Major Developmental Transitions Research Articles

A change of heart (mitochondria)

Heart Mitochondria Mitochondria provide an essential source of energy to drive cellular processes and are particularly important in heart muscle cells (see the Perspective by Gottlieb and Bernstein). After birth, the availability of oxygen and nutrients to organs and tissues changes. This invokes changes in metabolism. Gong et al. studied the developmental transitions in mouse heart mitochondria soon after birth. Mitochondria were replaced wholesale via mitophagy in cardiomyocytes over the first 3 weeks after birth. Preventing this turnover by interfering with parkin-mediated mitophagy specifically in cardiomyocytes prevented the normal metabolic transition and caused heart failure. Thus, the heart has coopted a quality-control pathway to facilitate a major developmental transition after birth. Wai et al. examined the role of mitochondrial fission and fusion in mouse cardiomyocytes. Disruption of these processes led to “middle-aged” death from a form of dilated cardiomyopathy. Mice destined to develop cardiomyopathy were protected by feeding with a high-fat diet, which altered cardiac metabolism. Science , this issue p. [10.1126/science.aad2459][1], p. [10.1126/science.aad0116][2]; see also p. [1162][3] [1]: /lookup/doi/10.1126/science.aad2459 [2]: /lookup/doi/10.1126/science.aad0116 [3]: /lookup/volpage/350/1162?iss=6265

Imbalanced OPA1 processing and mitochondrial fragmentation cause heart failure in mice.

Mitochondrial morphology is shaped by fusion and division of their membranes. Here, we found that adult myocardial function depends on balanced mitochondrial fusion and fission, maintained by processing of the dynamin-like guanosine triphosphatase OPA1 by the mitochondrial peptidases YME1L and OMA1. Cardiac-specific ablation of Yme1l in mice activated OMA1 and accelerated OPA1 proteolysis, which triggered mitochondrial fragmentation and altered cardiac metabolism. This caused dilated cardiomyopathy and heart failure. Cardiac function and mitochondrial morphology were rescued by Oma1 deletion, which prevented OPA1 cleavage. Feeding mice a high-fat diet or ablating Yme1l in skeletal muscle restored cardiac metabolism and preserved heart function without suppressing mitochondrial fragmentation. Thus, unprocessed OPA1 is sufficient to maintain heart function, OMA1 is a critical regulator of cardiomyocyte survival, and mitochondrial morphology and cardiac metabolism are intimately linked.

Parkin-mediated mitophagy directs perinatal cardiac metabolic maturation in mice.

In developing hearts, changes in the cardiac metabolic milieu during the perinatal period redirect mitochondrial substrate preference from carbohydrates to fatty acids. Mechanisms responsible for this mitochondrial plasticity are unknown. Here, we found that PINK1-Mfn2-Parkin-mediated mitophagy directs this metabolic transformation in mouse hearts. A mitofusin (Mfn) 2 mutant lacking PINK1 phosphorylation sites necessary for Parkin binding (Mfn2 AA) inhibited mitochondrial Parkin translocation, suppressing mitophagy without impairing mitochondrial fusion. Cardiac Parkin deletion or expression of Mfn2 AA from birth, but not after weaning, prevented postnatal mitochondrial maturation essential to survival. Five-week-old Mfn2 AA hearts retained a fetal mitochondrial transcriptional signature without normal increases in fatty acid metabolism and mitochondrial biogenesis genes. Myocardial fatty acylcarnitine levels and cardiomyocyte respiration induced by palmitoylcarnitine were concordantly depressed. Thus, instead of transcriptional reprogramming, fetal cardiomyocyte mitochondria undergo perinatal Parkin-mediated mitophagy and replacement by mature adult mitochondria. Mitophagic mitochondrial removal underlies developmental cardiomyocyte mitochondrial plasticity and metabolic transitioning of perinatal hearts.

Identification of novel tylosin analogues generated by a wblA disruption mutant of Streptomyces ansochromogenes.

