CTNND2 moderates the pace of synaptic maturation and links human evolution to synaptic neoteny.

Human-specific genes are potential drivers of brain evolution. Among them, SRGAP2C has contributed to the emergence of features characterizing human cortical synapses, including their extended period of maturation. SRGAP2C inhibits its ancestral copy, the postsynaptic protein SRGAP2A, but the synaptic molecular pathways differentially regulated in humans by SRGAP2 proteins remain largely unknown. Here, we identify CTNND2, a protein implicated in severe intellectual disability (ID) in Cri-du-Chat syndrome, as a major partner of SRGAP2. We demonstrate that CTNND2 slows synaptic maturation and promotes neuronal integrity. During postnatal development, CTNND2 moderates neuronal excitation and excitability. In adults, it supports synapse maintenance. While CTNND2 deficiency is deleterious and results in synaptic loss of SYNGAP1, another major ID-associated protein, the human-specific protein SRGAP2C, enhances CTNND2 synaptic accumulation in human neurons. Our findings suggest that CTNND2 regulation by SRGAP2C contributes to synaptic neoteny in humans and link human-specific and ID genes at the synapse.

Formation of multinucleated osteoclasts depends on an oxidized species of cell surface-associated La protein.

The bone-resorbing activity of osteoclasts plays a critical role in the life-long remodeling of our bones that is perturbed in many bone loss diseases. Multinucleated osteoclasts are formed by the fusion of precursor cells, and larger cells - generated by an increased number of cell fusion events - have higher resorptive activity. We find that osteoclast fusion and bone resorption are promoted by reactive oxygen species (ROS) signaling and by an unconventional low molecular weight species of La protein, located at the osteoclast surface. Here, we develop the hypothesis that La's unique regulatory role in osteoclast multinucleation and function is controlled by an ROS switch in La trafficking. Using antibodies that recognize reduced or oxidized species of La, we find that differentiating osteoclasts enrich an oxidized species of La at the cell surface, which is distinct from the reduced La species conventionally localized within cell nuclei. ROS signaling triggers the shift from reduced to oxidized La species, its dephosphorylation and delivery to the surface of osteoclasts, where La promotes multinucleation and resorptive activity. Moreover, intracellular ROS signaling in differentiating osteoclasts oxidizes critical cysteine residues in the C-terminal half of La, producing this unconventional La species that promotes osteoclast fusion. Our findings suggest that redox signaling induces changes in the location and function of La and may represent a promising target for novel skeletal therapies.

The role of Imp and Syp RNA-binding proteins in precise neuronal elimination by apoptosis through the regulation of transcription factors.

Neuronal stem cells generate a limited and consistent number of neuronal progenies, each possessing distinct morphologies and functions, which are crucial for optimal brain function. Our study focused on a neuroblast (NB) lineage in Drosophila known as Lin A/15, which generates motoneurons (MNs) and glia. Intriguingly, Lin A/15 NB dedicates 40% of its time to producing immature MNs (iMNs) that are subsequently eliminated through apoptosis. Two RNA-binding proteins, Imp and Syp, play crucial roles in this process. Imp+ MNs survive, while Imp-, Syp+ MNs undergo apoptosis. Genetic experiments show that Imp promotes survival, whereas Syp promotes cell death in iMNs. Late-born MNs, which fail to express a functional code of transcription factors (mTFs) that control their morphological fate, are subject to elimination. Manipulating the expression of Imp and Syp in Lin A/15 NB and progeny leads to a shift of TF code in late-born MNs toward that of early-born MNs, and their survival. Additionally, introducing the TF code of early-born MNs into late-born MNs also promoted their survival. These findings demonstrate that the differential expression of Imp and Syp in iMNs links precise neuronal generation and distinct identities through the regulation of mTFs. Both Imp and Syp are conserved in vertebrates, suggesting that they play a fundamental role in precise neurogenesis across species.

Population genomic analysis reveals a polygenic sex determination system in Apostichopus japonicus.

