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SUMMARYLung morphogenesis relies on diverse cell intrinsic and extrinsic mechanisms to ensure proper cellular differentiation and compartmentalization. Using genetic mouse models, tissue explants, and transcriptomic analysis, we demonstrate that the epigenetic reader Bromodomain Containing Protein 4 (BRD4) is required for lung morphogenesis and perinatal survival. Endodermal BRD4 deletion impairs epithelial-mesenchymal crosstalk, leading to disrupted proximal-distal patterning and branching morphogenesis. These early defects result in dilated airways and the formation of cystic distal airway structures, containing both airway and alveolar features. Moreover, BRD4 deficient lungs exhibit abnormal airway and alveolar epithelial cell lineage allocation and differentiation. Restoration of SHH signaling partially rescues defects due to loss of BRD4, suggesting a role for BRD4 in regulating the SHH-FGF signaling axis. Together, these data identify the essential role of Brd4 in lung development to ensure proper intercellular crosstalk to enable proper lineage specification, identity, and maturation.

The study of neuron morphology requires robust and comprehensive methods to quantify the differences between neurons of different subtypes and animal species. Several software packages have been developed for the analysis of neuron tracing results stored in the standard SWC format. The packages, however, provide relatively simple quantifications and their non-extendable architecture prohibit their use for advanced data analysis and visualization. We developed nGauge, a Python toolkit to support the parsing and analysis of neuron morphology data. As an application programming interface (API), nGauge can be referenced by other popular open-source software to create custom informatics analysis pipelines and advanced visualizations. nGauge defines an extendable data structure that handles volumetric constructions (e.g. soma), in addition to the SWC linear reconstructions, while remaining lightweight. This greatly extends nGauge’s data compatibility.

In recent years, advancements in microscope design have allowed faster, higher resolution imaging of all types of biological samples. Custom microscope designs have rapidly seen adoption, enabled by a rapid growth in open source software. In this report, we present a custom, open source, hardware design which provides 2 16-bit resolution high-speed analog outputs as well as breakouts for GPIO connections to the inexpensive and well-supported microcontroller (the $4 Raspberry Pi Pico). We provide firmware for interfacing the device with software and demonstrate the device performance when used to interface with a galvanometer. This device provides a working platform for development of custom microscope hardware.

IntroductionBacterial pulmonary infections are a major cause of morbidity and mortality in neonates, with less severity in older children. Previous studies demonstrated that the DNA of CD4+ T cells in the mouse lung, whose primary responsibility is to coordinate the immune response foreign pathogens, is differentially methylated in neonates compared with juveniles. Nevertheless, the effect of this differential DNA methylation on CD4+ T cell gene expression and response to infection remains unclear.MethodsWe treated E. coli-infected neonatal (4-day-old) and juvenile (13-day-old) mice with decitabine (DAC), a DNA methyltransferase inhibitor with broad-spectrum DNA demethylating activity, and performed simultaneous genome-wide DNA methylation and transcriptional profiling on lung CD4+ T cells.ResultsJuvenile and neonatal mice experienced differential demethylation in response to DAC treatment, with larger methylation differences observed in neonates. By cross-filtering differentially expressed genes between juveniles and neonates with those sites that were demethylated in neonates, we found that interferon-responsive genes such as Ifit1 are the most down-regulated methylation-sensitive genes in neonatal mice. DAC treatment shifted neonatal lung CD4+ T cells toward a gene expression program similar to that of juveniles.ConclusionFollowing lung infection with E. coli, lung CD4+ T cells in neonatal mice exhibit epigenetic repression of important host defense pathways, which are activated by inhibition of DNA methyltransferase activity to resemble a more mature profile.

