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The mammalian cochlea loses its ability to regenerate new hair cells prior to the onset of hearing. In contrast, the adult vestibular system can produce new hair cells in response to damage, or by reprogramming of supporting cells with the hair cell transcription factor Atoh1. We used RNA-seq and ATAC-seq to probe the transcriptional and epigenetic responses of utricle supporting cells to damage and Atoh1 transduction. We show that the regenerative response of the utricle correlates with a more accessible chromatin structure in utricle supporting cells compared to their cochlear counterparts. We also provide evidence that Atoh1 transduction of supporting cells is able to promote increased transcriptional accessibility of some hair cell genes. Our study offers a possible explanation for regenerative differences between sensory organs of the inner ear, but shows that additional factors to Atoh1 may be required for optimal reprogramming of hair cell fate.

The mechanisms that regulate post-natal growth of the craniofacial complex and that ultimately determine the size and shape of our faces are not well understood. Hippo signaling is a general mechanism to control tissue growth and organ size, and although it is known that Hippo signaling functions in neural crest specification and patterning during embryogenesis and before birth, its specific role in postnatal craniofacial growth remains elusive. We have identified the transcription factor FoxO6 as an activator of Hippo signaling regulating neonatal growth of the face. During late stages of mouse development, FoxO6 is expressed specifically in craniofacial tissues and FoxO6-/- mice undergo expansion of the face, frontal cortex, olfactory component and skull. Enlargement of the mandible and maxilla and lengthening of the incisors in FoxO6-/- mice are associated with increases in cell proliferation. In vitro and in vivo studies demonstrated that FoxO6 activates Lats1 expression, thereby increasing Yap phosphorylation and activation of Hippo signaling. FoxO6-/- mice have significantly reduced Hippo Signaling caused by a decrease in Lats1 expression and decreases in Shh and Runx2 expression, suggesting that Shh and Runx2 are also linked to Hippo signaling. In vitro, FoxO6 activates Hippo reporter constructs and regulates cell proliferation. Furthermore PITX2, a regulator of Hippo signaling is associated with Axenfeld-Rieger Syndrome causing a flattened midface and we show that PITX2 activates FoxO6 expression. Craniofacial specific expression of FoxO6 postnatally regulates Hippo signaling and cell proliferation. Together, these results identify a FoxO6-Hippo regulatory pathway that controls skull growth, odontogenesis and face morphology.

The diaphragm muscle is essential for breathing in mammals. Its asymmetric elevation during contraction correlates with morphological features suggestive of inherent left–right (L/R) asymmetry. Whether this asymmetry is due to L versus R differences in the muscle or in the phrenic nerve activity is unknown. Here, we have combined the analysis of genetically modified mouse models with transcriptomic analysis to show that both the diaphragm muscle and phrenic nerves have asymmetries, which can be established independently of each other during early embryogenesis in pathway instructed by Nodal, a morphogen that also conveys asymmetry in other organs. We further found that phrenic motoneurons receive an early L/R genetic imprint, with L versus R differences both in Slit/Robo signaling and MMP2 activity and in the contribution of both pathways to establish phrenic nerve asymmetry. Our study therefore demonstrates L–R imprinting of spinal motoneurons and describes how L/R modulation of axon guidance signaling helps to match neural circuit formation to organ asymmetry.

A microRNA regulates the expression of a network of genes in the heart to ensure that progenitor cells develop into strongly contractile cardiac muscle.

Among the most common human congenital anomalies, cleft lip and palate (CL/P) affects up to 1 in 700 live births. MicroRNA (miR)s are small, non-coding RNAs that repress gene expression post-transcriptionally. The miR-17-92 cluster encodes six miRs that have been implicated in human cancers and heart development. We discovered that miR-17-92 mutant embryos had severe craniofacial phenotypes, including incompletely penetrant CL/P and mandibular hypoplasia. Embryos that were compound mutant for miR-17-92 and the related miR-106b-25 cluster had completely penetrant CL/P. Expression of Tbx1 and Tbx3, the DiGeorge/velo-cardio-facial (DGS) and Ulnar-mammary syndrome (UMS) disease genes, was expanded in miR-17-92 mutant craniofacial structures. Both Tbx1 and Tbx3 had functional miR seed sequences that mediated gene repression. Analysis of miR-17-92 regulatory regions uncovered conserved and functional AP-2α recognition elements that directed miR-17-92 expression. Together, our data indicate that miR-17-92 modulates expression of critical T-box transcriptional regulators during midface development and is itself a target of Bmp-signaling and the craniofacial pioneer factor AP-2α. Our data are the first genetic evidence that an individual miR or miR cluster is functionally important in mammalian CL/P.

