tumor heterogeneity

Central nervous system tumors are marked with high intra- and inter-tumoral heterogeneity. For instance, an integrative large scale gene expression study performed in 2010 revealed 4 subtypes glioblastoma multiforme (GBM) with differential responses to treatment and prognosis [1]. A more comprehensive GBM classification based on combination of epigenetics, copy number variation, gene expression and genetic mutation analysis has led to identification of as many as 6 GBM subgroups [2]. Two, not mutually exclusive, general models have been proposed to explain tumor heterogeneity [3]. The genetic mutation model proposes that different genetic mutations lead to different tumor formation, while the cell of origin model explains different tumors as arising from different cell types. There is experimental evidence supporting both models. For example, 3 distinctly different CNS tumor types can be induced by infection of postnatal mouse neural stem cells with virus containing V12HRAS and c-MYC depending on the combination and sequence in which oncogenes are introduced [4]. Similarly, RNA interference (RNAi) knock down of NF1 and p53 in GFAP+ or SynI+ cells induces mesenchymal GBM, whereas the same RNAi in Nestin+ cells induced neural GBM[5]. GMB gene expression analysis also indicates that different GBM subtypes have transcipt profiles similar to different cell types [1]. To fully explore the causes of tumor diversity, it is desirable2 to have an animal model in which both the cell of origin and genetic insult can be conveniently and independently manipulated. To achieve this, we have recently developed a central nervous system tumor model in the rat in which multiple oncogenes can be expressed in selected cell populations at different times in brain development [6]. In this model, we used the piggyBac transposon system [7] to stably integrate oncogenes into defined cell populations by in utero electroporation (IUE). Using this model, we evaluated the contribution of cell of origin and genetic mutation in tumor heterogeneity. To test whether the same oncogenic event in different, but closely related, cell population gives rise to same or different tumors, we directed HRasV12/AKT expression in disparate cell populations in the radial glia lineage with promoters that are ubiquitously active (CAG promoter), astrocyte selective GFAP (glial fibrillary acidic protein) promoter or oligodendrocytes selective MBP (myelin basic protein) promoter. We showed that HRasV12/AKT expression under CAG or GFAP promoter induced similar tumors, glioblastoma multiforme (WHO grade 4). However, HRasV12/AKT expression controlled by MBP promoter induced anaplastic oligoastrocytoma (WHO grade 3). We further showed that these induced induced anaplastic oligoastrocytoma differed from glioblastoma multiforme both in histology and molecular signature. These results indicate that oncogenic events occurring in different cell types in the same cellular lineage can lead to different tumor types. We next investigated whether tumor phenotype could be modified by expression of neurogenic bHLH family protein Neurogenin2 (Ngn2) or Neural differentiation 1 (NeuroD1). Members of the bHLH gene family are well known to have important roles is cell-type determination in normal development. Expression of either Ngn2 or NeuroD1 along with HRasV12/AKT resulted in atypical teratoid rhabdoid tumor like (ATRT like) tumor, a tumor type not previously observed after expression of HRasV12/AKT oncogenes alone. We further tested whether this phenotypic transformation from GBM to ATRT like tumor was due to transient expression of bHLH factors in radial glia or due to expression in tumor cells. Our data showed that transient expression of Ngn2 in radial glia, prior to transformation by HRasV12/AKT was able to induce ATRT like tumor formation. These results may indicate that the same oncogenic events occurring in similar cell types expressing different levels of individual bHLH transcription factors can lead to very different tumor types. The main advantage of piggyBac IUE method is that it allows for introduction of multiple transgenes controlled by independent promoters. The high co-expression rates allowed us to direct expression in different subpopulations in sequence. In the same system, we also demonstrated use of multi-color fluorescent protein expression to produce a clonal readout of tumor growth and invasion. There are several additional features of the piggyBac system that make it useful for other applications in investigating tumor biology. For example, the piggyBac- IUE approach could be applied to other species other than rodent extending CNS tumor biology to other species, such as ferret. Combination of piggyBac-IUE with existing transgenic mouse models could potentially broaden the utility of both approaches. Also multicolor clonal labeling of tumor cells could facilitate in vivo imaging of clonally related tumor cells. These functionalities should make this approach a useful platform for screening potential modifiers of tumor development and for studying further how genetic modifiers and cell or origin are related to tumor development and heterogeneity.

