Lin28 paralogs regulate lung branching morphogenesis.

The molecular mechanisms that govern the choreographed timing of organ development remain poorly understood. Our investigation of the role of the Lin28a and Lin28b paralogs during the developmental process of branching morphogenesis establishes that dysregulation of Lin28a/b leads to abnormal branching morphogenesis in the lung and other tissues. Additionally, we find that the Lin28 paralogs, which regulate post-transcriptional processing of both mRNAs and microRNAs (miRNAs), predominantly control mRNAs during the initial phases of lung organogenesis. Target mRNAs include Sox2, Sox9, and Etv5, which coordinate lung development and differentiation. Moreover, we find that functional interactions between Lin28a and Sox9 are capable of bypassing branching defects in Lin28a/b mutant lungs. Here, we identify Lin28a and Lin28b as regulators of early embryonic lung development, highlighting the importance of the timing of post-transcriptional regulation of both miRNAs and mRNAs at distinct stages of organogenesis.

Evaluación de la variabilidad fenotípica en Simarouba amara Aubl., mediante descriptores cualitativos y cuantitativos / Assessment of phenotypic variability in Simarouba amara Aubl., through qualitative and quantitative descriptors / Avaliação da variabilidade fenotípica em Simarouba amara Aubl.,usando descritores qualitativos e quantitativos

Resumen Dentro de las estrategias de evaluación de especies forestales nativas por parte de la Corporación Colombiana de Investigación Agropecuaria - AGROSAVIA, se encuentra la caracterización fenotípica. Con el propósito de validar el uso de descriptores morfológicos y determinar la variabilidad fenotípica de la colección de trabajo del Centro de Investigación La Libertad ubicado en el piedemonte llanero (Villavicencio, Meta); se evaluaron 121 individuos de machaco (Simarouba amara Aubl.) de 6.2 años de edad, por medio de 34 descriptores de planta, hoja y fruto. Los datos cualitativos y cuantitativos se analizaron mediante análisis de correspondencias múltiples y análisis de componentes principales respectivamente, con el fin de reducir la dimensionalidad de los datos, seguido de análisis de conglomerados, utilizando el algoritmo de WARD para el agrupamiento de los individuos. Los resultados obtenidos permitieron identificar que el volumen de tronco presentó la mayor variabilidad (31.13%) y las variables cuantitativas asociadas al tamaño y diámetro de la copa, diámetro de fuste, volumen del tronco, longitud y ancho de la hoja, altura total y de copa presentaron las mayores correlaciones con los tres primeros componentes principales (57.82%). Se obtuvieron nueve conglomerados que reúnen el 95.73% de la variabilidad original y en los cuales se encontraron individuos potencialmente útiles con fines maderables, arboricultura y sistemas agroforestales. La forma del fuste, la altura de ramificación y de bifurcación, el tipo de corteza y el hábito de fructificación presentaron mínima variación en los individuos, contrario a la rectitud del fuste y la forma de la copa.

Abstract Phenotyping is one of the strategies used by Corporación Colombiana de Investigación Agropecuaria (AGROSAVIA) for evaluating native forest species. One hundred and twenty-one 6.2 year-old mountain damson/stavewood/bitterwood/paradise trees (Simarouba amara Aubl. commonly known as machaco in Colombia) were evaluated using 34 plant, leaf and fruit descriptors to validate the use of morphological descriptors and determine the phenotype variability of AGROSAVIA's La Libertad Research Centre's working collection. The centre is located in the foothills of Colombia's eastern plains (Villavicencio, Meta). Multiple correspondence analysis (MCA) was used for analysing qualitative data and principal component analysis (PCA) for +data to reduce dataset dimensionality; this was followed by cluster analysis, using Ward's method (minimum variance method or Ward's minimum - agglomerative algorithm) for hierarchical cluster analysis for grouping the trees. The results led to identifying that the wood volume descriptor had the greatest variability (31.13%) and the quantitative variables associated with crown size and diameter, stem diameter, trunk volume, leaf length and width, total and crown height had the greatest correlation with the first three components (57.82%). Nine clusters were obtained (accounting for 95.73% of original variability) and in which trees were found for timber, arboriculture and agroforestry system purposes. The trees' stem shape, branch height and bifurcation, the type of bark and fruiting habits had minimum variation, contrary to the that found regarding stem straightness and crown shape.

Resumo Dentro das estratégias de avaliação de espécies florestais nativas pela Colombian Agricultural Research Corporation - AGROSAVIA, está a caracterização fenotípica. A fim de validar o uso de descritores morfológicos e determinar a variabilidade fenotípica da coleção de trabalhos do Centro de Pesquisa La Libertad localizado no sopé da planície, (Villavicencio, Meta); 121 indivíduos de machaco (Simarouba amara Aubl.) de 6,2 anos de idade foram avaliados por meio de 34 descritores de planta, folha e fruto. Os dados qualitativos e quantitativos foram analisados por meio da análise de correspondência múltipla e da análise de componentes principais, respectivamente, a fim de reduzir a dimensionalidade dos dados, seguida da análise de cluster, por meio do algoritmo WARD, para agrupamento dos indivíduos. Os resultados obtidos permitiram identificar que o volume do tronco apresentou a maior variabilidade (31,13%) e as variáveis quantitativas associadas ao tamanho e diâmetro da copa, diâmetro do caule, volume do tronco, comprimento e largura da folha, total a altura e a copa apresentaram as maiores correlações com os três primeiros componentes principais (57,82%). Foram obtidos nove aglomerados que atendem a 95,73% da variabilidade original e nos quais foram encontrados indivíduos potencialmente úteis para fins madeireiros, arboricultura e sistemas agroflorestais. O formato do caule, a altura da ramificação e bifurcação, o tipo de casca e o hábito de frutificação apresentaram variação mínima nos indivíduos, ao contrário da retidão do caule e do formato da copa.