BackgroundStreptomyces, as the main source of antibiotics, has been intensively exploited for discovering new drug candidates to combat the evolving pathogens. Disruption of wblA, an actinobacteria-specific gene controlling major developmental transition, can cause the alteration of phenotype and morphology in many species of Streptomyces. One wblA homologue was found in Streptomyces ansochromogenes 7100 by using the Basic Local Alignment Search Tool. It is interesting to identify whether novel secondary metabolites could be produced by the wblA disruption mutant as evidenced in other Streptomyces.ResultsThe wblA disruption mutant of S. ansochromogenes 7100 (ΔwblA) was constructed by homologous recombination. ΔwblA failed to produce spores and nikkomycin, the major product of S. ansochromogenes 7100 (wild-type strain) during fermentation. Antibacterial activity against Staphylococcus aureus and Bacillus cereus was observed with fermentation broth of ΔwblA but not with that of the wild-type strain. To identify the antibacterial compounds, the two compounds (compound 1 and compound 2) produced by ΔwblA were characterized as 16-membered macrolides by mass spectrometry and nuclear magnetic resonance spectroscopy. The chemical structure of these compounds shows similarity with tylosin, and the bioassays indicated that the two compounds inhibited the growth of a number of gram-positive bacteria. It is intriguing that they displayed much higher activity than tylosin against Streptococcus pneumoniae.ConclusionsTwo novel tylosin analogues (compound 1 and 2) were generated by ΔwblA. Bioassays showed that compound 1 and 2 displayed much higher activity than tylosin against Streptococcus pneumoniae, implying that these two compounds might be used to widen the application of tylosin.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0359-5) contains supplementary material, which is available to authorized users.

Integration of Light and Photoperiodic Signaling in Transcriptional Nuclear Foci

Light regulates major plant developmental transitions by orchestrating a series of nuclear events. This study uncovers the molecular function of the natural variant, TZP (Tandem Zinc-finger-Plus3), as a signal integrator of light and photoperiodic pathways in transcriptional nuclear foci. We report that TZP acts as a positive regulator of photoperiodic flowering via physical interactions with the red-light receptor phytochrome B (phyB). We demonstrate that TZP localizes in dynamic nuclear domains regulated by light quality and photoperiod. This study shows that phyB is indispensable not only for localizing TZP to transcriptionally active nuclear photobodies, but also for recruiting TZP on the promoter of the floral inducer FLOWERING LOCUS T (FT). Our findings signify a unique transcriptional regulatory role to the highly enigmatic plant nuclear photobodies, where TZP directly activates FT gene expression and promotes flowering.

Analysis of gene–environment interactions in postnatal development of the mammalian intestine

Unlike mammalian embryogenesis, which takes place in the relatively predictable and stable environment of the uterus, postnatal development can be affected by a multitude of highly variable environmental factors, including diet, exposure to noxious substances, and microorganisms. Microbial colonization of the intestine is thought to play a particularly important role in postnatal development of the gastrointestinal, metabolic, and immune systems. Major changes in environmental exposure occur right after birth, upon weaning, and during pubertal maturation into adulthood. These transitions include dramatic changes in intestinal contents and require appropriate adaptations to meet changes in functional demands. Here, we attempt to both characterize and provide mechanistic insights into postnatal intestinal ontogeny. We investigated changes in global intestinal gene expression through postnatal developmental transitions. We report profound alterations in small and large intestinal transcriptional programs that accompany both weaning and puberty in WT mice. Using myeloid differentiation factor 88 (MyD88)/TIR-domain-containing adapter-inducing interferon-β (TRIF) double knockout littermates, we define the role of toll-like receptors (TLRs) and interleukin (IL)-1 receptor family member signaling in postnatal gene expression programs and select ontogeny-specific phenotypes, such as vascular and smooth muscle development and neonatal epithelial and mast cell homeostasis. Metaanalysis of the effect of the microbiota on intestinal gene expression allowed for mechanistic classification of developmentally regulated genes by TLR/IL-1R (TIR) signaling and/or indigenous microbes. We find that practically every aspect of intestinal physiology is affected by postnatal transitions. Developmental timing, microbial colonization, and TIR signaling seem to play distinct and specific roles in regulation of gene-expression programs throughout postnatal development.

Developmental regulation of ecdysone receptor (EcR) and EcR-controlled gene expression during pharate-adult development of honeybees (Apis mellifera).

