Dominique Baas

The Transcription Factor RFX3 Directs Nodal Cilium Development and Left-Right Asymmetry Specification

ABSTRACT There are five members of the RFX family of transcription factors in mammals. While RFX5 plays a well-defined role in the immune system, the functions of RFX1 to RFX4 remain largely unknown. We have generated mice with a deletion of the Rfx3 gene. RFX3-deficient mice exhibit frequent left-right (LR) asymmetry defects leading to a high rate of embryonic lethality and situs inversus in surviving adults. In vertebrates, specification of the LR body axis is controlled by monocilia in the embryonic node, and defects in nodal cilia consequently result in abnormal LR patterning. Consistent with this, Rfx3 is expressed in ciliated cells of the node and RFX3-deficient mice exhibit a pronounced defect in nodal cilia. In contrast to the case for wild-type embryos, for which we document for the first time a twofold increase in the length of nodal cilia during development, the cilia are present but remain markedly stunted in mutant embryos. Finally, we show that RFX3 regulates the expression of D2lic, the mouse orthologue of a Caenorhabditis elegans gene that is implicated in intraflagellar transport, a process required for the assembly and maintenance of cilia. In conclusion, RFX3 is essential for the differentiation of nodal monocilia and hence for LR body axis determination.

Mouse neuroblastoma cells release prion infectivity associated with exosomal vesicles

Background information. TSEs (transmissible spongiform encephalopathies) are neurodegenerative disorders affecting humans and animals. PrPSc, a conformationally altered isoform of the normal prion protein (PrPC), is thought to be the pathogenic agent. However, the biochemical composition of the prion agent is still matter of debate. The potential transmission risk of the prion agent through biological fluids has been shown, but the development of competitive diagnostic tests and treatment for TSEs requires a more comprehensive knowledge of the agent and the cellular mechanisms by which it is disseminated. With this aim, we initiated characterization of the prion agent and the pathways by which it can be propagated using the cellular model system neuroblastoma (N2a).

A deficiency in RFX3 causes hydrocephalus associated with abnormal differentiation of ependymal cells.

Ciliated ependymal cells play central functions in the control of cerebrospinal fluid homeostasis in the mammalian brain, and defects in their differentiation or ciliated properties can lead to hydrocephalus. Regulatory factor X (RFX) transcription factors regulate genes required for ciliogenesis in the nematode, drosophila and mammals. We show here that Rfx3‐deficient mice suffer from hydrocephalus without stenosis of the aqueduct of Sylvius. RFX3 is expressed strongly in the ciliated ependymal cells of the subcommissural organ (SCO), choroid plexuses (CP) and ventricular walls during embryonic and postnatal development. Ultrastructural analysis revealed that the hydrocephalus is associated with a general defect in CP differentiation and with severe agenesis of the SCO. The specialized ependymal cells of the CP show an altered epithelial organization, and the SCO cells lose their characteristic ultrastructural features and adopt aspects more typical of classical ependymal cells. These differentiation defects are associated with changes in the number of cilia, although no obvious ultrastructural defects of these cilia can be observed in adult mice. Moreover, agenesis of the SCO is associated with downregulation of SCO‐spondin expression as early as E14.5 of embryonic development. These results demonstrate that RFX3 is necessary for ciliated ependymal cell differentiation in the mouse.

Novel Function of the Ciliogenic Transcription Factor RFX3 in Development of the Endocrine Pancreas

The transcription factor regulatory factor X (RFX)-3 regulates the expression of genes required for the growth and function of cilia. We show here that mouse RFX3 is expressed in developing and mature pancreatic endocrine cells during embryogenesis and in adults. RFX3 expression already is evident in early Ngn3-positive progenitors and is maintained in all major pancreatic endocrine cell lineages throughout their development. Primary cilia of hitherto unknown function present on these cells consequently are reduced in number and severely stunted in Rfx3−/− mice. This ciliary abnormality is associated with a developmental defect leading to a uniquely altered cellular composition of the islets of Langerhans. Just before birth, Rfx3−/− islets contain considerably less insulin-, glucagon-, and ghrelin-producing cells, whereas pancreatic polypeptide–positive cells are markedly increased in number. In adult mice, the defect leads to small and disorganized islets, reduced insulin production, and impaired glucose tolerance. These findings suggest that RFX3 participates in the mechanisms that govern pancreatic endocrine cell differentiation and that the presence of primary cilia on islet cells may play a key role in this process.