The sea cucumber Apostichopus japonicus, a key species in Chinese aquaculture, plays a significant evolutionary role within the Echinodermata phylum. However, the sex determination mechanism in this species remains poorly understood. Here, we conducted extensive sex surveys and sampling of eight wild populations, investigating the sex-related SNPs and insertion or deletions (indels) through bulk segregation analysis (BSA) and genome-wide association study (GWAS) analysis. Our findings suggest that A. japonicus employs a polygenic sex determination (PSD) system, with solute carrier family 8 (SLC8A) being the candidate gene for sex determination, encoding sodium-calcium exchanger (NCX1). The analysis of normalized sequencing depth reveals variations across chromosomes 6, 13, 14, 16, and 18, supporting the PSD system. We also identified 541.656 kb of male-specific sequences and screened five markers (C77185, C98086, C64977, C125, and C876) for molecular sex identification. Overall, this study provides new insights into A. japonicus sex determination, highlighting a complex multi-gene mechanism rather than a simple XX/XY system.

Cell-cell interaction determines cell fate of mesoderm-derived cell in tongue development through Hh signaling.

Dysfunction of primary cilia leads to genetic disorder, ciliopathies, which shows various malformations in many vital organs such as brain. Multiple tongue deformities including cleft, hamartoma, and ankyloglossia are also seen in ciliopathies, which yield difficulties in fundamental functions such as mastication and vocalization. Here, we found these tongue anomalies in mice with mutation of ciliary protein. Abnormal cranial neural crest-derived cells (CNCC) failed to evoke Hh signal for differentiation of mesoderm-derived cells into myoblasts, which resulted in abnormal differentiation of mesoderm-derived cells into adipocytes. The ectopic adipose subsequently arrested tongue swelling formation. Ankyloglossia was caused by aberrant cell migration due to lack of non-canonical Wnt signaling. In addition to ciliopathies, these tongue anomalies are often observed as non-familial condition in human. We found that these tongue deformities could be reproduced in wild-type mice by simple mechanical manipulations to disturb cellular processes which were disrupted in mutant mice. Our results provide hints for possible future treatment in ciliopathies.

Volumetric analysis of the terminal ductal lobular unit architecture and cell phenotypes in the human breast.

The major lactiferous ducts of the human breast branch out and end at terminal ductal lobular units (TDLUs). Despite their functional and clinical importance, the three-dimensional (3D) architecture of TDLUs has remained undetermined. Our quantitative and volumetric imaging of healthy human breast tissue demonstrates that highly branched TDLUs, which exhibit increased proliferation, are uncommon in the resting tissue regardless of donor age, parity, or hormonal contraception. Overall, TDLUs have a consistent shape and branch parameters, and they contain a main subtree that dominates in bifurcation events and exhibits a more duct-like keratin expression pattern. Simulation of TDLU branching morphogenesis in three dimensions suggests that evolutionarily conserved mechanisms regulate mammary gland branching in humans and mice despite their anatomical differences. In all, our data provide structural insight into 3D anatomy and branching of the human breast and exemplify the power of volumetric imaging in gaining a deeper understanding of breast biology.

A protocol for time-resolved transcriptomics through metabolic labeling and combinatorial indexing.

The snapshot nature of single-cell transcriptomics presents a challenge for studying the dynamics of gene expression. Metabolic labeling, where nascent RNA is labeled with 4-thiouridine (4sU), captures temporal information at the single-cell level, providing greater insight into expression dynamics. Here, we present an optimized, automation-friendly protocol for the metabolic labeling of RNA alongside single-cell RNA sequencing through combinatorial indexing. We describe steps for 4sU labeling, cell fixation and chemical treatment, and automated two-level combinatorial indexing. For complete details on the use and execution of this protocol, please refer to Maizels et al.1.

The chromodomain protein CDYL confers forebrain identity to human cortical organoids by inhibiting neuronatin.

Fate determination of neural stem cells (NSCs) is crucial for cortex development and is closely linked to neurodevelopmental disorders when gene expression networks are disrupted. The transcriptional corepressor chromodomain Y-like (CDYL) is widely expressed across diverse cell populations within the human embryonic cortex. However, its precise role in cortical development remains unclear. Here, we show that CDYL is critical for human cortical neurogenesis and that its deficiency leads to a substantial increase in gamma-aminobutyric acid (GABA)-ergic neurons in cortical organoids. Subsequently, neuronatin (NNAT) is identified as a significant target of CDYL, and its abnormal expression obviously influences the fate commitment of cortical NSCs. Cross-species comparisons of CDYL targets unravel a distinct developmental trajectory between human cortical organoids and the mouse cortex at an analogous stage. Collectively, our data provide insight into the evolutionary roles of CDYL in human cortex development, emphasizing its critical function in maintaining the fate of human cortical NSCs.

