A comparison of frailty of primary neurons, embryonic, and aging mouse cortical layers.

Superficial layers I to III of the human cerebral cortex are more vulnerable toward Aβ peptides than deep layers V to VI in aging. Three models of layers were used to investigate this pattern of frailty. First, primary neurons from E14 and E17 embryonic murine cortices, corresponding respectively to future deep and superficial layers, were treated either with Aβ1-42, okadaic acid, or kainic acid. Second, whole E14 and E17 embryonic cortices, and third, in vitro separated deep and superficial layers of young and old C57BL/6J mice, were treated identically. We observed that E14 and E17 neurons in culture were prone to death after the Aβ and particularly the kainic acid treatment. This was also the case for the superficial layers of the aged cortex, but not for the embryonic, the young cortex, and the deep layers of the aged cortex. Thus, the aged superficial layers appeared to be preferentially vulnerable against Aβ and kainic acid. This pattern of vulnerability corresponds to enhanced accumulation of senile plaques in the superficial cortical layers with aging and Alzheimer's disease.

Loss of ATF2 function leads to cranial motoneuron degeneration during embryonic mouse development.

The AP-1 family transcription factor ATF2 is essential for development and tissue maintenance in mammals. In particular, ATF2 is highly expressed and activated in the brain and previous studies using mouse knockouts have confirmed its requirement in the cerebellum as well as in vestibular sense organs. Here we present the analysis of the requirement for ATF2 in CNS development in mouse embryos, specifically in the brainstem. We discovered that neuron-specific inactivation of ATF2 leads to significant loss of motoneurons of the hypoglossal, abducens and facial nuclei. While the generation of ATF2 mutant motoneurons appears normal during early development, they undergo caspase-dependent and independent cell death during later embryonic and foetal stages. The loss of these motoneurons correlates with increased levels of stress activated MAP kinases, JNK and p38, as well as aberrant accumulation of phosphorylated neurofilament proteins, NF-H and NF-M, known substrates for these kinases. This, together with other neuropathological phenotypes, including aberrant vacuolisation and lipid accumulation, indicates that deficiency in ATF2 leads to neurodegeneration of subsets of somatic and visceral motoneurons of the brainstem. It also confirms that ATF2 has a critical role in limiting the activities of stress kinases JNK and p38 which are potent inducers of cell death in the CNS.

GLUT8 is dispensable for embryonic development but influences hippocampal neurogenesis and heart function.

GLUT8 is a glucose transporter isoform expressed at high levels in testis; at intermediate levels in the brain, including the hippocampus; and at lower levels in the heart and several other tissues. GLUT8 is located in an intracellular compartment and does not appear to translocate to the cell surface, except in blastocysts, where insulin has been reported to induce its surface expression. Here, we generated mice with inactivation of the glut8 gene. We showed that expression of GLUT8 was not required for normal embryonic development and that glut8-/- mice had normal postnatal development, glucose homeostasis, and response to mild stress. Adult glut8-/- mice showed increased proliferation of hippocampal cells but no defect in memory acquisition and retention. Absence of GLUT8 from the heart did not alter heart size and morphology but led to an increase in P-wave duration, which was not associated with abnormal Nav1.5 Na+ channel or connexin expression. Thus, absence of GLUT8 expression in the mouse caused complex but mild physiological alterations.

PPARβ Interprets a Chromatin Signature of Pluripotency to Promote Embryonic Differentiation at Gastrulation.

Epigenetic post-transcriptional modifications of histone tails are thought to help in coordinating gene expression during development. An epigenetic signature is set in pluripotent cells and interpreted later at the onset of differentiation. In pluripotent cells, epigenetic marks normally associated with active genes (H3K4me3) and with silent genes (H3K27me3) atypically co-occupy chromatin regions surrounding the promoters of important developmental genes. However, it is unclear how these epigenetic marks are recognized when cell differentiation starts and what precise role they play. Here, we report the essential role of the nuclear receptor peroxisome proliferator-activated receptor β (PPARβ, NR1C2) in Xenopus laevis early development. By combining loss-of-function approaches, large throughput transcript expression analysis by the mean of RNA-seq and intensive chromatin immunoprecipitation experiments, we unveil an important cooperation between epigenetic marks and PPARβ. During Xenopus laevis gastrulation PPARβ recognizes H3K27me3 marks that have been deposited earlier at the pluripotent stage to activate early differentiation genes. Thus, PPARβis the first identified transcription factor that interprets an epigenetic signature of pluripotency, in vivo, during embryonic development. This work paves the way for a better mechanistic understanding of how the activation of hundreds of genes is coordinated during early development.

