During skeletal morphogenesis diverse mechanisms are used to support bone formation. This can be seen in the bones that require a cartilage template for their development. In mammals the cartilage ...template is removed, but in zebrafish the cartilage template persists and the bone mineralizes around the cartilage scaffold. Remodeling of unmineralized cartilage occurs via planar cell polarity (PCP) mediated cell rearrangements that contribute to lengthening of elements; however, the mechanisms that maintain the chondrocyte template that supports perichondral ossification remain unclear. We report double mutants disrupting two zebrafish kinesin-I genes (hereafter kif5Blof) that we generated using CRISPR/Cas9 mutagenesis. We show that zygotic Kif5Bs have a conserved function in maintaining muscle integrity, and are required for cartilage remodeling and maintenance during craniofacial morphogenesis by a PCP-distinct mechanism. Further, kif5Blof does not activate ER stress response genes, but instead disrupts lysosomal function, matrix secretion, and causes deregulated autophagic markers and eventual chondrocyte apoptosis. Ultrastructural and transplantation analysis reveal neighboring cells engulfing extruded kif5Blof chondrocytes. Initial cartilage specification is intact; however, during remodeling, kif5Blof chondrocytes die and the cartilage matrix devoid of hypertrophic chondrocytes remains and impedes normal ossification. Chimeric and mosaic analyses indicate that Kif5B functions cell-autonomously in secretion, nuclear position, cell elongation and maintenance of hypertrophic chondrocytes. Interestingly, large groups of wild-type cells can support elongation of neighboring mutant cells. Finally, mosaic expression of kif5Ba, but not kif5Aa in cartilage rescues the chondrocyte phenotype, further supporting a specific requirement for Kif5B. Cumulatively, we show essential Kif5B functions in promoting cartilage remodeling and chondrocyte maintenance during zebrafish craniofacial morphogenesis.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
•L1cam is required to curb premature differentiation of neural precursors in the DG.•L1cam restricts the dendritic arborization of new neurons in the adult DG.•Loss of L1cam in adult neural ...precursors results in increased anxiety-like behavior.
L1 is an immunoglobulin domain (Ig)-containing protein essential for a wide range of neurodevelopmental processes highly conserved across species from worms to humans. L1 can act as a cell adhesion molecule by binding to other Ig-containing proteins or as a ligand for certain tyrosine kinase receptors such as FGFRs and TRKs, which are required not only during neurodevelopment but also in hippocampal neurogenesis. Yet, the role of L1 itself in adult hippocampal neurogenesis remains unaddressed. Here, we used several Cre-driver lines in mice to conditionally delete a floxed allele of L1cam at different points along the differentiation lineage of new neurons and in surrounding neurons in the adult dentate gyrus of the hippocampus. We found that L1cam deletion in stem/progenitor cells increased: 1) the differentiation of progenitors into new neurons, 2) the complexity of dendritic arbors in immature neurons, and 3) anxiety-related behavior. In addition, deletion of L1cam in neurons leads to an earlier age-related decline in hippocampal neurogenesis. These data suggest that L1 is not only important for normal nervous system development, but also for maintaining certain neural processes in adulthood.
•Dissociated donor vascular cells in neural transplants form vessels.•Donor-derived vessels fuse with host vessels to circulate blood in neural grafts.•Including vascular cells in transplants at ...stroke sites is essential for graft growth.
Neural precursor cells (NPCs) transplanted into the adult neocortex generate neurons that synaptically integrate with host neurons, supporting the possibility of achieving functional tissue repair. However, poor survival and functional neuronal recovery of transplanted NPCs greatly limits engraftment. Here, we test the hypothesis that combining blood vessel-forming vascular cells with neuronal precursors improves engraftment. By transplanting mixed embryonic neocortical cells into adult mice with neocortical strokes, we show that transplant-derived neurons synapse with appropriate targets while donor vascular cells form vessels that fuse with the host vasculature to perfuse blood within the graft. Although all grafts became vascularized, larger grafts had greater contributions of donor-derived vessels that increased as a function of their distance from the host-graft border. Moreover, excluding vascular cells from the donor cell population strictly limited graft size. Thus, inclusion of vessel-forming vascular cells with NPCs is required for more efficient engraftment and ultimately for tissue repair.