Major developmental transitions in multicellular organisms are driven by steroid hormones. In insects, these, together with juvenile hormone (JH), control development, metamorphosis, reproduction and aging, and are also suggested to play an important role in caste differentiation of social insects. Here, we aimed to determine how EcR transcription and ecdysteroid titers are related during honeybee postembryonic development and what may actually be the role of EcR in caste development of this social insect. In addition, we expected that knocking-down EcR gene expression would give us information on the participation of the respective protein in regulating downstream targets of EcR. We found that in Apis mellifera females, EcR-A is the predominantly expressed variant in postembryonic development, while EcR-B transcript levels are higher in embryos, indicating an early developmental switch in EcR function. During larval and pupal stages, EcR-B expression levels are very low, while EcR-A transcripts are more variable and abundant in workers compared to queens. Strikingly, these transcript levels are opposite to the ecdysteroid titer profile. 20-hydroxyecdysone (20E) application experiments revealed that low 20E levels induce EcR expression during development, whereas high ecdysteroid titers seem to be repressive. By means of RNAi-mediated knockdown (KD) of both EcR transcript variants we detected the differential expression of 234 poly-A+ transcripts encoding genes such as CYPs, MRJPs and certain hormone response genes (Kr-h1 and ftz-f1). EcR-KD also promoted the differential expression of 70 miRNAs, including highly conserved ones (e.g., miR-133 and miR-375), as well honeybee-specific ones (e.g., miR-3745 and miR-3761). Our results put in evidence a broad spectrum of EcR-controlled gene expression during postembryonic development of honeybees, revealing new facets of EcR biology in this social insect.

Nuclear Size Scaling during Xenopus Early Development Contributes to Midblastula Transition Timing

Early Xenopus laevis embryogenesis is a robust system for investigating mechanisms of developmental timing. After a series of rapid cell divisions with concomitant reductions in cell size, the first major developmental transition is the midblastula transition (MBT), when zygotic transcription begins and cell cycles elongate. Whereas the maintenance of a constant nuclear-to-cytoplasmic (N/C) volume ratio is a conserved cellular property, it has long been recognized that the N/C volume ratio changes dramatically during early Xenopus development. We investigated how changes in nuclear size and the N/C volume ratio during early development contribute to the regulation of MBT timing. Whereas previous studies suggested a role for the N/C volume ratio in MBT timing, none directly tested the effects of altering nuclear size. In this study, we first quantify blastomere and nuclear sizes in X. laevis embryos, demonstrating that the N/C volume ratio increases prior to the MBT. We then manipulate nuclear volume in embryos by microinjecting different nuclear scaling factors, including import proteins, lamins, and reticulons. Using this approach, we show that increasing the N/C volume ratio in pre-MBT embryos leads to premature activation of zygotic gene transcription and early onset of longer cell cycles. Conversely, decreasing the N/C volume ratio delays zygotic transcription and leads to additional rapid cell divisions. Whereas the DNA-to-cytoplasmic ratio has been implicated in MBT timing, our data show that nuclear size also contributes to the regulation of MBT timing, demonstrating the functional significance of nuclear size during development.

The POU factor ventral veins lacking/Drifter directs the timing of metamorphosis through ecdysteroid and juvenile hormone signaling.

Although endocrine changes are known to modulate the timing of major developmental transitions, the genetic mechanisms underlying these changes remain poorly understood. In insects, two developmental hormones, juvenile hormone (JH) and ecdysteroids, are coordinated with each other to induce developmental changes associated with metamorphosis. However, the regulation underlying the coordination of JH and ecdysteroid synthesis remains elusive. Here, we examined the function of a homolog of the vertebrate POU domain protein, Ventral veins lacking (Vvl)/Drifter, in regulating both of these hormonal pathways in the red flour beetle, Tribolium castaneum (Tenebrionidae). RNA interference-mediated silencing of vvl expression led to both precocious metamorphosis and inhibition of molting in the larva. Ectopic application of a JH analog on vvl knockdown larvae delayed the onset of metamorphosis and led to a prolonged larval stage, indicating that Vvl acts upstream of JH signaling. Accordingly, vvl knockdown also reduced the expression of a JH biosynthesis gene, JH acid methyltransferase 3 (jhamt3). In addition, ecdysone titer and the expression of the ecdysone response gene, hormone receptor 3 (HR3), were reduced in vvl knockdown larvae. The expression of the ecdysone biosynthesis gene phantom (phm) and spook (spo) were reduced in vvl knockdown larvae in the anterior and posterior halves, respectively, indicating that Vvl might influence ecdysone biosynthesis in both the prothoracic gland and additional endocrine sources. Injection of 20-hydroxyecdysone (20E) into vvl knockdown larvae could restore the expression of HR3 although molting was never restored. These findings suggest that Vvl coordinates both JH and ecdysteroid biosynthesis as well as molting behavior to influence molting and the timing of metamorphosis. Thus, in both vertebrates and insects, POU factors modulate the production of major neuroendocrine regulators during sexual maturation.

