Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation.

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, a robust pipeline was designed to perform single-cell and nuclei analysis on mouse embryos from embryonic day E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to wild-type perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, a methodology is presented designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with a same-day genotyping protocol (3 h) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. This method further includes guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate that this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.

A Retinoic Acid:YAP1 signaling axis controls atrial lineage commitment.

Vitamin A/Retinoic Acid (Vit A/RA) signaling is essential for heart development. In cardiac progenitor cells (CPCs), RA signaling induces the expression of atrial lineage genes while repressing ventricular genes, thereby promoting the acquisition of an atrial cardiomyocyte cell fate. To achieve this, RA coordinates a complex regulatory network of downstream effectors that is not fully identified. To address this gap, we applied a functional genomics approach (i.e scRNAseq and snATACseq) to untreated and RA-treated human embryonic stem cells (hESCs)-derived CPCs. Unbiased analysis revealed that the Hippo effectors YAP1 and TEAD4 are integrated with the atrial transcription factor enhancer network, and that YAP1 is necessary for activation of RA-enhancers in CPCs. Furthermore, in vivo analysis of control and conditionally YAP1 KO mouse embryos (Sox2-cre) revealed that the expression of atrial lineage genes, such as NR2F2, is compromised by YAP1 deletion in the CPCs of the second heart field. Accordingly, we found that YAP1 is required for the formation of an atrial chamber but is dispensable for the formation of a ventricle, in hESC-derived patterned cardiac organoids. Overall, our findings revealed that YAP1 is a non-canonical effector of RA signaling essential for the acquisition of atrial lineages during cardiogenesis.

Single-cell RNA sequencing of mutant whole mouse embryos: from the epiblast to the end of gastrulation.

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, we designed a robust pipeline to perform single-cell and nuclei analysis on mouse embryos from E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to WT perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, we present a methodology designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with FAST genotyping protocol (3 hours) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. We also include guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula. SUMMARY: We establish a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

Altered lipid homeostasis is associated with cerebellar neurodegeneration in SNX14 deficiency.

Dysregulated lipid homeostasis is emerging as a potential cause of neurodegenerative disorders. However, evidence of errors in lipid homeostasis as a pathogenic mechanism of neurodegeneration remains limited. Here, we show that cerebellar neurodegeneration caused by Sorting Nexin 14 (SNX14) deficiency is associated with lipid homeostasis defects. Recent studies indicate that SNX14 is an interorganelle lipid transfer protein that regulates lipid transport, lipid droplet (LD) biogenesis, and fatty acid desaturation, suggesting that human SNX14 deficiency belongs to an expanding class of cerebellar neurodegenerative disorders caused by altered cellular lipid homeostasis. To test this hypothesis, we generated a mouse model that recapitulates human SNX14 deficiency at a genetic and phenotypic level. We demonstrate that cerebellar Purkinje cells (PCs) are selectively vulnerable to SNX14 deficiency while forebrain regions preserve their neuronal content. Ultrastructure and lipidomic studies reveal widespread lipid storage and metabolism defects in SNX14-deficient mice. However, predegenerating SNX14-deficient cerebella show a unique accumulation of acylcarnitines and depletion of triglycerides. Furthermore, defects in LD content and telolysosome enlargement in predegenerating PCs suggest lipotoxicity as a pathogenic mechanism of SNX14 deficiency. Our work shows a selective cerebellar vulnerability to altered lipid homeostasis and provides a mouse model for future therapeutic studies.

IMPDH2 filaments protect from neurodegeneration in AMPD2 deficiency.