Author Correction: A systems biology pipeline identifies regulatory networks for stem cell engineering.

In the version of this article initially published, the second NIH grant "R24-DK49216" to author George Q. Daley contained an error. The grant number should have read U54DK110805. The error has been corrected in the HTML and PDF versions of the article.

A systems biology pipeline identifies regulatory networks for stem cell engineering.

A major challenge for stem cell engineering is achieving a holistic understanding of the molecular networks and biological processes governing cell differentiation. To address this challenge, we describe a computational approach that combines gene expression analysis, previous knowledge from proteomic pathway informatics and cell signaling models to delineate key transitional states of differentiating cells at high resolution. Our network models connect sparse gene signatures with corresponding, yet disparate, biological processes to uncover molecular mechanisms governing cell fate transitions. This approach builds on our earlier CellNet and recent trajectory-defining algorithms, as illustrated by our analysis of hematopoietic specification along the erythroid lineage, which reveals a role for the EGF receptor family member, ErbB4, as an important mediator of blood development. We experimentally validate this prediction and perturb the pathway to improve erythroid maturation from human pluripotent stem cells. These results exploit an integrative systems perspective to identify new regulatory processes and nodes useful in cell engineering.

The developmental stage of the hematopoietic niche regulates lineage in MLL-rearranged leukemia.

Leukemia phenotypes vary with age of onset. Delineating mechanisms of age specificity in leukemia could improve disease models and uncover new therapeutic approaches. Here, we used heterochronic transplantation of leukemia driven by MLL/KMT2A translocations to investigate the contribution of the age of the hematopoietic microenvironment to age-specific leukemia phenotypes. When driven by MLL-AF9, leukemia cells in the adult microenvironment sustained a myeloid phenotype, whereas the neonatal microenvironment supported genesis of mixed early B cell/myeloid leukemia. In MLL-ENL leukemia, the neonatal microenvironment potentiated B-lymphoid differentiation compared with the adult. Ccl5 elaborated from adult marrow stroma inhibited B-lymphoid differentiation of leukemia cells, illuminating a mechanism of age-specific lineage commitment. Our study illustrates the contribution of the developmental stage of the hematopoietic microenvironment in defining the age specificity of leukemia.

A CLK3-HMGA2 Alternative Splicing Axis Impacts Human Hematopoietic Stem Cell Molecular Identity throughout Development.

While gene expression dynamics have been extensively cataloged during hematopoietic differentiation in the adult, less is known about transcriptome diversity of human hematopoietic stem cells (HSCs) during development. To characterize transcriptional and post-transcriptional changes in HSCs during development, we leveraged high-throughput genomic approaches to profile miRNAs, lincRNAs, and mRNAs. Our findings indicate that HSCs manifest distinct alternative splicing patterns in key hematopoietic regulators. Detailed analysis of the splicing dynamics and function of one such regulator, HMGA2, identified an alternative isoform that escapes miRNA-mediated targeting. We further identified the splicing kinase CLK3 that, by regulating HMGA2 splicing, preserves HMGA2 function in the setting of an increase in let-7 miRNA levels, delineating how CLK3 and HMGA2 form a functional axis that influences HSC properties during development. Collectively, our study highlights molecular mechanisms by which alternative splicing and miRNA-mediated post-transcriptional regulation impact the molecular identity and stage-specific developmental features of human HSCs.

Regulation of embryonic haematopoietic multipotency by EZH1.

All haematopoietic cell lineages that circulate in the blood of adult mammals derive from multipotent haematopoietic stem cells (HSCs). By contrast, in the blood of mammalian embryos, lineage-restricted progenitors arise first, independently of HSCs, which only emerge later in gestation. As best defined in the mouse, 'primitive' progenitors first appear in the yolk sac at 7.5 days post-coitum. Subsequently, erythroid-myeloid progenitors that express fetal haemoglobin, as well as fetal lymphoid progenitors, develop in the yolk sac and the embryo proper, but these cells lack HSC potential. Ultimately, 'definitive' HSCs with long-term, multilineage potential and the ability to engraft irradiated adults emerge at 10.5 days post-coitum from arterial endothelium in the aorta-gonad-mesonephros and other haemogenic vasculature. The molecular mechanisms of this reverse progression of haematopoietic ontogeny remain unexplained. We hypothesized that the definitive haematopoietic program might be actively repressed in early embryogenesis through epigenetic silencing, and that alleviating this repression would elicit multipotency in otherwise lineage-restricted haematopoietic progenitors. Here we show that reduced expression of the Polycomb group protein EZH1 enhances multi-lymphoid output from human pluripotent stem cells. In addition, Ezh1 deficiency in mouse embryos results in precocious emergence of functional definitive HSCs in vivo. Thus, we identify EZH1 as a repressor of haematopoietic multipotency in the early mammalian embryo.