Comparative transcriptomic analysis of the regulation of flowering in temperate and tropical lotus (Nelumbo nucifera) by RNA‐Seq

AbstractThe switch from vegetative to reproductive growth is a major developmental transition in flowering plants, which depends on the balanced expression of the genes within a complex network that is controlled by both environmental and endogenous factors. Molecular regulation of flower development has been investigated extensively in model plants, particularly Arabidopsis. However, little is known about the mechanisms that regulate flowering in lotus. To analyse the molecular regulation of flowering in lotus, comparative transcript profiling was performed at two stages of bud development – the initial developmental stage (T1) and the fast developing stage (T2) – in the lotus cultivars ‘BG’ (B, temperate lotus) and ‘WR1’ (W, tropical lotus). A total of 140 504 368 high‐quality 100‐bp reads were obtained and aligned against the lotus reference genome. Of the 23 361 genes assembled, at least 88% of these transcripts were detected in each sample. These genes were significantly enriched in 40 Gene Ontology terms and 236 Kyoto Encyclopedia of Genes and Genomes pathways. Further comparisons of the transcripts in the four libraries revealed that 1808, 1330, 785, 702, 1954, and 2050 genes were differentially expressed between BT1 and BT2, WT1 and WT2, BT1 and WT1, BT2 and WT2, BT1 and WT2 and BT2 and WT1 samples, respectively. Analysis of the four libraries identified 147 lotus flowering‐time genes homologous to genes that control flowering‐time pathways in other plants. Differential regulation of the COP1, CCA1, LHY, CO‐LIKE, VIN3, GAI, and FT genes, which participate in the photoperiod, vernalization and gibberellic acid pathways, suggested that they might control the early flowering of lotus. The extensive transcriptome dataset should provide a foundation for comparative gene expression studies on the regulation of flowering in lotus. Comparative transcriptomic analysis detected several differentially expressed genes and potential candidate genes required for early flowering in lotus. These results provide new insight into the molecular mechanisms that regulate flowering in lotus.

INO80-dependent regression of ecdysone-induced transcriptional responses regulates developmental timing in Drosophila

Sequential pulses of the steroid hormone ecdysone regulate the major developmental transitions in Drosophila, and the duration of each developmental stage is determined by the length of time between ecdysone pulses. Ecdysone regulates biological responses by directly initiating target gene transcription. In turn, these transcriptional responses are known to be self-limiting, with mechanisms in place to ensure regression of hormone-dependent transcription. However, the biological significance of these transcriptional repression mechanisms remains unclear. Here we show that the chromatin remodeling protein INO80 facilitates transcriptional repression of ecdysone-regulated genes during prepupal development. In ino80 mutant animals, inefficient repression of transcriptional responses to the late larval ecdysone pulse delays the onset of the subsequent prepupal ecdysone pulse, resulting in a significantly longer prepupal stage. Conversely, increased expression of ino80 is sufficient to shorten the prepupal stage by increasing the rate of transcriptional repression. Furthermore, we demonstrate that enhancing the rate of regression of the mid-prepupal competence factor βFTZ-F1 is sufficient to determine the timing of head eversion and thus the duration of prepupal development. Although ino80 is conserved from yeast to humans, this study represents the first characterization of a bona fide ino80 mutation in any metazoan, raising the possibility that the functions of ino80 in transcriptional repression and developmental timing are evolutionarily conserved.

