Cilia-localized GID/CTLH ubiquitin ligase complex regulates protein homeostasis of sonic hedgehog signaling components.

Cilia are evolutionarily conserved organelles that orchestrate a variety of signal transduction pathways, such as sonic hedgehog (SHH) signaling, during embryonic development. Our recent studies have shown that loss of GID ubiquitin ligase function results in aberrant AMP-activated protein kinase (AMPK) activation and elongated primary cilia, which suggests a functional connection to cilia. Here, we reveal that the GID complex is an integral part of the cilium required for primary cilia-dependent signal transduction and the maintenance of ciliary protein homeostasis. We show that GID complex subunits localize to cilia in both Xenopus laevis and NIH3T3 cells. Furthermore, we report SHH signaling pathway defects that are independent of AMPK and mechanistic target of rapamycin (MTOR) activation. Despite correct localization of SHH signaling components at the primary cilium and functional GLI3 processing, we find a prominent reduction of some SHH signaling components in the cilium and a significant decrease in SHH target gene expression. Since our data reveal a critical function of the GID complex at the primary cilium, and because suppression of GID function in X. laevis results in ciliopathy-like phenotypes, we suggest that GID subunits are candidate genes for human ciliopathies that coincide with defects in SHH signal transduction.

Hedgehog-dependent E3-ligase Midline1 regulates ubiquitin-mediated proteasomal degradation of Pax6 during visual system development.

Pax6 is a key transcription factor involved in eye, brain, and pancreas development. Although pax6 is expressed in the whole prospective retinal field, subsequently its expression becomes restricted to the optic cup by reciprocal transcriptional repression of pax6 and pax2 However, it remains unclear how Pax6 protein is removed from the eyestalk territory on time. Here, we report that Mid1, a member of the RBCC/TRIM E3 ligase family, which was first identified in patients with the X-chromosome-linked Opitz BBB/G (OS) syndrome, interacts with Pax6. We found that the forming eyestalk is a major domain of mid1 expression, controlled by the morphogen Sonic hedgehog (Shh). Here, Mid1 regulates the ubiquitination and proteasomal degradation of Pax6 protein. Accordantly, when Mid1 levels are knocked down, Pax6 expression is expanded and eyes are enlarged. Our findings indicate that remaining or misaddressed Pax6 protein is cleared from the eyestalk region to properly set the border between the eyestalk territory and the retina via Mid1. Thus, we identified a posttranslational mechanism, regulated by Sonic hedgehog, which is important to suppress Pax6 activity and thus breaks pax6 autoregulation at defined steps during the formation of the visual system.

Suppression of vascular network formation by chronic hypoxia and prolyl-hydroxylase 2 (phd2) deficiency during vertebrate development.

In the adult, new vessels and red blood cells form in response to hypoxia. Here, the oxygen-sensing system (PHD-HIF) has recently been put into focus, since the prolyl-hydroxylase domain proteins (PHD) and hypoxia-inducible factors (HIF) are considered as potential therapeutic targets to treat ischemia, cancers or age-related macula degeneration. While the oxygen-sensing system (PHD-HIF) has been studied intensively in this respect, only little is known from developing vertebrate embryos since mutations within this pathway led to an early decease of embryos due to placental defects. During vertebrate embryogenesis, a progenitor cell called hemangioblast is assumed to give rise to blood cells and blood vessels in a process called hematopoiesis and vasculogenesis, respectively. Xenopus provides an ideal experimental system to address these processes in vivo, as its development does not depend on a functional placenta and thus allows analyzing the role of oxygen directly. To this end, we adopted a computer-controlled four-channel system, which allowed us to culture Xenopus embryos under defined oxygen concentrations. Our data show that the development of vascular structures and blood cells is strongly impaired under hypoxia, while general development is less compromised. Interestingly, suppression of Phd2 function using specific antisense morpholinos or a chemical inhibitor resulted in mostly overlapping vascular defects; nevertheless, blood cell was formed almost normally. Our results provide the first evidence that oxygen via Phd2 has a decisive influence on the formation of the vascular network during vertebrate embryogenesis. These findings may be considered in certain potential treatment concepts.

OTUD3: A Lys6 and Lys63 specific deubiquitinase in early vertebrate development.

Ubiquitination and deubiquitylation regulate essential cellular processes and involve hundreds of sequentially acting enzymes, many of which are barely understood. OTUD3 is an evolutionarily highly conserved deubiquitinase involved in many aspects of cellular homeostasis. However, its biochemical properties and physiological role during development are poorly understood. Here, we report on the expression of OTUD3 in human tissue samples where it appears prominently in those of neuronal origin. In cells, OTUD3 is present in the cytoplasm where it can bind to microtubules. Interestingly, we found that OTUD3 cleaves preferentially at K6 and K63, i.e., poly-ubiquitin linkages that are not primarily involved in protein degradation. We employed Xenopus embryos to study the consequences of suppressing otud3 function during early neural development. We found that Otud3 deficiency led to impaired formation of cranial and particularly of cranial neural crest-derived structures as well as movement defects. Thus, OTUD3 appears as a neuronally enriched deubiquitinase that is involved in the proper development of the neural system.

