Genes regulated by the transcription factor Xrx1: a microarray analysis

Eye development is a multistep process that requires specific inductive signals and precise morphogenetic movements, starting in a well-definite region of the anterior neural plate, the eye field. Recently, it was demonstrated that an elaborate gene network of eye field transcription factors (EFTFs) contributes to specify the neural and retinal fate of the eye field. Among these EFTFs, Xrx1 is involved in proliferation and neurogenesis in the eye field and is necessary for the correct development of the retina . By means of Affymetrix microarrays, a high throughput screening was performed, looking for genes that can mediate the function of Xrx1, by comparison of the expression profiles of whole embryos in which the Xrx1 function was either overexpressed or down-regulated. A suitable experimental design has been designed, and a PCR-screening procedure was developed to correctly select embryos microinjected either with Xrx1 mRNA (gain of function) or Morpholino antisense oligonucleotides (loss of function). The selected injected embryos were used in the microarray experiments in five biological replicates for each condition for higher statistical consistency. The intensity data were resumed, normalized and analyzed via the GC-RMA algorithm: a row by row T-test between control and injected groups revealed 793 differentially expressed transcripts for the gain of function experiment and 1893 differentially expressed transcripts for the loss of function experiment. 122 transcripts were present in both gene lists. A complete reannotation, based on the Xenopus laevis Unigene #73 was performed for each transcript via newly developed algorithm in a fraction of the time needed by other applications. The analysis of the resulting datalists showed that more than 30% of the identified transcripts was poorly or not annotated. An initial exploration of annotated transcripts in the in the gain of function gene list showed several transcripts involved in patterning, cell proliferation and cell signaling. Attention was focused as well on genes whose expression was coherently affected in the experiments (i.e. genes activated in gain of function and repressed in loss of function experiments, and vice-versa). An initial pool of candidate targets was selected among the common list of 122 genes : the relative EST clones were transcribed into antisense probes and used for whole mount in situ experiments on embryos at different developmental stages (13, 16, 19, 27, 33/34 and fully developed sectioned eyes) for a first validation of the microarray data. Whole mount hybridizations of non annotated ESTs provided as well confirmations for the consistency of the microarray data: many ESTs resulted actually transcribed within the Xrx1 expression domain, including the eye, eye-field or diencephalic structures. Selected candidate transcripts from the microarray experiments, showing an overlapping expression domain with Xrx1 in the developing retina and/or in the ciliary marginal zone will be tested for the ability to respond to Xrx1 overexpression by in situ hybridization. Their identity and predicted molecular function will be defined based on BLAST analysis, Gene Ontology and NCBI Unigene annotation. An inducible GR-Xrx1 construct will be used to assess if the candidate transcripts are direct targets of Xrx1

Kdm7a expression is spatiotemporally regulated in developing Xenopus laevis embryos, and its overexpression influences late retinal development

Background: Post-translational histone modifications are among the most common epigenetic modifications that orchestrate gene expression, playing a pivotal role during embryonic development and in various pathological conditions. Among histone lysine demethylases, KDM7A, also known as KIAA1718 or JHDM1D, catalyzes the demethylation of H3K9me1/2 and H3K27me1/2, leading to transcriptional regulation. Previous data suggest that KDM7A plays a central role in several biological processes, including cell proliferation, commitment, differentiation, apoptosis, and maintenance. However, information on the expression pattern of KDM7A in whole organisms is limited, and its functional role is still unclear. Results: In Xenopus development, kdm7a is expressed early, undergoing spatiotemporal regulation in various organs and tissues, including the central nervous system and the eye. Focusing on retinal development, we found that kdm7a overexpression does not affect the expression of genes critically involved in early neural development and eye-field specification, whereas unbalances the distribution of neural cell subtypes in the mature retina by disfavoring the development of ganglion cells while promoting that of horizontal cells. Conclusions: Kdm7a is dynamically expressed during embryonic development, and its overexpression influences late retinal development, suggesting a potential involvement in the molecular machinery regulating the spatiotemporally ordered generation of retinal neuronal subtypes

Preferential Enhancement of Sensory and Motor Axon Regeneration by Combining Extracellular Matrix Components with Neurotrophic Factors