An overview of the gene regulatory network controlling trichome development in the model plant, Arabidopsis.

Trichomes are specialized epidermal cells located on aerial parts of plants and are associated with a wide array of biological processes. Trichomes protect plants from adverse conditions including UV light and herbivore attack and are also an important source of a number of phytochemicals. The simple unicellular trichomes of Arabidopsis serve as an excellent model to study molecular mechanism of cell differentiation and pattern formation in plants. The emerging picture suggests that the developmental process is controlled by a transcriptional network involving three major groups of transcription factors (TFs): the R2R3 MYB, basic helix-loop-helix (bHLH), and WD40 repeat (WDR) protein. These regulatory proteins form a trimeric activator complex that positively regulates trichome development. The single repeat R3 MYBs act as negative regulators of trichome development. They compete with the R2R3 MYBs to bind the bHLH factor and form a repressor complex. In addition to activator–repressor mechanism, a depletion mechanism may operate in parallel during trichome development. In this mechanism, the bHLH factor traps the WDR protein which results in depletion of WDR protein in neighboring cells. Consequently, the cells with high levels of bHLH and WDR proteins are developed into trichomes. A group of C2H2 zinc finger TFs has also been implicated in trichome development. Phytohormones, including gibberellins and jasmonic acid, play significant roles in this developmental process. Recently, microRNAs have been shown to be involved in trichome development. Furthermore, it has been demonstrated that the activities of the key regulatory proteins involved in trichome development are controlled by the 26S/ubiquitin proteasome system (UPS), highlighting the complexity of the regulatory network controlling this developmental process. To complement several excellent recent relevant reviews, this review focuses on the transcriptional network and hormonal interplay controlling trichome development in Arabidopsis.

Cell elongation is regulated through a central circuit of interacting transcription factors in the Arabidopsis hypocotyl.

As the major mechanism of plant growth and morphogenesis, cell elongation is controlled by many hormonal and environmental signals. How these signals are coordinated at the molecular level to ensure coherent cellular responses remains unclear. In this study, we illustrate a molecular circuit that integrates all major growth-regulating signals, including auxin, brassinosteroid, gibberellin, light, and temperature. Analyses of genome-wide targets, genetic and biochemical interactions demonstrate that the auxin-response factor ARF6, the light/temperature-regulated transcription factor PIF4, and the brassinosteroid-signaling transcription factor BZR1, interact with each other and cooperatively regulate large numbers of common target genes, but their DNA-binding activities are blocked by the gibberellin-inactivated repressor RGA. In addition, a tripartite HLH/bHLH module feedback regulates PIFs and additional bHLH factors that interact with ARF6, and thereby modulates auxin sensitivity according to developmental and environmental cues. Our results demonstrate a central growth-regulation circuit that integrates hormonal, environmental, and developmental controls of cell elongation in Arabidopsis hypocotyl.

BHLH Factors in Self-Renewal, Multipotency, and Fate Choice of Neural Progenitor Cells

Multipotent neural progenitor cells (NPCs) undergo self-renewal while producing neurons, astrocytes, and oligodendrocytes. These processes are controlled by multiple basic helix-loop-helix (bHLH) fate determination factors, which exhibit different functions by posttranslational modifications. Furthermore, depending on the expression dynamics, each bHLH factor seems to have two contradictory functions, promoting NPC proliferation and cell-cycle exit for differentiation. The oscillatory expression of multiple bHLH factors correlates with the multipotent and proliferative state, whereas sustained expression of a selected single bHLH factor regulates the fate determination. bHLH factors also regulate direct reprogramming of adult somatic cells into neurons and oligodendrocytes. Thus, bHLH factors play key roles in development and regeneration of the nervous system. Here, we review versatile functions of bHLH factors, focusing on telencephalic development.

