Keeping the Balance Between Proliferation and Differentiation: The Primary Cilium

Primary cilia are post-mitotic cellular organelles that are present in the vast majority of cell types in the human body. An extensive body of data gathered in recent years is demonstrating a crucial role for this organelle in a number of cellular processes that include mechano and chemo-sensation as well as the transduction of signaling cascades critical for the development and maintenance of different tissues and organs. Consequently, cilia are currently viewed as cellular antennae playing a critical role at the interphase between cells and their environment, integrating a range of stimuli to modulate cell fate decisions including cell proliferation, migration and differentiation. Importantly, this regulatory role is not just a consequence of their participation in signal transduction but is also the outcome of both the tight synchronization/regulation of ciliogenesis with the cell cycle and the role of individual ciliary proteins in cilia-dependent and independent processes. Here we review the role of primary cilia in the regulation of cell proliferation and differentiation and illustrate how this knowledge has provided insight to understand the phenotypic consequences of ciliary dysfunction

Neuron's little helper: the role of primary cilia in neurogenesis

The generation of new neurons involves a great variety of cell-extrinsic and cell-intrinsic signals. The primary cilium, long regarded as an "evolutionary vestige," has emerged as an essential signaling hub in many cells, including neural progenitors and differentiating neurons. Most progenitors harbor an apically-localized primary cilium, which is assembled and disassembled following the cell cycle, while the presence, position and length of this organelle appears to be even more variable in differentiating neurons. One of the main extracellular cues acting through the cilium is Sonic Hedgehog, which modulates spatial patterning, the progression of the cell cycle and the timing of neurogenesis. Other extracellular signals appear to bind to cilia-localized receptors and affect processes such as dendritogenesis. All the observed dynamics, as well as the many signaling pathways depending on cilia, indicate this organelle as an important structure involved in neurogenesis.Agencia Nacional de Investigación e InnovaciónPEDECIBAInstitut Pasteur de Montevideo–FOCEM Mercosu

Insights into in vivo adipocyte differentiation through cell-specific labeling in zebrafish.

White adipose tissue hyperplasia has been shown to be crucial for handling excess energy in healthy ways. Though adipogenesis mechanisms have been underscored in vitro, we lack information on how tissue and systemic factors influence the differentiation of new adipocytes. While this could be studied in zebrafish, adipocyte identification currently relies on neutral lipid labeling, thus precluding access to cells in early stages of differentiation. Here we report the generation and analysis of a zebrafish line with the transgene fabp4a(- 2.7):EGFPcaax. In vivo confocal microscopy of the pancreatic and abdominal visceral depots of transgenic larvae, revealed the presence of labeled mature adipocytes as well as immature cells in earlier stages of differentiation. Through co-labeling for blood vessels, we observed a close interaction of differentiating adipocytes with endothelial cells through cell protrusions. Finally, we implemented hyperspectral imaging and spectral phasor analysis in Nile Red-labeled transgenic larvae and revealed the lipid metabolic transition towards neutral lipid accumulation of differentiating adipocytes. Altogether our work presents the characterization of a novel adipocyte-specific label in zebrafish and uncovers previously unknown aspects of in vivo adipogenesis.Agencia Nacional de Investigación e InnovaciónPrograma de Desarrollo de las Ciencias BásicasFondo de Convergencia Estructural del Mercosu

Characterization of primary cilia during the differentiation of retinal ganglion cells in the zebrafish

BACKGROUND: Retinal ganglion cell (RGC) differentiation in vivo is a highly stereotyped process, likely resulting from the interaction of cell type-specific transcription factors and tissue-derived signaling factors. The primary cilium, as a signaling hub in the cell, may have a role during this process but its presence and localization during RGC generation, and its contribution to the process of cell differentiation, have not been previously assessed in vivo. METHODS: In this work we analyzed the distribution of primary cilia in vivo using laser scanning confocal microscopy, as well as their main ultrastructural features by transmission electron microscopy, in the early stages of retinal histogenesis in the zebrafish, around the time of RGC generation and initial differentiation. In addition, we knocked-down ift88 and elipsa, two genes with an essential role in cilia generation and maintenance, a treatment that caused a general reduction in organelle size. The effect on retinal development and RGC differentiation was assessed by confocal microscopy of transgenic or immunolabeled embryos. RESULTS: Our results show that retinal neuroepithelial cells have an apically-localized primary cilium usually protruding from the apical membrane. We also found a small proportion of sub-apical cilia, before and during the neurogenic period. This organelle was also present in an apical position in neuroblasts during apical process retraction and dendritogenesis, although between these stages cilia appeared highly dynamic regarding both presence and position. Disruption of cilia caused a decrease in the proliferation of retinal progenitors and a reduction of neural retina volume. In addition, retinal histogenesis was globally delayed albeit RGC layer formation was preferentially reduced with respect to the amacrine and photoreceptor cell layers. CONCLUSIONS: These results indicate that primary cilia exhibit a highly dynamic behavior during early retinal differentiation, and that they are required for the proliferation and survival of retinal progenitors, as well as for neuronal generation, particularly of RGCs.Agencia Nacional de Investigación e InnovaciónPEDECIBAInstitut Pasteur de Montevide

Haploinsufficiency of ALX4 as a Potential Cause of Parietal Foramina in the 11p11.2 Contiguous Gene–Deletion Syndrome

