Genome-wide screening identifies Trim33 as an essential regulator of dendritic cell differentiation.

The development of dendritic cells (DCs), including antigen-presenting conventional DCs (cDCs) and cytokine-producing plasmacytoid DCs (pDCs), is controlled by the growth factor Flt3 ligand (Flt3L) and its receptor Flt3. We genetically dissected Flt3L-driven DC differentiation using CRISPR-Cas9-based screening. Genome-wide screening identified multiple regulators of DC differentiation including subunits of TSC and GATOR1 complexes, which restricted progenitor growth but enabled DC differentiation by inhibiting mTOR signaling. An orthogonal screen identified the transcriptional repressor Trim33 (TIF-1γ) as a regulator of DC differentiation. Conditional targeting in vivo revealed an essential role of Trim33 in the development of all DCs, but not of monocytes or granulocytes. In particular, deletion of Trim33 caused rapid loss of DC progenitors, pDCs, and the cross-presenting cDC1 subset. Trim33-deficient Flt3+ progenitors up-regulated pro-inflammatory and macrophage-specific genes but failed to induce the DC differentiation program. Collectively, these data elucidate mechanisms that control Flt3L-driven differentiation of the entire DC lineage and identify Trim33 as its essential regulator.

Essential transcription factors for induced neuron differentiation.

Neurogenins are proneural transcription factors required to specify neuronal identity. Their overexpression in human pluripotent stem cells rapidly produces cortical-like neurons with spiking activity and, because of this, they have been widely adopted for human neuron disease models. However, we do not fully understand the key downstream regulatory effectors responsible for driving neural differentiation. Here, using inducible expression of NEUROG1 and NEUROG2, we identify transcription factors (TFs) required for directed neuronal differentiation by combining expression and chromatin accessibility analyses with a pooled in vitro CRISPR-Cas9 screen targeting all ~1900 TFs in the human genome. The loss of one of these essential TFs (ZBTB18) yields few MAP2-positive neurons. Differentiated ZBTB18-null cells have radically altered gene expression, leading to cytoskeletal defects and stunted neurites and spines. In addition to identifying key downstream TFs for neuronal differentiation, our work develops an integrative multi-omics and TFome-wide perturbation platform to rapidly characterize essential TFs for the differentiation of any human cell type.

Bee Assemblage in the Southern Chihuahuan Desert: The Role of Season, Year, and Trap Color in Abundance.

Recognizing how populations fluctuate over time is a crucial factor in determining the environmental elements affecting population persistence. However, the limited information on wild bee populations complicates the estimation of the impact of anthropogenic threats leading to changes in population size. To address this, we conducted a study capturing and monitoring nine species of wild bees through monthly samplings over four years. Tray traps were placed in permanent plots, and capture records were used to determine population size (N) and density (D). A generalized linear model (GLM) was employed to determine how the use of traps affected bee species captures. The families Apidae and Halictidae represented the most captures. Apis mellifera, the Lasioglossum (Dialictus spp.) complex, and Macrotera sinaloana exhibited the largest number of captures and highest population density. Most species (77.7%) showed a tendency to remain constant over the years and to have a higher number of captures in the spring months. Moreover, yellow traps were the most effective in capturing bee individuals. We suggest that the availability of essential resources and the reduction in environmental stressors positively affected the capture of wild bee populations.

Effective index model as a reliable tool for the design of nanostructured thin-film solar cells.

Nanostructured anti-reflection coatings (ARC) are used to reduce the reflectivity of the front surface of solar cells. Computational electromagnetism helps to evaluate the spectral reflectivity of of this type of ARC using several approaches. They typically require large computational resources both in time and hardware elements (memory allocation, speed of processors, etc.). Long computational times may jeopardize optimization processes based on the iterative evaluation of a given merit function that depends on several parameters. Then, simplified analytic methods can speed up this evaluation with moderate computational resources. In this contribution we adapt an Effective Index Model (EIM) to the case of the design of an ARC made with nanoparticles (NP) embedded in a medium at the front surface of a thin-film silicon solar cell. Our approach modifies the discrete dipole approximation method to adapt it to the geometric and material properties of the NPs. The results obtained from the analytic method are compared with those evaluated through a Finite Element Method (FEM) for several cases involving variations in the size and geometry of the NP arrangement, obtaining reflectances that differ less than 10[Formula: see text] for the worst case analyzed but bieng about 100 times faster than the FEM.

