A Humanized and Viable Animal Model for Congenital Adrenal Hyperplasia-CYP21A2-R484Q Mutant Mouse.

Congenital Adrenal Hyperplasia (CAH) is an autosomal recessive disorder impairing cortisol synthesis due to reduced enzymatic activity. This leads to persistent adrenocortical overstimulation and the accumulation of precursors before the blocked enzymatic step. The predominant form of CAH arises from mutations in CYP21A2, causing 21-hydroxylase deficiency (21-OHD). Despite emerging treatment options for CAH, it is not always possible to physiologically replace cortisol levels and counteract hyperandrogenism. Moreover, there is a notable absence of an effective in vivo model for pre-clinical testing. In this work, we developed an animal model for CAH with the clinically relevant point mutation p.R484Q in the previously humanized CYP21A2 mouse strain. Mutant mice showed hyperplastic adrenals and exhibited reduced levels of corticosterone and 11-deoxycorticosterone and an increase in progesterone. Female mutants presented with higher aldosterone concentrations, but blood pressure remained similar between wildtype and mutant mice in both sexes. Male mutant mice have normal fertility with a typical testicular appearance, whereas female mutants are infertile, exhibit an abnormal ovarian structure, and remain in a consistent diestrus phase. Conclusively, we show that the animal model has the potential to contribute to testing new treatment options and to prevent comorbidities that result from hormone-related derangements and treatment-related side effects in CAH patients.

The growth factor EPIREGULIN promotes basal progenitor cell proliferation in the developing neocortex.

Neocortex expansion during evolution is linked to higher numbers of neurons, which are thought to result from increased proliferative capacity and neurogenic potential of basal progenitor cells during development. Here, we show that EREG, encoding the growth factor EPIREGULIN, is expressed in the human developing neocortex and in gorilla cerebral organoids, but not in the mouse neocortex. Addition of EPIREGULIN to the mouse neocortex increases proliferation of basal progenitor cells, whereas EREG ablation in human cortical organoids reduces proliferation in the subventricular zone. Treatment of cortical organoids with EPIREGULIN promotes a further increase in proliferation of gorilla but not of human basal progenitor cells. EPIREGULIN competes with the epidermal growth factor (EGF) to promote proliferation, and inhibition of the EGF receptor abrogates the EPIREGULIN-mediated increase in basal progenitor cells. Finally, we identify putative cis-regulatory elements that may contribute to the observed inter-species differences in EREG expression. Our findings suggest that species-specific regulation of EPIREGULIN expression may contribute to the increased neocortex size of primates by providing a tunable pro-proliferative signal to basal progenitor cells in the subventricular zone.

Longer metaphase and fewer chromosome segregation errors in modern human than Neanderthal brain development.

Since the ancestors of modern humans separated from those of Neanderthals, around 100 amino acid substitutions spread to essentially all modern humans. The biological significance of these changes is largely unknown. Here, we examine all six such amino acid substitutions in three proteins known to have key roles in kinetochore function and chromosome segregation and to be highly expressed in the stem cells of the developing neocortex. When we introduce these modern human-specific substitutions in mice, three substitutions in two of these proteins, KIF18a and KNL1, cause metaphase prolongation and fewer chromosome segregation errors in apical progenitors of the developing neocortex. Conversely, the ancestral substitutions cause shorter metaphase length and more chromosome segregation errors in human brain organoids, similar to what we find in chimpanzee organoids. These results imply that the fidelity of chromosome segregation during neocortex development improved in modern humans after their divergence from Neanderthals.

A set of gene knockouts as a resource for global lipidomic changes.

Enzyme specificity in lipid metabolic pathways often remains unresolved at the lipid species level, which is needed to link lipidomic molecular phenotypes with their protein counterparts to construct functional pathway maps. We created lipidomic profiles of 23 gene knockouts in a proof-of-concept study based on a CRISPR/Cas9 knockout screen in mammalian cells. This results in a lipidomic resource across 24 lipid classes. We highlight lipid species phenotypes of multiple knockout cell lines compared to a control, created by targeting the human safe-harbor locus AAVS1 using up to 1228 lipid species and subspecies, charting lipid metabolism at the molecular level. Lipid species changes are found in all knockout cell lines, however, some are most apparent on the lipid class level (e.g., SGMS1 and CEPT1), while others are most apparent on the fatty acid level (e.g., DECR2 and ACOT7). We find lipidomic phenotypes to be reproducible across different clones of the same knockout and we observed similar phenotypes when two enzymes that catalyze subsequent steps of the long-chain fatty acid elongation cycle were targeted.