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•In newborn adult hippocampal neurons, FGFR signaling through FRS is required to promote proximal dendrite length.•In contrast, FGFRs are required to inhibit distal dendrite length ...through PLCγ in newborn adult hippocampal neurons.•FGFRs through as yet unidentified mediators promote or counterbalance inhibition of dendrite length by FGFR-PLCγ.
Fibroblast Growth Factor Receptors (FGFRs) play crucial roles in promoting dendrite growth and branching during development. In mice, three FGFR genes, Fgfr1, Fgfr2, and Fgfr3, remain expressed in the adult neurogenic niche of the hippocampal dentate gyrus. However, the function of FGFRs in the dendritic maturation of adult-born neurons remains largely unexplored. Here, using conditional alleles of Fgfr1, Fgfr2, and Fgfr3 as well as Fgfr1 alleles lacking binding sites for Phospholipase-Cγ (PLCγ) and FGF Receptor Substrate (FRS) proteins, we test the requirement for FGFRs in dendritogenesis of adult-born granule cells. We find that deleting all three receptors results in a small decrease in proximal dendrite elaboration. In contrast, specifically mutating Tyr766 in FGFR1 (a PLCγ binding site) in an Fgfr2;Fgfr3 deficient background results in a dramatic increase of overall dendrite elaboration, while blocking FGFR1-FRS signaling causes proximal dendrite deficits and, to a lesser extent than Tyr766 mutants, increases distal dendrite elaboration. These findings reveal unexpectedly complex roles for FGFRs and their intracellular mediators in regulating proximal and distal dendrite elaboration, with the most notable role in suppressing distal elaboration through the PLCγbinding site.
Detecting mutations from single DNA molecules is crucial in many fields but challenging. Next-generation sequencing (NGS) affords tremendous throughput but cannot directly sequence double-stranded ...DNA molecules ('single duplexes') to discern the true mutations on both strands. Here we present Concatenating Original Duplex for Error Correction (CODEC), which confers single duplex resolution to NGS. CODEC affords 1,000-fold higher accuracy than NGS, using up to 100-fold fewer reads than duplex sequencing. CODEC revealed mutation frequencies of 2.72 × 10
in sperm of a 39-year-old individual, and somatic mutations acquired with age in blood cells. CODEC detected genome-wide, clonal hematopoiesis mutations from single DNA molecules, single mutated duplexes from tumor genomes and liquid biopsies, microsatellite instability with 10-fold greater sensitivity and mutational signatures, and specific tumor mutations with up to 100-fold fewer reads. CODEC enables more precise genetic testing and reveals biologically significant mutations, which are commonly obscured by NGS errors.
The addition of new neurons to existing neural circuits in the adult brain remains of great interest to neurobiology because of its therapeutic implications. The premier model for studying this ...process has been the hippocampal dentate gyrus in mice, where new neurons are added to mature circuits during adulthood. Notably, external factors such as an enriched environment (EE) and exercise markedly increase hippocampal neurogenesis. Here, we demonstrate that EE acts by increasing fibroblast growth factor receptor (FGFR) function autonomously within neurogenic cells to expand their numbers in adult male and female mice. FGFRs activated by EE signal through their mediators, FGFR substrate (FRS), to induce stem cell proliferation, and through FRS and phospholipase Cγ to increase the number of adult-born neurons, providing a mechanism for how EE promotes adult neurogenesis.
How the environment we live in affects cognition remains poorly understood. In the current study, we explore the mechanism underlying the effects of an enriched environment on the production of new neurons in the adult hippocampal dentate gyrus, a brain area integral in forming new memories. A mechanism is provided for how neural precursor cells in the adult mammalian dentate gyrus respond to an enriched environment to increase their neurogenic output. Namely, an enriched environment acts on stem and progenitor cells by activating fibroblast growth factor receptor signaling through phospholipase Cγ and FGF receptor substrate proteins to expand the pool of precursor cells.
