Analysis of cilia dysfunction phenotypes in zebrafish embryos depleted of origin recognition complex factors

Meier–Gorlin syndrome (MGS) is a rare, congenital primordial microcephalic dwarfism disorder. MGS is caused by genetic variants of components of the origin recognition complex (ORC) consisting of ORC1–6 and the pre-replication complex, which together enable origin firing and hence genome replication. In addition, ORC1 has previously been shown to play a role in ciliogenesis. Here, we extend this work and investigate the function of ORC1 and two other members of the complex on cilia at an organismal level. Knockdown experiments in zebrafish confirmed the impact of ORC1 on cilia. ORC1-deficiency confers defects anticipated to arise from impaired cilia function such as formation of oedema, kidney cysts, curved bodies and left–right asymmetry defects. We found ORC1 furthermore required for cilium formation in zebrafish and demonstrate that ciliopathy phenotypes in ORC1-depleted zebrafish could not be rescued by reconstitution with ORC1 bearing a genetic variant previously identified in MGS patients. Loss-of-function of Orc4 and Orc6, respectively, conferred similar ciliopathy phenotypes and cilium shortening in zebrafish, suggesting that several, if not all, components of the ORC regulate ciliogenesis downstream to or in addition to their canonical function in replication initiation. This study presents the first in vivo evidence of an influence of the MGS genes of the ORC family on cilia, and consolidates the possibility that cilia dysfunction could contribute to the clinical manifestation of ORC-deficient MGS

Resting cells rely on the DNA helicase component MCM2 to build cilia

Minichromosome maintenance (MCM) proteins facilitate replication by licensing origins and unwinding the DNA double strand. Interestingly, the number of MCM hexamers greatly exceeds the number of firing origins suggesting additional roles of MCMs. Here we show a hitherto unanticipated function of MCM2 in cilia formation in human cells and zebrafish that is uncoupled from replication. Zebrafish depleted of MCM2 develop ciliopathy-phenotypes including microcephaly and aberrant heart looping due to malformed cilia. In non-cycling human fibroblasts, loss of MCM2 promotes transcription of a subset of genes, which cause cilia shortening and centriole overduplication. Chromatin immunoprecipitation experiments show that MCM2 binds to transcription start sites of cilia inhibiting genes. We propose that such binding may block RNA polymerase II-mediated transcription. Depletion of a second MCM (MCM7), which functions in complex with MCM2 during its canonical functions, reveals an overlapping cilia-deficiency phenotype likely unconnected to replication, although MCM7 appears to regulate a distinct subset of genes and pathways. Our data suggests that MCM2 and 7 exert a role in ciliogenesis in post-mitotic tissues

Nucleolar Stress Functions Upstream to Stimulate Expression of Autophagy Regulators

Ribosome biogenesis is essential for protein synthesis, cell growth and survival. The process takes places in nucleoli and is orchestrated by various proteins, among them RNA polymerases I–III as well as ribosome biogenesis factors. Perturbation of ribosome biogenesis activates the nucleolar stress response, which classically triggers cell cycle arrest and apoptosis. Nucleolar stress is utilized in modern anti-cancer therapies, however, also contributes to the development of various pathologies, including cancer. Growing evidence suggests that nucleolar stress stimulates compensatory cascades, for instance bulk autophagy. However, underlying mechanisms are poorly understood. Here we demonstrate that induction of nucleolar stress activates expression of key autophagic regulators such as ATG7 and ATG16L1, essential for generation of autophagosomes. We show that knockdown of the ribosomopathy factor SBDS, or of key ribosome biogenesis factors (PPAN, NPM, PES1) is associated with enhanced levels of ATG7 in cancer cells. The same holds true when interfering with RNA polymerase I function by either pharmacological inhibition (CX-5461) or depletion of the transcription factor UBF-1. Moreover, we demonstrate that RNA pol I inhibition by CX-5461 stimulates autophagic flux. Together, our data establish that nucleolar stress affects transcriptional regulation of autophagy. Given the contribution of both axes in propagation or cure of cancer, our data uncover a connection that might be targeted in future

Vertical Signalling Involves Transmission of Hox Information from Gastrula Mesoderm to Neurectoderm