Heterozygous mutations in MSX2 are responsible for an autosomal dominant form of parietal foramina (PFM). PFM are oval defects of the parietal bones that are also a characteristic feature of a contiguous gene–deletion syndrome caused by a proximal deletion in the short arm of chromosome 11 (Potocki-Shaffer syndrome). We have identified a human bacterial artificial chromosome (BAC) clone mapping to chromosome 11, containing a region homologous to the human homeobox gene MSX2. Further sequence analysis demonstrated that the human orthologue (ALX4) of the mouse Aristaless-like 4 gene (Alx4) is contained within this 11p clone. We used FISH to test for the presence—or for the heterozygous deletion—of this clone in two patients with the 11p11.2-deletion syndrome and showed that this clone is deleted in these patients. ALX4 and Alx4 were shown to be expressed in bone and to be absent from all other tissues tested. The involvement of Alx4 in murine skull development, its bone-specific expression pattern, the fact that Alx4 is a dosage-sensitive gene in mice, and the localization of a human genomic clone containing ALX4 to 11p11.2, with hemizygosity in patients with deletion of 11p11.2 who have biparietal foramina, support the contention that ALX4 is a candidate gene for the PFM in the 11p11.2-deletion syndrome

Ribonomic analysis of human DZIP1 reveals its involvement in ribonucleoprotein complexes and stress granules

Submitted by Renata Fontoura ([email protected]) on 2014-11-26T12:39:22Z No. of bitstreams: 1 artigo 1.pdf: 1716335 bytes, checksum: 17480039c8f2b4b9a96d1131ed066e83 (MD5)Approved for entry into archive by Renata Fontoura ([email protected]) on 2014-11-26T13:11:26Z (GMT) No. of bitstreams: 1 artigo 1.pdf: 1716335 bytes, checksum: 17480039c8f2b4b9a96d1131ed066e83 (MD5)Made available in DSpace on 2014-11-26T13:11:26Z (GMT). No. of bitstreams: 1 artigo 1.pdf: 1716335 bytes, checksum: 17480039c8f2b4b9a96d1131ed066e83 (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Básica de Células Tronco. Curitiba, PR, Brasil.Instituto de Investigaciones Biológicas Clemente Estable. Genomics Department. Montevideo, Uruguay. / Universidad de la República Uruguay. Facultad de Ciencias. Departamento de Biología Celular y Molecular. Montevideo, Uruguay.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Básica de Células Tronco. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Básica de Células Tronco. Curitiba, PR, Brasil.Instituto Pasteur. Montevideo, Uruguay. / Universidad de la República Uruguay. Facultad de Ciencias. Departamento de Biología Celular y Molecular. Montevideo, Uruguay.Instituto Pasteur. Montevideo, Uruguay.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Básica de Células Tronco. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Básica de Células Tronco. Curitiba, PR, Brasil.National Laboratory for Cancer Research. Leidos Biomedical Research, Inc. Cancer Research Technology Program. Frederick, USA.Background: DZIP1 (DAZ-interacting protein 1) has been described as a component of the Hh signaling pathway with a putative regulatory role in ciliogenesis. DZIP1 interacts with DAZ RNA binding proteins in embryonic stem cells and human germ cells suggesting a role in mRNA regulation. Results: We investigated DZIP1 function in HeLa cells and its involvement in ribonucleoprotein complexes. DZIP1 was predominantly located in granules in the cytoplasm. Under oxidative stress conditions, DZIP1 re-localized to stress granules. DZIP appears to be important for the formation of stress granules during the stress response. We used immunoprecipitation assays with antibodies against DZIP1 and microarray hybridization to identify mRNAs associated with DZIP1. The genetic networks formed by the DZIP1-associated mRNAs were involved in cell cycle and gene expression regulation. DZIP1 is involved in the Hedgehog signaling pathway. We used cyclopamine, a specific inhibitor of this pathway, to analyze the expression of DZIP1 and its associated mRNAs. The abundance of DZIP1-associated mRNAs increased with treatment; however, the silencing or overexpression of DZIP1 in HeLa cells had no effect on the accumulation of the associated mRNAs. Polysomal profile analysis by sucrose gradient centrifugation demonstrated the presence of DZIP1 in the polysomal fraction. Conclusions: Our results suggest that DZIP1 is part of an RNP complex that occupies various subcellular locations. The diversity of the mRNAs associated with DZIP1 suggests that this protein is a component of different RNPs associated with translating polysomes and with RNA granules

Tricuspid regurgitation: recent advances in understanding pathophysiology, severity grading and outcome

International audienceHeightened interest in tricuspid regurgitation (TR) stems from the consistent association of mortality with greater severity of TR, and a low use of surgical solutions in the setting of high in-hospital mortality attributed to the late presentation of the disease. The delay in intervention is likely related to a limited understanding of the valvular/ventricular anatomy and disease pathophysiology, along with an underestimation of TR severity by standard imaging modalities. With the rapid development of transcatheter solutions which have shown early safety and efficacy, there is a growing need to understand and accurately diagnose the valvular disease process in order to determine appropriate management solutions. The current review will describe both normal and pathologic tricuspid valvular anatomy, the classification of these anatomic substrates of TR, the strengths and limitations of the current guidelines-recommended multi-parametric echocardiographic approach and the role of multi-modality imaging, as well as the role of transcatheter device therapy in the management of the disease