Summary
CRISPR/Cas9‐based strategies are widely used for genome editing in many organisms, including zebrafish. Although most applications consist in introducing double strand break (DSB)‐induced ...mutations, it is also possible to use CRISPR/Cas9 to enhance homology directed repair (HDR) at a chosen genomic location to create knock‐ins with optimally controlled precision. Here, we describe the use of CRISPR/Cas9‐targeted DSB followed by HDR to generate zebrafish transgenic lines where exogenous coding sequences are added in the nefma gene, in frame with the endogenous coding sequence. The resulting knock‐in embryos express the added gene (fluorescent reporter or KalTA4 transactivator) specifically in the populations of neurons that express nefma, making them convenient tools for research on these populations.
Development of the forebrain critically depends on the Sonic Hedgehog (Shh) signaling pathway, as illustrated in humans by the frequent perturbation of this pathway in holoprosencephaly, a condition ...defined as a defect in the formation of midline structures of the forebrain and face. The Shh pathway requires functional primary cilia, microtubule-based organelles present on virtually every cell and acting as cellular antennae to receive and transduce diverse chemical, mechanical or light signals. The dysfunction of cilia in humans leads to inherited diseases called ciliopathies, which often affect many organs and show diverse manifestations including forebrain malformations for the most severe forms.
The purpose of this review is to provide the reader with a framework to understand the developmental origin of the forebrain defects observed in severe ciliopathies with respect to perturbations of the Shh pathway. We propose that many of these defects can be interpreted as an imbalance in the ratio of activator to repressor forms of the Gli transcription factors, which are effectors of the Shh pathway.
We also discuss the complexity of ciliopathies and their relationships with forebrain disorders such as holoprosencephaly or malformations of cortical development, and emphasize the need for a closer examination of forebrain defects in ciliopathies, not only through the lens of animal models but also taking advantage of the increasing potential of the research on human tissues and organoids.
•Primary cilia are sensory and signaling organelles with central role in brain development.•Severe human ciliopathies encompass forebrain developmental malformations.•Forebrain malformations in ciliopathies overlap with microcephaly and holoprosencephaly.•Several forebrain developmental defects in ciliopathies can be attributed to Hedgehog/Gli perturbations.
Ciliopathies are life‐threatening human diseases caused by defective cilia. They can often be traced back to mutations of genes encoding transition zone (TZ) proteins demonstrating that the ...understanding of TZ organisation is of paramount importance. The TZ consists of multimeric protein modules that are subject to a stringent assembly hierarchy. Previous reports place Rpgrip1l at the top of the TZ assembly hierarchy in Caenorhabditis elegans. By performing quantitative immunofluorescence studies in RPGRIP1L−/− mouse embryos and human embryonic cells, we recognise a different situation in vertebrates in which Rpgrip1l deficiency affects TZ assembly in a cell type‐specific manner. In cell types in which the loss of Rpgrip1l alone does not affect all modules, additional truncation or removal of vertebrate‐specific Rpgrip1 results in an impairment of all modules. Consequently, Rpgrip1l and Rpgrip1 synergistically ensure the TZ composition in several vertebrate cell types, revealing a higher complexity of TZ assembly in vertebrates than in invertebrates.
Synopsis
The transition zone (TZ) compartment within the cilium controls the ciliary entry and exit of proteins, and its disruption is linked to dysfunctional cilia and ciliopathies. Analyses in various mouse and human cell types reveal a higher complexity of TZ assembly in vertebrates as compared to the simpler hierarchy known from Caenorhabditis elegans.
A Rpgrip1l‐Cep290‐Nphp4‐Invs‐Nphp1 axis defines the hierarchy of TZ assembly in mice.
Assembly of a functional TZ depends on sequential recruitment of TZ proteins in appropriate amounts.
Rpgrip1l and Rpgrip1 synergistically ensure proper TZ assembly in a cell type‐specific manner.
Synergistic roles of paralogous proteins Rpgrip1 and Rpgrip1l suggest a higher complexity of ciliary transition zone assembly in mouse embryos and human embryonic cells as compared to the simpler hierarchy known from invertebrates.