Impaired enteroendocrine cells differentiation signaling pathways through microbiota transfer (1107.12)

Recently, we showed that susceptibility to obesity is characterized by an unfavorable microbiome and that the gut chemosensory signature can be transferred via microbiota. To examine whether changes in the signaling pathways controlling differentiation of enteroendocrine cells (EEC) are transmitted through gut microbiota, we assessed gene and protein expression of intestinal bHLH transcription factors, in germ free mice conventionalized with microbiota from obese prone and resistant rats. Gut permeability, intestinal lipid and carbohydrates transporters as well as circulating levels and intestinal protein expression of gut peptides were determined. We showed that obese animals exhibited decreased expression of bHLH transcription factors controlling EECs differentiation (MATH1, NGN3, NEUROD1), increased expression of bHLH factors modulating absorptive enterocyte expression. and decreased number of total EECs L‐, I‐ and S‐cells. This was accompanied by increased gut permeability and expression of lipid and carbohydrates transporters. Transfer of obese gut microbiota replicated obesity‐associated decreased in bHLH transcription factors in germ free mice. These findings demonstrate that gut microbiota modulates complex molecular signaling machinery responsible for EECs differentiation pathways. It shows that susceptibility to obesity and associated microbiome impacts EEC differentiation through downregulation of bHLH transcription factors resulting in reduced EEC number and satiety gut hormone levels.

The bHLH Factors Extramacrochaetae and Daughterless Control Cell Cycle in Drosophila Imaginal Discs through the Transcriptional Regulation of the cdc25 Phosphatase string

One of the major issues in developmental biology is about having a better understanding of the mechanisms that regulate organ growth. Identifying these mechanisms is essential to understand the development processes that occur both in physiological and pathological conditions, such as cancer. The E protein family of basic helix-loop helix (bHLH) transcription factors, and their inhibitors the Id proteins, regulate cell proliferation in metazoans. This notion is further supported because the activity of these factors is frequently deregulated in cancerous cells. The E protein orthologue Daughterless (Da) and the Id orthologue Extramacrochaetae (Emc) are the only members of these classes of bHLH proteins in Drosophila. Although these factors are involved in controlling proliferation, the mechanism underlying this regulatory activity is poorly understood. Through a genetic analysis, we show that during the development of epithelial cells in the imaginal discs, the G2/M transition, and hence cell proliferation, is controlled by Emc via Da. In eukaryotic cells, the main activator of this transition is the Cdc25 phosphatase, string. Our genetic analyses reveal that the ectopic expression of string in cells with reduced levels of Emc or high levels of Da is sufficient to rescue the proliferative defects seen in these mutant cells. Moreover, we present evidence demonstrating a role of Da as a transcriptional repressor of string. Taken together, these findings define a mechanism through which Emc controls cell proliferation by regulating the activity of Da, which transcriptionally represses string.

Ascl1 as a Novel Player in the Ptf1a Transcriptional Network for GABAergic Cell Specification in the Retina

In contrast with the wealth of data involving bHLH and homeodomain transcription factors in retinal cell type determination, the molecular bases underlying neurotransmitter subtype specification is far less understood. Using both gain and loss of function analyses in Xenopus, we investigated the putative implication of the bHLH factor Ascl1 in this process. We found that in addition to its previously characterized proneural function, Ascl1 also contributes to the specification of the GABAergic phenotype. We showed that it is necessary for retinal GABAergic cell genesis and sufficient in overexpression experiments to bias a subset of retinal precursor cells towards a GABAergic fate. We also analysed the relationships between Ascl1 and a set of other bHLH factors using an in vivo ectopic neurogenic assay. We demonstrated that Ascl1 has unique features as a GABAergic inducer and is epistatic over factors endowed with glutamatergic potentialities such as Neurog2, NeuroD1 or Atoh7. This functional specificity is conferred by the basic DNA binding domain of Ascl1 and involves a specific genetic network, distinct from that underlying its previously demonstrated effects on catecholaminergic differentiation. Our data show that GABAergic inducing activity of Ascl1 requires the direct transcriptional regulation of Ptf1a, providing therefore a new piece of the network governing neurotransmitter subtype specification during retinogenesis.