CYP21A2 Gene Expression in a Humanized 21-Hydroxylase Mouse Model Does Not Affect Adrenocortical Morphology and Function.

Steroid 21-hydroxylase is an enzyme of the steroid pathway that is involved in the biosynthesis of cortisol and aldosterone by hydroxylation of 17α-hydroxyprogesterone and progesterone at the C21 position. Mutations in CYP21A2, the gene encoding 21-hydroxylase, cause the most frequent form of the autosomal recessive disorder congenital adrenal hyperplasia (CAH). In this study, we generated a humanized 21-hydroxylase mouse model as the first step to the generation of mutant mice with different CAH-causing mutations. We replaced the mouse Cyp21a1 gene with the human CYP21A2 gene using homologous recombination in combination with CRISPR/Cas9 technique. The aim of this study was to characterize the new humanized mouse model. All results described are related to the homozygous animals in comparison with wild-type mice. We show analogous expression patterns of human 21-hydroxylase by the murine promoter and regulatory elements in comparison to murine 21-hydroxylase in wild-type animals. As expected, no Cyp21a1 transcript was detected in homozygous CYP21A2 adrenal glands. Alterations in adrenal gene expression were observed for Cyp11a1, Star, and Cyb11b1. These differences, however, were not pathological. Outward appearance, viability, growth, and fertility were not affected in the humanized CYP21A2 mice. Plasma steroid levels of corticosterone and aldosterone showed no pathological reduction. In addition, adrenal gland morphology and zonation were similar in both the humanized and the wild-type mice. In conclusion, humanized homozygous CYP21A2 mice developed normally and showed no differences in histological analyses, no reduction in adrenal and gonadal gene expression, or in plasma steroids in comparison with wild-type littermates.

Expression of human-specific ARHGAP11B in mice leads to neocortex expansion and increased memory flexibility.

Neocortex expansion during human evolution provides a basis for our enhanced cognitive abilities. Yet, which genes implicated in neocortex expansion are actually responsible for higher cognitive abilities is unknown. The expression of human-specific ARHGAP11B in embryonic/foetal mouse, ferret and marmoset neocortex was previously found to promote basal progenitor proliferation, upper-layer neuron generation and neocortex expansion during development, features commonly thought to contribute to increased cognitive abilities. However, a key question is whether this phenotype persists into adulthood and if so, whether cognitive abilities are indeed increased. Here, we generated a transgenic mouse line with physiological ARHGAP11B expression that exhibits increased neocortical size and upper-layer neuron numbers persisting into adulthood. Adult ARHGAP11B-transgenic mice showed altered neurobehaviour, notably increased memory flexibility and a reduced anxiety level. Our data are consistent with the notion that neocortex expansion by ARHGAP11B, a gene implicated in human evolution, underlies some of the altered neurobehavioural features observed in the transgenic mice, such as the increased memory flexibility, a neocortex-associated trait, with implications for the increase in cognitive abilities during human evolution.

Cooperative genetic networks drive embryonic stem cell transition from naïve to formative pluripotency.

In the mammalian embryo, epiblast cells must exit the naïve state and acquire formative pluripotency. This cell state transition is recapitulated by mouse embryonic stem cells (ESCs), which undergo pluripotency progression in defined conditions in vitro. However, our understanding of the molecular cascades and gene networks involved in the exit from naïve pluripotency remains fragmentary. Here, we employed a combination of genetic screens in haploid ESCs, CRISPR/Cas9 gene disruption, large-scale transcriptomics and computational systems biology to delineate the regulatory circuits governing naïve state exit. Transcriptome profiles for 73 ESC lines deficient for regulators of the exit from naïve pluripotency predominantly manifest delays on the trajectory from naïve to formative epiblast. We find that gene networks operative in ESCs are also active during transition from pre- to post-implantation epiblast in utero. We identified 496 naïve state-associated genes tightly connected to the in vivo epiblast state transition and largely conserved in primate embryos. Integrated analysis of mutant transcriptomes revealed funnelling of multiple gene activities into discrete regulatory modules. Finally, we delineate how intersections with signalling pathways direct this pivotal mammalian cell state transition.

Recapitulating Evolutionary Divergence in a Single Cis-Regulatory Element Is Sufficient to Cause Expression Changes of the Lens Gene Tdrd7.