Microarray and RNAi Analysis of P450s in Anopheles gambiae Male and Female Steroidogenic Tissues: CYP307A1 Is Required for Ecdysteroid Synthesis

In insects, the steroid hormone 20-hydroxyecdysone (20E) coordinates major developmental transitions. While the first and the final steps of 20E biosynthesis are characterized, the pathway from 7-dehydrocholesterol to 5β-ketodiol, commonly referred as the “black box”, remains hypothetical and whether there are still unidentified enzymes is unknown. The black box would include some oxidative steps, which are believed to be mediated by P450 enzymes. To identify new enzyme(s) involved in steroid synthesis, we analyzed by small-scale microarray the expression of all the genes encoding P450 enzymes of the malaria mosquito Anopheles gambiae in active steroidogenic organs of adults, ovaries from blood-fed females and male reproductive tracts, compared to inactive steroidogenic organs, ovaries from non-blood-fed females. Some genes encoding P450 enzymes were specifically overexpressed in female ovaries after a blood-meal or in male reproductive tracts but only three genes were found to be overexpressed in active steroidogenic organs of both females and males: cyp307a1, cyp4g16 and cyp6n1. Among these genes, only cyp307a1 has an expression pattern similar to other mosquito steroidogenic genes. Moreover, loss-of-function by transient RNAi targeting cyp307a1 disrupted ecdysteroid production demonstrating that this gene is required for ecdysteroid biosynthesis in Anopheles gambiae.

Genome-wide transcriptomic analysis of the sporophyte of the moss Physcomitrella patens.

Bryophytes, the most basal of the extant land plants, diverged at least 450 million years ago. A major feature of these plants is the biphasic alternation of generations between a dominant haploid gametophyte and a minor diploid sporophyte phase. These dramatic differences in form and function occur in a constant genetic background, raising the question of whether the switch from gametophyte-to-sporophyte development reflects major changes in the spectrum of genes being expressed or alternatively whether only limited changes in gene expression occur and the differences in plant form are due to differences in how the gene products are put together. This study performed replicated microarray analyses of RNA from several thousand dissected and developmentally staged sporophytes of the moss Physcomitrella patens, allowing analysis of the transcriptomes of the sporophyte and early gametophyte, as well as the early stages of moss sporophyte development. The data indicate that more significant changes in transcript profile occur during the switch from gametophyte to sporophyte than recently reported, with over 12% of the entire transcriptome of P. patens being altered during this major developmental transition. Analysis of the types of genes contributing to these differences supports the view of the early sporophyte being energetically and nutritionally dependent on the gametophyte, provides a profile of homologues to genes involved in angiosperm stomatal development and physiology which suggests a deeply conserved mechanism of stomatal control, and identifies a novel series of transcription factors associated with moss sporophyte development.

Metabolic and Transcriptional Reprogramming in Developing Soybean (Glycine max) Embryos

Soybean (Glycine max) seeds are an important source of seed storage compounds, including protein, oil, and sugar used for food, feed, chemical, and biofuel production. We assessed detailed temporal transcriptional and metabolic changes in developing soybean embryos to gain a systems biology view of developmental and metabolic changes and to identify potential targets for metabolic engineering. Two major developmental and metabolic transitions were captured enabling identification of potential metabolic engineering targets specific to seed filling and to desiccation. The first transition involved a switch between different types of metabolism in dividing and elongating cells. The second transition involved the onset of maturation and desiccation tolerance during seed filling and a switch from photoheterotrophic to heterotrophic metabolism. Clustering analyses of metabolite and transcript data revealed clusters of functionally related metabolites and transcripts active in these different developmental and metabolic programs. The gene clusters provide a resource to generate predictions about the associations and interactions of unknown regulators with their targets based on “guilt-by-association” relationships. The inferred regulators also represent potential targets for future metabolic engineering of relevant pathways and steps in central carbon and nitrogen metabolism in soybean embryos and drought and desiccation tolerance in plants.

Epigenetic and small RNA regulation of senescence

Leaf senescence is regulated through a complex regulatory network triggered by internal and external signals for the reprogramming of gene expression. In plants, the major developmental phase transitions and stress responses are under epigenetic control. In this review, the underlying molecular mechanisms are briefly discussed and evidence is shown that epigenetic processes are also involved in the regulation of leaf senescence. Changes in the chromatin structure during senescence, differential histone modifications determining active and inactive sites at senescence-associated genes and DNA methylation are addressed. In addition, the role of small RNAs in senescence regulation is discussed.

Conserved roles for Polycomb Repressive Complex 2 in the regulation of lateral organ development in Aquilegia x coerulea 'Origami'.