Zac1 Regulates Cell Cycle Arrest in Neuronal Progenitors via Tcf4

Imprinted genes play a critical role in brain development and mental health, although the underlying molecular and cellular mechanisms remain incompletely understood. The family of basic helix-loop-helix (bHLH) proteins directs the proliferation, differentiation, and specification of distinct neuronal progenitor populations. Here, we identified the bHLH factor gene Tcf4 as a direct target gene of Zac1/Plagl1, a maternally imprinted transcriptional regulator, during early neurogenesis. Zac1 and Tcf4 expression levels concomitantly increased during neuronal progenitor differentiation; moreover, Zac1 interacts with two cis-regulatory elements in the Tcf4 gene locus, and these elements together confer synergistic activation of the Tcf4 gene. Tcf4 upregulation enhances the expression of the cyclin-dependent kinase inhibitor gene p57(Kip2), a paternally imprinted Tcf4 target gene, and increases the number of cells in G1 phase. Overall, we show that Zac1 controls cell cycle arrest function in neuronal progenitors through induction of p57(Kip2) via Tcf4 and provide evidence for cooperation between imprinted genes and a bHLH factor in early neurodevelopment.

Impact of high-fat feeding on basic helix–loop–helix transcription factors controlling enteroendocrine cell differentiation

Gut hormones secreted by enteroendocrine cells (EECs) play a major role in energy regulation. Differentiation of EEC is controlled by the expression of basic helix-loop-helix (bHLH) transcription factors. High-fat (HF) feeding alters gut hormone levels; however, the impact of HF feeding on bHLH transcription factors in mediating EEC differentiation and subsequent gut hormone secretion and expression is not known. Outbred Sprague-Dawley rats were maintained on chow or HF diet for 12 weeks. Gene and protein expression of intestinal bHLH transcription factors, combined with immunofluorescence studies, were analyzed for both groups in the small intestine and colon. Gut permeability, intestinal lipid and carbohydrate transporters as well as circulating levels and intestinal protein expression of gut peptides were determined. We showed that HF feeding resulted in hyperphagia and increased adiposity. HF-fed animals exhibited decreased expression of bHLH transcription factors controlling EEC differentiation (MATH1, NGN3, NEUROD1) and increased expression of bHLH factors modulating enterocyte expression. Furthermore, HF-fed animals had decreased number of total EECs and L-cells. This was accompanied by increased gut permeability and expression of lipid and carbohydrate transporters, and a decrease in circulating and intestinal gut hormone levels. Taken together, our results demonstrate that HF feeding caused decreased secretory lineage (that is, EECs) differentiation through downregulation of bHLH transcription factors, resulting in reduced EEC number and gut hormone levels. Thus, impaired EEC differentiation pathways by HF feeding may promote hyperphagia and subsequent obesity.

The Prdm13 histone methyltransferase encoding gene is a Ptf1a–Rbpj downstream target that suppresses glutamatergic and promotes GABAergic neuronal fate in the dorsal neural tube

The basic helix–loop–helix (bHLH) transcriptional activator Ptf1a determines inhibitory GABAergic over excitatory glutamatergic neuronal cell fate in progenitors of the vertebrate dorsal spinal cord, cerebellum and retina. In an in situ hybridization expression survey of PR domain containing genes encoding putative chromatin-remodeling zinc finger transcription factors in Xenopus embryos, we identified Prdm13 as a histone methyltransferase belonging to the Ptf1a synexpression group. Gain and loss of Ptf1a function analyses in both frog and mice indicates that Prdm13 is positively regulated by Ptf1a and likely constitutes a direct transcriptional target. We also showed that this regulation requires the formation of the Ptf1a–Rbp-j complex. Prdm13 knockdown in Xenopus embryos and in Ptf1a overexpressing ectodermal explants lead to an upregulation of Tlx3/Hox11L2, which specifies a glutamatergic lineage and a reduction of the GABAergic neuronal marker Pax2. It also leads to an upregulation of Prdm13 transcription, suggesting an autonegative regulation. Conversely, in animal caps, Prdm13 blocks the ability of the bHLH factor Neurog2 to activate Tlx3. Additional gain of function experiments in the chick neural tube confirm that Prdm13 suppresses Tlx3+/glutamatergic and induces Pax2+/GABAergic neuronal fate. Thus, Prdm13 is a novel crucial component of the Ptf1a regulatory pathway that, by modulating the transcriptional activity of bHLH factors such as Neurog2, controls the balance between GABAergic and glutamatergic neuronal fate in the dorsal and caudal part of the vertebrate neural tube.