Abstract A critical challenge of single-cell spatial transcriptomics (sc-ST) technologies is their panel size. Being based on fluorescence in situ hybridization, they are typically limited to panels of about a thousand genes. This constrains researchers to build panels from only the marker genes of different cell types and forgo other genes of interest, e.g., genes encoding ligand-receptor complexes or those in specific pathways. We propose scGIST, a constrained feature selection tool that designs sc-ST panels prioritizing user-specified genes without compromising cell type detection accuracy. We demonstrate scGIST’s efficacy in diverse use cases, highlighting it as a valuable addition to sc-ST’s algorithmic toolbox.

Abstract Background Myeloid cells play critical roles in the regulation of myocardial injury and repair. Clonal hematopoiesis (CH)‐related mutations in genes, such as Ten‐eleven Translocation 2 (TET2), can impair myeloid cells and are associated with increased risk of cardiovascular disease (CVD). How Tet2 loss‐of‐function (LOF) impacts myeloid cells and disrupts normal myocardial repair remains unclear. Methods We established ischemia‐induced myocardial infarction (MI) in a myeloid‐specific Tet2‐deficient mouse model. The echocardiographic assessment was conducted to evaluate the cardiac function. Histological analysis was performed to evaluate morphological changes in infarcted areas and fibrosis. To monitor the dynamic changes of myeloid cells in cardiac tissues during cardiac remodeling after MI, we performed longitudinal analysis on subsets of myeloid cells using flow cytometry. We performed immunofluorescence (IF) staining to examine the DNA damage and genome instability caused by Tet2 LOF. Gene expression was assessed by real‐time qRT‐PCR. Stimulator of Interferon Genes (STING) pathway activation was assessed using various methods, including Western blotting, flow cytometry, ELISA and IF staining of key signaling proteins involved in this pathway. Additionally, H‐151 was used as a pharmacological tool to antagonize augmented STING activation in the murine MI model. Results We observed a substantial increase of neutrophils in the post‐MI mice, which contributes to adverse outcomes during heart repair. Mechanistically, Tet2‐deficient myeloid cells exhibited increased genome instability, accompanied with augmented activation of the STING pathway. Furthermore, the use of H‐151 a covalent STING binder that targets the cysteine residue at position 91 and functions as a potent STING antagonist, led to a substantial decrease in neutrophil populations in Tet2‐deficient mice following myocardial infarction, thereby reversing adverse cardiac outcomes. Conclusion Our novel findings establish the rationale for targeting the cGAS‐STING pathway as a promising therapeutic strategy to mitigate cardiovascular disease risk in individuals with clonal hematopoiesis harboring TET2 loss‐of‐function mutations. Highlights Myeloid‐specific Tet2 depletion promotes neutrophil expansion upon myocardium infarction (MI); Tet2‐deficient myeloid cells exhibit increased genome instability and cGAS‐STING overactivation; STING antagonist H‐151 treatment reduces neutrophil expansion in Tet2‐deficient mice after MI and mitigates deleterious cardiac outcomes.

Abstract Hematopoietic stem cells (HSCs) are capable of regenerating the blood system, but the instructive cues that direct HSCs to regenerate particular lineages lost to the injury remain elusive. Here, we show that iron is increasingly taken up by HSCs during anemia and induces erythroid gene expression and regeneration in a Tet2-dependent manner. Lineage tracing of HSCs reveals that HSCs respond to hemolytic anemia by increasing erythroid output. The number of HSCs in the spleen, but not bone marrow, increases upon anemia and these HSCs exhibit enhanced proliferation, erythroid differentiation, iron uptake, and TET2 protein expression. Increased iron in HSCs promotes DNA demethylation and expression of erythroid genes. Suppressing iron uptake or TET2 expression impairs erythroid genes expression and erythroid differentiation of HSCs; iron supplementation, however, augments these processes. These results establish that the physiological level of iron taken up by HSCs has an instructive role in promoting erythroid-biased differentiation of HSCs.