ISSCR Education Committee syllabus and learning guide for enhancing stem cell literacy.

The rapidly evolving stem cell field puts much stress on developing educational resources. The ISSCR Education Committee has created a flexible stem cell syllabus rooted in core concepts to facilitate stem cell literacy. The free syllabus will be updated regularly to maintain accuracy and relevance.

Foxa2 and Pet1 Direct and Indirect Synergy Drive Serotonergic Neuronal Differentiation.

Neuronal programming by forced expression of transcription factors (TFs) holds promise for clinical applications of regenerative medicine. However, the mechanisms by which TFs coordinate their activities on the genome and control distinct neuronal fates remain obscure. Using direct neuronal programming of embryonic stem cells, we dissected the contribution of a series of TFs to specific neuronal regulatory programs. We deconstructed the Ascl1-Lmx1b-Foxa2-Pet1 TF combination that has been shown to generate serotonergic neurons and found that stepwise addition of TFs to Ascl1 canalizes the neuronal fate into a diffuse monoaminergic fate. The addition of pioneer factor Foxa2 represses Phox2b to induce serotonergic fate, similar to in vivo regulatory networks. Foxa2 and Pet1 appear to act synergistically to upregulate serotonergic fate. Foxa2 and Pet1 co-bind to a small fraction of genomic regions but mostly bind to different regulatory sites. In contrast to the combinatorial binding activities of other programming TFs, Pet1 does not strictly follow the Foxa2 pioneer. These findings highlight the challenges in formulating generalizable rules for describing the behavior of TF combinations that program distinct neuronal subtypes.

Synthetic regulatory reconstitution reveals principles of mammalian Hox cluster regulation.

Precise Hox gene expression is crucial for embryonic patterning. Intra-Hox transcription factor binding and distal enhancer elements have emerged as the major regulatory modules controlling Hox gene expression. However, quantifying their relative contributions has remained elusive. Here, we introduce "synthetic regulatory reconstitution," a conceptual framework for studying gene regulation, and apply it to the HoxA cluster. We synthesized and delivered variant rat HoxA clusters (130 to 170 kilobases) to an ectopic location in the mouse genome. We found that a minimal HoxA cluster recapitulated correct patterns of chromatin remodeling and transcription in response to patterning signals, whereas the addition of distal enhancers was needed for full transcriptional output. Synthetic regulatory reconstitution could provide a generalizable strategy for deciphering the regulatory logic of gene expression in complex genomes.

Capybara: A computational tool to measure cell identity and fate transitions.

Measuring cell identity in development, disease, and reprogramming is challenging as cell types and states are in continual transition. Here, we present Capybara, a computational tool to classify discrete cell identity and intermediate "hybrid" cell states, supporting a metric to quantify cell fate transition dynamics. We validate hybrid cells using experimental lineage tracing data to demonstrate the multi-lineage potential of these intermediate cell states. We apply Capybara to diagnose shortcomings in several cell engineering protocols, identifying hybrid states in cardiac reprogramming and off-target identities in motor neuron programming, which we alleviate by adding exogenous signaling factors. Further, we establish a putative in vivo correlate for induced endoderm progenitors. Together, these results showcase the utility of Capybara to dissect cell identity and fate transitions, prioritizing interventions to enhance the efficiency and fidelity of stem cell engineering.

The BTB transcription factors ZBTB11 and ZFP131 maintain pluripotency by repressing pro-differentiation genes.