BackgroundEpigenetic regulation is necessary for maintaining gene expression patterns in multicellular organisms. The Polycomb Group (PcG) proteins form several complexes with important and deeply conserved epigenetic functions in both the plant and animal kingdoms. One such complex, the Polycomb Repressive Complex 2 (PRC2), is critical to many developmental processes in plants including the regulation of major developmental transitions. In addition, PRC2 restricts the expression domain of various transcription factor families in Arabidopsis, including the class I KNOX genes and several of the ABCE class MADS box genes. While the functions of these transcription factors are known to be deeply conserved, whether or not their regulation by PRC2 is similarly conserved remains an open question.ResultsHere we use virus-induced gene silencing (VIGS) to characterize the function of the PRC2 complex in lateral organ development of Aquilegia x coerulea ‘Origami’, a member of the lower eudicot order Ranunculales. Leaves with PRC2 down-regulation displayed a range of phenotypes including ruffled or curled laminae, additional lobing, and an increased frequency of higher order branching. Sepals and petals were also affected, being narrowed, distorted, or, in the case of the sepals, exhibiting partial homeotic transformation. Many of the petal limbs also had a particularly intense yellow coloration due to an accumulation of carotenoid pigments. We show that the A. x coerulea floral MADS box genes AGAMOUS1 (AqAG1), APETALA3-3 (AqAP3-3) and SEPALLATA3 (AqSEP3) are up-regulated in many tissues, while expression of the class I KNOX genes and several candidate genes involved in carotenoid production or degradation are largely unaffected.ConclusionsPRC2 targeting of several floral MADS box genes may be conserved in dicots, but other known targets do not appear to be. In the case of the type I KNOX genes, this may reflect a regulatory shift associated with the evolution of compound leaves.

Conversion of 3-oxo steroids into ecdysteroids triggers molting and expression of 20E-inducible genes in Drosophila melanogaster

Ecdysteroids, steroid hormones in insects, coordinate major developmental transitions. During postembryonic development, ecdysone is biosynthesized from dietary cholesterol in the prothoracic gland (PG). Despite extensive studies, the initial conversion process, the so-called “Black Box”, has not been characterized. A cytochrome P450 enzyme, Spookier (Spok), is speculated as a rate limiting enzyme in the Black Box during larval-pupal transitions in Drosophila melanogaster. RNAi mediated knockdown of spok expression in the PG results in arrest of molting. Because the developmental arrest can be rescued by application of an appropriate intermediate, we examined potential activities of candidate intermediates in the RNAi-treated larvae. We found that two 3-oxo steroids, cholesta-4,7-diene-3,6-dione-14α-ol (Δ4-diketol) and 5β [H]cholesta-7-ene-3,6-dione-14α-ol (diketol), triggered molting of the RNAi-treated larvae. We also detected an enhancement of the amounts of ecdysteroids in the RNAi-treated larvae by feeding the Δ4-diketol or diketol, indicating that the dietary 3-oxo steroids were incorporated and converted into ecdysteroids in vivo. Furthermore, 20-hydroxyecdysone inducible genes were induced in the RNAi-treated larvae by feeding the Δ4-diketol or diketol. These results indicate that Δ4-diketol and diketol are components of the ecdysteroid biosynthetic pathway and lie downstream of a step catalyzed by Spok.

Flowering time and transcriptome variation in Capsella bursa‐pastoris (Brassicaceae)

• Flowering is a major developmental transition and its timing in relation to environmental conditions is of crucial importance to plant fitness. Understanding the genetic basis of flowering time variation is important to determining how plants adapt locally. • Here, we investigated flowering time variation of Capsella bursa-pastoris collected from different latitudes in China. We also used a digital gene expression (DGE) system to generate partial gene expression profiles for 12 selected samples. • We found that flowering time was highly variable and most strongly correlated with day length and winter temperature. Significant differences in gene expression between early- and late-flowering samples were detected for 72 candidate genes for flowering time. Genes related to circadian rhythms were significantly overrepresented among the differentially expressed genes. • Our data suggest that circadian rhythms and circadian clock genes play an important role in the evolution of flowering time, and C. bursa-pastoris plants exhibit expression differences for candidate genes likely to affect flowering time across the broad range of environments they face in China.