Abundant Occurrence of Basal Radial Glia in the Subventricular Zone of Embryonic Neocortex of a Lissencephalic Primate, the Common Marmoset Callithrix jacchus.

Subventricular zone (SVZ) progenitors are a hallmark of the developing neocortex. Recent studies described a novel type of SVZ progenitor that retains a basal process at mitosis, sustains expression of radial glial markers, and is capable of self-renewal. These progenitors, referred to here as basal radial glia (bRG), occur at high relative abundance in the SVZ of gyrencephalic primates (human) and nonprimates (ferret) but not lissencephalic rodents (mouse). Here, we analyzed the occurrence of bRG cells in the embryonic neocortex of the common marmoset Callithrix jacchus, a near-lissencephalic primate. bRG cells, expressing Pax6, Sox2 (but not Tbr2), glutamate aspartate transporter, and glial fibrillary acidic protein and retaining a basal process at mitosis, occur at similar relative abundance in the marmoset SVZ as in human and ferret. The proportion of progenitors in M-phase was lower in embryonic marmoset than developing ferret neocortex, raising the possibility of a longer cell cycle. Fitting the gyrification indices of 26 anthropoid species to an evolutionary model suggested that the marmoset evolved from a gyrencephalic ancestor. Our results suggest that a high relative abundance of bRG cells may be necessary, but is not sufficient, for gyrencephaly and that the marmoset's lissencephaly evolved secondarily by changing progenitor parameters other than progenitor type.

Dimeric recombinant IgA directed against carcino-embryonic antigen, a novel tool for carcinoma localization.

Carcinoembryonic antigen (CEA) has been shown to be one of the best markers for in vivo tumor targeting of radiolabeled antibodies, despite the fact that it is localized predominantly at the apical side of human colon carcinoma cells within the fairly closed pseudolumen structures formed by these tumors. Due to this particular histological localization, a large proportion of the CEA molecules may remain inaccessible to the intravenously injected radiolabeled anti-CEA antibodies of IgG isotype, which are widely used in the clinic. In order to improve targeting, we made a recombinant dimeric IgA, which should have the capacity to translocate from the basolateral to the apical side of the pseudolumen formed by colon carcinoma cells after binding to the polyIg receptor (pIgR). A genomic chimeric mouse-human IgA2 construct was made using one of our most specific anti-CEA hybridomas, CE-25. The chimeric IgA (chIgA) was expressed in the Sp2/0 myeloma cell line. The secreted recombinant antibody was found to consist mostly of a dimeric form of IgA with a molecular weight of about 350 kDa. The dimeric chIgA was shown to translocate efficiently in vitro across a monolayer of epithelial cells expressing the pIgR and to retain full CEA binding activity.

Role of retinoic acid signaling pathway in endoderm antero-posterior patterning

Summary : During vertebrate embryonic development, the endoderm gives rise to the digestive tract and associated organs such as thyroid, lung, liver and pancreas. Earlier studies have shown that extracellular signals coming from the lateral plate mesoderm pattern the endoderm along the antero-posterior axis specifying different organ primordia. An early sign of patterning is the expression of organ-specific genes in restricted endoderm domains. In this study, we focused on the role of the retinoic acid (RA) signaling pathway in the regionalization of the future gut tube along the main body axis. We show that the RA-synthesizing enzyme Raldh2 is expressed in mesoderm close to the endoderm during gastrulation and during somitogenesis. During the same period, all retinoic acid receptors (RARs), which directly activate gene transcription, are expressed in endoderm suggesting that endoderm can be responsive to RA. Activation or inhibition of RA signaling was achieved by adding RA or RAR inhibitors tither on beads or in the medium to cultured chick embryos. Branchial arch (BA) endoderm markers were shifted posteriorly upon depletion of RA at gastrulation, but were not shifted after this stage. Conversely, exposure to exogenous RA repressed the most-anterior BA markers and shifted more posterior BA markers anteriorly. This suggests that graded levels of RA activity in the foregut define gene boundaries and expression levels. The posterior foregut and midget markers Pdxl and CdxA require RA for their expression, but elevated RA does not shift their expression domain along the antero-posterior axis. In addition, we investigated if RA signaling pathway interacts with other signaling pathways to pattern the endoderm. Although both RA and FGFs block anterior foregut marker expression, our experiments suggest that FGF signaling does not depend on RA in anterior endoderm. To validate our chick data in mammalians and evaluate whether RA acts directly on endoderm, we have further generated a conditional loss-of-function system in the mouse, which is still under examination.