In pluripotent cells, a delicate activation-repression balance maintains pro-differentiation genes ready for rapid activation. The identity of transcription factors (TFs) that specifically repress pro-differentiation genes remains obscure. By targeting ∼1,700 TFs with CRISPR loss-of-function screen, we found that ZBTB11 and ZFP131 are required for embryonic stem cell (ESC) pluripotency. ESCs without ZBTB11 or ZFP131 lose colony morphology, reduce proliferation rate, and upregulate transcription of genes associated with three germ layers. ZBTB11 and ZFP131 bind proximally to pro-differentiation genes. ZBTB11 or ZFP131 loss leads to an increase in H3K4me3, negative elongation factor (NELF) complex release, and concomitant transcription at associated genes. Together, our results suggest that ZBTB11 and ZFP131 maintain pluripotency by preventing premature expression of pro-differentiation genes and present a generalizable framework to maintain cellular potency.

CRISPR and biochemical screens identify MAZ as a cofactor in CTCF-mediated insulation at Hox clusters.

CCCTC-binding factor (CTCF) is critical to three-dimensional genome organization. Upon differentiation, CTCF insulates active and repressed genes within Hox gene clusters. We conducted a genome-wide CRISPR knockout (KO) screen to identify genes required for CTCF-boundary activity at the HoxA cluster, complemented by biochemical approaches. Among the candidates, we identified Myc-associated zinc-finger protein (MAZ) as a cofactor in CTCF insulation. MAZ colocalizes with CTCF at chromatin borders and, similar to CTCF, interacts with the cohesin subunit RAD21. MAZ KO disrupts gene expression and local contacts within topologically associating domains. Similar to CTCF motif deletions, MAZ motif deletions lead to derepression of posterior Hox genes immediately after CTCF boundaries upon differentiation, giving rise to homeotic transformations in mouse. Thus, MAZ is a factor contributing to appropriate insulation, gene expression and genomic architecture during development.

PRC1 sustains the integrity of neural fate in the absence of PRC2 function.

Polycomb repressive complexes (PRCs) 1 and 2 maintain stable cellular memories of early fate decisions by establishing heritable patterns of gene repression. PRCs repress transcription through histone modifications and chromatin compaction, but their roles in neuronal subtype diversification are poorly defined. We found that PRC1 is essential for the specification of segmentally restricted spinal motor neuron (MN) subtypes, while PRC2 activity is dispensable to maintain MN positional identities during terminal differentiation. Mutation of the core PRC1 component Ring1 in mice leads to increased chromatin accessibility and ectopic expression of a broad variety of fates determinants, including Hox transcription factors, while neuronal class-specific features are maintained. Loss of MN subtype identities in Ring1 mutants is due to the suppression of Hox-dependent specification programs by derepressed Hox13 paralogs (Hoxa13, Hoxb13, Hoxc13, Hoxd13). These results indicate that PRC1 can function in the absence of de novo PRC2-dependent histone methylation to maintain chromatin topology and postmitotic neuronal fate.

An expansion of the non-coding genome and its regulatory potential underlies vertebrate neuronal diversity.

Proper assembly and function of the nervous system requires the generation of a uniquely diverse population of neurons expressing a cell-type-specific combination of effector genes that collectively define neuronal morphology, connectivity, and function. How countless partially overlapping but cell-type-specific patterns of gene expression are controlled at the genomic level remains poorly understood. Here we show that neuronal genes are associated with highly complex gene regulatory systems composed of independent cell-type- and cell-stage-specific regulatory elements that reside in expanded non-coding genomic domains. Mapping enhancer-promoter interactions revealed that motor neuron enhancers are broadly distributed across the large chromatin domains. This distributed regulatory architecture is not a unique property of motor neurons but is employed throughout the nervous system. The number of regulatory elements increased dramatically during the transition from invertebrates to vertebrates, suggesting that acquisition of new enhancers might be a fundamental process underlying the evolutionary increase in cellular complexity.

Macular and choroidal thickness in Down syndrome by swept-source optical coherence tomography (SS-OCT).