Astrogliosis and neuroinflammation underlie scoliosis upon cilia dysfunction.

Cilia defects lead to scoliosis in zebrafish, but the underlying pathogenic mechanisms are poorly understood and may diverge depending on the mutated gene. Here, we dissected the mechanisms of scoliosis onset in a zebrafish mutant for the rpgrip1l gene encoding a ciliary transition zone protein. rpgrip1l mutant fish developed scoliosis with near-total penetrance but asynchronous onset in juveniles. Taking advantage of this asynchrony, we found that curvature onset was preceded by ventricle dilations and was concomitant to the perturbation of Reissner fiber polymerization and to the loss of multiciliated tufts around the subcommissural organ. Rescue experiments showed that Rpgrip1l was exclusively required in foxj1a-expressing cells to prevent axis curvature. Genetic interactions investigations ruled out Urp1/2 levels as a main driver of scoliosis in rpgrip1 mutants. Transcriptomic and proteomic studies identified neuroinflammation associated with increased Annexin levels as a potential mechanism of scoliosis development in rpgrip1l juveniles. Investigating the cell types associated with annexin2 over-expression, we uncovered astrogliosis, arising in glial cells surrounding the diencephalic and rhombencephalic ventricles just before scoliosis onset and increasing with time in severity. Anti-inflammatory drug treatment reduced scoliosis penetrance and severity and this correlated with reduced astrogliosis and macrophage/microglia enrichment around the diencephalic ventricle. Mutation of the cep290 gene encoding another transition zone protein also associated astrogliosis with scoliosis. Thus, we propose astrogliosis induced by perturbed ventricular homeostasis and associated with immune cell activation as a novel pathogenic mechanism of zebrafish scoliosis caused by cilia dysfunction.

Cell density couples tissue mechanics to control the elongation speed of the body axis.

The vertebrate body forms by the addition of new tissues at the posterior end. This elongates the body axis, allowing continued anterior segmentation to produce the stereotypic body plan. This balance requires the elongation speed to be constrained. Here we utilized modeling and tissue force microscopy on chicken embryos to show that cell density of the posterior presomitic mesoderm (pPSM) dynamically modulates elongation speed in a negative feedback loop. Elongation alters the cell density in the pPSM, which in turn controls progenitor cell influx through the mechanical coupling of body axis tissues. This enables responsive cell dynamics in over- and under-elongated axes that consequently self-adjust speed to achieve long-term robustness in axial length. Our simulations and experiments further suggest that cell density and FGF activity synergistically drive elongation. Our work supports a simple mechanism of morphogenetic speed control where the cell density relates negatively to progress, and positively to force generation.

A MSTNDel73C mutation with FGF5 knockout sheep by CRISPR/Cas9 promotes skeletal muscle myofiber hyperplasia.

Mutations in the well-known Myostatin (MSTN) produce a 'double-muscle' phenotype, which makes it commercially invaluable for improving livestock meat production and providing high-quality protein for humans. However, mutations at different loci of the MSTN often produce a variety of different phenotypes. In the current study, we increased the delivery ratio of Cas9 mRNA to sgRNA from the traditional 1:2 to 1:10, which improves the efficiency of the homozygous mutation of biallelic gene. Here, a MSTNDel73C mutation with FGF5 knockout sheep, in which the MSTN and FGF5 dual-gene biallelic homozygous mutations were produced via the deletion of 3-base pairs of AGC in the third exon of MSTN, resulting in cysteine-depleted at amino acid position 73, and the FGF5 double allele mutation led to inactivation of FGF5 gene. The MSTNDel73C mutation with FGF5 knockout sheep highlights a dominant 'double-muscle' phenotype, which can be stably inherited. Both F0 and F1 generation mutants highlight the excellent trait of high-yield meat with a smaller cross-sectional area and higher number of muscle fibers per unit area. Mechanistically, the MSTNDel73C mutation with FGF5 knockout mediated the activation of FOSL1 via the MEK-ERK-FOSL1 axis. The activated FOSL1 promotes skeletal muscle satellite cell proliferation and inhibits myogenic differentiation by inhibiting the expression of MyoD1, and resulting in smaller myotubes. In addition, activated ERK1/2 may inhibit the secondary fusion of myotubes by Ca2+-dependent CaMKII activation pathway, leading to myoblasts fusion to form smaller myotubes.