Metabolic dysregulation is one of the most common causes of pediatric neurodegenerative disorders. However, how the disruption of ubiquitous and essential metabolic pathways predominantly affect neural tissue remains unclear. Here we use mouse models of AMPD2 deficiency to study cellular and molecular mechanisms that lead to selective neuronal vulnerability to purine metabolism imbalance. We show that AMPD deficiency in mice primarily leads to hippocampal dentate gyrus degeneration despite causing a generalized reduction of brain GTP levels. Remarkably, we found that neurodegeneration resistant regions accumulate micron sized filaments of IMPDH2, the rate limiting enzyme in GTP synthesis. In contrast, IMPDH2 filaments are barely detectable in the hippocampal dentate gyrus, which shows a progressive neuroinflammation and neurodegeneration. Furthermore, using a human AMPD2 deficient neural cell culture model, we show that blocking IMPDH2 polymerization with a dominant negative IMPDH2 variant, impairs AMPD2 deficient neural progenitor growth. Together, our findings suggest that IMPDH2 polymerization prevents detrimental GTP deprivation in neurons with available GTP precursor molecules, providing resistance to neurodegeneration. Our findings open the possibility of exploring the involvement of IMPDH2 assembly as a therapeutic intervention for neurodegeneration.

Targeted therapy improves cellular dysfunction, ataxia, and seizure susceptibility in a model of a progressive myoclonus epilepsy.

The recurrent variant KCNC1-p.Arg320His causes progressive myoclonus epilepsy (EPM) type 7, defined by progressive myoclonus, epilepsy, and ataxia, and is without effective treatment. KCNC1 encodes the voltage-gated potassium channel subunit Kv3.1, specifically expressed in high-frequency-firing neurons. Variant subunits act via loss of function; hence, EPM7 pathogenesis may involve impaired excitability of Kv3.1-expressing neurons, while enhancing Kv3 activity could represent a viable therapeutic strategy. We generate a mouse model, Kcnc1-p.Arg320His/+, which recapitulates the core features of EPM7, including progressive ataxia and seizure susceptibility. Kv3.1-expressing cerebellar granule cells and neocortical parvalbumin-positive GABAergic interneurons exhibit abnormalities consistent with Kv3 channel dysfunction. A Kv3-specific positive modulator (AUT00206) selectively enhances the firing frequency of Kv3.1-expressing neurons and improves motor function and seizure susceptibility in Kcnc1-Arg320His/+ mice. This work identifies a cellular and circuit basis of dysfunction in EPM7 and demonstrates that Kv3 positive modulators such as AUT00206 have therapeutic potential for the treatment of EPM7.

High density SNP array and reanalysis of genome sequencing uncovers CNVs associated with neurodevelopmental disorders in KOLF2.1J iPSCs.

The KOLF2.1J iPSC line was recently proposed as a reference iPSC to promote the standardization of research studies in the stem cell field. Due to overall good performance differentiating to neural cell lineages, high gene editing efficiency, and absence of genetic variants associated to neurological disorders KOLF2.1J iPSC line was particularly recommended for neurodegenerative disease modeling. However, our work uncovers that KOLF2.1J hPSCs carry heterozygous small copy number variants (CNVs) that cause DTNBP1, JARID2 and ASTN2 haploinsufficiencies, all of which are associated with neurological disorders. We further determine that these CNVs arose in vitro over the course of KOLF2.1J iPSC generation from a healthy donor-derived KOLF2 iPSC line and affect the expression of DNTBP1, JARID2 and ASTN2 proteins in KOLF2.1J iPSCs and neural progenitors. Therefore, our study suggests that KOLF2.1J iPSCs carry genetic variants that may be deleterious for neural cell lineages. This data is essential for a careful interpretation of neural cell studies derived from KOLF2.1J iPSCs and highlights the need for a catalogue of iPSC lines that includes a comprehensive genome characterization analysis.

Mechanism of KMT5B haploinsufficiency in neurodevelopment in humans and mice.

Pathogenic variants in KMT5B, a lysine methyltransferase, are associated with global developmental delay, macrocephaly, autism, and congenital anomalies (OMIM# 617788). Given the relatively recent discovery of this disorder, it has not been fully characterized. Deep phenotyping of the largest (n = 43) patient cohort to date identified that hypotonia and congenital heart defects are prominent features that were previously not associated with this syndrome. Both missense variants and putative loss-of-function variants resulted in slow growth in patient-derived cell lines. KMT5B homozygous knockout mice were smaller in size than their wild-type littermates but did not have significantly smaller brains, suggesting relative macrocephaly, also noted as a prominent clinical feature. RNA sequencing of patient lymphoblasts and Kmt5b haploinsufficient mouse brains identified differentially expressed pathways associated with nervous system development and function including axon guidance signaling. Overall, we identified additional pathogenic variants and clinical features in KMT5B-related neurodevelopmental disorder and provide insights into the molecular mechanisms of the disorder using multiple model systems.