Neuronal circuits, the functional building blocks of the nervous system, assemble during development through a series of dynamic processes including the migration of neurons to their final position, ...the growth and navigation of axons and their synaptic connection with target cells. While the role of chemical cues in guiding neuronal migration and axonal development has been extensively analysed, the contribution of mechanical inputs, such as forces and stiffness, has received far less attention. In this article, we review the in vitro and more recent in vivo studies supporting the notion that mechanical signals are critical for multiple aspects of neuronal circuit assembly, from the emergence of axons to the formation of functional synapses. By combining live imaging approaches with tools designed to measure and manipulate the mechanical environment of neurons, the emerging field of neuromechanics will add a new paradigm in our understanding of neuronal development and potentially inspire novel regenerative therapies.
Review: Neurons develop in complex 3D environments in which they are subjected to mechanical forces and heterogeneous rigidities. This review summarises the reported in vitro and more recent in vivo roles of these mechanical cues in the sculpting of neuronal shape and of the connection of neurons into functional circuits. We further discuss potential mechanisms of neuronal mechanotransduction and how neurons integrate mechanical information with chemical cues to establish neuronal networks.
A range of severe human diseases called ciliopathies is caused by the dysfunction of primary cilia. Primary cilia are cytoplasmic protrusions consisting of the basal body (BB), the axoneme, and the ...transition zone (TZ). The BB is a modified mother centriole from which the axoneme, the microtubule-based ciliary scaffold, is formed. At the proximal end of the axoneme, the TZ functions as the ciliary gate governing ciliary protein entry and exit. Since ciliopathies often develop due to mutations in genes encoding proteins that localize to the TZ, the understanding of the mechanisms underlying TZ function is of eminent importance. Here, we show that the ciliopathy protein Rpgrip1l governs ciliary gating by ensuring the proper amount of Cep290 at the vertebrate TZ. Further, we identified the flavonoid eupatilin as a potential agent to tackle ciliopathies caused by mutations in
as it rescues ciliary gating in the absence of Rpgrip1l.
Ciliary shedding occurs from unicellular organisms to metazoans. Although required during the cell cycle and during neurogenesis, the process remains poorly understood. In all cellular models, this ...phenomenon occurs distal to the transition zone (TZ), suggesting conserved molecular mechanisms. The TZ module proteins (Meckel Gruber syndrome MKS/Nephronophtysis NPHP/Centrosomal protein of 290 kDa CEP290/Retinitis pigmentosa GTPase regulator-Interacting Protein 1-Like Protein RPGRIP1L) are known to cooperate to establish TZ formation and function. To determine whether they control deciliation, we studied the function of 5 of them (Transmembrane protein 107 TMEM107, Transmembrane protein 216 TMEM216, CEP290, RPGRIP1L, and NPHP4) in Paramecium. All proteins are recruited to the TZ of growing cilia and localize with 9-fold symmetry at the level of the most distal part of the TZ. We demonstrate that depletion of the MKS2/TMEM216 and TMEM107 proteins induces constant deciliation of some cilia, while depletion of either NPHP4, CEP290, or RPGRIP1L prevents Ca2+/EtOH deciliation. Our results constitute the first evidence for a role of conserved TZ proteins in deciliation and open new directions for understanding motile cilia physiology.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Background
Neurogenesis in the murine cerebral cortex involves the coordinated divisions of two main types of progenitor cells, whose numbers, division modes and cell cycle durations set up ...the final neuronal output. To understand the respective roles of these factors in the neurogenesis process, we combine experimental in vivo studies with mathematical modeling and numerical simulations of the dynamics of neural progenitor cells. A special focus is put on the population of intermediate progenitors (IPs), a transit amplifying progenitor type critically involved in the size of the final neuron pool.