<div><p>Development and patterning of neural tissue in the vertebrate embryo involves a set of molecules and processes whose relationships are not fully understood. Classical embryology revealed a remarkable phenomenon known as vertical signalling, a gastrulation stage mechanism that copies anterior-posterior positional information from mesoderm to prospective neural tissue. Vertical signalling mediates unambiguous copying of complex information from one tissue layer to another. In this study, we report an investigation of this process in recombinates of mesoderm and ectoderm from gastrulae of <i>Xenopus laevis</i>. Our results show that copying of positional information involves non cell autonomous autoregulation of particular <i>Hox</i> genes whose expression is copied from mesoderm to neurectoderm in the gastrula. Furthermore, this information sharing mechanism involves unconventional translocation of the homeoproteins themselves. This conserved primitive mechanism has been known for three decades but has only recently been put into any developmental context. It provides a simple, robust way to pattern the neurectoderm using the <i>Hox</i> pattern already present in the mesoderm during gastrulation. We suggest that this mechanism was selected during evolution to enable unambiguous copying of rather complex information from cell to cell and that it is a key part of the original ancestral mechanism mediating axial patterning by the highly conserved <i>Hox</i> genes.</p></div

Uptake of <i>d1-HD-GFP</i> and <i>GFP</i> by <i>Drosophila</i> imaginal wing discs.

<p><i>Drosphila</i> imaginal wing discs were incubated with <i>Hoxd1-HD-GFP (d1-HD-gfp)</i> recombinant protein (<b>B</b>) or wild type GFP protein (<b>A</b>). or mutated <i>mut</i>-<i>d1-HD-GFP.</i> Recombinant <i>d1-HD-gfp</i> was taken up by the discs while wild type GFP and <i>mut-d1-HD-GFP</i> were not. Each photo in this figure represents 10 imaginal discs giving the same result. These data clearly show the cargo function of Hoxd1 homeodomain and suggest that this uptake is by a species independent mechanism.</p

Craniofacial structures of <i>Xenopus laevis</i> tadpoles upon injection of <i>Hoxd1</i> mRNA or protein.

<p><b>a</b>: Schematic ventral view of an uninjected untreated control embryo. <b>b</b>: uninjected embryo. <b>c</b>: embryo injected with wild type GFP into the blastoecel at blastula stage. <b>d</b>: Embryo injected with <i>Hoxd1</i> mRNA at 4 cell stage. <b>e</b>: Embryo injected with recombinant HOXD1 protein into 4 cell stage embryo. <b>f</b>: injection of recombinant HOXD1 protein into the blastoecel. Please note that standard injection of Hoxd1 mRNA or its protein counterpart injection in the cytoplasm or in the extracellular matrix leads to similar phenotypes in <b>d, e</b> and <b>f</b>. The embryos are strongly posteriorised as shown by the reduction (or deletion) of anterior structures. These data strongly suggest that the Hoxd1 protein successfully crossed the cellular membranes and retained its function as it leads to a severe truncation of anterior cartilage structures. Infrarostrale (in), Meckel’s cartilage (me), palatoquadrate (pa), ceratohyale (ce), basibranchiale (ba), branchial arches (br), eye (ey), intestine (in). Each photo represents 10 identically treated embryos giving the same result.</p

Mesodermal <i>Hox</i> loss of function of a single <i>Hox</i> gene prevents neurectodermal <i>Hox</i> expression of the same <i>Hox</i> gene.

<p><b>Aa</b>, Wrap assay consists of a piece of non-organiser mesoderm (NOM) and a piece of Spemann organiser mesoderm (SO) combined between two ectodermal animal caps. All tissues are excised from early gastrulae (st. 10a) <b>Ab</b>, <b>Ba</b>, <b>Ca:</b> External views of late gastrula stage <i>Xenopus laevis</i> expressing <i>Hoxd1, Hoxb4</i> and <i>Hoxb9</i> respectively. Note that the midline of the embryo, overlying the SO, does not express any <i>Hox</i> gene. <b>Ac</b>, <b>Bb</b>, <b>Cb:</b> wraps containing only SO explants [AC(SO/SO)AC]. <b>Ad</b>, <b>Bc</b>, <b>Cc:</b> wraps containing SO and NOM treated with control morpholino (ctMO) [AC(SO/NOM+ctMO)AC]. <b>Ae</b>, <b>Bd</b>, <b>Cd</b>, wrap with NOM treated with <i>Hoxd1-, Hoxb4</i> and <i>Hoxb9</i> MO’s respectively. Please note that in each case, only the wraps containing NOM and SO show <i>Hox</i> expression in the neurectoderm (<b>Ad</b>, <b>Bc</b>, <b>Cc</b>) and those containing only SO do not show any expression in accordance with the embryo’s lack of <i>Hox</i> expression in SO (<b>Ab</b>, <b>Ba</b>, <b>Ca</b>, and <b>Ac</b>, <b>Bb</b>, <b>Cb</b>). In each case, <i>Hox</i> MO treatment of NOM mesoderm also prevents the expression of the homologous <i>Hox</i> gene in neurectoderm (<b>Ae</b>, <b>Bd</b>, <b>Cd</b>). These wraps were fixed and analysed 6–8 hrs after they were made. Each photo of two recombinates or an embryo in this figure is representative of at least 20 recombinates or embryos, all showing the same result.</p

Mesodermal ectopic expression of a single <i>Ho</i>x gene copies expression of the same <i>Hox</i> gene from mesoderm to neurectoderm.