Molecular and Functional Characterization of Early Lineage Commitment of Human Hematopoietic Stem Cells

Abstract 907The hematopoietic system is a highly regulated cellular hierarchy, responsible for the day-to-day production of mature blood cells which can be divided in two major lineages, myeloid and lymphoid. Hematopoietic stem cells (HSCs) have the unique ability to give rise to all hematopoietic cell types, by first generating lineage-commited progenitors which in turn will produce terminally differentiated cells. HSCs are characterized by their extensive self-renewal and differentiation capacities. While in mice the mechanisms underlying early HSC differentiation and lineage determination are well understood at the molecular level, very few transcription factors regulating lineage decisions have been identified in human hematopoiesis. Our group has recently established a novel cell sorting strategy for human HSCs and early lineage committed progenitors (Doulatov et al., Nature Immunology, 2010; Notta et al., Science, 2011) which uncovered the existence of a novel human multilymphoid progenitor (MLP). MLPs give rise to all lymphoid cell types, as well as dendritic cells and monocytic cells.Here we report a comprehensive analysis of gene expression at each developmental stage of the early human hematopoietic hierarchy, ranging from the long-term repopulating stem cells to lineage-restricted progenitors through multipotent progenitors such as MLP, CMP (common myeloid progenitor), GMP (granulocyte-monocyte progenitor) and MEP (megakaryocyte-erythroid progenitor). We show that hematopoietic specification is defined by a small number of global gene expression clusters that correspond to major biological lineages and that lineage programs in committed progenitors are paired with HSC-shared priming programs. HSCs display most extensive priming along the lympho-myeloid branch (MLP). In contrast early progenitors of the megakaryocytic/erythrocytic lineage form a distinct cluster, highly enriched for cell cycle genes.To identify regulators of each major developmental transition, we computationally extracted population-specific gene-sets (“signatures”). We then integrated transcription factor expression data and enrichment of transcription factors binding sites in the promoters of each “signature” to obtain a map of transcriptional regulators in the context of the developmental hierarchy. Based on this model, we selected more than 15 candidate genes for functional validation. We chose genes predicted to act either on lymphoid (MLP), myeloid (MLP, CMP) or erythroid (MEP) commitment. Among these, we investigated the function of BCL11a, a C2H2 zinc finger transcriptional repressor, which expression is primed in HSCs then peaks in the newly discovered MLP population, indicating a putative role in lymphocyte specification. Consistent with this hypothesis, BCL11a has been implicated in the development of B cell progenitors in mouse. When BCL11a was knocked down in cord blood derived hematopoietic stem cells and early progenitors, we observed reduced formation of cells committed to the B cell fate both in vitro and in an in vivo xenograft assay. BCL11a knock-down resulted in a partial block of B cell maturation at the proB to preB cell transition, that was accompanied by a decrease in the key B cell maturation transcription factor, Pax5. These preliminary results suggest that BCL11a directs B cell specification in human and that our genome-wide strategy not only provides a valuable resource for the hematology community but also allows identification of key regulators of early human lineage commitment. Disclosures:No relevant conflicts of interest to declare.

Short Day–Mediated Cessation of Growth Requires the Downregulation of AINTEGUMENTALIKE1 Transcription Factor in Hybrid Aspen

Day length is a key environmental cue regulating the timing of major developmental transitions in plants. For example, in perennial plants such as the long-lived trees of the boreal forest, exposure to short days (SD) leads to the termination of meristem activity and bud set (referred to as growth cessation). The mechanism underlying SD–mediated induction of growth cessation is poorly understood. Here we show that the AIL1-AIL4 (AINTEGUMENTALIKE) transcription factors of the AP2 family are the downstream targets of the SD signal in the regulation of growth cessation response in hybrid aspen trees. AIL1 is expressed in the shoot apical meristem and leaf primordia, and exposure to SD signal downregulates AIL1 expression. Downregulation of AIL gene expression by SDs is altered in transgenic hybrid aspen plants that are defective in SD perception and/or response, e.g. PHYA or FT overexpressors. Importantly, SD–mediated regulation of growth cessation response is also affected by overexpression or downregulation of AIL gene expression. AIL1 protein can interact with the promoter of the key cell cycle genes, e.g. CYCD3.2, and downregulation of the expression of D-type cyclins after SD treatment is prevented by AIL1 overexpression. These data reveal that execution of SD–mediated growth cessation response requires the downregulation of AIL gene expression. Thus, while early acting components like PHYA and the CO/FT regulon are conserved in day-length regulation of flowering time and growth cessation between annual and perennial plants, signaling pathways downstream of SD perception diverge, with AIL transcription factors being novel targets of the CO/FT regulon connecting the perception of SD signal to the regulation of meristem activity.