Nuclear and cytosolic fractions of SOX2 synergize as transcriptional and translational co-regulators of cell fate.

Stemness and pluripotency are mediated by transcriptional master regulators that promote self-renewal and repress cell differentiation, among which is the high-mobility group (HMG) box transcription factor SOX2. Dysregulated SOX2 expression, by contrast, leads to transcriptional aberrations relevant to oncogenic transformation, cancer progression, metastasis, therapy resistance, and relapse. Here, we report a post-transcriptional mechanism by which the cytosolic pool of SOX2 contributes to these events in an unsuspected manner. Specifically, a low-complexity region within SOX2's C-terminal segment connects to the ribosome to modulate the expression of cognate downstream factors. Independent of nuclear structures or DNA, this C-terminal functionality alone changes metabolic properties and induces non-adhesive growth when expressed in the cytosol of SOX2 knockout cells. We thus propose a revised model of SOX2 action where nuclear and cytosolic fractions cooperate to impose cell fate decisions via both transcriptional and translational mechanisms.

Negative cell cycle regulation by calcineurin is necessary for proper beta cell regeneration in zebrafish.

Stimulation of pancreatic beta cell regeneration could be a therapeutic lead to treat diabetes. Unlike humans, the zebrafish can efficiently regenerate beta cells, notably from ductal pancreatic progenitors. To gain insight into the molecular pathways involved in this process, we established the transcriptomic profile of the ductal cells after beta cell ablation in the adult zebrafish. These data highlighted the protein phosphatase calcineurin (CaN) as a new potential modulator of beta cell regeneration. We showed that CaN overexpression abolished the regenerative response, leading to glycemia dysregulation. On the opposite, CaN inhibition increased ductal cell proliferation and subsequent beta cell regeneration. Interestingly, the enhanced proliferation of the progenitors was paradoxically coupled with their exhaustion. This suggests that the proliferating progenitors are next entering in differentiation. CaN appears as a guardian which prevents an excessive progenitor proliferation to preserve the pool of progenitors. Altogether, our findings reveal CaN as a key player in the balance between proliferation and differentiation to enable a proper beta cell regeneration.

DDI2 protease controls embryonic development and inflammation via TCF11/NRF1.

DDI2 is an aspartic protease that cleaves polyubiquitinated substrates. Upon proteotoxic stress, DDI2 activates the transcription factor TCF11/NRF1 (NFE2L1), crucial for maintaining proteostasis in mammalian cells, enabling the expression of rescue factors, including proteasome subunits. Here, we describe the consequences of DDI2 ablation in vivo and in cells. DDI2 knock-out (KO) in mice caused embryonic lethality at E12.5 with severe developmental failure. Molecular characterization of embryos showed insufficient proteasome expression with proteotoxic stress, accumulation of high molecular weight ubiquitin conjugates and induction of the unfolded protein response (UPR) and cell death pathways. In DDI2 surrogate KO cells, proteotoxic stress activated the integrated stress response (ISR) and induced a type I interferon (IFN) signature and IFN-induced proliferative signaling, possibly ensuring survival. These results indicate an important role for DDI2 in the cell-tissue proteostasis network and in maintaining a balanced immune response.

Protocol for high-resolution 3D visualization of insect regenerating legs through micro-computed tomography.

Appendage regeneration occurs within the opaque exoskeleton in arthropods, making it challenging to visualize the regenerative processes dynamically. In this protocol, we present a strategy to scan and capture the high-resolution details of microstructural tissues at certain regeneration points through micro-computed tomography (micro-CT). We describe steps for tissue preparation, fixation, critical point drying, micro-CT scanning, and 3D visualization. This approach promises significant utility in the field of regeneration, particularly in studies involving arthropods such as insects and crustaceans. For complete details on the use and execution of this protocol, please refer to Ren et al.1.

EDC-3 and EDC-4 regulate embryonic mRNA clearance and biomolecular condensate specialization.

