Simona Casarosa

Xrx1 controls proliferation and neurogenesis in Xenopus anterior neural plate

In Xenopus neuroectoderm, posterior cells start differentiating at the end of gastrulation, while anterior cells display an extended proliferative period and undergo neurogenesis only at tailbud stage. Recent studies have identified several important components of the molecular pathways controlling posterior neurogenesis, but little is known about those controlling the timing and positioning of anterior neurogenesis. We investigate the role of Xrx1, a homeobox gene required for eye and anterior brain development, in the control of proliferation and neurogenesis of the anterior neural plate. Xrx1 is expressed in the entire proliferative region of the anterior neural plate delimited by cells expressing the neuronal determination gene X-ngnr-1, the neurogenic gene X-Delta-1, and the cell cycle inhibitor p27Xic1. Positive and negative signals position Xrx1 expression to this region. Xrx1 is activated by chordin and Hedgehog gene signaling, which induce anterior and proliferative fate, and is repressed by the differentiation-promoting activity of neurogenin and retinoic acid. Xrx1 is required for anterior neural plate proliferation and, when overexpressed, induces proliferation, inhibits X-ngnr-1, X-Delta-1 and N-tubulin and counteracts X-ngnr-1- and retinoic acid-mediated differentiation. We find that Xrx1 does not act by increasing lateral inhibition but by inducing the antineurogenic transcriptional repressors Xhairy2 and Zic2, and by repressing p27Xic1. The effects of Xrx1 on proliferation, neurogenesis and gene expression are restricted to the most rostral region of the embryo, implicating this gene as an anterior regulator of neurogenesis.

Epilepsy as a neurodevelopmental disorder

Epilepsy is characterized by spontaneous recurrent seizures and comprises a diverse group of syndromes with different etiologies. Epileptogenesis refers to the process whereby the brain becomes epileptic and can be related to several factors, such as acquired structural brain lesions, inborn brain malformations, alterations in neuronal signaling, and defects in maturation and plasticity of neuronal networks. In this review, we will focus on alterations of brain development that lead to an hyperexcitability phenotype in adulthood, providing examples from both animal and human studies. Malformations of cortical development (including focal cortical dysplasia, lissencephaly, heterotopia, and polymicrogyria) are frequently epileptogenic and result from defects in cell proliferation in the germinal zone and/or impaired neuronal migration and differentiation. Delayed or reduced arrival of inhibitory interneurons into the cortical plate is another possible cause of epileptogenesis. GABAergic neurons are generated during early development in the ganglionic eminences, and failure to pursue migration toward the cortex alters the excitatory/inhibitory balance resulting in aberrant network hyperexcitability. More subtle defects in the developmental assembly of excitatory and inhibitory synapses are also involved in epilepsy. For example, mutations in the presynaptic proteins synapsins and SNAP-25 cause derangements of synaptic transmission and plasticity which underlie appearance of an epileptic phenotype. Finally, there is evidence that defects in synapse elimination and remodeling during early “critical periods” can trigger hyperexcitability later in life. Further clarification of the developmental pathways to epilepsy has important implications for disease prevention and therapy.

Cloning and developmental expression of the Xenopus homeobox gene Xvsx1

In contrast to the high degree of evolutionary conservation of the Vsx2/Chx10 gene family, vertebrate orthologues of Vsx1 display more divergent sequences and spatio-temporal expression patterns. Here, we report the cloning and expression pattern of Xenopus laevis Vsx1. Differently from the mouse and zebrafish orthologues, Xvsx1 transcription is activated at early neurula both in the evaginating eye vesicles and in the presumptive spinal cord. Compared to other retinal homeobox genes, such as Xrx1, Xsix3 and Xpax6, Xvsx1 is activated at a later stage; in addition, its anterior expression appears to be more specifically restricted to the retina. At tail bud stage, Xvsx1 expression in retinal progenitors persists, and its neural tube expression, which in the spinal cord corresponds to interneurons, progressively expands anteriorly reaching the midbrain–hindbrain boundary. During retinal neurogenesis, Xvsx1 expression is maintained in retinal progenitors and in a peripheral region of the ciliary marginal zone, while in the central retina, it becomes restricted to differentiated bipolar cells.