Drug discovery for Diamond-Blackfan anemia using reprogrammed hematopoietic progenitors.

Diamond-Blackfan anemia (DBA) is a congenital disorder characterized by the failure of erythroid progenitor differentiation, severely curtailing red blood cell production. Because many DBA patients fail to respond to corticosteroid therapy, there is considerable need for therapeutics for this disorder. Identifying therapeutics for DBA requires circumventing the paucity of primary patient blood stem and progenitor cells. To this end, we adopted a reprogramming strategy to generate expandable hematopoietic progenitor cells from induced pluripotent stem cells (iPSCs) from DBA patients. Reprogrammed DBA progenitors recapitulate defects in erythroid differentiation, which were rescued by gene complementation. Unbiased chemical screens identified SMER28, a small-molecule inducer of autophagy, which enhanced erythropoiesis in a range of in vitro and in vivo models of DBA. SMER28 acted through autophagy factor ATG5 to stimulate erythropoiesis and up-regulate expression of globin genes. These findings present an unbiased drug screen for hematological disease using iPSCs and identify autophagy as a therapeutic pathway in DBA.

Gfap-positive radial glial cells are an essential progenitor population for later-born neurons and glia in the zebrafish spinal cord.

Radial glial cells are presumptive neural stem cells (NSCs) in the developing nervous system. The direct requirement of radial glia for the generation of a diverse array of neuronal and glial subtypes, however, has not been tested. We employed two novel transgenic zebrafish lines and endogenous markers of NSCs and radial glia to show for the first time that radial glia are essential for neurogenesis during development. By using the gfap promoter to drive expression of nuclear localized mCherry we discerned two distinct radial glial-derived cell types: a major nestin+/Sox2+ subtype with strong gfap promoter activity and a minor Sox2+ subtype lacking this activity. Fate mapping studies in this line indicate that gfap+ radial glia generate later-born CoSA interneurons, secondary motorneurons, and oligodendroglia. In another transgenic line using the gfap promoter-driven expression of the nitroreductase enzyme, we induced cell autonomous ablation of gfap+ radial glia and observed a reduction in their specific derived lineages, but not Blbp+ and Sox2+/gfap-negative NSCs, which were retained and expanded at later larval stages. Moreover, we provide evidence supporting classical roles of radial glial in axon patterning, blood-brain barrier formation, and locomotion. Our results suggest that gfap+ radial glia represent the major NSC during late neurogenesis for specific lineages, and possess diverse roles to sustain the structure and function of the spinal cord. These new tools will both corroborate the predicted roles of astroglia and reveal novel roles related to development, physiology, and regeneration in the vertebrate nervous system. GLIA 2016;64:1170-1189.

Kif11 dependent cell cycle progression in radial glial cells is required for proper neurogenesis in the zebrafish neural tube.

Radial glia serve as the resident neural stem cells in the embryonic vertebrate nervous system, and their proliferation must be tightly regulated to generate the correct number of neuronal and glial cell progeny in the neural tube. During a forward genetic screen, we recently identified a zebrafish mutant in the kif11 loci that displayed a significant increase in radial glial cell bodies at the ventricular zone of the spinal cord. Kif11, also known as Eg5, is a kinesin-related, plus-end directed motor protein responsible for stabilizing and separating the bipolar mitotic spindle. We show here that Gfap+ radial glial cells express kif11 in the ventricular zone and floor plate. Loss of Kif11 by mutation or pharmacological inhibition with S-trityl-L-cysteine (STLC) results in monoastral spindle formation in radial glial cells, which is characteristic of mitotic arrest. We show that M-phase radial glia accumulate over time at the ventricular zone in kif11 mutants and STLC treated embryos. Mathematical modeling of the radial glial accumulation in kif11 mutants not only confirmed an ~226× delay in mitotic exit (likely a mitotic arrest), but also predicted two modes of increased cell death. These modeling predictions were supported by an increase in the apoptosis marker, anti-activated Caspase-3, which was also found to be inversely proportional to a decrease in cell proliferation. In addition, treatment with STLC at different stages of neural development uncovered two critical periods that most significantly require Kif11 function for stem cell progression through mitosis. We also show that loss of Kif11 function causes specific reductions in oligodendroglia and secondary interneurons and motorneurons, suggesting these later born populations require proper radial glia division. Despite these alterations to cell cycle dynamics, survival, and neurogenesis, we document unchanged cell densities within the neural tube in kif11 mutants, suggesting that a mechanism of compensatory regulation may exist to maintain overall proportions in the neural tube. We propose a model in which Kif11 normally functions during mitotic spindle formation to facilitate the progression of radial glia through mitosis, which leads to the maturation of progeny into specific secondary neuronal and glial lineages in the developing neural tube.