Down syndrome (DS) is associated with certain structural and functional disorders in the whole visual system. The purpose was to compare retinal and choroidal thickness using swept-source optical coherence tomography (SS-OCT) in DS subjects with controls. This cross-sectional study included 100 eyes of 52 DS subjects and 78 eyes of 39 matching age and axial length controls. Our results showed that inner or outer retinal and ganglionar thickness showed no significant differences between DS and control group (p > 0.05). However, retinal foveal thickness (rFT), ganglion foveal thickness (gFT) were significantly higher in DS group than in controls, whereas choroidal foveal thickness (cFT) and some choroidal quadrants of inner and outer rings were significantly lower (p < 0.05). This the first pilot study to provide information about macular and choroidal thicknesses in SD using SS-OCT compared to controls. Further analyses with larger numbers of subjects are needed to confirm our results.

Espesor macular y coroideo en el síndrome de Down mediante swept-source tomografía de coherencia óptica (SS-OCT) / Macular and choroidal thickness in Down syndrome by swept-source optical coherence tomography (SS-OCT)

El síndrome de Down (SD) se asocia a diversos trastornos estructurales y funcionales en la totalidad del sistema visual. El propósito del estudio fue comparar el grosor retiniano y coroideo utilizando tomografía de coherencia óptica swept-source (SS-OCT) en sujetos con SD y controles. Este estudio transversal incluye 100 ojos de 52 sujetos SD y 78 ojos de 39 controles de edad y longitud axial concordantes. Los resultados mostraron que el grosor retiniano interno o externo y el grosor ganglionar no presentaron diferencias significativas entre los sujetos con SD y el grupo de control (p>0,05). Sin embargo, el grosor retiniano foveal (rFT) y el grosor ganglionar foveal (gFT) fueron significativamente superiores en el grupo SD que en los controles, mientras que el grosor foveal coroideo (cFT) y algunos cuadrantes coroideos de los anillos interno y externo fueron significativamente inferiores (p<0,05). Este es el primer estudio piloto que proporciona información sobre los grosores macular y coroideo en SD utilizando SS-OCT comparado con controles. Es necesario realizar análisis adicionales con grupos mayores de sujetos para confirmar estos resultados (AU)

Down syndrome (DS) is associated with certain structural and functional disorders in the whole visual system. The purpose was to compare retinal and choroidal thickness using swept-source optical coherence tomography (SS-OCT) in DS subjects with controls. This cross-sectional study included 100 eyes of 52 DS subjects and 78 eyes of 39 matching age and axial length controls. Our results showed that inner or outer retinal and ganglionar thickness showed no significant differences between DS and control group (p>0.05). However, retinal foveal thickness (rFT), ganglion foveal thickness (gFT) were significantly higher in DS group than in controls, whereas choroidal foveal thickness (cFT) and some choroidal quadrants of inner and outer rings were significantly lower (p<0.05). This the first pilot study to provide information about macular and choroidal thicknesses in SD using SS-OCT compared to controls. Further analyses with larger numbers of subjects are needed to confirm our results (AU)

Performance enhancement of an ultrafast all-fiber laser based on an InN saturable absorber using GRIN coupling.

Indium nitride (InN)-based semiconductor saturable absorbers have previously shown advantages for application in near-IR fiber lasers due to their broad modulation depth, ultrafast nonlinear response and thermal stability. However, up to now all demonstrated saturable absorber elements based on InN (either transmissive or reflective) have shown limited performance due to poor coupling and insertion losses. We present here a simple mode-locking device based on a GRIN-rod lens in conjunction with an InN semiconductor saturable absorber mirror (SESAM) for its use in a passively mode-locked all-fiber laser system operating at telecom wavelengths. Our results demonstrate that this coupling element ensures not only a compact, turnkey and alignment-free design but also a highly-stable optical femtosecond pulse train. The reduction of insertion losses (3.5 dB) enables the generation of 90-fs ultrafast pulses with an average power of 40 mW and up to 7 nJ of pulse energy without the need for additional amplification.

Macular and choroidal thickness in Down syndrome by swept-source optical coherence tomography (SS-OCT). / Espesor macular y coroideo en el síndrome de Down mediante swept-source tomografía de coherencia óptica (SS-OCT).