Mutations in cis-regulatory elements play important roles for phenotypic changes during evolution. Eye degeneration in the blind mole rat (BMR; Nannospalax galili) and other subterranean mammals is significantly associated with widespread divergence of eye regulatory elements, but the effect of these regulatory mutations on eye development and function has not been explored. Here, we investigate the effect of mutations observed in the BMR sequence of a conserved noncoding element upstream of Tdrd7, a pleiotropic gene required for lens development and spermatogenesis. We first show that this conserved element is a transcriptional repressor in lens cells and that the BMR sequence partially lost repressor activity. Next, we recapitulated evolutionary changes in this element by precisely replacing the endogenous regulatory element in a mouse line by the orthologous BMR sequence with CRISPR-Cas9. Strikingly, this repressor replacement caused a more than 2-fold upregulation of Tdrd7 in the developing lens; however, increased mRNA level does not result in a corresponding increase in TDRD7 protein nor an obvious lens phenotype, possibly explained by buffering at the posttranscriptional level. Our results are consistent with eye degeneration in subterranean mammals having a polygenic basis where many small-effect mutations in different eye-regulatory elements collectively contribute to phenotypic differences.

Cellular Expression and Functional Roles of All 26 Neurotransmitter GPCRs in the C. elegans Egg-Laying Circuit.

Maps of the synapses made and neurotransmitters released by all neurons in model systems, such as Caenorhabditis elegans have left still unresolved how neural circuits integrate and respond to neurotransmitter signals. Using the egg-laying circuit of C. elegans as a model, we mapped which cells express each of the 26 neurotransmitter GPCRs of this organism and also genetically analyzed the functions of all 26 GPCRs. We found that individual neurons express many distinct receptors, epithelial cells often express neurotransmitter receptors, and receptors are often positioned to receive extrasynaptic signals. Receptor knockouts reveal few egg-laying defects under standard laboratory conditions, suggesting that the receptors function redundantly or regulate egg-laying only in specific conditions; however, increasing receptor signaling through overexpression more efficiently reveals receptor functions. This map of neurotransmitter GPCR expression and function in the egg-laying circuit provides a model for understanding GPCR signaling in other neural circuits.SIGNIFICANCE STATEMENT Neurotransmitters signal through GPCRs to modulate activity of neurons, and changes in such signaling can underlie conditions such as depression and Parkinson's disease. To determine how neurotransmitter GPCRs together help regulate function of a neural circuit, we analyzed the simple egg-laying circuit in the model organism C. elegans We identified all the cells that express every neurotransmitter GPCR and genetically analyzed how each GPCR affects the behavior the circuit produces. We found that many neurotransmitter GPCRs are expressed in each neuron, that neurons also appear to use these receptors to communicate with other cell types, and that GPCRs appear to often act redundantly or only under specific conditions to regulate circuit function.

A Family of Argonaute-Interacting Proteins Gates Nuclear RNAi.

Nuclear RNA interference (RNAi) pathways work together with histone modifications to regulate gene expression and enact an adaptive response to transposable RNA elements. In the germline, nuclear RNAi can lead to trans-generational epigenetic inheritance (TEI) of gene silencing. We identified and characterized a family of nuclear Argonaute-interacting proteins (ENRIs) that control the strength and target specificity of nuclear RNAi in C. elegans, ensuring faithful inheritance of epigenetic memories. ENRI-1/2 prevent misloading of the nuclear Argonaute NRDE-3 with small RNAs that normally effect maternal piRNAs, which prevents precocious nuclear translocation of NRDE-3 in the early embryo. Additionally, they are negative regulators of nuclear RNAi triggered from exogenous sources. Loss of ENRI-3, an unstable protein expressed mostly in the male germline, misdirects the RNAi response to transposable elements and impairs TEI. The ENRIs determine the potency and specificity of nuclear RNAi responses by gating small RNAs into specific nuclear Argonautes.

Non-canonical Caspase-1 Signaling Drives RIP2-Dependent and TNF-α-Mediated Inflammation In Vivo.