Results
A multiscale formalism describing IP dynamics allows one to track the progression of cells along the subsequent phases of the cell cycle, as well as the temporal evolution of the different cell numbers. Our model takes into account the dividing apical progenitors (AP) engaged into neurogenesis, both neurogenic and proliferative IPs, and the newborn neurons. The transfer rates from one population to another are subject to the mode of division (proliferative, or neurogenic) and may be time-varying. The model outputs are successfully fitted to experimental cell numbers from mouse embryos at different stages of cortical development, taking into account IPs and neurons, in order to adjust the numerical parameters. We provide additional information on cell kinetics, such as the mitotic and S phase indexes, and neurogenic fraction.
Conclusions
Applying the model to a mouse mutant for
Ftm/Rpgrip1l
, a gene involved in human ciliopathies with severe brain abnormalities, reveals a shortening of the neurogenic period associated with an increased influx of newborn IPs from apical progenitors at mid-neurogenesis. Our model can be used to study other mouse mutants with cortical neurogenesis defects and can be adapted to study the importance of progenitor dynamics in cortical evolution and human diseases.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cerebello-oculo-renal syndrome (CORS), also called Joubert syndrome type B, and Meckel (MKS) syndrome belong to the group of developmental autosomal recessive disorders that are associated with ...primary cilium dysfunction. Using SNP mapping, we identified missense and truncating mutations in RPGRIP1L (KIAA1005) in both CORS and MKS, and we show that inactivation of the mouse ortholog Rpgrip1l (Ftm) recapitulates the cerebral, renal and hepatic defects of CORS and MKS. In addition, we show that RPGRIP1L colocalizes at the basal body and centrosomes with the protein products of both NPHP6 and NPHP4, known genes associated with MKS, CORS and nephronophthisis (a related renal disorder and ciliopathy). In addition, the RPGRIP1L missense mutations found in CORS individuals diminishes the interaction between RPGRIP1L and nephrocystin-4. Our findings show that mutations in RPGRIP1L can cause the multiorgan phenotypic abnormalities found in CORS or MKS, which therefore represent a continuum of the same underlying disorder.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
During the development of the cerebral cortex, neurons are generated directly from radial glial cells or indirectly via basal progenitors. The balance between these division modes determines the ...number and types of neurons formed in the cortex thereby affecting cortical functioning. Here, we investigate the role of primary cilia in controlling the decision between forming neurons directly or indirectly. We show that a mutation in the ciliary gene
leads to a transient increase in direct neurogenesis and subsequently to an overproduction of layer V neurons in newborn mice. Loss of
also affects ciliary structure coinciding with reduced Gli3 repressor levels. Genetically restoring Gli3 repressor rescues the decreased indirect neurogenesis in
mutants. Overall, our analyses reveal how primary cilia determine neuronal subtype composition of the cortex by controlling direct versus indirect neurogenesis. These findings have implications for understanding cortical malformations in ciliopathies with
mutations.
Primary cilia have essential functions in vertebrate development and signaling. However, little is known about cilia function in brain morphogenesis, a process that is severely affected in human ...ciliopathies. Here, we study telencephalic morphogenesis in a mouse mutant for the ciliopathy gene Ftm (Rpgrip1l). We show that the olfactory bulbs are present in an ectopic location in the telencephalon of Ftm(-/-) fetuses and do not display morphological outgrowth at the end of gestation. Investigating the developmental origin of this defect, we have established that E12.5 Ftm(-/-) telencephalic neuroepithelial cells lack primary cilia. Moreover, in the anterior telencephalon, the subpallium is expanded at the expense of the pallium, a phenotype reminiscent of Gli3 mutants. This phenotype indeed correlates with a decreased production of the short form of the Gli3 protein. Introduction of a Gli3 mutant allele encoding the short form of Gli3 into Ftm mutants rescues both telencephalic patterning and olfactory bulb morphogenesis, despite the persistence of cilia defects. Together, our results show that olfactory bulb morphogenesis depends on primary cilia and that the essential role of cilia in this process is to produce processed Gli3R required for developmental patterning. Our analysis thus provides the first in vivo demonstration that primary cilia control a developmental process via production of the short, repressor form of Gli3. Moreover, our findings shed light on the developmental origin of olfactory bulb agenesis and of other brain morphogenetic defects found in human diseases affecting the primary cilium.
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