Xenopus er71 is involved in vascular development.

Vasculogenesis and hematopoiesis are closely linked in developing vertebrates. Recently, the existence of a common progenitor of these two tissues, the hemangioblast, has been demonstrated in different organisms. In Xenopus early vascular and hematopoietic cells differentiate in a region called the anterior ventral blood island (aVBI). Differentiating cells from this region migrate out to form embryonic blood and part of the vascular structures of the early frog embryo. A number of members of the ETS family of transcription factors are expressed in endothelial cells and some of them play important roles at various stages of vascular development. The loss of ER71 function in mice led to a complete loss of blood and vascular structures. Similarly, knock down of the zebrafish homolog of er71, etsrp, greatly affected development of vascular structures and myeloid cells. We have identified the Xenopus ortholog of er71 and could show that er71 function in Xenopus is required for vasculogenesis, but not for the development of hematopoietic cells.

Interplay of TRIM2 E3 Ubiquitin Ligase and ALIX/ESCRT Complex: Control of Developmental Plasticity During Early Neurogenesis.

Tripartite motif 2 (TRIM2) drives neurite outgrowth and polarization, is involved in axon specification, and confers neuroprotective functions during rapid ischemia. The mechanisms controlling neuronal cell fate determination and differentiation are fundamental for neural development. Here, we show that in Xenopus, trim2 knockdown affects primary neurogenesis and neural progenitor cell survival. Embryos also suffer from severe craniofacial malformation, a reduction in brain volume, and the loss of motor sensory function. Using a high-throughput LC-MS/MS approach with GST-Trim2 as bait, we pulled down ALG-2 interacting protein X (Alix) from Xenopus embryonic lysates. We demonstrate that the expression of trim2/TRIM2 and alix/ALIX overlap during larval development and on a cellular level in cell culture. Interestingly, trim2 morphants showed a clustering and apoptosis of neural progenitors, which are phenotypic hallmarks that are also observed in Alix KO mice. Therefore, we propose that the interaction of Alix and Trim2 plays a key role in the determination and differentiation of neural progenitors via the modulation of cell proliferation/apoptosis during neurogenesis.

A multichannel computer-driven system to raise aquatic embryos under selectable hypoxic conditions.

The formation of a functional cardiovascular system is an essential step in the early vertebrate embryo. Nevertheless, the effect of hypoxia on the developmental program of organisms was studied rarely. In particular, this holds true for vertebrate embryos that depend on a functional placenta for proper development and had not been studied in this respect due to the obvious limitation. We established a protocol to culture aquatic embryos, which enabled us to culture a high number of Xenopus embryos until tadpole stage under defined hypoxic conditions in four hypoxia chambers simultaneously, employing a computerized system. In general, our results show that hypoxia results in delayed development and, in particular, we could show that oxygen availability was most crucial during gastrulation and organogenesis (early tailbud) phases during embryonic development of Xenopus laevis.

lrpap1 as a specific marker of proximal pronephric kidney tubuli in Xenopus laevis embryos.

LRPAP1, also known as receptor associated protein (RAP) is a small protein of 40 kDa associated with six of the seven members of the evolutionary conserved family of LDL receptors. Numerous studies showed that LRPAP1 has a dual function, initially as a chaperone insuring proper formation of intermolecular disulfide bonds during biogenesis of low density lipoprotein (LDL) receptors and later as an escort protein during trafficking through the endoplasmic reticulum and the early Golgi compartment, preventing premature interaction of receptor and ligand. Because of the general influence of LRPAP1 protein on lipid metabolism, we analyzed the temporal and spatial expression of the Xenopus laevis ortholog of lrpap1. Here, we show that lrpap1 was expressed in the developing neural system, the eye and ear anlagen, the branchial arches, the developing skin and the pronephric kidney. The very high expression level of lrpap1 specifically in the proximal tubules of the developing pronephros establishes this gene as a novel marker for the analysis of pronephros formation.

Divergent siblings: E2F2 and E2F4 but not E2F1 and E2F3 induce DNA synthesis in cardiomyocytes without activation of apoptosis.