Multimerization of Zika Virus-NS5 Causes Ciliopathy and Forces Premature Neurogenesis.

Zika virus (ZikV) is a flavivirus that infects neural tissues, causing congenital microcephaly. ZikV has evolved multiple mechanisms to restrict proliferation and enhance cell death, although the underlying cellular events involved remain unclear. Here we show that the ZikV-NS5 protein interacts with host proteins at the base of the primary cilia in neural progenitor cells, causing an atypical non-genetic ciliopathy and premature neuron delamination. Furthermore, in human microcephalic fetal brain tissue, ZikV-NS5 persists at the base of the motile cilia in ependymal cells, which also exhibit a severe ciliopathy. Although the enzymatic activity of ZikV-NS5 appears to be dispensable, the amino acids Y25, K28, and K29 that are involved in NS5 oligomerization are essential for localization and interaction with components of the cilium base, promoting ciliopathy and premature neurogenesis. These findings lay the foundation for therapies that target ZikV-NS5 multimerization and prevent the developmental malformations associated with congenital Zika syndrome.

Loss of Protocadherin-12 Leads to Diencephalic-Mesencephalic Junction Dysplasia Syndrome.

OBJECTIVE: To identify causes of the autosomal-recessive malformation, diencephalic-mesencephalic junction dysplasia (DMJD) syndrome. METHODS: Eight families with DMJD were studied by whole-exome or targeted sequencing, with detailed clinical and radiological characterization. Patient-derived induced pluripotent stem cells were derived into neural precursor and endothelial cells to study gene expression. RESULTS: All patients showed biallelic mutations in the nonclustered protocadherin-12 (PCDH12) gene. The characteristic clinical presentation included progressive microcephaly, craniofacial dysmorphism, psychomotor disability, epilepsy, and axial hypotonia with variable appendicular spasticity. Brain imaging showed brainstem malformations and with frequent thinned corpus callosum with punctate brain calcifications, reflecting expression of PCDH12 in neural and endothelial cells. These cells showed lack of PCDH12 expression and impaired neurite outgrowth. INTERPRETATION: DMJD patients have biallelic mutations in PCDH12 and lack of protein expression. These patients present with characteristic microcephaly and abnormalities of white matter tracts. Such pathogenic variants predict a poor outcome as a result of brainstem malformation and evidence of white matter tract defects, and should be added to the phenotypic spectrum associated with PCDH12-related conditions. Ann Neurol 2018;84:646-655.

Biallelic mutations in the 3' exonuclease TOE1 cause pontocerebellar hypoplasia and uncover a role in snRNA processing.

Deadenylases are best known for degrading the poly(A) tail during mRNA decay. The deadenylase family has expanded throughout evolution and, in mammals, consists of 12 Mg2+-dependent 3'-end RNases with substrate specificity that is mostly unknown. Pontocerebellar hypoplasia type 7 (PCH7) is a unique recessive syndrome characterized by neurodegeneration and ambiguous genitalia. We studied 12 human families with PCH7, uncovering biallelic, loss-of-function mutations in TOE1, which encodes an unconventional deadenylase. toe1-morphant zebrafish displayed midbrain and hindbrain degeneration, modeling PCH-like structural defects in vivo. Surprisingly, we found that TOE1 associated with small nuclear RNAs (snRNAs) incompletely processed spliceosomal. These pre-snRNAs contained 3' genome-encoded tails often followed by post-transcriptionally added adenosines. Human cells with reduced levels of TOE1 accumulated 3'-end-extended pre-snRNAs, and the immunoisolated TOE1 complex was sufficient for 3'-end maturation of snRNAs. Our findings identify the cause of a neurodegenerative syndrome linked to snRNA maturation and uncover a key factor involved in the processing of snRNA 3' ends.

