Multiple Differentiation Processes Research Articles

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors.

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology has emerged recently. It is still in its infancy and has some limitations unsolved yet. The current protocols do not allow obtaining organoids to be consistent enough for drug discovery and preclinical studies. The maturation of organoids can take up to a year, pushing the researchers to launch multiple differentiation processes simultaneously. It imposes additional costs for the laboratory in terms of space and equipment. In addition, brain organoids often have a necrotic zone in the center, which suffers from nutrient and oxygen deficiency. Hence, most current protocols use a circulating system for culture medium to improve nutrition. Meanwhile, there are no inexpensive dynamic systems or bioreactors for organoid cultivation. This paper describes a protocol for producing brain organoids in compact and inexpensive home-made mini bioreactors. This protocol allows obtaining high quality organoids in large quantities.

PML-RARα interferes with erythropoiesis by repressing LMO2 in acute promyelocytic leukaemia.

The PML‐RARα fusion gene, generated by the t(15;17) chromosome translocation, is regarded as the initiating factor of acute promyelocytic leukaemia (APL). In addition to the well‐known effects on blocking myeloid differentiation at the promyelocytic stage, promyelocytic leukaemia‐retinoic acid receptor α (PML‐RARα) has also been reported to interfere with multiple differentiation processes, including erythroid differentiation. However, the detailed molecular mechanism by which PML‐RARα impairs erythropoiesis has not yet been fully addressed. By chromatin immunoprecipitation‐PCR assay, we found that PML‐RARα bound to the distal promoter region of LMO2 (LIM‐only protein 2), a critical erythroid‐specific transcription factor. Luciferase reporter assays and qRT‐PCR results demonstrated that PML‐RARα down‐regulated the expression of the LMO2 distal transcript through transrepressing its promoter activity. Analysis of gene expression profiling data from large cohorts of acute myeloid leukaemia (AML) patients confirmed that LMO2 expressed at a markedly lower level in APL patients in comparison to non‐APL AML patients. Further flow cytometry analysis demonstrated that PML‐RARα inhibited erythropoietin‐induced erythroid differentiation by down‐regulating LMO2 expression. Our findings reveal a previously unidentified mechanism, by which PML‐RARα interferes with erythropoiesis through directly targeting and transrepressing LMO2 expression in the development of APL.

A Linc1405/Eomes Complex Promotes Cardiac Mesoderm Specification and Cardiogenesis

Large intergenic non-coding RNAs (lincRNAs) play widespread roles in epigenetic regulation during multiple differentiation processes, but little is known about their mode of action in cardiac differentiation. Here, we identified the key roles of a lincRNA, termedlinc1405, in modulating the core network of cardiac differentiation by functionally interacting with Eomes. Chromatin- and RNA-immunoprecipitation assays showed that exon 2 of linc1405 physically mediates a complex consisting of Eomes, trithorax group (TrxG) subunit WDR5, and histone acetyltransferase GCN5 binding at the enhancer region of Mesp1 gene and activates its expression during cardiac mesoderm specification of embryonic stem cells. Importantly, linc1405 co-localizes with Eomes, WDR5, and GCN5 at the primitive streak, and linc1405 depletion impairs heart development and function invivo. In summary, linc1405 mediates a Eomes/WDR5/GCN5 complex that contributes to cardiogenesis, highlighting the critical roles of lincRNA-based complexes in the epigenetic regulation of cardiogenesis invitro and invivo.

DLK1 is a novel inflammatory inhibitor which interferes with NOTCH1 signaling in TLR-activated murine macrophages.