Down syndrome (DS) is associated with certain structural and functional disorders in the whole visual system. The purpose was to compare retinal and choroidal thickness using swept-source optical coherence tomography (SS-OCT) in DS subjects with controls. This cross-sectional study included 100 eyes of 52 DS subjects and 78 eyes of 39 matching age and axial length controls. Our results showed that inner or outer retinal and ganglionar thickness showed no significant differences between DS and control group (p>0.05). However, retinal foveal thickness (rFT), ganglion foveal thickness (gFT) were significantly higher in DS group than in controls, whereas choroidal foveal thickness (cFT) and some choroidal quadrants of inner and outer rings were significantly lower (p<0.05). This the first pilot study to provide information about macular and choroidal thicknesses in SD using SS-OCT compared to controls. Further analyses with larger numbers of subjects are needed to confirm our results.

De novo assembly and delivery to mouse cells of a 101 kb functional human gene.

Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here, we describe a workflow for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kilobase pairs (kb). The DNA assembly step is supported by an integrated robotic workcell. We demonstrate assembly of the 101 kb human HPRT1 gene in yeast from 3 kb building blocks, precision delivery of the resulting construct to mouse embryonic stem cells, and subsequent expression of the human protein from its full-length human gene in mouse cells. This workflow provides a framework for mammalian genome writing. We envision utility in producing designer variants of human genes linked to disease and their delivery and functional analysis in cell culture or animal models.

An interpretable bimodal neural network characterizes the sequence and preexisting chromatin predictors of induced transcription factor binding.

BACKGROUND: Transcription factor (TF) binding specificity is determined via a complex interplay between the transcription factor's DNA binding preference and cell type-specific chromatin environments. The chromatin features that correlate with transcription factor binding in a given cell type have been well characterized. For instance, the binding sites for a majority of transcription factors display concurrent chromatin accessibility. However, concurrent chromatin features reflect the binding activities of the transcription factor itself and thus provide limited insight into how genome-wide TF-DNA binding patterns became established in the first place. To understand the determinants of transcription factor binding specificity, we therefore need to examine how newly activated transcription factors interact with sequence and preexisting chromatin landscapes. RESULTS: Here, we investigate the sequence and preexisting chromatin predictors of TF-DNA binding by examining the genome-wide occupancy of transcription factors that have been induced in well-characterized chromatin environments. We develop Bichrom, a bimodal neural network that jointly models sequence and preexisting chromatin data to interpret the genome-wide binding patterns of induced transcription factors. We find that the preexisting chromatin landscape is a differential global predictor of TF-DNA binding; incorporating preexisting chromatin features improves our ability to explain the binding specificity of some transcription factors substantially, but not others. Furthermore, by analyzing site-level predictors, we show that transcription factor binding in previously inaccessible chromatin tends to correspond to the presence of more favorable cognate DNA sequences. CONCLUSIONS: Bichrom thus provides a framework for modeling, interpreting, and visualizing the joint sequence and chromatin landscapes that determine TF-DNA binding dynamics.

Differential abilities to engage inaccessible chromatin diversify vertebrate Hox binding patterns.

Although Hox genes encode for conserved transcription factors (TFs), they are further divided into anterior, central and posterior groups based on their DNA-binding domain similarity. The posterior Hox group expanded in the deuterostome clade and patterns caudal and distal structures. We aimed to address how similar Hox TFs diverge to induce different positional identities. We studied Hox TF DNA-binding and regulatory activity during an in vitro motor neuron differentiation system that recapitulates embryonic development. We found diversity in the genomic binding profiles of different Hox TFs, even among the posterior group paralogs that share similar DNA-binding domains. These differences in genomic binding were explained by differing abilities to bind to previously inaccessible sites. For example, the posterior group HOXC9 had a greater ability to bind occluded sites than the posterior HOXC10, producing different binding patterns and driving differential gene expression programs. From these results, we propose that the differential abilities of posterior Hox TFs to bind to previously inaccessible chromatin drive patterning diversification.This article has an associated 'The people behind the papers' interview.