EZH2 regulates neuroepithelium structure and neuroblast proliferation by repressing p21.

The function of EZH2 as a transcription repressor is well characterized. However, its role during vertebrate development is still poorly understood, particularly in neurogenesis. Here, we uncover the role of EZH2 in controlling the integrity of the neural tube and allowing proper progenitor proliferation. We demonstrate that knocking down the EZH2 in chick embryo neural tubes unexpectedly disrupts the neuroepithelium (NE) structure, correlating with alteration of the Rho pathway, and reduces neural progenitor proliferation. Moreover, we use transcriptional profiling and functional assays to show that EZH2-mediated repression of p21(WAF1/CIP1) contributes to both processes. Accordingly, overexpression of cytoplasmic p21(WAF1/CIP1) induces NE structural alterations and p21(WAF1/CIP1) suppression rescues proliferation defects and partially compensates for the structural alterations and the Rho activity. Overall, our findings describe a new role of EZH2 in controlling the NE integrity in the neural tube to allow proper progenitor proliferation.

EZH2 orchestrates apicobasal polarity and neuroepithelial cell renewal.

During early stages of neural development, neuroepithelial cells translocate their nuclei along the apicobasal axis in a harmonized manner with the cell cycle. How cell cycle progression and neuroepithelium polarity are coordinated remains unclear. It has been proposed that developmental cues, epigenetic mechanisms and cell cycle regulators must be linked in order to orchestrate these processes. We have recently discovered that a master epigenetic factor, EZH2 is essential to coordinate these events. EZH2 directly represses the cell cycle regulator p21WAF1/CIP in the chicken spinal cord. By doing so, EZH2 controls neural progenitor cell renewal and fine-tunes Rho signaling pathway, which is essential to maintain neuroepithelial structure. Our findings point to a new role of EZH2 during development that could have potential implication in other areas as cancer.

Inactivating mutations in MFSD2A, required for omega-3 fatty acid transport in brain, cause a lethal microcephaly syndrome.

Docosahexanoic acid (DHA) is the most abundant omega-3 fatty acid in brain, and, although it is considered essential, deficiency has not been linked to disease. Despite the large mass of DHA in phospholipids, the brain does not synthesize it. DHA is imported across the blood-brain barrier (BBB) through the major facilitator superfamily domain-containing 2a (MFSD2A) protein. MFSD2A transports DHA as well as other fatty acids in the form of lysophosphatidylcholine (LPC). We identify two families displaying MFSD2A mutations in conserved residues. Affected individuals exhibited a lethal microcephaly syndrome linked to inadequate uptake of LPC lipids. The MFSD2A mutations impaired transport activity in a cell-based assay. Moreover, when expressed in mfsd2aa-morphant zebrafish, mutants failed to rescue microcephaly, BBB breakdown and lethality. Our results establish a link between transport of DHA and LPCs by MFSD2A and human brain growth and function, presenting the first evidence of monogenic disease related to transport of DHA in humans.

Biallelic mutations in SNX14 cause a syndromic form of cerebellar atrophy and lysosome-autophagosome dysfunction.

Pediatric-onset ataxias often present clinically as developmental delay and intellectual disability, with prominent cerebellar atrophy as a key neuroradiographic finding. Here we describe a new clinically distinguishable recessive syndrome in 12 families with cerebellar atrophy together with ataxia, coarsened facial features and intellectual disability, due to truncating mutations in the sorting nexin gene SNX14, encoding a ubiquitously expressed modular PX domain-containing sorting factor. We found SNX14 localized to lysosomes and associated with phosphatidylinositol (3,5)-bisphosphate, a key component of late endosomes/lysosomes. Patient-derived cells showed engorged lysosomes and a slower autophagosome clearance rate upon autophagy induction by starvation. Zebrafish morphants for snx14 showed dramatic loss of cerebellar parenchyma, accumulation of autophagosomes and activation of apoptosis. Our results characterize a unique ataxia syndrome due to biallelic SNX14 mutations leading to lysosome-autophagosome dysfunction.