Animal development is dictated by the selective and timely decay of mRNAs in developmental transitions, but the impact of mRNA decapping scaffold proteins in development is unclear. This study unveils the roles and interactions of the DCAP-2 decapping scaffolds EDC-3 and EDC-4 in the embryonic development of C. elegans. EDC-3 facilitates the timely removal of specific embryonic mRNAs, including cgh-1, car-1, and ifet-1 by reducing their expression and preventing excessive accumulation of DCAP-2 condensates in somatic cells. We further uncover a role for EDC-3 in defining the boundaries between P bodies, germ granules, and stress granules. Finally, we show that EDC-4 counteracts EDC-3 and engenders the assembly of DCAP-2 with the GID (CTLH) complex, a ubiquitin ligase involved in maternal-to-zygotic transition (MZT). Our findings support a model where multiple RNA decay mechanisms temporally clear maternal and zygotic mRNAs throughout embryonic development.

Protocol for the efficient and inducible generation of CRISPR-Cas9-edited human cortical neurons from the iCas9-iNgn2 hPSCs.

Generation of CRISPR-Cas9-edited cortical neurons from human pluripotent stem cells (hPSCs) enables the study of gene functions and neural disease mechanisms. Here, we present a protocol for developing iCas9-iNgn2 hPSC, an inducible cell line that allows the simultaneous induction of the neuralizing transcription factor Ngn2 and the Cas9 nuclease to rapidly generate edited human cortical neurons. We describe the steps of the protocol from transducing iCas9-iNgn2 with guide RNA-containing lentivirus to producing edited cortical neurons in about 2 weeks. For complete details on the use and execution of this protocol, please refer to Dhaliwal et al.1.

Development of pial collaterals by extension of pre-existing artery tips.

Pial collaterals provide protection from ischemic damage and improve the prognosis of stroke patients. The origin or precise sequence of events underlying pial collateral development is unclear and has prevented clinicians from adapting new vascularization and regeneration therapies. We use genetic lineage tracing and intravital imaging of mouse brains at cellular resolution to show that during embryogenesis, pial collateral arteries develop from extension and anastomoses of pre-existing artery tips in a VegfR2-dependent manner. This process of artery tip extension occurs on pre-determined microvascular tracks. Our data demonstrate that an arterial receptor, Cxcr4, earlier shown to drive artery cell migration and coronary collateral development, is dispensable for the formation and maintenance of pial collateral arteries. Our study shows that collateral arteries of the brain are built by a mechanism distinct from that of the heart.

Placenta-derived SOD3 deletion impairs maternal behavior via alterations in FGF/FGFR-prolactin signaling axis.

Offspring growth requires establishing maternal behavior associated with the maternal endocrine profile. Placentae support the adaptations of the mother, producing bioactive molecules that affect maternal organs. We recently reported that placentae produce superoxide dismutase 3 (SOD3) that exerts sustained effects on the offspring liver via epigenetic modifications. Here, we demonstrate that placenta-specific Sod3 knockout (Sod3-/-) dams exhibited impaired maternal behavior and decreased prolactin levels. Most fibroblast growth factor (FGF)-regulated pathways were downregulated in the pituitary tissues from Sod3-/- dams. FGF1-, FGF2-, and FGF4-induced prolactin expression and signaling via the phosphoinositide 3-kinase (PI3K)-phospholipase C-γ1 (PLCγ1)-protein kinase-Cδ (PKC)δ axis were reduced in primary pituitary cells from Sod3-/- dams. Mechanistically, FGF1/FGF receptor (FGFR)2 expressions were inhibited by the suppression of the ten-eleven translocation (TET)/isocitrate dehydrogenase (IDH)/α-ketoglutarate pathway and DNA demethylation levels at the zinc finger and BTB domain containing 18 (ZBTB18)-targeted promoters of Fgf1/Fgfr2. Importantly, offspring from Sod3-/- dams also showed impaired nurturing behavior to their grandoffspring. Collectively, placenta-derived SOD3 promotes maternal behavior via epigenetic programming of the FGF/FGFR-prolactin axis.

Protocol for generating a 3D culture of epiblast stem cells.

Mouse gastrulation entails concomitant changes in cell fate, tissue shape, and embryo size. The use of a reproducible in vitro system is crucial for dissecting the mechanisms that coordinate these events. Here, we present a protocol for generating a 3D culture of epiblast stem cells (3D EpiSCs), which grow as epithelial spheroids mimicking key features of the gastrulating mouse embryonic epiblast. We describe steps for spheroid formation, growth, and passaging, followed by imaging or further downstream analyses. For complete details on the use and execution of this protocol, please refer to Sato et al.1.