There is a growing amount of data uncovering the cellular diversity of the pulmonary circulation and mechanisms governing vascular repair after injury. However, the molecular and cellular mechanisms contributing to the morphogenesis and growth of the pulmonary vasculature during embryonic development are less clear. Importantly, deficits in vascular development lead to significant pediatric lung diseases, indicating a need to uncover fetal programs promoting vascular growth. To address this, we used a transgenic mouse reporter for expression of Cxcl12, an arterial endothelial hallmark gene, and performed single-cell RNA sequencing on isolated Cxcl12-DsRed+ endothelium to assess cellular heterogeneity within pulmonary endothelium. Combining cell annotation with gene ontology and histological analysis allowed us to segregate the developing artery endothelium into functionally and spatially distinct subpopulations. Expression of Cxcl12 is highest in the distal arterial endothelial subpopulation, a compartment enriched in genes for vascular development. Accordingly, disruption of CXCL12 signaling led to, not only abnormal branching, but also distal vascular hypoplasia. These data provide evidence for arterial endothelial functional heterogeneity and reveal conserved signaling mechanisms essential for pulmonary vascular development.

In adults, there is a growing amount of data uncovering the cellular diversity of the pulmonary circulation and mechanisms governing vascular repair after injury, however, molecular and cellular mechanisms contributing to the morphogenesis and growth of the pulmonary vasculature during embryonic development are less clear. Importantly, deficits in vascular development lead to a large number of lung diseases in children, indicating a need to uncover fetal programs that promote pulmonary vascular growth. To address this, we used a transgenic mouse reporter for expression of Cxcl12, an arterial hallmark gene, and performed single-cell RNA sequencing on isolated Cxcl12-DsRed+ endothelium to assess cellular heterogeneity within pulmonary endothelium. Combining cell annotation, gene ontology analysis, and spatial transcriptomics allowed us to segregate the developing artery into spatially and functionally distinct novel subpopulations. In addition, expression of Cxcl12 suggests a morphogen gradient from arteries to capillaries, suggesting directed cell migration for pulmonary vascular development. Disruption of this gradient led to abnormal branching and pulmonary vascular hypoplasia. These data provide evidence for arterial endothelial functional heterogeneity and reveal conserved signaling mechanisms essential for pulmonary vascular development.

SUMMARY Elucidating cell lineages provides crucial understanding of development. Recently developed sequencing-based techniques enhance the scale of lineage tracing but eliminate the spatial information offered by conventional approaches. Multi-spectral labeling techniques, such as Brainbow, have the potential to identify lineage-related cells in situ. Here, we report nuclear Bitbow (nBitbow), a “digital” version of Brainbow that greatly expands the color diversity for scoring cells, and a suite of statistical methods for quantifying the lineage relationship of any two cells. Applying these tools to the Drosophila peripheral nervous system (PNS), we determined lineage relationship between all neuronal pairs. This study demonstrates nBitbow as an efficient tool for in situ lineage mapping, and the complete lineage relationship among larval PNS neurons opens new possibilities for studying how neurons gain specific features and circuit connectivity., In Brief Veling et al. report a multi-color labeling system and statistical methods for mapping cell lineages. They identify the lineage relationship of all neurons in the peripheral nervous system of Drosophila larvae and show the utility of this technique in mapping neurons in the CNS., Graphical Abstract

The study of neuron morphology requires robust and comprehensive methods to quantify the differences between neurons of different subtypes and animal species. Several software packages have been developed for the analysis of neuron tracing results stored in the standard SWC format. However, providing relatively simple quantifications and their non-extendable architecture prohibit their use for advanced data analysis and visualization. We developed nGauge, a Python toolkit to support the parsing and analysis of neuron morphology data. As an application programming interface (API), nGauge can be referenced by other popular open-source software to create custom informatics analysis pipelines and advanced visualizations. nGauge defines an extendable data structure that handles volumetric constructions (e.g. soma), in addition to the SWC linear reconstructions, while remaining light-weight. This greatly extends nGauge’s data compatibility.