Pro-inflammatory caspase-1 is a key player in innate immunity. Caspase-1 processes interleukin (IL)-1ß and IL-18 to their mature forms and triggers pyroptosis. These caspase-1 functions are linked to its enzymatic activity. However, loss-of-function missense mutations in CASP1 do not prevent autoinflammation in patients, despite decreased IL-1ß production. In vitro data suggest that enzymatically inactive caspase-1 drives inflammation via enhanced nuclear factor κB (NF-κB) activation, independent of IL-1ß processing. Here, we report two mouse models of enzymatically inactive caspase-1-C284A, demonstrating the relevance of this pathway in vivo. In contrast to Casp1-/- mice, caspase-1-C284A mice show pronounced hypothermia and increased levels of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-6 when challenged with lipopolysaccharide (LPS). Caspase-1-C284A signaling is RIP2 dependent and mediated by TNF-α but independent of the NLRP3 inflammasome. LPS-stimulated whole blood from patients carrying loss-of-function missense mutations in CASP1 secretes higher amounts of TNF-α. Taken together, these results reveal non-canonical caspase-1 signaling in vivo.

CRISPR/Cas9-based knockout pipeline for reverse genetics in mammalian cell culture.

We present a straightforward protocol for reverse genetics in cultured mammalian cells, using CRISPR/Cas9-mediated homology-dependent repair (HDR) based insertion of a protein trap cassette, resulting in a termination of the endogenous gene expression. Complete loss of function can be achieved with monoallelic trap cassette insertion, as the second allele is frequently disrupted by an error-prone non-homologous end joining (NHEJ) mechanism. The method should be applicable to any expressed gene in most cell lines, including those with low HDR efficiency, as the knockout alleles can be directly selected for.

A novel population of Hopx-dependent basal radial glial cells in the developing mouse neocortex.

A specific subpopulation of neural progenitor cells, the basal radial glial cells (bRGCs) of the outer subventricular zone (OSVZ), are thought to have a key role in the evolutionary expansion of the mammalian neocortex. In the developing lissencephalic mouse neocortex, bRGCs exist at low abundance and show significant molecular differences from bRGCs in developing gyrencephalic species. Here, we demonstrate that the developing mouse medial neocortex (medNcx), in contrast to the canonically studied lateral neocortex (latNcx), exhibits an OSVZ and an abundance of bRGCs similar to that in developing gyrencephalic neocortex. Unlike bRGCs in developing mouse latNcx, the bRGCs in medNcx exhibit human bRGC-like gene expression, including expression of Hopx, a human bRGC marker. Disruption of Hopx expression in mouse embryonic medNcx and forced Hopx expression in mouse embryonic latNcx demonstrate that Hopx is required and sufficient, respectively, for bRGC abundance as found in the developing gyrencephalic neocortex. Taken together, our data identify a novel bRGC subpopulation in developing mouse medNcx that is highly related to bRGCs of developing gyrencephalic neocortex.

Extracellular glucose level regulates dependence on GRP78 for cell surface localization of multipass transmembrane proteins in HeLa cells.

Many human-cultured cell lines survive glucose starvation, but the underlying mechanisms remain unclear. Here, we searched for proteins required for cellular adaptation to glucose-limited conditions and identified several endoplasmic reticulum chaperones in the glucose-regulated protein (GRP) family as proteins enriched in the cellular membrane. Surprisingly, these proteins, which are required for cell surface localization of GLUT1 under high-glucose conditions, become dispensable for targeting GLUT1 to the surface upon glucose starvation. In marked contrast, cell surface localization of single-pass transmembrane proteins, such as epidermal growth factor receptor and CD98, is not disturbed by GRP78 depletion regardless of the extracellular glucose level. These results indicate that the extracellular glucose level regulates dependence on the GRPs for cell surface localization of multipass transmembrane proteins.

Protein Dynamics in Complex DNA Lesions.

A single mutagen can generate multiple different types of DNA lesions. How different repair pathways cooperate in complex DNA lesions, however, remains largely unclear. Here we measured, clustered, and modeled the kinetics of recruitment and dissociation of 70 DNA repair proteins to laser-induced DNA damage sites in HeLa cells. The precise timescale of protein recruitment reveals that error-prone translesion polymerases are considerably delayed compared to error-free polymerases. We show that this is ensured by the delayed recruitment of RAD18 to double-strand break sites. The time benefit of error-free polymerases disappears when PARP inhibition significantly delays PCNA recruitment. Moreover, removal of PCNA from complex DNA damage sites correlates with RPA loading during 5'-DNA end resection. Our systematic study of the dynamics of DNA repair proteins in complex DNA lesions reveals the multifaceted coordination between the repair pathways and provides a kinetics-based resource to study genomic instability and anticancer drug impact.

