Background: Cytokines are small proteins that regulate immunity in vertebrate species. Marsupial and eutherian mammals last shared a common ancestor more than 180 million years ago, so it is not surprising that attempts to isolate many key marsupial cytokines using traditional laboratory techniques have been unsuccessful. This paucity of molecular data has led some authors to suggest that the marsupial immune system is 'primitive' and not on par with the sophisticated immune system of eutherian (placental) mammals.

Background Clozapine is the most effective medication for treatment-refractory schizophrenia but is associated with significant adverse drug reactions, including nocturnal enuresis and urinary incontinence. This side effect can be burdensome and lead to medication nonadherence and psychotic relapse. Evidence to guide treatment of clozapine-induced nocturnal enuresis and urinary incontinence is sparse. We therefore aimed to synthesize the evidence base to guide management for clinicians, patients, and their carers. Methods We systematically searched PubMed, Embase, PsycInfo, CINAHL, and the Cochrane Trial Registry databases from inception to May 2021 for publications on management of clozapine-induced nocturnal enuresis and urinary incontinence using a PROSPERO preregistered search strategy. Results We identified 22 case reports and case series describing 74 patients. Interventions included clozapine dose reduction, nonpharmacological treatment, and pharmacological treatments. Among pharmacological treatments, desmopressin, oxybutynin, trihexyphenidyl, tolterodine, imipramine, amitriptyline, ephedrine, pseudoephedrine, aripiprazole, and verapamil were associated with complete resolution of nocturnal enuresis and urinary incontinence. Balancing evidence for effectiveness against risk of adverse effects, we developed a management framework for clozapine-induced nocturnal enuresis and urinary incontinence. Conclusions Following assessment of urological, psychiatric, pharmacological, and common comorbid medical issues, first-line treatments should be nonpharmacological, including bathroom alarms, voiding before bedtime, and nocturnal fluid restriction. If these interventions do not provide adequate relief, aripiprazole should be trialed. Desmopressin may be considered for severe refractory cases, but monitoring for hyponatremia is essential.

45Differentiation into diverse cell lineages requires orchestration of gene regulatory networks guiding cell fate choices. Here, we present the dissection of cellular composition and gene networks from transcriptomic data of 43,168 cells across five discrete time points during cardiac-directed differentiation. We utilize unsupervised clustering and implement a lineage trajectory prediction algorithm that integrates transcription factor networks to predict cell fate progression of 15 subpopulations that correlate with germ 50 layer and cardiovascular differentiation in vivo. These data reveal transcriptional networks underlying lineage derivation of mesoderm, definitive endoderm, vascular endothelium, cardiac precursors, and definitive cell types that comprise cardiomyocytes and a previously unrecognized cardiac outflow tract population. Single cell analysis of genetic regulators governing cardiac fate diversification identified the non-DNA binding homeodomain protein, HOPX, as functionally necessary for endothelial specification. 55Our findings further implicate dysregulation of HOPX during in vitro cardiac-directed differentiation underlying the molecular and physiological immaturity of stem cell-derived cardiomyocytes. 65Key words: human pluripotent stem cells, cardiomyocytes, transcriptomic profiling, outflow tract, heart, development, CRISPR, engineered heart tissue, RNA-sequencing, HOPX, scdiff 70 75. CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/229294 doi: bioRxiv preprint first posted online Dec. 7, 2017; Introduction 80Studies of cardiac development at single-cell resolution have provided valuable new insights into cell diversity and genetic regulation of cell types revealing mechanisms underlying cardiovascular differentiation and morphogenesis. Single-cell analysis of in vivo mouse heart development have revealed chamber-specific and temporal changes in gene expression underlying embryonic heart development from E9.5 to postnatal day 21 establishing anatomical patterns of gene expression in the heart (Li et al., 2016) 85and new insights into transcriptional programs underlying cardiac maturation (DeLaughter et al., 2016). These studies provide a valuable resource by which to understand transcriptional mechanisms underlying diverse fate choices involved in cardiac development and morphogenesis in vivo. Like many single-cell transcriptomic studies, they further highlight the importance of dissecting cell heterogeneity to understand mechanisms underlying the identity and fate of cells in health and disease. 90Human pluripotent stem cells are a key model system to study human cardiovascular developmental biology (Murry and Keller, 2008). However, the fidelity by which cardiac directed differentiation in vitro recapitulates the transcriptional programs governing the diversity of cell fates generated in vivo is not well understood. Analyzing differentiation efficiency...

Precise patterns of gene expression are driven by interactions between transcription factors, regulatory DNA sequence, and chromatin. How DNA mutations affecting any one of these regulatory ‘layers’ is buffered or propagated to gene expression remains unclear. To address this, we quantified allele-specific changes in chromatin accessibility, histone modifications, and gene expression in F1 embryos generated from eight Drosophila crosses, at three embryonic stages, yielding a comprehensive dataset of 240 samples spanning multiple regulatory layers. Genetic variation in cis-regulatory elements is common, highly heritable, and surprisingly consistent in its effects across embryonic stages. Much of this variation does not propagate to gene expression. When it does, it acts through H3K4me3 or alternatively through chromatin accessibility and H3K27ac. The magnitude and evolutionary impact of mutations is influenced by a genes’ regulatory complexity (i.e. enhancer number), with transcription factors being most robust to cis-acting, and most influenced by trans-acting, variation. Overall, the impact of genetic variation on regulatory phenotypes appears context-dependent even within the constraints of embryogenesis.

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