DHPR α1S subunit controls skeletal muscle mass and morphogenesis

The α1S subunit has a dual function in skeletal muscle: it forms the L‐type Ca2+ channel in T‐tubules and is the voltage sensor of excitation–contraction coupling at the level of triads. It has been proposed that L‐type Ca2+ channels might also be voltage‐gated sensors linked to transcriptional activity controlling differentiation. By using the U7‐exon skipping strategy, we have achieved long‐lasting downregulation of α1S in adult skeletal muscle. Treated muscles underwent massive atrophy while still displaying significant amounts of α1S in the tubular system and being not paralysed. This atrophy implicated the autophagy pathway, which was triggered by neuronal nitric oxide synthase redistribution, activation of FoxO3A, upregulation of autophagy‐related genes and autophagosome formation. Subcellular investigations showed that this atrophy was correlated with the disappearance of a minor fraction of α1S located throughout the sarcolemma. Our results reveal for the first time that this sarcolemmal fraction could have a role in a signalling pathway determining muscle anabolic or catabolic state and might act as a molecular sensor of muscle activity.

Role for the pleckstrin homology domain-containing protein CKIP-1 in phosphatidylinositol 3-kinase-regulated muscle differentiation.

ABSTRACT In this work, we report the implication of the pleckstrin homology (PH) domain-containing protein CKIP-1 in phosphatidylinositol 3-kinase (PI3-K)-regulated muscle differentiation. CKIP-1 is upregulated during muscle differentiation in C2C12 cells. We show that CKIP-1 binds to phosphatidylinositol 3-phosphate through its PH domain and localizes to the plasma membrane in a PI3-K-dependent manner. Activation of PI3-K by insulin or expression of an active form of PI3-K p110 induces a rapid translocation of CKIP-1 to the plasma membrane. Conversely, expression of the 3-phosphoinositide phosphatase myotubularin or PI3-K inhibition by LY294002, wortmannin, or mutant p85 abolishes CKIP-1 binding to the membrane. Upon induction of differentiation in low-serum medium, CKIP-1 overexpression in C2C12 myoblasts first promotes proliferation and then stimulates the expression of myogenin and cell fusion in a manner reminiscent of the dual positive effect of insulin-like growth factors on muscle cells. Interference with the PI3-K pathway impedes the effect of CKIP-1 on C2C12 cell differentiation. Finally, silencing of CKIP-1 by RNA interference abolishes proliferation and delays myogenin expression. Altogether, these data strongly implicate CKIP-1 as a new component of PI3-K signaling in muscle differentiation.

RFX2 Is a Major Transcriptional Regulator of Spermiogenesis

Spermatogenesis consists broadly of three phases: proliferation of diploid germ cells, meiosis, and finally extensive differentiation of the haploid cells into effective delivery vehicles for the paternal genome. Despite detailed characterization of many haploid developmental steps leading to sperm, only fragmentary information exists on the control of gene expression underlying these processes. Here we report that the RFX2 transcription factor is a master regulator of genes required for the haploid phase. A targeted mutation of Rfx2 was created in mice. Rfx2-/- mice are perfectly viable but show complete male sterility. Spermatogenesis appears to progress unperturbed through meiosis. However, haploid cells undergo a complete arrest in spermatid development just prior to spermatid elongation. Arrested cells show altered Golgi apparatus organization, leading to a deficit in the generation of a spreading acrosomal cap from proacrosomal vesicles. Arrested cells ultimately merge to form giant multinucleated cells released to the epididymis. Spermatids also completely fail to form the flagellar axoneme. RNA-Seq analysis and ChIP-Seq analysis identified 139 genes directly controlled by RFX2 during spermiogenesis. Gene ontology analysis revealed that genes required for cilium function are specifically enriched in down- and upregulated genes showing that RFX2 allows precise temporal expression of ciliary genes. Several genes required for cell adhesion and cytoskeleton remodeling are also downregulated. Comparison of RFX2-regulated genes with those controlled by other major transcriptional regulators of spermiogenesis showed that each controls independent gene sets. Altogether, these observations show that RFX2 plays a major and specific function in spermiogenesis.