The molecular mechanisms behind adult hippocampal neurogenesis are beginning to be explored. The generation of new neurons in adulthood provides a unique opportunity to study maturation and ...development of adult-born neurons and their integration into existing hippocampal circuits. Similarly to developmentally derived neurons, adult-born neurons require a series of developmental milestones to survive, reach full maturity, and properly integrate into circuits, all of which are mediated by intercellular signaling. Proper adult-born neuron development and survival involve responding to molecular signals through the action of various receptors. Fibroblast growth factor receptors (FGFRs) are a family of tyrosine kinases implicated in proper functioning of adult neurogenesis, as well as survival and proliferation of adult subgranular zone stem cells. Yet, the intracellular mechanisms mediating the various functions of FGFRs are poorly understood. Thus, I aimed to address what roles FGFRs play in adult hippocampal neurogenesis and which downstream signaling pathways are involved. To address these questions, we pursued a series of studies to assess how disruption of FGFRs impacts cell proliferation, development of proper dendritic arbors, and the response of neurogenesis to environmental conditions. First, we addressed the roles of FGFRs and downstream signaling pathways mediated through Phospholipase-c γ (PLCγ) and FGF Receptor Substrate proteins 2 and 3 (FRS) in adult-born neuron dendritogenesis. We conditionally deleted FGFR1, 2, and 3 from stem cells and early progenitors in adult mice and assessed the dendritic tree branching. The results showed that loss of FGFRs in stem cells and early progenitors, as well as late progenitors showed a very mild dendritic phenotype. However, using alleles that specifically abrogate FGFR1-signaling through FRS2/3 and PLCγ, we showed that loss of signaling mediated through the FGFR1-PLC and FGFR1-FRS lead to dendritic overgrowth and branching defects. Second, adult hippocampal neurogenesis is heavily modulated by environmental factors. It is well established that increased voluntary exercise and enriched environment increase adult neurogenesis. The current literature strongly points to the neurothrophic factor, brain derived neurotrophic factor (BDNF), and its tyrosine receptor kinase (TrkB) receptor as being the main regulators of this phenomenon. Yet, the evidence fully supporting this conclusion is lacking. As an alternative signaling pathway, we tested the roles of FGFRs in mediating the effects of the enriched environment and exercise on neural stem/progenitor cell proliferation and on numbers of total new-born neurons, as well as the roles of the FGFR-PLCγ and FGFR1-FRS pathways. The results showed that FGFRs are required for mediating the effects of enriched environment and exercise by promoting proliferation and increasing total cell numbers; while the FGFR-PLCγ and FGFR1-FRS pathways played distinct roles in promoting proliferation and total cell numbers. Third, the L1cam adhesion molecule has been implicated in many developmentally driven neuronal deficits. Yet, its roles in adult neurogenesis remained unexplored. We tested the roles of L1cam in adult-born stem cells, progenitors, mature cells, and surrounding cells by conditionally deleting L1cam from these multiple specific cell populations during adulthood. The results showed that L1cam is required for proper dendritic development when lost in stem cells, and total numbers of immature cells when lost in late progenitors and immature neurons. L1cam loss also led to anxiogenic behavior. In conclusion, this thesis determined the roles of FGFRs in adult hippocampal neurogenesis and revealed novel molecular mechanisms modulating various aspects of adult-born neuron proliferation, cell expansion, and anxiogenic behavior, as well as mediating the effects of enriched environment and exercise. This thesis also tested the roles of the L1cam adhesion molecule in the proliferation of adult-born precursors, on numbers of total cells, and on anxiety-like behaviors. Together these results reveal important molecular mechanisms used by stem and progenitor cells during adult neurogenesis and how environmental factors specifically act through the FGFRs to promote maturation and integration of adult-born neurons into the hippocampal circuitry.
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