The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans

The C. elegans heterochronic gene pathway consists of a cascade of regulatory genes that are temporally controlled to specify the timing of developmental events. Mutations in heterochronic genes cause temporal transformations in cell fates in which stage-specific events are omitted or reiterated. Here we show that let-7 is a heterochronic switch gene. Loss of let-7 gene activity causes reiteration of larval cell fates during the adult stage, whereas increased let-7 gene dosage causes precocious expression of adult fates during larval stages. let-7 encodes a temporally regulated 21-nucleotide RNA that is complementary to elements in the 3' untranslated regions of the heterochronic genes lin-14, lin-28, lin-41, lin-42 and daf-12, indicating that expression of these genes may be directly controlled by let-7. A reporter gene bearing the lin-41 3' untranslated region is temporally regulated in a let-7-dependent manner. A second regulatory RNA, lin-4, negatively regulates lin-14 and lin-28 through RNA-RNA interactions with their 3' untranslated regions. We propose that the sequential stage-specific expression of the lin-4 and let-7 regulatory RNAs triggers transitions in the complement of heterochronic regulatory proteins to coordinate developmental timing.     

The stem-cell niche as an entity of action

Stem-cell populations are established in 'niches'--specific anatomic locations that regulate how they participate in tissue generation, maintenance and repair. The niche saves stem cells from depletion, while protecting the host from over-exuberant stem-cell proliferation. It constitutes a basic unit of tissue physiology, integrating signals that mediate the balanced response of stem cells to the needs of organisms. Yet the niche may also induce pathologies by imposing aberrant function on stem cells or other targets. The interplay between stem cells and their niche creates the dynamic system necessary for sustaining tissues, and for the ultimate design of stem-cell therapeutics.     

Dominant role of the niche in melanocyte stem-cell fate determination

Stem cells which have the capacity to self-renew and generate differentiated progeny are thought to be maintained in a specific environment known as a niche. The localization of the niche, however, remains largely obscure for most stem-cell systems. Melanocytes (pigment cells) in hair follicles proliferate and differentiate closely coupled to the hair regeneration cycle. Here we report that stem cells of the melanocyte lineage can be identified, using Dct-lacZ transgenic mice, in the lower permanent portion of mouse hair follicles throughout the hair cycle. It is only the population in this region that fulfils the criteria for stem cells, being immature, slow cycling, self-maintaining and fully competent in regenerating progeny on activation at early anagen (the growing phase of hair follicles). Induction of the re-pigmentation process in K14-steel factor transgenic mice demonstrates that a portion of amplifying stem-cell progeny can migrate out from the niche and retain sufficient self-renewing capability to function as stem cells after repopulation into vacant niches. Our data indicate that the niche has a dominant role in the fate determination of melanocyte stem-cell progeny.     

In vivo imaging of Treg cells providing immune privilege to the haematopoietic stem-cell niche

Stem cells reside in a specialized regulatory microenvironment or niche, where they receive appropriate support for maintaining self-renewal and multi-lineage differentiation capacity. The niche may also protect stem cells from environmental insults including cytotoxic chemotherapy and perhaps pathogenic immunity. The testis, hair follicle and placenta are all sites of residence for stem cells and are immune-suppressive environments, called immune-privileged sites, where multiple mechanisms cooperate to prevent immune attack, even enabling prolonged survival of foreign allografts without immunosuppression. We sought to determine if somatic stem-cell niches more broadly are immune-privileged sites by examining the haematopoietic stem/progenitor cell (HSPC) niche in the bone marrow, a site where immune reactivity exists. We observed persistence of HSPCs from allogeneic donor mice (allo-HSPCs) in non-irradiated recipient mice for 30?days without immunosuppression with the same survival frequency compared to syngeneic HSPCs. These HSPCs were lost after the depletion of FoxP3 regulatory T (T(reg)) cells. High-resolution in vivo imaging over time demonstrated marked co-localization of HSPCs with T(reg) cells that accumulated on the endosteal surface in the calvarial and trabecular bone marrow. T(reg) cells seem to participate in creating a localized zone where HSPCs reside and where T(reg) cells are necessary for allo-HSPC persistence. In addition to processes supporting stem-cell function, the niche will provide a relative sanctuary from immune attack.     