SOST expression is restricted to the great arteries during embryonic and neonatal cardiovascular development.

Spatial-temporal regulation of bone morphogenetic protein (BMP) and Wnt activity is essential for normal cardiovascular development, and altered activity of these growth factors causes maldevelopment of the cardiac outflow tract and great arteries. In the present study, we show that SOST, a Dan family member reported to antagonize BMP and Wnt activity, is expressed within the medial vessel wall of the great arteries containing smooth muscle cells. The ascending aorta, aortic arch, brachiocephalic artery, common carotids, and pulmonary trunk were all associated with SOST expressing smooth muscle cells, while the heart itself, including the valves, and more distal arteries, that is, pulmonary arteries, subclavian arteries, and descending aorta, were negative. SOST was expressed from embryonic day 15.5 up to the neonatal period. SOST expression, however, did not correspond with inhibition of Smad-dependent BMP activity or beta-catenin-dependent Wnt activity in the great arteries. Activity of both signaling pathways was already down-regulated before induction of SOST expression.

Involvement of the CXCR7/CXCR4/CXCL12 Axis in the Malignant Progression of Human Neuroblastoma.

Neuroblastoma (NB) is a typical childhood and heterogeneous neoplasm for which efficient targeted therapies for high-risk tumors are not yet identified. The chemokine CXCL12, and its receptors CXCR4 and CXCR7 have been involved in tumor progression and dissemination. While CXCR4 expression is associated to undifferentiated tumors and poor prognosis, the role of CXCR7, the recently identified second CXCL12 receptor, has not yet been elucidated in NB. In this report, CXCR7 and CXCL12 expressions were evaluated using a tissue micro-array including 156 primary and 56 metastatic NB tissues. CXCL12 was found to be highly associated to NB vascular and stromal structures. In contrast to CXCR4, CXCR7 expression was low in undifferentiated tumors, while its expression was stronger in matured tissues and specifically associated to differentiated neural tumor cells. As determined by RT-PCR, CXCR7 expression was mainly detected in N-and S-type NB cell lines, and was slightly induced upon NB cell differentiation in vitro. The relative roles of the two CXCL12 receptors were further assessed by overexpressing CXCR7 or CXCR4 receptor alone, or in combination, in the IGR-NB8 and the SH-SY5Y NB cell lines. In vitro functional analyses indicated that, in response to their common ligand, both receptors induced activation of ERK1/2 cascade, but not Akt pathway. CXCR7 strongly reduced in vitro growth, in contrast to CXCR4, and impaired CXCR4/CXCL12-mediated chemotaxis. Subcutaneous implantation of CXCR7-expressing NB cells showed that CXCR7 also significantly reduced in vivo growth. Moreover, CXCR7 affected CXCR4-mediated orthotopic growth in a CXCL12-producing environment. In such model, CXCR7, in association with CXCR4, did not induce NB cell metastatic dissemination. In conclusion, the CXCR7 and CXCR4 receptors revealed specific expression patterns and distinct functional roles in NB. Our data suggest that CXCR7 elicits anti-tumorigenic functions, and may act as a regulator of CXCR4/CXCL12-mediated signaling in NB.

In vitro functionality of isolated embryonic hypothalamic vasopressinergic and oxytocinergic neurons: modulatory effects of brain-derived neurotrophic factor and angiotensin II.