A continuum of mRNP complexes in embryonic microRNA-mediated silencing.

MicroRNAs (miRNAs) impinge on the translation and stability of their target mRNAs, and play key roles in development, homeostasis and disease. The gene regulation mechanisms they instigate are largely mediated through the CCR4­NOT deadenylase complex, but the molecular events that occur on target mRNAs are poorly resolved. We observed a broad convergence of interactions of germ granule and P body mRNP components on AIN-1/GW182 and NTL-1/CNOT1 in Caenorhabditis elegans embryos. We show that the miRISC progressively matures on the target mRNA from a scanning form into an effector mRNP particle by sequentially recruiting the CCR4­NOT complex, decapping and decay, or germ granule proteins. Finally, we implicate intrinsically disordered proteins, key components in mRNP architectures, in the embryonic function of lsy-6 miRNA. Our findings define dynamic steps of effector mRNP assembly in miRNA-mediated silencing, and identify a functional continuum between germ granules and P bodies in the C. elegans embryo.

A genome-wide resource for the analysis of protein localisation in Drosophila.

The Drosophila genome contains >13000 protein-coding genes, the majority of which remain poorly investigated. Important reasons include the lack of antibodies or reporter constructs to visualise these proteins. Here, we present a genome-wide fosmid library of 10000 GFP-tagged clones, comprising tagged genes and most of their regulatory information. For 880 tagged proteins, we created transgenic lines, and for a total of 207 lines, we assessed protein expression and localisation in ovaries, embryos, pupae or adults by stainings and live imaging approaches. Importantly, we visualised many proteins at endogenous expression levels and found a large fraction of them localising to subcellular compartments. By applying genetic complementation tests, we estimate that about two-thirds of the tagged proteins are functional. Moreover, these tagged proteins enable interaction proteomics from developing pupae and adult flies. Taken together, this resource will boost systematic analysis of protein expression and localisation in various cellular and developmental contexts.

Proteome-wide dataset supporting the study of ancient metazoan macromolecular complexes.

Our analysis examines the conservation of multiprotein complexes among metazoa through use of high resolution biochemical fractionation and precision mass spectrometry applied to soluble cell extracts from 5 representative model organisms Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, Strongylocentrotus purpuratus, and Homo sapiens. The interaction network obtained from the data was validated globally in 4 distant species (Xenopus laevis, Nematostella vectensis, Dictyostelium discoideum, Saccharomyces cerevisiae) and locally by targeted affinity-purification experiments. Here we provide details of our massive set of supporting biochemical fractionation data available via ProteomeXchange (PXD002319-PXD002328), PPIs via BioGRID (185267); and interaction network projections via (http://metazoa.med.utoronto.ca) made fully accessible to allow further exploration. The datasets here are related to the research article on metazoan macromolecular complexes in Nature [1].

CRISPR/Cas9-induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo.

We have applied the CRISPR/Cas9 system in vivo to disrupt gene expression in neural stem cells in the developing mammalian brain. Two days after in utero electroporation of a single plasmid encoding Cas9 and an appropriate guide RNA (gRNA) into the embryonic neocortex of Tis21::GFP knock-in mice, expression of GFP, which occurs specifically in neural stem cells committed to neurogenesis, was found to be nearly completely (≈ 90%) abolished in the progeny of the targeted cells. Importantly, upon in utero electroporation directly of recombinant Cas9/gRNA complex, near-maximal efficiency of disruption of GFP expression was achieved already after 24 h. Furthermore, by using microinjection of the Cas9 protein/gRNA complex into neural stem cells in organotypic slice culture, we obtained disruption of GFP expression within a single cell cycle. Finally, we used either Cas9 plasmid in utero electroporation or Cas9 protein complex microinjection to disrupt the expression of Eomes/Tbr2, a gene fundamental for neocortical neurogenesis. This resulted in a reduction in basal progenitors and an increase in neuronal differentiation. Thus, the present in vivo application of the CRISPR/Cas9 system in neural stem cells provides a rapid, efficient and enduring disruption of expression of specific genes to dissect their role in mammalian brain development.

TransgeneOmics--A transgenic platform for protein localization based function exploration.

The localization of a protein is intrinsically linked to its role in the structural and functional organization of the cell. Advances in transgenic technology have streamlined the use of protein localization as a function discovery tool. Here we review the use of large genomic DNA constructs such as bacterial artificial chromosomes as a transgenic platform for systematic tag-based protein function exploration.