Tet-mediated DNA demethylation is intimately involved in reguatling embryonic development. Here the authors characterise DNA methylation and hydroxymethylation dynamics during early cardiac development in both human and mice and provide evidence that Tet-mediated DNA demethylation plays a role in regulating chromatin organization during early heart development.

Endocrine progenitors form early in pancreatic development but the diversity of this cell population is unclear. Here, the authors use single cell RNA sequencing of the mouse pancreas at e14.5 and e16.5 to show that endocrine progenitors are temporally distinct and those formed later are more likely to become beta cells

Abstract Background Left-sided lesions (LSLs) account for an important fraction of severe congenital cardiovascular malformations (CVMs). The genetic contributions to LSLs are complex, and the mutations that cause these malformations span several diverse biological signaling pathways: TGFB, NOTCH, SHH, and more. Here, we use whole exome sequence data generated in 342 LSL cases to identify likely damaging variants in putative candidate CVM genes. Methods Using a series of bioinformatics filters, we focused on genes harboring population-rare, putative loss-of-function (LOF), and predicted damaging variants in 1760 CVM candidate genes constructed a priori from the literature and model organism databases. Gene variants that were not observed in a comparably sequenced control dataset of 5492 samples without severe CVM were then subjected to targeted validation in cases and parents. Whole exome sequencing data from 4593 individuals referred for clinical sequencing were used to bolster evidence for the role of candidate genes in CVMs and LSLs. Results Our analyses revealed 28 candidate variants in 27 genes, including 17 genes not previously associated with a human CVM disorder, and revealed diverse patterns of inheritance among LOF carriers, including 9 confirmed de novo variants in both novel and newly described human CVM candidate genes (ACVR1, JARID2, NR2F2, PLRG1, SMURF1) as well as established syndromic CVM genes (KMT2D, NF1, TBX20, ZEB2). We also identified two genes (DNAH5, OFD1) with evidence of recessive and hemizygous inheritance patterns, respectively. Within our clinical cohort, we also observed heterozygous LOF variants in JARID2 and SMAD1 in individuals with cardiac phenotypes, and collectively, carriers of LOF variants in our candidate genes had a four times higher odds of having CVM (odds ratio = 4.0, 95% confidence interval 2.5–6.5). Conclusions Our analytical strategy highlights the utility of bioinformatic resources, including human disease records and model organism phenotyping, in novel gene discovery for rare human disease. The results underscore the extensive genetic heterogeneity underlying non-syndromic LSLs, and posit potential novel candidate genes and complex modes of inheritance in this important group of birth defects.

Summary: In response to retinal damage, the Müller glial cells (MGs) of the zebrafish retina have the ability to undergo a cellular reprogramming event in which they enter the cell cycle and divide asymmetrically, thereby producing multipotent retinal progenitors capable of regenerating lost retinal neurons. However, mammalian MGs do not exhibit such a proliferative and regenerative ability. Here, we identify Hippo pathway-mediated repression of the transcription cofactor YAP as a core regulatory mechanism that normally blocks mammalian MG proliferation and cellular reprogramming. MG-specific deletion of Hippo pathway components Lats1 and Lats2, as well as transgenic expression of a Hippo non-responsive form of YAP (YAP5SA), resulted in dramatic Cyclin D1 upregulation, loss of adult MG identity, and attainment of a highly proliferative, progenitor-like cellular state. Our results reveal that mammalian MGs may have latent regenerative capacity that can be stimulated by repressing Hippo signaling. : Rueda et al. identify the Hippo pathway as an endogenous molecular mechanism normally preventing mammalian Müller glial reprogramming to a proliferative, progenitor-like state. Keywords: retina, Hippo pathway, reprogramming, Müller glia, YAP, LATS, regeneration

Expression of Pitx2 on the left side of the embryo patterns left-right (LR) organs including the dorsal mesentery (DM), whose asymmetric cell behavior directs gut looping. Despite the importance of organ laterality, chromatin-level regulation of Pitx2 remains undefined. Here, we show that genes immediately neighboring Pitx2 in chicken and mouse, including a long noncoding RNA (Pitx2 locus-asymmetric regulated RNA or Playrr), are expressed on the right side and repressed by Pitx2. CRISPR/Cas9 genome editing of Playrr, 3D fluorescent in situ hybridization (FISH), and variations of chromatin conformation capture (3C) demonstrate that mutual antagonism between Pitx2 and Playrr is coordinated by asymmetric chromatin interactions dependent on Pitx2 and CTCF. We demonstrate that transcriptional and morphological asymmetries driving gut looping are mirrored by chromatin architectural asymmetries at the Pitx2 locus. We propose a model whereby Pitx2 auto-regulation directs chromatin topology to coordinate LR transcription of this locus essential for LR organogenesis.