Delta-like protein 1 (DLK1) is a noncanonical ligand that inhibits NOTCH1 receptor activity and regulates multiple differentiation processes. In macrophages, NOTCH signaling increases TLR-induced expression of key pro-inflammatory mediators. We have investigated the role of DLK1 in macrophage activation and inflammation using Dlk1-deficient mice and Raw 264.7 cells overexpressing Dlk1. In the absence of Dlk1, NOTCH1 expression is increased and the activation of macrophages with TLR3 or TLR4 agonists leads to higher production of IFN-β and other pro-inflammatory cytokines, including TNF-α, IL-12, and IL-23. The expression of key proteins involved in IFN-β signaling, such as IRF3, IRF7, IRF1, or STAT1, as well as cRel, or RelB, which are responsible for the generation of IL-12 and IL-23, is enhanced in Dlk1 KO macrophages. Consistently, Dlk1 KO mice are more sensitive to LPS-induced endotoxic shock. These effects seem to be mediated through the modulation of NOTCH1 signaling. TLR4 activation reduces DLK1 expression, whereas increases NOTCH1 levels. In addition, DLK1 expression diminishes during differentiation of human U937 cells to macrophages. Overall, these results reveal a novel role for DLK1 as a regulator of NOTCH-mediated, pro-inflammatory macrophage activation, which could help to ensure a baseline level preventing constant tissue inflammation.

LAMMER Kinase LkhA Plays Multiple Roles in the Vegetative Growth and Asexual and Sexual Development of Aspergillus nidulans

LAMMER kinase plays pivotal roles in various physiological processes in eukaryotes; however, its function in filamentous fungi is not known. We performed molecular studies on the function of the Aspergillus nidulans LAMMER kinase, LkhA, and report its involvement in multiple developmental processes. The gene for LkhA was highly expressed during reproductive organ development, such as that of conidiophores and cleistothecia. During vegetative growth, the patterns of germ tube emergence and hyphal polarity were changed and septation was increased by lkhA deletion. Northern analyses showed that lkhA regulated the transcription of brlA, csnD, and ppoA, which supported the detrimental effect of lkhA-deletion on asexual and sexual differentiation. LkhA also affected expression of cyclin-dependent kinase NimXcdc2, a multiple cell cycle regulator, and StuA, an APSES family of fungal transcription factors that play pivotal roles in multiple differentiation processes. Here, for the first time, we present molecular evidence showing that LAMMER kinase is involved in A. nidulans development by modulating the expression of key regulators of developmental processes.

The non-canonical NOTCH ligand DLK1 exhibits a novel vascular role as a strong inhibitor of angiogenesis

The epidermal growth factor-like protein Delta-like 1 (DLK1) regulates multiple differentiation processes. It resembles NOTCH ligands structurally and is considered a non-canonical ligand. Given the crucial role of the NOTCH pathway in angiogenesis, we hypothesized that DLK1 could regulate angiogenesis by interfering with NOTCH. We therefore investigated the expression and function of DLK1 in the vascular endothelium and its role in the regulation of angiogenesis. We report DLK1 expression in the endothelium of different species, including human, cow, pig, and mouse. Angiogenesis was studied by using in vitro and in vivo models of angiotube formation in endothelial cells, retinal phenotypes in Dlk1-null mice, and vessel development in zebrafish. DLK1 overexpression strongly inhibited angiotube formation, whereas lung endothelial cells from Dlk1-null mice were highly angiogenic. In vivo studies demonstrated DLK1-mediated inhibition of neovessel formation and revealed an altered pattern of angiogenesis in the retinas of Dlk1-null mice. The expression of human DLK1 in zebrafish embryos severely altered the formation of intersegmental vessels, while knockdown of the orthologous gene was associated with ectopic and increased tumour-induced angiogenesis. NOTCH-dependent signalling as determined by gene expression reporters was inhibited by the presence of DLK1 in vascular endothelial cells. In contrast, Dlk1-null mice showed increased levels of NOTCH downstream targets, such as Snail and Slug. Our results unveil a novel inhibitory role for DLK1 in the regulation of angiogenesis, mediated by antagonism of the NOTCH pathway, and establish the basis for investigating its action in pathological settings.