SUMMARYTheDrosophilatype-II neuroblast (NB) lineages present an attractive model to investigate the neural differentiation process. With only 16 stem cells, the type-II NB lineages generate many intermediate neural progenitors (INPs) to rapidly expand the neuron and glia pool, similar to those in the human outer subventricular zone (OSVZ). We performed targeted single-cell mRNA sequencing (scRNA-seq) in 3rd instar larval brains and created MiCV, an scRNA-seq data visualization web tool to integrate results from multiple bioinformatics analyses, display co-expression patterns of multiple genes simultaneously, and retrieve gene function and ortholog annotations. We identified novel markers that label distinct neural subsets using MiCV and subsequentlyin situprofiled them to recover the spatial information lacking in the scRNA-seq data. These new markers further enabled us to build novel neural developmental trajectories that lead to unique neuronal cell fates. Combining prior knowledge,in silicoanalyses, andin situevidence, this multi-informatic investigation describes the molecular landscape of neural differentiation from a single developmental snapshot inDrosophila, and provides an experimental and analytical roadmap for navigating the differentiation process of more complex brains.

Identifying the cellular origins and mapping the dendritic and axonal arbors of neurons have been century old quests to understand the heterogeneity among these brain cells. Classical chemical and genetic methods take advantage of light microscopy and sparse labeling to unambiguously, albeit inefficiently, trace a few neuronal lineages or reconstruct their morphologies in each sampled brain. To improve the analysis throughput, we designed Bitbow, a digital format of Brainbow which exponentially expands the color palette to provide tens of thousands of spectrally resolved unique labels. We generated transgenic Bitbow Drosophila lines, established statistical tools, and streamlined sample preparation, image processing and data analysis pipelines to allow conveniently mapping neural lineages, studying neuronal morphology and revealing neural network patterns with an unprecedented speed, scale and resolution.

SUMMARYElucidating cell lineages provides crucial understanding of development. Recently developed sequencing-based techniques enhance the scale of lineage tracing but eliminate the spatial information offered by conventional approaches. Multispectral labeling techniques, such asBrainbow, have the potential to identify lineage-related cellsin situ. Here, we reportLineage Tracker Bitbow, a “digital” version ofBrainbowthat greatly expands the color diversity, and a suite of statistical methods for quantifying the lineage relationship of any two cells. Applying these tools toDrosophilaperipheral nervous system, we determined lineage relationship between all neuronal pairs. Based on the refined lineage map, we explored whether distinctcis-regulatory elements are used in controlling the expression of a terminal selector gene in distinct lineage patterns. This study demonstratesLT-Bitbowas an efficient tool forin-situlineage mapping and its potential in studying molecular mechanisms in the lineage context.

SUMMARY The Drosophila type II neuroblast lineages present an attractive model to investigate the neurogenesis and differentiation process as they adapt to a process similar to that in the human outer subventricular zone. We perform targeted single-cell mRNA sequencing in third instar larval brains to study this process of the type II NB lineage. Combining prior knowledge, in silico analyses, and in situ validation, our multi-informatic investigation describes the molecular landscape from a single developmental snapshot. 17 markers are identified to differentiate distinct maturation stages. 30 markers are identified to specify the stem cell origin and/or cell division numbers of INPs, and at least 12 neuronal subtypes are identified. To foster future discoveries, we provide annotated tables of pairwise gene-gene correlation in single cells and MiCV, a web tool for interactively analyzing scRNA-seq datasets. Taken together, these resources advance our understanding of the neural differentiation process at the molecular level., In brief Using a combination of targeted scRNA-seq, in situ RNA staining, and a multi-informatic analysis paradigm, Michki et al. characterize the transcriptome landscape of thousands of type II neurons and their progenitors in the developing larval fruit fly brain., Graphical Abstract

Modeling has predicted that intramolecular structural vibrations enables proteins to access functionally important structural change. We show that the vibrational density of states and the isotropic absorption in the terahertz range are only weakly dependent on the protein functional state for several bench marking proteins. At the same time the direction of motions changes dramatically with functional state and with a resulting impact on the anisotropic absorption. Our anisotropic THz microscopy (ATM) measurements confirm this sensitivity. Here we apply the technique to the question of whether the protein dynamical transition (DT) is important to protein function. We find a strong anisotropic resonance at 70 cm−1 rapidly increases in strength at temperatures above the DT. As these intramolecular vibrations enable protein structure to change conformation, the results suggest function will cease below DT for those proteins that require large scale conformational change.