Expression of Six bHLH Superfamily Members in Mouse Uterus During Early Pregnancy

bHLH transcript factor superfamily members involve in various bio-events in mice. According to existing researches, there are six members of bHLH superfamily likely to participate in the uterine development during early pregnancy in mice. Expression of six members of bHLH superfamily Hes6, Tcf23, Twist1, Twist2, Mitf and Tead4 in mouse uterus in early pregnancy was detected by RT-qPCR. All the six genes were expressed in early pregnancy mouse uterus, and expression pattern of each one was different from others. In situ hybridization results showed that Tead4 was expressed in decidual tissues of the 8th day pregnancy mouse. Tead4 might be involved in the regulation of embryo implantation in mice.

Evolutionary Aspects of Variability in bHLH Orthologous Families: Insights from the Pearl Oyster, Pinctada fucata

Basic helix-loop-helix (bHLH) transcription factors play significant roles in multiple biological processes in metazoan cells. In recent work, we showed that three orthologous HLH families, pearl, amber, and peridot, have apparently been lost in the Drosophila melanogaster, Caenorhabditis elegans, and Homo sapiens lineages. To further address the gain and loss of bHLH proteins during bilaterian evolution, we examined the genome of the pearl oyster, Pinctada fucata, which has recently been sequenced. We characterized the putative full set 65 bHLH genes and showed that genes previously categorized into the orthologous family PTFb, actually fall into two distinct orthologous families, 48-related-1 and 48-related-2. We also identified a novel orthologous family, clockwork orange. Based on these newly identified orthologous family members and on orphan bHLH factors, we propose that genes encoding bHLH factors in bilaterians are not as evolutionarily stable as previously thought.

Uncovering the roadmap to hematopoietic stem cells

Hematopoietic stem cells (HSC) emerge from hemogenic endothelium during development and then become responsible for sustaining lifelong hematopoiesis. To succeed in generating HSCs from pluripotent stem cells, it is critical to define the intrinsic and extrinsic mechanisms that establish hemogenic endothelium and direct these precursors to multipotent, self-renewing HSCs rather than transient embryonic progenitors. Hematopoietic stem/progenitor cells (HS/PC) are generated in multiple anatomical locations, including the yolk sac, the AGM and the placenta. The bHLH factor Scl/tal1 dictates the specification of embryonic endothelium to hematopoietic fate; in the absence of Scl, the prospective hemogenic endothelium becomes mis-specified to cardiac fate. Scl binds to poised enhancers in lateral plate mesoderm and both activates a broad gene regulatory network required for HS/PC development and represses regulators of cardiogenesis. Surprisingly, Scl partners Gata 1 and 2 are dispensable for hematopoietic vs. cardiac specification although they enable Scl to activate transcription factors required for HS/PC emergence from hemogenic endothelium. Several signaling pathways promote the generation of HS/PCs form hemogenic endothelium. Our work suggests that the establishment of lymphoid potential in hemogenic endothelium is sensitive to the dosage of VEGF. Although VEGF haploinsufficient embryos can generate primitive erythroid and transient erythro-myeloid progenitors in the yolk sac, the generation of multipotent HS/PCs with lymphoid potential becomes severely impaired in all hemogenic tissues. Modulation of VEGF levels may thus have an impact on the types of hematopoietic cells that are generated from pluripotent cells. Work by us and others indicate that human ES cells can generate myeloerythroid and lymphoid (at least T) cells in culture, evidencing establishment of multipotent hemogenic endothelium. However, these progenitors lack self-renewal ability and lose proliferative potential prematurely. Our data suggest that the inability to induce critical self-renewal regulators such as the HOXA cluster genes during HS/PC differentiation from hESCs compromises developmental maturation and self-renewal of these HS/PCs. Studies focusing on the mechanisms that establish HSC self-renewal during development will be critical for succeeding in the generation of self-renewing HSCs from pluripotent cells for therapeutic applications. Hematopoietic stem cells (HSC) emerge from hemogenic endothelium during development and then become responsible for sustaining lifelong hematopoiesis. To succeed in generating HSCs from pluripotent stem cells, it is critical to define the intrinsic and extrinsic mechanisms that establish hemogenic endothelium and direct these precursors to multipotent, self-renewing HSCs rather than transient embryonic progenitors. Hematopoietic stem/progenitor cells (HS/PC) are generated in multiple anatomical locations, including the yolk sac, the AGM and the placenta. The bHLH factor Scl/tal1 dictates the specification of embryonic endothelium to hematopoietic fate; in the absence of Scl, the prospective hemogenic endothelium becomes mis-specified to cardiac fate. Scl binds to poised enhancers in lateral plate mesoderm and both activates a broad gene regulatory network required for HS/PC development and represses regulators of cardiogenesis. Surprisingly, Scl partners Gata 1 and 2 are dispensable for hematopoietic vs. cardiac specification although they enable Scl to activate transcription factors required for HS/PC emergence from hemogenic endothelium. Several signaling pathways promote the generation of HS/PCs form hemogenic endothelium. Our work suggests that the establishment of lymphoid potential in hemogenic endothelium is sensitive to the dosage of VEGF. Although VEGF haploinsufficient embryos can generate primitive erythroid and transient erythro-myeloid progenitors in the yolk sac, the generation of multipotent HS/PCs with lymphoid potential becomes severely impaired in all hemogenic tissues. Modulation of VEGF levels may thus have an impact on the types of hematopoietic cells that are generated from pluripotent cells. Work by us and others indicate that human ES cells can generate myeloerythroid and lymphoid (at least T) cells in culture, evidencing establishment of multipotent hemogenic endothelium. However, these progenitors lack self-renewal ability and lose proliferative potential prematurely. Our data suggest that the inability to induce critical self-renewal regulators such as the HOXA cluster genes during HS/PC differentiation from hESCs compromises developmental maturation and self-renewal of these HS/PCs. Studies focusing on the mechanisms that establish HSC self-renewal during development will be critical for succeeding in the generation of self-renewing HSCs from pluripotent cells for therapeutic applications.