There are only a few studies on the ontogeny and differentiation process of the hypothalamic supraoptic-paraventriculo-neurohypophysial neurosecretory system. In vitro neuron survival improves if cells are of embryonic origin; however, surviving hypothalamic neurons in culture were found to express small and minimal amounts of arginine-vasopressin (AVP) and oxytocin (OT), respectively. The aim of this study was to develop a primary neuronal culture design applicable to the study of magnocellular hypothalamic system functionality. For this purpose, a primary neuronal culture was set up after mechanical dissociation of sterile hypothalamic blocks from 17-day-old Sprague-Dawley rat embryos (E17) of both sexes. Isolated hypothalamic cells were cultured with supplemented (B27)-NeuroBasal medium containing an agent inhibiting non-neuron cell proliferation. The neurosecretory process was characterized by detecting AVP and OT secreted into the medium on different days of culture. Data indicate that spontaneous AVP and OT release occurred in a culture day-dependent fashion, being maximal on day 13 for AVP, and on day 10 for OT. Interestingly, brain-derived neurotrophic factor (BDNF) and Angiotensin II (A II) were able to positively modulate neuropeptide output. Furthermore, on day 17 of culture, non-specific (high-KCl) and specific (Angiotensin II) stimuli were able to significantly (P < 0.05) enhance the secretion of both neuropeptides over respective baselines. This study suggests that our experimental design is useful for the study of AVP- and OT-ergic neuron functionality and that BDNF and A II are positive modulators of embryonic hypothalamic cell development.

Activation of Transient Receptor Potential Canonical 3 (TRPC3)-mediated Ca2+ Entry by A1 Adenosine Receptor in Cardiomyocytes Disturbs Atrioventricular Conduction.

Although the activation of the A(1)-subtype of the adenosine receptors (A(1)AR) is arrhythmogenic in the developing heart, little is known about the underlying downstream mechanisms. The aim of this study was to determine to what extent the transient receptor potential canonical (TRPC) channel 3, functioning as receptor-operated channel (ROC), contributes to the A(1)AR-induced conduction disturbances. Using embryonic atrial and ventricular myocytes obtained from 4-day-old chick embryos, we found that the specific activation of A(1)AR by CCPA induced sarcolemmal Ca(2+) entry. However, A(1)AR stimulation did not induce Ca(2+) release from the sarcoplasmic reticulum. Specific blockade of TRPC3 activity by Pyr3, by a dominant negative of TRPC3 construct, or inhibition of phospholipase Cs and PKCs strongly inhibited the A(1)AR-enhanced Ca(2+) entry. Ca(2+) entry through TRPC3 was activated by the 1,2-diacylglycerol (DAG) analog OAG via PKC-independent and -dependent mechanisms in atrial and ventricular myocytes, respectively. In parallel, inhibition of the atypical PKCζ by myristoylated PKCζ pseudosubstrate inhibitor significantly decreased the A(1)AR-enhanced Ca(2+) entry in both types of myocytes. Additionally, electrocardiography showed that inhibition of TRPC3 channel suppressed transient A(1)AR-induced conduction disturbances in the embryonic heart. Our data showing that A(1)AR activation subtly mediates a proarrhythmic Ca(2+) entry through TRPC3-encoded ROC by stimulating the phospholipase C/DAG/PKC cascade provide evidence for a novel pathway whereby Ca(2+) entry and cardiac function are altered. Thus, the A(1)AR-TRPC3 axis may represent a potential therapeutic target.

Ventricular but not atrial electro-mechanical delay of the embryonic heart is altered by anoxia-reoxygenation and improved by nitric oxide.