The ageing systemic milieu negatively regulates neurogenesis and cognitive function

In the central nervous system, ageing results in a precipitous decline in adult neural stem/progenitor cells and neurogenesis, with concomitant impairments in cognitive functions. Interestingly, such impairments can be ameliorated through systemic perturbations such as exercise. Here, using heterochronic parabiosis we show that blood-borne factors present in the systemic milieu can inhibit or promote adult neurogenesis in an age-dependent fashion in mice. Accordingly, exposing a young mouse to an old systemic environment or to plasma from old mice decreased synaptic plasticity, and impaired contextual fear conditioning and spatial learning and memory. We identify chemokines--including CCL11 (also known as eotaxin)--the plasma levels of which correlate with reduced neurogenesis in heterochronic parabionts and aged mice, and the levels of which are increased in the plasma and cerebrospinal fluid of healthy ageing humans. Lastly, increasing peripheral CCL11 chemokine levels in vivo in young mice decreased adult neurogenesis and impaired learning and memory. Together our data indicate that the decline in neurogenesis and cognitive impairments observed during ageing can be in part attributed to changes in blood-borne factors.     

mTORC1 in the Paneth cell niche couples intestinal stem-cell function to calorie intake

How adult tissue stem and niche cells respond to the nutritional state of an organism is not well understood. Here we find that Paneth cells, a key constituent of the mammalian intestinal stem-cell (ISC) niche, augment stem-cell function in response to calorie restriction. Calorie restriction acts by reducing mechanistic target of rapamycin complex 1 (mTORC1) signalling in Paneth cells, and the ISC-enhancing effects of calorie restriction can be mimicked by rapamycin. Calorie intake regulates mTORC1 in Paneth cells, but not ISCs, and forced activation of mTORC1 in Paneth cells during calorie restriction abolishes the ISC-augmenting effects of the niche. Finally, increased expression of bone stromal antigen 1 (Bst1) in Paneth cells—an ectoenzyme that produces the paracrine factor cyclic ADP ribose—mediates the effects of calorie restriction and rapamycin on ISC function. Our findings establish that mTORC1 non-cell-autonomously regulates stem-cell self-renewal, and highlight a significant role of the mammalian intestinal niche in coupling stem-cell function to organismal physiology.     

Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA

Two small RNAs regulate the timing of Caenorhabditis elegans development. Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA, and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA. The lin-4 and let-7 RNA genes are not homologous to each other, but are each complementary to sequences in the 3' untranslated regions of a set of protein-coding target genes that are normally negatively regulated by the RNAs. Here we have detected let-7 RNAs of approximately 21 nucleotides in samples from a wide range of animal species, including vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from several cnidarian and poriferan species, Saccharomyces cerevisiae, Escherichia coli or Arabidopsis. We did not detect lin-4 RNA in these species. We found that let-7 temporal regulation is also conserved: let-7 RNA expression is first detected at late larval stages in C. elegans and Drosophila, at 48 hours after fertilization in zebrafish, and in adult stages of annelids and molluscs. The let-7 regulatory RNA may control late temporal transitions during development across animal phylogeny.     

The cell-surface proteoglycan Dally regulates Wingless signalling in Drosophila.