Protein structural motion is necessary for function. A typical measure of protein flexibility is the average mean squared atomic displacement (MSD). Both X-ray and neutron scattering have found a strong increase in the MSD at 200-220 K for a number of proteins. This increase is often referred to as the dynamical transition (DT) [1]. Function ceases for many proteins below the DT. It was suggested that the DT indicated the temperature at which the motions necessary for function are frozen out. A question has been raised as to how long-range motions are affected by the transition. Specifically, would the long-range intramolecular vibrations associated with conformational changes be present at low frequencies, and then at higher temperatures red shift due to anharmonicity, as is seen in conventional solids? The different DT measurement techniques do not distinguish between local particle excitations and coherent long-range motions. Direct optical measurement of the long-range intramolecular vibrations requires spectroscopy within the energy range of 1-100 cm−1 (ν = 0.03-3 THz). Our recently developed technique, CATM (Crystal Anisotropy Terahertz Microscopy) [2], uses polarization dependent absorption on aligned molecules to isolate the long-range intramolecular vibrations from the local excitations. Temperature dependent CATM from 180K to 295K is used to investigate how the DT effects long-range vibrations. We find that a sharp 70 cm-1 band grows in as the temperature increases. We suggest that this new band above DT demonstrates that in fact the surrounding solvent acts as a frozen cage preventing long-range correlated motions, and as the surrounding solvent becomes more mobile, the large-scale motions necessary for function can occur.1.Doster, W., et al. Physical Review Letters, 2010. 104(9): p. 098101.2.Acbas, G., et al. Nature Communications, 2014. 5, 3076 http://dx.doi.org/10.1038/ncomms4076.

The protein dynamical transition is the remarkable increase in the average atomic root mean squared displacement (RMSD) in the 180-220 K range. The effect is associated with the onset of anharmonic motions critical to biological function. However evidence of the actual biological relevance of the dynamical transition is inconsistent. While for some proteins, function ceases below the dynamical transition, for other proteins the dynamical transition appears to have no effect. Here we report measurements that suggest the difference arises from the dependence of function on large scale conformational change, and specifically the reliance on long range vibrations to access these structural changes. The dynamical transition has been extensively observed using X-ray, neutron scattering, NMR and terahertz absorption spectroscopy [1,2], with the results indicating it arises from thermally activated solvent motions. Those techniques measure all motions contributing to the RMSD including both localized motions and intramolecular vibrations. To isolate the vibrations and examine how the dynamical transition impacts them, we use a new technique, anisotropy terahertz microscopy (ATM) [3]. This unique method suppresses the background from the localized motions giving unprecedented access to the long range motions that enable large scale conformational changes. ATM measurements of lysozyme anisotropic optical absorbance in the 150-300 K temperature range show that the resonant vibrational bands rapidly increase in intensity at the dynamical transition, and surprisingly blue shift with increasing temperature, in contrast to the expected anharmonicity. The measurements demonstrate that the surrounding solvent below the dynamical transition acts as a frozen cage preventing the vibrations necessary for functional conformational change. This solvent slaving of the long range vibrations potentially impacts protein structural stability and vulnerability to structural disorder. This work was supported by NSF (DBI 1556359 and MCB 1616529), and DOE DE-SC0016317. 1. Doster,W., et al. Phys.Rev.Lett., 2010.104(9):098101. 2. Niessen,K., et al. Biophys.Rev., 2015.7,201. 3. Acbas,G., et al. Nat.Commun., 2014.5,3076.