Regulation of CBP and Tip60 coordinates histone acetylation at local and global levels during Ras-induced transformation.

Cell transformation is clearly linked to epigenetic changes. However, the role of the histone-modifying enzymes in this process is still poorly understood. In this study, we investigated the contribution of the histone acetyltransferase (HAT) enzymes to Ras-mediated transformation. Our results demonstrated that lysine acetyltransferase 5, also known as Tip60, facilitates histone acetylation of bulk chromatin in Ras-transformed cells. As a consequence, global H4 acetylation (H4K8ac and H4K12ac) increases in Ras-transformed cells, rendering a more decompacted chromatin than in parental cells. Furthermore, low levels of CREB-binding protein (CBP) lead to hypoacetylation of retinoblastoma 1 (Rb1) and cyclin-dependent kinase inhibitor 1B (Cdkn1b or p27Kip1) tumour suppressor gene promoters to facilitate Ras-mediated transformation. In agreement with these data, overexpression of Cbp counteracts Ras transforming capability in a HAT-dependent manner. Altogether our results indicate that CBP and Tip60 coordinate histone acetylation at both local and global levels to facilitate Ras-induced transformation.

An increase in MECP2 dosage impairs neural tube formation.

Epigenetic mechanisms are fundamental for shaping the activity of the central nervous system (CNS). Methyl-CpG binding protein 2 (MECP2) acts as a bridge between methylated DNA and transcriptional effectors responsible for differentiation programs in neurons. The importance of MECP2 dosage in CNS is evident in Rett Syndrome and MECP2 duplication syndrome, which are neurodevelopmental diseases caused by loss-of-function mutations or duplication of the MECP2 gene, respectively. Although many studies have been performed on Rett syndrome models, little is known about the effects of an increase in MECP2 dosage. Herein, we demonstrate that MECP2 overexpression affects neural tube formation, leading to a decrease in neuroblast proliferation in the neural tube ventricular zone. Furthermore, an increase in MECP2 dose provokes premature differentiation of neural precursors accompanied by greater cell death, resulting in a loss of neuronal populations. Overall, our data indicate that correct MECP2 expression levels are required for proper nervous system development.

Exome sequencing links corticospinal motor neuron disease to common neurodegenerative disorders.

Hereditary spastic paraplegias (HSPs) are neurodegenerative motor neuron diseases characterized by progressive age-dependent loss of corticospinal motor tract function. Although the genetic basis is partly understood, only a fraction of cases can receive a genetic diagnosis, and a global view of HSP is lacking. By using whole-exome sequencing in combination with network analysis, we identified 18 previously unknown putative HSP genes and validated nearly all of these genes functionally or genetically. The pathways highlighted by these mutations link HSP to cellular transport, nucleotide metabolism, and synapse and axon development. Network analysis revealed a host of further candidate genes, of which three were mutated in our cohort. Our analysis links HSP to other neurodegenerative disorders and can facilitate gene discovery and mechanistic understanding of disease.

Mutations in CSPP1 lead to classical Joubert syndrome.

Joubert syndrome and related disorders (JSRDs) are genetically heterogeneous and characterized by a distinctive mid-hindbrain malformation. Causative mutations lead to primary cilia dysfunction, which often results in variable involvement of other organs such as the liver, retina, and kidney. We identified predicted null mutations in CSPP1 in six individuals affected by classical JSRDs. CSPP1 encodes a protein localized to centrosomes and spindle poles, as well as to the primary cilium. Despite the known interaction between CSPP1 and nephronophthisis-associated proteins, none of the affected individuals in our cohort presented with kidney disease, and further, screening of a large cohort of individuals with nephronophthisis demonstrated no mutations. CSPP1 is broadly expressed in neural tissue, and its encoded protein localizes to the primary cilium in an in vitro model of human neurogenesis. Here, we show abrogated protein levels and ciliogenesis in affected fibroblasts. Our data thus suggest that CSPP1 is involved in neural-specific functions of primary cilia.