Wingless (Wg) is a member of the Wnt family of growth factors, secreted proteins that control proliferation and differentiation during development. Studies in Drosophila have shown that responses to Wg require cell-surface heparan sulphate, a glycosaminoglycan component of proteoglycans. These findings suggest that a cell-surface proteoglycan is a component of a Wg/Wnt receptor complex. We demonstrate here that the protein encoded by the division abnormally delayed (dally) gene is a cell-surface, heparan-sulphate-modified proteoglycan. dally partial loss-of-function mutations compromise Wg-directed events, and disruption of dally function with RNA interference produces phenotypes comparable to those found with RNA interference of wg or frizzled (fz)/Dfz2. Ectopic expression of Dally potentiates Wg signalling without altering levels of Wg and can rescue a wg partial loss-of-function mutant. We also show that dally, a regulator of Decapentaplegic (Dpp) signalling during post-embryonic development, has tissue-specific effects on Wg and Dpp signalling. Dally can therefore differentially influence signalling mediated by two growth factors, and may form a regulatory component of both Wg and Dpp receptor complexes.     

The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation

Vascular development is a complex but orderly process that is tightly regulated. A number of secreted factors produced by surrounding cells regulate endothelial cell (EC) differentiation, proliferation, migration and coalescence into cord-like structures. Vascular cords then undergo tubulogenesis to form vessels with a central lumen. But little is known about how tubulogenesis is regulated in vivo. Here we report the identification and characterization of a new EC-derived secreted factor, EGF-like domain 7 (Egfl7). Egfl7 is expressed at high levels in the vasculature associated with tissue proliferation, and is downregulated in most of the mature vessels in normal adult tissues. Loss of Egfl7 function in zebrafish embryos specifically blocks vascular tubulogenesis. We uncover a dynamic process during which gradual separation and proper spatial arrangement of the angioblasts allow subsequent assembly of vascular tubes. This process fails to take place in Egfl7 knockdown embryos, leading to the failure of vascular tube formation. Our study defines a regulator that controls a specific and important step in vasculogenesis.     

The future of ageing

Advances in our knowledge of age-associated diseases have far outpaced advances in our understanding of the fundamental ageing processes that underlie the vulnerability to these pathologies. If we are to increase human life expectancy beyond the fifteen-year limit that would result if today's leading causes of death were resolved, more attention must be paid to basic research on ageing. Determination of longevity must be distinguished from ageing to take us from the common question of why we age to a more revealing question that is rarely posed: why do we live as long as we do? But if the ability to intervene in ageing ever becomes a reality, it will be rife with unintended and undesirable consequences.     

Postnatal loss of Dlk1 imprinting in stem cells and niche astrocytes regulates neurogenesis

The gene for the atypical NOTCH ligand delta-like homologue 1 (Dlk1) encodes membrane-bound and secreted isoforms that function in several developmental processes in vitro and in vivo. Dlk1, a member of a cluster of imprinted genes, is expressed from the paternally inherited chromosome. Here we show that mice that are deficient in Dlk1 have defects in postnatal neurogenesis in the subventricular zone: a developmental continuum that results in depletion of mature neurons in the olfactory bulb. We show that DLK1 is secreted by niche astrocytes, whereas its membrane-bound isoform is present in neural stem cells (NSCs) and is required for the inductive effect of secreted DLK1 on self-renewal. Notably, we find that there is a requirement for Dlk1 to be expressed from both maternally and paternally inherited chromosomes. Selective absence of Dlk1 imprinting in both NSCs and niche astrocytes is associated with postnatal acquisition of DNA methylation at the germ-line-derived imprinting control region. The results emphasize molecular relationships between NSCs and the niche astrocyte cells of the microenvironment, identifying a signalling system encoded by a single gene that functions coordinately in both cell types. The modulation of genomic imprinting in a stem-cell environment adds a new level of epigenetic regulation to the establishment and maintenance of the niche, raising wider questions about the adaptability, function and evolution of imprinting in specific developmental contexts.     