Noggin Elicits Retinal Fate in Xenopus Animal Cap Embryonic Stem Cells

Driving specific differentiation pathways in multipotent stem cells is a main goal of cell therapy. Here we exploited the differentiating potential of Xenopus animal cap embryonic stem (ACES) cells to investigate the factors necessary to drive multipotent stem cells toward retinal fates. ACES cells are multipotent, and can be diverged from their default ectodermal fate to give rise to cell types from all three germ layers. We found that a single secreted molecule, Noggin, is sufficient to elicit retinal fates in ACES cells. Reverse‐transcription polymerase chain reaction, immunohistochemistry, and in situ hybridization experiments showed that high doses of Noggin are able to support the expression of terminal differentiation markers of the neural retina in ACES cells in vitro. Following in vivo transplantation, ACES cells expressing high Noggin doses form eyes, both in the presumptive eye field region and in ectopic posterior locations. The eyes originating from the transplants in the eye field region are functionally equivalent to normal eyes, as seen by electrophysiology and c‐fos expression in response to light. Our data show that in Xenopus embryos, proper doses of a single molecule, Noggin, can drive ACES cells toward retinal cell differentiation without additional cues. This makes Xenopus ACES cells a suitable model system to direct differentiation of stem cells toward retinal fates and encourages further studies on the role of Noggin in the retinal differentiation of mammalian stem cells. STEM CELLS 2009;27:2146–2152

Genipin-crosslinked gelatin–silk fibroin hydrogels for modulating the behaviour of pluripotent cells

Different hydrogel materials have been prepared to investigate the effects of culture substrate on the behaviour of pluripotent cells. In particular, genipin‐crosslinked gelatin–silk fibroin hydrogels of different compositions have been prepared, physically characterized and used as substrates for the culture of pluripotent cells. Pluripotent cells cultured on hydrogels remained viable and proliferated. Gelatin and silk fibroin promoted the proliferation of cells in the short and long term, respectively. Moreover, cells cultured on genipin‐crosslinked gelatin–silk fibroin blended hydrogels were induced to an epithelial ectodermal differentiation fate, instead of the neural ectodermal fate obtained by culturing on tissue culture plates. This work confirms that specific culture substrates can be used to modulate the behaviour of pluripotent cells and that our genipin‐crosslinked gelatin–silk fibroin blended hydrogels can induce pluripotent cells differentiation to an epithelial ectodermal fate. Copyright

Viability and neuronal differentiation of neural stem cells encapsulated in silk fibroin hydrogel functionalized with an IKVAV peptide

Three‐dimensional (3D) porous scaffolds combined with therapeutic stem cells play vital roles in tissue engineering. The adult brain has very limited regeneration ability after injuries such as trauma and stroke. In this study, injectable 3D silk fibroin‐based hydrogel scaffolds with encapsulated neural stem cells were developed, aiming at supporting brain regeneration. To improve the function of the hydrogel towards neural stem cells, silk fibroin was modified by an IKVAV peptide through covalent binding. Both unmodified and modified silk fibroin hydrogels were obtained, through sonication, with mechanical stiffness comparable to that of brain tissue. Human neural stem cells were encapsulated in both hydrogels and the effects of IKVAV peptide conjugation on cell viability and neural differentiation were assessed. The silk fibroin hydrogel modified by IKVAV peptide showed increased cell viability and an enhanced neuronal differentiation capability, which contributed to understanding the effects of IKVAV peptide on the behaviour of neural stem cells. For these reasons, IKVAV‐modified silk fibroin is a promising material for brain tissue engineering. Copyright

The positional identity of mouse ES cell-generated neurons is affected by BMP signaling

We investigated the effects of bone morphogenetic proteins (BMPs) in determining the positional identity of neurons generated in vitro from mouse embryonic stem cells (ESCs), an aspect that has been neglected thus far. Classical embryological studies in lower vertebrates indicate that BMPs inhibit the default fate of pluripotent embryonic cells, which is both neural and anterior. Moreover, mammalian ESCs generate neurons more efficiently when cultured in a minimal medium containing BMP inhibitors. In this paper, we show that mouse ESCs produce, secrete, and respond to BMPs during in vitro neural differentiation. After neuralization in a minimal medium, differentiated ESCs show a gene expression profile consistent with a midbrain identity, as evaluated by the analysis of a number of markers of anterior–posterior and dorsoventral identity. We found that BMPs endogenously produced during neural differentiation mainly act by inhibiting the expression of a telencephalic gene profile, which was revealed by the treatment with Noggin or with other BMP inhibitors. To better characterize the effect of BMPs on positional fate, we compared the global gene expression profiles of differentiated ESCs with those of embryonic forebrain, midbrain, and hindbrain. Both Noggin and retinoic acid (RA) support neuronal differentiation of ESCs, but they show different effects on their positional identity: whereas RA supports the typical gene expression profile of hindbrain neurons, Noggin induces a profile characteristic of dorsal telencephalic neurons. Our findings show that endogenously produced BMPs affect the positional identity of the neurons that ESCs spontaneously generate when differentiating in vitro in a minimal medium. The data also support the existence of an intrinsic program of neuronal differentiation with dorsal telencephalic identity. Our method of ESC neuralization allows for fast differentiation of neural cells via the same signals found during in vivo embryonic development and for the acquisition of cortical identity by the inhibition of BMP alone.