<p><b>A– A’’’, E, F</b> Localisation of mesoderm and neurectoderm in the wrap assay shown by expression of the mesodermal markers <i>Chordin (Chd,</i> A<i>)</i> and <i>Brachyury (Bra,</i><b>A’</b><i>)</i>, and the neural marker <i>Nrp1</i> (<b>A”</b>). In <b>A’’’</b>, lineage labelling by ectopic expression of GFP in the NOM only. These recombinants were analyzed 6 to 8 h after tissue healing. These data show that there is no tissue intermingling during wrap culture. Mesodermal <i>Chd</i> (expressed in SO, <b>A</b>) and GFP (within NOM, <b>A’’’</b>) do not mix with each other within the time course of wrap culture. Consistently, <i>Bra</i>, a pan mesodermal marker, is expressed in both types of mesoderm in accordance with its expression domain in the embryo (<b>A’</b>).The neural marker <i>Nrp-1</i> is expressed in the space between mesoderm and the outermost ectodermal layer of the wrap, consistent with its known pattern of expression within the embryo (<b>A’’</b>). <b>B–B’</b>: Induction of <i>Hoxd1</i><b>B:</b> A wrap containing SO and NOM and ectoderm [AC(SO/NOM)AC] shows induction of <i>Hoxd1</i> in the neurectoderm as well as mesoderm. <b>B’, A</b> wrap containing normal SO and SO ectopically expressing <i>Hoxd1</i> also shows the induction of endogenous <i>Hoxd1</i> in the neurectoderm as well as in the mesoderm. Endogenous Hoxd1 expression was detected using a 3′UTR probe that recognizes only the endogenous messenger. <b>C</b>, ectopic <i>Hoxb4</i> in SO induces its own expression within the neurectoderm and in the mesoderm as in <b>B’. D</b>, wrap as in <b>B’</b> and <b>C</b> but with ectopic <i>Hoxc6</i> expression. This shows induction of <i>Hoxc6</i> in neurectoderm and in the mesoderm. We used 3′UTR probes to detect expression of the endogenous mRNA’s in each of these experiments. <b>E, F</b> sections showing expression of <i>Nrp1</i> (neural) and <i>Bra</i> (mesodermal) in control or standard [AC(SO/NOM)AC] recombinant. <b>E</b><i>Nrp1</i> expression is internal in the recombinant but excluded from an internal cell mass that is clearly the mesoderm. It is particularly strong around one end of the cell mass which is the neural inducing SO. Expression is also absent from the very outer layer of the recombinant, which represents the outer non neural layer of the neurectoderm. <b>F:</b><i>Bra</i> expression is in an internal cell mass (the mesoderm). Please note that the germ layer markers <i>Bra, Ch</i>, and the mesodermal lineage label GFP are confined to an internal cell mass excluding tissue intermingling and that Hox expression is detected in neurectoderm as well as the mesodermal cell mass. Each photo in this figure represents at least 20 recombinants and embryos with consistently the same results.</p

Imbalanced mitochondrial function provokes heterotaxy via aberrant ciliogenesis

About 1% of all newborns are affected by congenital heart disease (CHD). Recent findings identify aberrantly functioning cilia as a possible source for CHD. Faulty cilia also prevent the development of proper left-right asymmetry and cause heterotaxy, the incorrect placement of visceral organs. Intriguingly, signaling cascades such as mTor that influence mitochondrial biogenesis also affect ciliogenesis, and can cause heterotaxy-like phenotypes in zebrafish. Here, we identify levels of mitochondrial function as a determinant for ciliogenesis and a cause for heterotaxy. We detected reduced mitochondrial DNA content in biopsies of heterotaxy patients. Manipulation of mitochondrial function revealed a reciprocal influence on ciliogenesis and affected cilia-dependent processes in zebrafish, human fibroblasts and Tetrahymena thermophila. Exome analysis of heterotaxy patients revealed an increased burden of rare damaging variants in mitochondria-associated genes as compared to 1000 Genome controls. Knockdown of such candidate genes caused cilia elongation and ciliopathy-like phenotypes in zebrafish, which could not be rescued by RNA encoding damaging rare variants identified in heterotaxy patients. Our findings suggest that ciliogenesis is coupled to the abundance and function of mitochondria. Our data further reveal disturbed mitochondrial function as an underlying cause for heterotaxy-linked CHD and provide a mechanism for unexplained phenotypes of mitochondrial disease.publishersversionpublishe