Proliferation of mammalian cardiomyocytes ceases around birth when a transition from hyperplastic to hypertrophic myocardial growth occurs. Previous studies demonstrated that directed expression of the transcription factor E2F1 induces S-phase entry in cardiomyocytes along with stimulation of programmed cell death. Here, we show that directed expression of E2F2 and E2F4 by adenovirus mediated gene transfer in neonatal cardiomyocytes induced S-phase entry but did not result in an onset of apoptosis whereas directed expression of E2F1 and E2F3 strongly evoked programmed cell death concomitant with cell cycle progression. Although both E2F2 and E2F4 induced S-phase entry only directed expression of E2F2 resulted in mitotic cell division of cardiomyocytes. Expression of E2F5 or a control LacZ-Adenovirus had no effects on cell cycle progression. Quantitative real time PCR revealed that E2F1, E2F2, E2F3, and E2F4 alleviate G0 arrest by induction of cyclinA and E cyclins. Furthermore, directed expression of E2F1, E2F3, and E2F5 led to a transcriptional activation of several proapoptotic genes, which were mitigated by E2F2 and E2F4. Our finding that expression of E2F2 induces cell division of cardiomyocytes along with a suppression of proapoptotic genes might open a new access to improve the regenerative capacity of cardiomyocytes.

Xenopus cadherin 5 is specifically expressed in endothelial cells of the developing vascular system.

Vasculogenesis is an important, multistep process leading to the formation of a functional primary network of blood vessels in the developing embryo. A series of interactions between secreted growth factors and their specific receptors leads to the specification of mesodermal cells to become hemangioblasts, which then differentiate into angioblasts. These subsequently proliferate, coalesce into cords and finally form tubular vascular structures. For proper function of these primary blood vessels, the close connection of endothelial cells is required. This is conferred by the interaction of an endothelium specific cadherin (Cadherin-5), starting during early vascular development. However, this interaction remains important throughout life and ageing. Therefore, cadherin-5 is a useful marker for late stages of vasculogenesis in several vertebrate species. To establish cadherin-5 as a marker for vascular studies in Xenopus, we cloned the Xenopus laevis ortholog and analyzed its expression pattern during embryogenesis.

Treatment with bone morphogenetic protein 2 limits infarct size after myocardial infarction in mice.

Various strategies have been devised to reduce the clinical consequences of myocardial infarction, including acute medical care, revascularization, stem cell transplantations, and more recently, prevention of cardiomyocyte cell death. Activation of embryonic signaling pathways is a particularly interesting option to complement these strategies and to improve the functional performance and survival rate of cardiomyocytes. Here, we have concentrated on bone morphogenetic protein 2 (BMP-2), which induces ectopic formation of beating cardiomyocytes during development in the mesoderm and protects neonatal cardiomyocytes from ischemia-reperfusion injury. In a mouse model of acute myocardial infarction, an i.v. injection of BMP-2 reduced infarct size in mice when given after left anterior descending artery ligation. Mice treated with BMP-2 are characterized by a reduced rate of apoptotic cardiomyocytes both in the border zone of the infarcts and in the remote myocardium. In vitro, BMP-2 increases the frequency of spontaneously beating neonatal cardiomyocytes and the contractile performance under electrical pacing at 2 Hz, preserves cellular adenosine triphosphate stores, and decreases the rate of apoptosis despite the increased workload. In addition, BMP-2 specifically induced phosphorylation of Smad1/5/8 proteins and protected adult cardiomyocytes from long-lasting hypoxia-induced cellular damage and oxidative stress without activation of the cardiodepressant transforming growth factor-ß pathway. Our data suggest that BMP-2 treatment may have considerable therapeutic potential in individuals with acute and chronic myocardial ischemia by improving the contractility of cardiomyocytes and preventing cardiomyocyte cell death.

Kidney specific expression of cTPTE during development of the chick embryo.

Here we report the cloning and expression of chick TPTE, the avian ortholog of the mammalian pTEN2 gene. In the chick embryo, cTPTE expression begins at HH (Hamburger and Hamilton) stage 18 and is restricted to the tubular structures of the developing kidney. In the mesonephros, cTPTE expression localizes to the proximal tubules. In the adult kidney cTPTE expression is no longer detectable. The data presented here suggest that cTPTE plays an important role in the regulation of embryonic kidney function.

Overexpression of Kelch domain containing-2 (mKlhdc2) inhibits differentiation and directed migration of C2C12 myoblasts.

Targeted migration of muscle precursor cells to the anlagen of limb muscles is a complex process, which is only partially understood. We have used Lbx1 mutant mice, which are unable to establish correct migration paths of muscle precursor cells into the limbs to identify new genes involved in the accurate placement of myogenic cells in developing muscles. We found that mKlhdc2 (Kelch domain containing-2), a novel member of the family of Kelch domain containing proteins, is significantly downregulated in Lbx1 homozygous mutant embryos. Functional characterization of mKlhdc2 by targeted overexpression in 10T1/2 fibroblasts and C2C12 muscle cells rendered these cells unable to respond to chemoattractants such as HGF. Furthermore, C2C12 myoblasts overexpressing mKlhdc2 display altered cellular morphology and are unable to differentiate into mature myotubes. Our results suggest that a tightly controlled expression of mKlhdc2 is essential for a faithful execution of the myogenic differentiation and migration program.