BACKGROUND/AIM: Excitation-contraction coupling is modulated by nitric oxide (NO) which otherwise has either beneficial or detrimental effects on myocardial function during hypoxia-reoxygenation. This work aimed at characterizing the variations of electromechanical delay (EMD) induced by anoxia-reoxygenation within the developing heart and determining whether atrial and ventricular EMD are modulated by NO to the same extent. METHODS: Hearts of 4 or 4.5-day-old chick embryos were excised and submitted in vitro to normoxia (45 min), anoxia (30 min) and reoxygenation (60 min). Electrocardiogram and atrial and ventricular contractions were simultaneously recorded throughout experiment. Anoxia-reoxygenation-induced chrono-, dromo-and inotropic disturbances and changes in EMD in atrium (EMDa) and ventricle (EMDv) were investigated in control hearts and in hearts exposed to 0.1, 1, 10, 50 and 100 microM of DETA-NONOate (a NO donating agent) or to 50 microM of L-NAME (a NOS inhibitor). RESULTS: Under normoxia, heart rate, PR interval, ventricular shortening velocity, EMDa and EMDv were similar in control, L-NAME-treated and DETA-NONOate-treated hearts. Under anoxia, cardiac activity became markedly erratic within less than 10 min in all groups. At the onset of reoxygenation, EMDv was increased by about 300% with respect to the preanoxic value while EMDa did not vary significatively. Compared to control conditions, L-NAME or DETA-NONOate had no influence on the negative chrono-, dromo- and inotropic effects induced by anoxia-reoxygenation. However, L-NAME prolonged EMDv during anoxia and delayed EMDv recovery during reoxygenation while 100 microM DETA-NONOate had the opposite effects. EMDa was neither affected by NOS inhibitor nor NO donor. At the end of reoxygenation, all the investigated parameters returned to their basal values. CONCLUSION: This work provides evidence that a NO-dependent pathway is involved in regulation of the ventricular excitation-contraction coupling in the anoxic-reoxygenated developing heart.

Activation of a HIF1alpha-PPARgamma axis underlies the integration of glycolytic and lipid anabolic pathways in pathologic cardiac hypertrophy.

Development of cardiac hypertrophy and progression to heart failure entails profound changes in myocardial metabolism, characterized by a switch from fatty acid utilization to glycolysis and lipid accumulation. We report that hypoxia-inducible factor (HIF)1alpha and PPARgamma, key mediators of glycolysis and lipid anabolism, respectively, are jointly upregulated in hypertrophic cardiomyopathy and cooperate to mediate key changes in cardiac metabolism. In response to pathologic stress, HIF1alpha activates glycolytic genes and PPARgamma, whose product, in turn, activates fatty acid uptake and glycerolipid biosynthesis genes. These changes result in increased glycolytic flux and glucose-to-lipid conversion via the glycerol-3-phosphate pathway, apoptosis, and contractile dysfunction. Ventricular deletion of Hif1alpha in mice prevents hypertrophy-induced PPARgamma activation, the consequent metabolic reprogramming, and contractile dysfunction. We propose a model in which activation of the HIF1alpha-PPARgamma axis by pathologic stress underlies key changes in cell metabolism that are characteristic of and contribute to common forms of heart disease.

Vascular continuity, cell axialization and auxin.

Phytohormones have been implicated in vascular development in various ways, but their precise function and the extent of their influence is still controversial. Recent results from experimental manipulation of developing organs and Arabidopsis developmental genetics support a role for polar auxin flow in cell axis formation within the vascular system and, interestingly, also in the embryonic establishment of the plant body axis. Vascular responses to auxin transport inhibition indicate patterns of auxin distribution during leaf development and new technologies may enable these predictions to be tested within the near future. Moreover, recently discovered Arabidopsis axialisation mutants seem to identify essential genes that relay auxin signals in vascular development. A first gene in this class, MONOPTEROS (MP) has been cloned and encodes a transcription factor capable of binding to auxin response elements in the control regions of auxin regulated genes. Molecular identification of further axialisation genes may provide access to a mechanistic understanding of plant cell axis formation.

Differential expression of peroxisome proliferator-activated receptor-alpha, -beta, and -gamma during rat embryonic development.

The expression patterns of the three different peroxisome proliferator-activated receptor (PPAR) isotypes have been determined during rat embryonic development by in situ hybridization. The expression of PPARalpha starts late in development, with increasing levels in organs such as liver, kidney, intestine, and pancreas, in which it will also be present later in adulthood to regulate its specific target genes. PPARalpha is also transiently expressed in the embryonic epidermis and central nervous system. PPARgamma presents a very restricted pattern of expression, being strongly expressed in brown adipose tissue, in which differentiation it has been shown to participate. Like PPARalpha, it is also expressed transiently in the central nervous system. Interestingly, PPARalpha, -beta and -gamma are coexpressed at high levels in brown adipose tissue. Finally, the high and ubiquitous expression of PPARbeta suggests some fundamental role(s) that this receptor might play throughout development.