The coiled-coil domain containing protein CCDC151 is required for the function of IFT-dependent motile cilia in animals

Cilia are evolutionarily conserved organelles endowed with essential physiological and developmental functions. In humans, disruption of cilia motility or signaling leads to complex pleiotropic genetic disorders called ciliopathies. Cilia motility requires the assembly of multi-subunit motile components such as dynein arms, but mechanisms underlying their assembly pathway and transport into the axoneme are still largely unknown. We identified a previously uncharacterized coiled-coil domain containing protein CCDC151, which is evolutionarily conserved in motile ciliated species and shares ancient features with the outer dynein arm-docking complex 2 of Chlamydomonas. In Drosophila, we show that CG14127/CCDC151 is associated with motile intraflagellar transport (IFT)-dependent cilia and required for geotaxis behavior of adult flies. In zebrafish, Ccdc151 is expressed in tissues with motile cilia, and morpholino-induced depletion of Ccdc151 leads to left-right asymmetry defects and kidney cysts. We demonstrate that Ccdc151 is required for proper motile function of cilia in the Kupffers vesicle and in the pronephros by controlling dynein arm assembly, showing that Ccdc151 is a novel player in the control of IFT-dependent dynein arm assembly in animals. However, we observed that CCDC151 is also implicated in other cellular functions in vertebrates. In zebrafish, ccdc151 is involved in proper orientation of cell divisions in the pronephros and genetically interacts with prickle1 in this process. Furthermore, knockdown experiments in mammalian cells demonstrate that CCDC151 is implicated in the regulation of primary cilium length. Hence, CCDC151 is required for motile cilia function in animals but has acquired additional non-motile functions in vertebrates.

mTOR inactivation in myocardium from infant mice rapidly leads to dilated cardiomyopathy due to translation defects and p53/JNK-mediated apoptosis.

Mechanistic target of rapamycin (mTOR) is a central regulator of cell growth, proliferation, survival and metabolism, as part of mTOR complex 1 (mTORC1) and mTORC2. While partial inhibition of mTORC1 using rapamycin was shown to be cardioprotective, genetic studies in mouse models revealed that mTOR is essential for embryonic heart development and cardiac function in adults. However, the physiological role of mTOR during postnatal cardiac maturation is not fully elucidated. We have therefore generated a mouse model in which cardiac mTOR was inactivated at an early postnatal stage. Mutant mTORcmKO mice rapidly developed a dilated cardiomyopathy associated with cardiomyocyte growth defects, apoptosis and fibrosis, and died during their third week. Here, we show that reduced cardiomyocyte growth results from impaired protein translation efficiency through both 4E-BP1-dependent and -independent mechanisms. In addition, infant mTORcmKO hearts displayed markedly increased apoptosis linked to stretch-induced ANKRD1 (Ankyrin repeat-domain containing protein 1) up-regulation, JNK kinase activation and p53 accumulation. Pharmacological inhibition of p53 with pifithrin-α attenuated caspase-3 activation. Cardiomyocyte death did not result from activation of the MST1/Hippo pro-apoptotic pathway as reported in adult rictor/mTORC2 KO hearts. As well, mTORcmKO hearts showed a strong downregulation of myoglobin content, thereby leading to a hypoxic environment. Nevertheless, they lacked a HIF1α-mediated adaptive response, as mTOR is required for hypoxia-induced HIF-1α activation. Altogether, our results demonstrate that mTOR is critically required for cardiomyocyte growth, viability and oxygen supply in early postnatal myocardium and provide insight into the molecular mechanisms involved in apoptosis of mTOR-depleted cardiomyocytes.

CKIP-1 regulates mammalian and zebrafish myoblast fusion

Summary Multinucleated muscle fibres arise by fusion of precursor cells called myoblasts. We previously showed that CKIP-1 ectopic expression in C2C12 myoblasts increased cell fusion. In this work, we report that CKIP-1 depletion drastically impairs C2C12 myoblast fusion in vitro and in vivo during zebrafish muscle development. Within developing fast-twich myotome, Ckip-1 localises at the periphery of fast precursor cells, closed to the plasma membrane. Unlike wild-type myoblasts that form spatially arrayed multinucleated fast myofibres, Ckip-1-deficient myoblasts show a drastic reduction in fusion capacity. A search for CKIP-1 binding partners identified the ARPC1 subunit of Arp2/3 actin nucleation complex essential for myoblast fusion. We demonstrate that CKIP-1, through binding to plasma membrane phosphoinositides via its PH domain, regulates cell morphology and lamellipodia formation by recruiting the Arp2/3 complex at the plasma membrane. These results establish CKIP-1 as a regulator of cortical actin that recruits the Arp2/3 complex at the plasma membrane essential for muscle precursor elongation and fusion.