After peripheral nerve injury, motor and sensory axons are able to regenerate but inaccuracy of target reinnervation leads to poor functional recovery. Extracellular matrix (ECM) components and neurotrophic factors (NTFs) exert their effect on different neuronal populations creating a suitable environment to promote axonal growth. Here, we assessed in vitro and in vivo the selective effects of combining different ECM components with NTFs on motor and sensory axons regeneration and target reinnervation. Organotypic cultures with collagen, laminin and nerve growth factor (NGF)/neurotrophin-3 (NT3) or collagen, fibronectin and brain-derived neurotrophic factor (BDNF) selectively enhanced sensory neurite outgrowth of DRG neurons and motor neurite outgrowth from spinal cord slices respectively. For in vivo studies, the rat sciatic nerve was transected and repaired with a silicone tube filled with a collagen and laminin matrix with NGF/NT3 encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres (MP) (LM + MP.NGF/NT3), or a collagen and fibronectin matrix with BDNF in PLGA MPs (FN + MP.BDNF). Retrograde labeling and functional tests showed that LM + MP.NGF/NT3 increased the number of regenerated sensory neurons and improved sensory functional recovery, whereas FN + MP.BDNF preferentially increased regenerated motoneurons and enhanced motor functional recovery. Therefore, combination of ECM molecules with NTFs may be a good approach to selectively enhance motor and sensory axons regeneration and promote appropriate target reinnervation

Preferential enhancement of sensory and motor axon regeneration by combining extracellular matrix components with neurotrophic factors

After peripheral nerve injury, motor and sensory axons are able to regenerate but inaccuracy of target reinnervation leads to poor functional recovery. Extracellular matrix (ECM) components and neurotrophic factors (NTFs) exert their effect on different neuronal populations creating a suitable environment to promote axonal growth. Here, we assessed in vitro and in vivo the selective effects of combining different ECM components with NTFs on motor and sensory axons regeneration and target reinnervation. Organotypic cultures with collagen, laminin and nerve growth factor (NGF)/neurotrophin-3 (NT3) or collagen, fibronectin and brain-derived neurotrophic factor (BDNF) selectively enhanced sensory neurite outgrowth of DRG neurons and motor neurite outgrowth from spinal cord slices respectively. For in vivo studies, the rat sciatic nerve was transected and repaired with a silicone tube filled with a collagen and laminin matrix with NGF/NT3 encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres (MP) (LM + MP.NGF/NT3), or a collagen and fibronectin matrix with BDNF in PLGA MPs (FN + MP.BDNF). Retrograde labeling and functional tests showed that LM + MP.NGF/NT3 increased the number of regenerated sensory neurons and improved sensory functional recovery, whereas FN + MP.BDNF preferentially increased regenerated motoneurons and enhanced motor functional recovery. Therefore, combination of ECM molecules with NTFs may be a good approach to selectively enhance motor and sensory axons regeneration and promote appropriate target reinnervatio

Ruolo del gene homeobox Xrx1 nella piastra neurale anteriore: analisi funzionale mediante inattivazione con oligonucleotidi antisenso morpholino.