Cellular Trans-Differentiation and Morphogenesis Toward the Lymphatic Lineage in Regenerative Medicine

Lymphedema is a medically irresolvable condition. The lack of therapies addressing lymphatic vessel dysfunction suggests that improved understanding of lymphatic cell differentiation and vessel maturation processes is key to the development of novel, regenerative medicine, and tissue engineering approaches. In this review we provide an overview of lymphatic characterization markers and morphology in development. Further, we describe multiple differentiation processes of the lymphatic system during embryonic, postnatal, and pathogenic development. Using the example of pathogenic Kaposi's sarcoma-associated herpes infection, we illustrate the involvement of the Notch and PI3K pathways for lymphatic transdifferentiation. We also discuss the plasticity of certain cell types and biofactors that enable transdifferentiation toward the lymphatic lineage. Here we argue the importance of pathway-associated induction factors for lymphatic transdifferentiation, including growth factors such as vascular endothelial growth factor receptor-C and interleukins, and the involvement of extracellular matrix characteristics and dynamics for morphological functionality.

Network-based comparison of temporal gene expression patterns

In the pursuits of mechanistic understanding of cell differentiation, it is often necessary to compare multiple differentiation processes triggered by different external stimuli and internal perturbations. Available methods for comparing temporal gene expression patterns are limited to a gene-by-gene approach, which ignores co-expression information and thus is sensitive to measurement noise. We present a method for co-expression network based comparison of temporal expression patterns (NACEP). NACEP compares the temporal patterns of a gene between two experimental conditions, taking into consideration all of the possible co-expression modules that this gene may participate in. The NACEP program is available at http://biocomp.bioen.uiuc.edu/nacep. We applied NACEP to analyze retinoid acid (RA)-induced differentiation of embryonic stem (ES) cells. The analysis suggests that RA may facilitate neural differentiation by inducing the shh and insulin receptor pathways. NACEP was also applied to compare the temporal responses of seven RNA inhibition (RNAi) experiments. As proof of concept, we demonstrate that the difference in the temporal responses to RNAi treatments can be used to derive interaction relationships of transcription factors (TFs), and therefore infer regulatory modules within a transcription network. In particular, the analysis suggested a novel regulatory relationship between two pluripotency regulators, Esrrb and Tbx3, which was supported by in vivo binding of Esrrb to the promoter of Tbx3. The NACEP program and the supplementary documents are available at http://biocomp.bioen.uiuc.edu/nacep. szhong@illinois.edu Supplementary data are available at Bioinformatics online.

Robust Generation and Decoding of Morphogen Gradients

Morphogen gradients play a key role in multiple differentiation processes. Both the formation of the gradient and its interpretation by the receiving cells need to occur at high precision to ensure reproducible patterning. This need for quantitative precision is challenged by fluctuations in the environmental conditions and by variations in the genetic makeup of the developing embryos. We discuss mechanisms that buffer morphogen profiles against variations in gene dosage. Self-enhanced morphogen degradation and pre-steady-state decoding provide general means for buffering the morphogen profile against fluctuations in morphogen production rate. A more specific "shuttling" mechanism, which establishes a sharp and robust activation profile of a widely expressed morphogen, and enables the adjustment of morphogen profile with embryo size, is also described. Finally, we consider the transformation of the smooth gradient profile into sharp borders of gene expression in the signal-receiving cells. The integration theory and experiments are increasingly used, providing key insights into the system-level functioning of the developmental system.

Multiple roles of mouse Numb in tuning developmental cell fates

Background: Notch signaling regulates multiple differentiation processes and cell fate decisions during both invertebrate and vertebrate development. Numb encodes an intracellular protein that was shown in Drosophila to antagonize Notch signaling at binary cell fate decisions of certain cell lineages. Although overexpression experiments suggested that Numb might also antagonize some Notch activity in vertebrates, the developmental processes in which Numb is involved remained elusive.Results: We generated mice with a homozygous inactivation of Numb. These mice died before embryonic day E11.5, probably because of defects in angiogenic remodeling and placental dysfunction. Mutant embryos had an open anterior neural tube and impaired neuronal differentiation within the developing cranial central nervous system (CNS). In the developing spinal cord, the number of differentiated motoneurons was reduced. Within the peripheral nervous system (PNS), ganglia of cranial sensory neurons were formed. Trunk neural crest cells migrated and differentiated into sympathetic neurons. In contrast, a selective differentiation anomaly was observed in dorsal root ganglia, where neural crest–derived progenitor cells had migrated normally to form ganglionic structures, but failed to differentiate into sensory neurons.Conclusions: Mouse Numb is involved in multiple developmental processes and required for cell fate tuning in a variety of lineages. In the nervous system, Numb is required for the generation of a large subset of neuronal lineages. The restricted requirement of Numb during neural development in the mouse suggests that in some neuronal lineages, Notch signaling may be regulated independently of Numb.