Identification of the haematopoietic stem cell niche and control of the niche size

Haematopoietic stem cells (HSCs) are a subset of bone marrow cells that are capable of self-renewal and of forming all types of blood cells (multi-potential). However, the HSC 'niche'--the in vivo regulatory microenvironment where HSCs reside--and the mechanisms involved in controlling the number of adult HSCs remain largely unknown. The bone morphogenetic protein (BMP) signal has an essential role in inducing haematopoietic tissue during embryogenesis. We investigated the roles of the BMP signalling pathway in regulating adult HSC development in vivo by analysing mutant mice with conditional inactivation of BMP receptor type IA (BMPRIA). Here we show that an increase in the number of spindle-shaped N-cadherin+CD45- osteoblastic (SNO) cells correlates with an increase in the number of HSCs. The long-term HSCs are found attached to SNO cells. Two adherens junction molecules, N-cadherin and beta-catenin, are asymmetrically localized between the SNO cells and the long-term HSCs. We conclude that SNO cells lining the bone surface function as a key component of the niche to support HSCs, and that BMP signalling through BMPRIA controls the number of HSCs by regulating niche size.     

Mei-P26 regulates microRNAs and cell growth in the Drosophila ovarian stem cell lineage

Drosophila neuroblasts and ovarian stem cells are well characterized models for stem cell biology. In both cell types, one daughter cell self-renews continuously while the other undergoes a limited number of divisions, stops to proliferate mitotically and differentiates. Whereas neuroblasts segregate the Trim-NHL (tripartite motif and Ncl-1, HT2A and Lin-41 domain)-containing protein Brain tumour (Brat) into one of the two daughter cells, ovarian stem cells are regulated by an extracellular signal from the surrounding stem cell niche. After division, one daughter cell looses niche contact. It undergoes 4 transit-amplifying divisions to form a cyst of 16 interconnected cells that reduce their rate of growth and stop to proliferate mitotically. Here we show that the Trim-NHL protein Mei-P26 (refs 7, 8) restricts growth and proliferation in the ovarian stem cell lineage. Mei-P26 expression is low in stem cells but is strongly induced in 16-cell cysts. In mei-P26 mutants, transit-amplifying cells are larger and proliferate indefinitely leading to the formation of an ovarian tumour. Like brat, mei-P26 regulates nucleolar size and can induce differentiation in Drosophila neuroblasts, suggesting that these genes act through the same pathway. We identify Argonaute-1, a component of the RISC complex, as a common binding partner of Brat and Mei-P26, and show that Mei-P26 acts by inhibiting the microRNA pathway. Mei-P26 and Brat have a similar domain composition that is also found in other tumour suppressors and might be a defining property of a new family of microRNA regulators that act specifically in stem cell lineages.     

brinker is a target of Dpp in Drosophila that negatively regulates Dpp-dependent genes

Growth and patterning of the Drosophila wing is controlled in part by the long-range organizing activities of the Decapentaplegic protein (Dpp). Dpp is synthesized by cells that line the anterior side of the anterior/posterior compartment border of the wing imaginal disc. From this source, Dpp is thought to generate a concentration gradient that patterns both anterior and posterior compartments. Among the gene targets that it regulates are optomotor blind (omb), spalt (sal), and daughters against dpp (dad). We report here the molecular cloning of brinker (brk), and show that brk expression is repressed by dpp. brk encodes, a protein that negatively regulates Dpp-dependent genes. Expression of brk in Xenopus embryos indicates that brk can also repress the targets of a vertebrate homologue of Dpp, bone morphogenetic protein 4 (BMP-4). The evolutionary conservation of Brk function underscores the importance of its negative role in proportioning Dpp activity.     

双级时效对7A52铝合金组织与性能的影响

采用拉伸力学性能测试、电导率测定、透射电镜分析等手段研究双级时效处理条件下7A52铝合金的力学性能、电导率和显微组织结构。研究结果表明:7A52铝合金适宜的双级时效工艺为105℃/8 h 130℃/14 h。在此条件下,合金的抗拉强度、屈服强度、伸长率和电导率分别为500 MPa,444 MPa,11.1%和18.44 S/m。与单级峰值时效相比,强度并未明显降低而电导率有明显提高,表明合金抗应力腐蚀性能得到改善。双级时效后,合金的晶内组织为细小弥散分布的η′(MgZn2)相,晶界上有断续分布的晶界析出相MgZn2和较明显的无沉淀析出带。