Abstract 356: Scl Represses Cardiogenesis via Distant Enhancers during Hemogenic Endothelium Specification

Understanding the mechanisms directing mesoderm specification holds a great potential to advance the development of cell-based therapies for cardiovascular and blood disorders. The bHLH transcription factor Scl is known as the master regulator of the hematopoietic fate. We recently discovered that, in addition to its critical function in promoting the establishment of hemogenic endothelium during hematopoietic stem/progenitor cell (HS/PC) development, Scl is also required to repress cardiomyogenesis in endothelium in hematopoietic tissues and endocardium in the heart. However, the mechanisms for the cardiac repression have remained unknown. Using ChIP-sequencing and microarray analysis of Flk+ mesoderm differentiated from mouse ES cells, we show that Scl both directly activates a broad gene regulatory network required for hemogenic endothelium and HS/PC development (e.g. Runx1, cMyb, Lyl1, Mef2C, Sox7 etc.), and directly represses transcriptional regulators required for cardiogenesis (e.g. Gata4, Gata6, Myocd, etc.) and mesoderm development (Eomes, Mixl1, Etv2, etc.). Repression of cardiac and mesodermal programs occurs during a short developmental window through Scl binding to distant enhancers, while binding to hematopoietic regulators extends throughout HS/PC and red blood cell development and encompasses both distant and proximal binding sites. We also discovered that, surprisingly, Scl complex partners Gata 1 and 2 are dispensable for hematopoietic vs. cardiac specification and Scl binding to majority of its target genes. Nevertheless, Gata factors co-operate with Scl to activate selected transcription factors that facilitate HS/PC emergence from hemogenic endothelium. These results denote Scl as a true master regulator of hematopoietic vs. cardiac fate choice and suggest a mechanism by which lineage-specific bHLH factors direct the divergence of competing fates.