Parathyroid hormone related peptide mRNA expression during murine postimplantation development: evidence for involvement in multiple differentiation processes.

In this study we describe the spatio-temporal expression of Parathyroid Hormone related Peptide (PTHrP) mRNA during murine postimplantation development from day 5.5 post coitum (pc) until day 12.5 pc. From day 5.5 pc and onwards PTHrP mRNA was detected in the trophoblast. In addition, at day 5.5 and 6.5 pc epithelial cells of the antimesometrial crypt and cells of the inner zone of the decidua directly adjacent to the implanted embryo expressed PTHrP mRNA. This supported a previous model in which parietal endoderm formation is regulated by a paracrine mechanism involving PTHrP expressing trophoblast cells and receptor expressing extra-embryonal endoderm cells. The first embryonal PTHrP mRNA expression was detected in the roof of the hindbrain at gestation day 10.5 pc. From day 11.5 pc and onwards PTHrP mRNA was detected in the otic vesicle, the semilateral channels, the roof of the hindbrain and later in the choroid plexus, in epithelial cells of the lung and heart ventricle, mesenchymal cells lining the nasal pit, the dermis of the snout and at all sites of endochondral bone formation. The widespread expression of PTHrP mRNA during embryogenesis in extra-embryonic and embryonic tissues suggests the involvement of the peptide in multiple growth and differentiation processes.

Evolution of silicic magma in the upper crust: the mid-Tertiary Latir volcanic field and its cogenetic granitic batholith, northern New Mexico, U.S.A.

ABSTRACTStructural and topographic relief along the eastern margin of the Rio Grande rift, northern New Mexico, provides a remarkable cross-section through the 26-Ma Questa caldera and cogenetic volcanic and plutonic rocks of the Latir field. Exposed levels increase in depth from mid-Tertiary depositional surfaces in northern parts of the igneous complex to plutonic rocks originally at 3–5 km depths in the S. Erosional remnants of an ash-flow sheet of weakly peralkaline rhyolite (Amalia Tuff) and andesitic to dacitic precursor lavas, disrupted by rift-related faults, are preserved as far as 45 km beyond their sources at the Questa caldera. Broadly comagmatic 26 Ma batholithic granitic rocks, exposed over an area of 20 by 35 km, range from mesozonal granodiorite to epizonal porphyritic granite and aplite; shallower and more silicic phases are mostly within the caldera. Compositionally and texturally distinct granites define resurgent intrusions within the caldera and discontinuous ring dikes along its margins; a batholithic mass of granodiorite extends 20 km S of the caldera and locally grades vertically to granite below its flat-lying roof. A negative Bouguer gravity anomaly (15–20 mgal), which encloses exposed granitic rocks and coincides with boundaries of the Questa caldera, defines boundaries of the shallow batholith, emplaced low in the volcanic sequence and in underlying Precambrian rocks. Palaeomagnetic pole positions indicate that successively crystallised granitic plutons cooled through Curie temperatures during the time of caldera formation, initial regional extension, and rotational tilting of the volcanic rocks. Isotopic ages for most intrusions are indistinguishable from the volcanic rocks. These relations indicate that the batholithic complex broadly represents the source magma for the volcanic rocks, into which the Questa caldera collapsed, and that the magma was largely liquid during regional tectonic disruption.Volcanic and plutonic magmas (1) changed from early high-K calc-alkaline to alkalic prior to caldera eruptions; (2) differentiated to a weakly peralkaline rhyolite and equivalent acmiteartvedsonite granite cap (underlain by calc-alkaline granite) when the caldera formed at 26·5 Ma; then (3) reverted to calc-alkaline compositions. Concentrations of alkalis and minor elements such as Rb, Th, U, Nb, Zr, and Y reached maxima at the caldera stage. The volcanic rocks constitute intermittently quenched samples of upper parts of Questa magma bodies at early stages of crystallisation; in contrast, the comagmatic granitic rocks preserve an integrated record of protracted crystallisation of the magmatic residue as eruptions diminished. Multiple differentiation processes were active during evolution of the Questa magmatic system: crystal fractionation, replenishment by mantle and lower crustal melts of varying chemical and isotopic character, mixing of evolved with more primitive magmas, upper crustal assimilation, and perhaps volatile-transfer processes. As a result, an evolving batholithic cluster of coalesced magma chambers generated diverse assemblages of broadly cogenetic rocks within a few million years. Evolution of the Questa magmatic system and similar high-level Tertiary granitic batholiths nearby in the southern Rocky Mountains provides broad insights into magmatic processes in continental regions such as the overall shapes of batholiths, time and compositional relations between cogenetic volcanic and plutonic rocks, density equilibration of magmas with country rocks, and thermal evolution of continental crust.