Combined methylmalonic acidemia (MMA) and hyperhomocysteinemias are inborn errors of vitamin B12 metabolism, and mutations in the transcriptional regulators HCFC1 and RONIN (THAP11) underlie some forms of these disorders. Here the authors generated mouse models of a human syndrome due to mutations in RONIN (THAP11) and HCFC1, and show that this syndrome is both an inborn error of vitamin B12 metabolism and displays some features of ribosomopathy.

Atrial fibrillation (AF), the most common sustained cardiac arrhythmia and a major risk factor for stroke, often arises through ectopic electrical impulses derived from the pulmonary veins (PVs). Sequence variants in enhancers controlling expression of the transcription factor PITX2, which is expressed in the cardiomyocytes (CMs) of the PV and left atrium (LA), have been implicated in AF predisposition. Single nuclei multiomic profiling of RNA and analysis of chromatin accessibility combined with spectral clustering uncovered distinct PV- and LA-enriched CM cell states. Pitx2-mutant PV and LA CMs exhibited gene expression changes consistent with cardiac dysfunction through cell type–distinct, PITX2-directed, cis-regulatory grammars controlling target gene expression. The perturbed network targets in each CM were enriched in distinct human AF predisposition genes, suggesting combinatorial risk for AF genesis. Our data further reveal that PV and LA Pitx2-mutant CMs signal to endothelial and endocardial cells through BMP10 signaling with pathogenic potential. This work provides a multiomic framework for interrogating the basis of AF predisposition in the PVs of humans.

Background The conversion of fibroblasts into induced cardiomyocytes may regenerate myocardial tissue from cardiac scar through in situ cell transdifferentiation. The efficiency transdifferentiation is low, especially for human cells. We explored the leveraging of Hippo pathway intermediates to enhance induced cardiomyocyte generation. Methods and Results We screened Hippo effectors Yap (yes‐associated protein), Taz (transcriptional activator binding domain), and Tead1 (TEA domain transcription factor 1; Td) for their reprogramming efficacy with cardio‐differentiating factors Gata4, Mef2C, and Tbx5 (GMT). Td induced nearly 3‐fold increased expression of cardiomyocyte marker cTnT (cardiac troponin T) by mouse embryonic and adult rat fibroblasts versus GMT administration alone (P

The majority of disease-associated genetic variants lie in non-coding regions. Here the authors generated and compiled human transcriptomic, epigenomic and chromatin conformation datasets, to identify genes associated with atrial fibrillation and functional non-coding variants.

Summary: Cellular heterogeneity within the mammalian brain poses a challenge toward understanding its complex functions. Within the olfactory bulb, odor information is processed by subtypes of inhibitory interneurons whose heterogeneity and functionality are influenced by ongoing adult neurogenesis. To investigate this cellular heterogeneity and better understand adult-born neuron development, we utilized single-cell RNA sequencing and computational modeling to reveal diverse and transcriptionally distinct neuronal and nonneuronal cell types. We also analyzed molecular changes during adult-born interneuron maturation and uncovered developmental programs within their gene expression profiles. Finally, we identified that distinct neuronal subtypes are differentially affected by sensory experience. Together, these data provide a transcriptome-based foundation for investigating subtype-specific neuronal function in the olfactory bulb (OB), charting the molecular profiles that arise during the maturation and integration of adult-born neurons and how they dynamically change in an activity-dependent manner. : Using single-cell sequencing, Tepe et al. describe cellular heterogeneity in the mouse olfactory bulb, uncover markers for each cell type, and reveal differentially regulated genes in adult-born neurons. These findings provide a framework for studying cell-type-specific functions and circuit integration in the mammalian brain. Keywords: single-cell RNA-seq, adult neurogenesis, olfactory bulb, circuit integration, olfaction, lineage, development, interneuron, transcriptome