Abstract 136: Distinct Temporal Requirements for Scl to Repress Cardiomyogenesis in Endothelial-derived Cells in Hemogenic Tissues and the Heart

Identification of precursors with the capacity to generate cardiomyocytes is critical for advancing cardiac regenerative medicine. By analyzing knockout embryos for the bHLH factor Scl, we demonstrated that endothelial cells in hematopoietic tissues and the heart possess latent cardiomyogenic capacity. Furthermore, analysis of tamoxifen-inducible Rosa26-Cre ERT2 Scl fl/fl embryos suggested that the time window during which Scl is required for cardiac repression extends later in the heart versus the yolk sac. However, the cell types in which Scl acts remained elusive. We then deleted Scl in a cell-type specific manner in early mesoderm using Mesp1-Cre and in endothelial cells using Tie2-Cre. Lineage tracing in Mesp1-Cre Rosa26-YFP embryos demonstrated that at E9.5, a large majority of hematopoietic and endothelial cells in the yolk sac and heart were labeled. Moreover, deletion of Scl in Mesp1-Cre Scl fl/fl embryos phenocopied the germline knockout, essentially abrogating hematopoiesis and promoting the emergence of CD31 + PDGFRα + cardiomyogenic precursors and ectopic expression of the cardiomyocyte genes Myl7 and Tnnt2 in yolk sac vasculature. In contrast, deletion of Scl after endothelium had been specified in Tie2-Cre Scl fl/fl embryos did not grossly affect yolk sac hematopoiesis, nor did it induce ectopic cardiomyogenesis in hemogenic tissues. However, endothelial-derived cells in the hearts of Tie2-Cre Scl fl/fl embryos evidenced profound expansion of CD31 + PDGFRα + cardiogenic precursors at E11.5 and E13.5, as well as displayed dramatic upregulation of Myl7 and Tnnt2 , showing that the requirement for Scl to repress the cardiomyogenic program extends longer in endothelial derivatives in the heart than in the yolk sac. These data demonstrate that endocardial-derived cells in the heart retain latent cardiomyogenic potential until mid-gestation and nominate Scl as a critical regulator of endocardial fate.

The bHLH Subgroup IIId Factors Negatively Regulate Jasmonate-Mediated Plant Defense and Development

Plants have evolved sophisticated systems for adaptation to their natural habitat. In response to developmental and environmental cues, plants produce and perceive jasmonate (JA) signals, which induce degradation of JASMONATE-ZIM-Domain (JAZ) proteins and derepress the JAZ-repressed transcription factors to regulate diverse aspects of defense responses and developmental processes. Here, we identified the bHLH subgroup IIId transcription factors (bHLH3, bHLH13, bHLH14 and bHLH17) as novel targets of JAZs. These bHLH subgroup IIId transcription factors act as transcription repressors and function redundantly to negatively regulate JA responses. The quadruple mutant bhlh3 bhlh13 bhlh14 bhlh17 showed severe sensitivity to JA-inhibited root growth and JA-induced anthocyanin accumulation, and exhibited obvious increase in JA-regulated plant defense against pathogen infection and insect attack. Transgenic plants overexpressing bHLH13 or bHLH17 displayed reduced JA responses. Furthermore, these bHLH factors functioned as transcription repressors to antagonize the transcription activators, such as MYC2 and the WD-repeat/bHLH/MYB complex, through binding to their target sequences. Coordinated regulation of JA responses by transcription activators and repressors would benefit plants by allowing fine regulation of defense and development, and survival in their frequently changing environment.

RICE SALT SENSITIVE3 Forms a Ternary Complex with JAZ and Class-C bHLH Factors and Regulates Jasmonate-Induced Gene Expression and Root Cell Elongation

Plasticity of root growth in response to environmental cues and stresses is a fundamental characteristic of land plants. However, the molecular basis underlying the regulation of root growth under stressful conditions is poorly understood. Here, we report that a rice nuclear factor, RICE SALT SENSITIVE3 (RSS3), regulates root cell elongation during adaptation to salinity. Loss of function of RSS3 only moderately inhibits cell elongation under normal conditions, but it provokes spontaneous root cell swelling, accompanied by severe root growth inhibition, under saline conditions. RSS3 is preferentially expressed in the root tip and forms a ternary complex with class-C basic helix-loop-helix (bHLH) transcription factors and JASMONATE ZIM-DOMAIN proteins, the latter of which are the key regulators of jasmonate (JA) signaling. The mutated protein arising from the rss3 allele fails to interact with bHLH factors, and the expression of a significant portion of JA-responsive genes is upregulated in rss3. These results, together with the known roles of JAs in root growth regulation, suggest that RSS3 modulates the expression of JA-responsive genes and plays a crucial role in a mechanism that sustains root cell elongation at appropriate rates under stressful conditions.