Multiple differentiation processes in early‐rift calc‐alkaline volcanics, northern Rio Grande Rift, New Mexico

Structurally elevated segments of a north trending intrarift horst in northern New Mexico expose Late Oligocene/Early Miocene volcanic rocks erupted during the inception of the Rio Grande rift. Postcaldera rocks of the Latir volcanic field on the intrarift horst (Timber Mountain and Brushy Mountain) include two distinct calc‐alkaline sequences differing in age by up to 3 m.y. The lower sequence (25 Ma) is dominated by dacite (plag + cpx + opx + Fe‐Ti oxides ± hbld) but includes basaltic andesite (ol + cpx + plag) and low‐silica rhyolite (plag + sanidine + qtz + biotite + Fe‐Ti oxides). Dacites contain quenched andesitic micropillows, high‐silica rhyolite glass, and sanidine and oligoclase xenocrysts. Incompatible element concentrations increase with silica content throughout the sequence and are accompanied by concomitant decreases in Sr, Sc, and Cr. The upper sequence (22 Ma) includes basaltic andesite (ol + cpx + plag), dacite lava flows (plag + hbld + cpx + Fe‐Ti oxides + opx + sanidine + sphene + biotite), and a basal lava dome of high‐silica rhyolite (qtz + sanidine ± biotite ± Fe‐Ti oxides ± sphene + hornblende) overlying remnants of the outflow sheet of the 26‐Ma Amalia Tuff. Upper sequence dacites are distinguished from lower sequence dacites by their hydrous mineralogy and lower relative K2O, TiO2, Zr, Y, Nb, Y/Zr, and total rare earth element concentrations for any given silica content. Major and trace element models, to predict fractionation paths from an andesite parent, indicate that suppressed K, Sr, and elevated Mg, Y, and Lu concentrations in the lower sequence dacites resulted from fractionation of phenocryst phases, plus mixing in proportions approaching one to one, with high‐silica rhyolite similar in composition to the Amalia Tuff. Low‐silica rhyolites may have been derived through fractionation of dacites plus mixing with the high‐silica rhyolite. Positive correlations of 87/86Sr0 with SiO2 are interpreted to reflect the increasing proportions of crustally contaminated high‐silica rhyolite in lower sequence, intermediate‐composition melts. These differentiation models support a petrogenic link between the mildly peralkaline Amalia Tuff of the caldera‐forming phase of the Questa magmatic system and early postcaldera lavas. Subsequent eruption of upper sequence volcanics marks the regeneration of more typical metaluminous magmas characteristic of both precaldera volcanics and postcaldera intrusions.