Transcriptome profiling in engrailed-2 mutant mice reveals common molecular pathways associated with autism spectrum disorders

BackgroundTranscriptome analysis has been used in autism spectrum disorder (ASD) to unravel common pathogenic pathways based on the assumption that distinct rare genetic variants or epigenetic modifications affect common biological pathways. To unravel recurrent ASD-related neuropathological mechanisms, we took advantage of the En2-/- mouse model and performed transcriptome profiling on cerebellar and hippocampal adult tissues.MethodsCerebellar and hippocampal tissue samples from three En2-/- and wild type (WT) littermate mice were assessed for differential gene expression using microarray hybridization followed by RankProd analysis. To identify functional categories overrepresented in the differentially expressed genes, we used integrated gene-network analysis, gene ontology enrichment and mouse phenotype ontology analysis. Furthermore, we performed direct enrichment analysis of ASD-associated genes from the SFARI repository in our differentially expressed genes.ResultsGiven the limited number of animals used in the study, we used permissive criteria and identified 842 differentially expressed genes in En2-/- cerebellum and 862 in the En2-/- hippocampus. Our functional analysis revealed that the molecular signature of En2-/- cerebellum and hippocampus shares convergent pathological pathways with ASD, including abnormal synaptic transmission, altered developmental processes and increased immune response. Furthermore, when directly compared to the repository of the SFARI database, our differentially expressed genes in the hippocampus showed enrichment of ASD-associated genes significantly higher than previously reported. qPCR was performed for representative genes to confirm relative transcript levels compared to those detected in microarrays.ConclusionsDespite the limited number of animals used in the study, our bioinformatic analysis indicates the En2-/- mouse is a valuable tool for investigating molecular alterations related to ASD.

Neurobiological bases of autism–epilepsy comorbidity: a focus on excitation/inhibition imbalance

Autism spectrum disorders (ASD) and epilepsy are common neurological diseases of childhood, with an estimated incidence of approximately 0.5–1% of the worldwide population. Several genetic, neuroimaging and neuropathological studies clearly showed that both ASD and epilepsy have developmental origins and a substantial degree of heritability. Most importantly, ASD and epilepsy frequently coexist in the same individual, suggesting a common neurodevelopmental basis for these disorders. Genome‐wide association studies recently allowed for the identification of a substantial number of genes involved in ASD and epilepsy, some of which are mutated in syndromes presenting both ASD and epilepsy clinical features. At the cellular level, both preclinical and clinical studies indicate that the different genetic causes of ASD and epilepsy may converge to perturb the excitation/inhibition (E/I) balance, due to the dysfunction of excitatory and inhibitory circuits in various brain regions. Metabolic and immune dysfunctions, as well as environmental causes also contribute to ASD pathogenesis. Thus, an E/I imbalance resulting from neurodevelopmental deficits of multiple origins might represent a common pathogenic mechanism for both diseases. Here, we will review the most significant studies supporting these hypotheses. A deeper understanding of the molecular and cellular determinants of autism–epilepsy comorbidity will pave the way to the development of novel therapeutic strategies.

Hippocampal Dysregulation of Neurofibromin-Dependent Pathways Is Associated with Impaired Spatial Learning in Engrailed 2 Knock-Out Mice

Genome-wide association studies indicated the homeobox-containing transcription factor Engrailed-2 (En2) as a candidate gene for autism spectrum disorders (ASD). Accordingly, En2 knock-out (En2−/−) mice show anatomical and behavioral “ASD-like” features, including decreased sociability and learning deficits. The molecular pathways underlying these deficits in En2−/− mice are not known. Deficits in signaling pathways involving neurofibromin and extracellular-regulated kinase (ERK) have been associated with impaired learning. Here we investigated the neurofibromin-ERK cascade in the hippocampus of wild-type (WT) and En2−/− mice before and after spatial learning testing. When compared with WT littermates, En2−/− mice showed impaired performance in the Morris water maze (MWM), which was accompanied by lower expression of the activity-dependent gene Arc. Quantitative RT-PCR, immunoblotting, and immunohistochemistry experiments showed a marked downregulation of neurofibromin expression in the dentate gyrus of both naive and MWM-treated En2−/− mice. ERK phosphorylation, known to be induced in the presence of neurofibromin deficiency, was increased in the dentate gyrus of En2−/− mice after MWM. Treatment of En2−/− mice with lovastatin, an indirect inhibitor of ERK phosphorylation, markedly reduced ERK phosphorylation in the dentate gyrus, but was unable to rescue learning deficits in MWM-trained mutant mice. Further investigation is needed to unravel the complex molecular mechanisms linking dysregulation of neurofibromin-dependent pathways to spatial learning deficits in the En2 mouse model of ASD.