Lo sviluppo dell’ occhio segue una serie di eventi sequenziali che richiede segnali induttivi spazialmente e temporalmente specifici e precisi movimenti morfogenetici: nello specifico, la prima evidenza morfologica si ha nel momento in cui le pareti laterali del cervello anteriore evaginano per dare origine bilateralmente alle vescicole ottiche, dalle quali deriveranno tutte le strutture neurali dell’ occhio, quali sono la retina, l’ epitelio pigmentato e il peduncolo ottico. Attraverso l’induzione cooperativa di segnali planari e verticali, l’ectoderma che sovrasta la vescicola ottica differenzia in cornea e placode e del cristallino, il quale a sua volta induce la vescicola ottica stessa a invaginarsi in una coppa ottica costituita da uno strato interno e uno esterno, destinati a diventare, al termine dei rispettivi processi differenziativi, retina ed epitelio pigmentato. In Xenopus laevis è dimostrato che le cellule posteriori della piastra neurale iniziano a differenziare alla fine della gastrulazione, laddove quelle più anteriori,ossia il territorio che viene determinato dai seguenti eventi di neurulazione a formare retina e cervello anteriore, manifestano un periodo proliferativo più esteso e vanno incontro a neurogenesi solo allo stadio di bottone caudale. Xrx1 è un gene appartenente alla famiglia degli homeobox paired-like: dotato di un dominio di legame al DNA di tipo paired e di un dominio carbossiterminale OAR di transattivazione, è espresso nella piastra neurale anteriore a partire dallo stadio di gastrula tardiva e a seguire in retina neurale, epitelio pigmentato, ghiandola pineale, diencefalo ed epifisi. Gli esperimenti di sovraespressione e inattivazione funzionale dimostrano che Xrx1 è necessario per un corretto sviluppo dell’ occhio e del cervello anteriore. In particolare, non essendo ancora possibile eliminare selettivamente singoli geni in Xenopus, l’inattivazione funzionale di Xrx1 è stata finora realizzata mediante la microiniezione in singoli blastomeri di mRNA del costrutto dominante negativo Xrx1-Enr. I fenotipi generati manifestano riduzioni di gravità variabile nelle strutture cefaliche anteriori, fino a giungere alla loro completa ablazione. Questo approccio risente tuttavia di importanti limitazioni, dato che la proteina endogena di Xrx1 continua a essere regolarmente prodotta, edentra in competizione funzionale con il dominante negativo iniettato. Lo scopo di questa tesi è la validazione di un nuovo approccio per l’inattivazione funzionale di Xrx1, mediante l’utilizzo di un oligonucleotide antisenso Morpholino. La peculiare costituzione chimica fa si che un oligo Morpholino accuratamente disegnato blocchi la traduzione dell’RNA messaggero a cui si appaia causando un impedimento sterico al cammino del ribosoma lungo il filamento di mRNA. La reale efficacia e specificità dell’oligonucleotide Morpholino MoXrx1, diretto contro la regione immediatamente a valle del codone di inizio del gene Xrx1, è stata studiata sia quantitativamente che funzionalmente mediante differenti approcci sperimentali. Tramite PCR e successivo clonaggio in vettore plasmidico, sono stati realizzati due costrutti, denominati Xrx1-Mt e Mt-Xrx1, ognuno dotato di un Myctag rispettivamente posto al 5’ oppure al 3’ della sequenza codificante del cDNA di Xrx1. Nel caso del costrutto Xrx1-Mt la sequenza bersaglio per MoXrx1 contiene il primo codone di inizio della proteina chimerica, mentre nel costrutto Mt-Xrx1 la sequenza bersaglio e’ in una regione interna del cDNA. Dopo trascrizione in vitro, l’ mRNA di tali costrutti è stato microiniettato in embrioni di Xenopus laevis, in singoli blastomeri dorsali. Sono state effettuate microiniezioni di ciascun singolo trascritto, oppure co-iniezioni dei trascritti insieme a MoXrx1 o alternativamente a un oligonucleotide di controllo negativo. La traduzione dei singoli costrutti è stata valutata mediante SDS-PAGE seguita da western blotting di estratti proteici dei differenti embrioni microiniettati. La rivelazione delle bande proteiche per chemoluminescenza mostra che la coiniezione di MoXrx1 abolisce la traduzione di Xrx1-Mt, ma non quella di Mt-Xrx1, e dimostra l’efficacia e la specificità di azione di MoXrx1 dipendente dalla posizione della sequenza bersaglio. Mediante ibridazione in situ su embrioni interi (whole mount) sono state inoltre studiate le alterazioni indotte dalla microiniezione di MoXrx1 sull’espressione di geni coinvolti nel ciclo cellulare (Cyclin D1, P27, Gadd-45), nella neurogenesi (Xhairy2, Xngn-r,Elr-C) e nel patterning (Fez, XBf-1, Xpax6, Xrx1,Xotx2,Xanf-1,Pax2,Xotx5, FGF-8) della piastra neurale anteriore. I risultati ottenuti, confermano le proprietà dell’oligonucleotide MoXrx1 di inibitore efficace e specifico della traduzione di Xrx1 e ne rendono possibile un suo futuro utilizzo nella costruzione di una libreria sottrattiva finalizzata alla ricerca di geni bersaglio a valle nella cascata d attivazione trascrizionale di Xrx1stesso

Focal release of neurotrophic factors by biodegradable microspheres enhance motor and sensory axonal regeneration in vitro and in vivo

Neurotrophic factors (NTFs) promote nerve regeneration and neuronal survival after peripheral nerve injury. However, drawbacks related with administration and bioactivity during long periods limit their therapeutic application. In this study, PLGA microspheres (MPs) were used to locally release different NTFs and evaluate whether they accelerate axonal regeneration in comparison with free NTFs or controls. ELISA, SEM, UV/visible light microscopy, organotypic cultures of DRG explants and spinal cord slices were used to characterize MP properties and the bioactivity of the released NTFs. Results of organotypic cultures showed that encapsulated NTFs maintain longer bioactivity and enhance neurite regeneration of both sensory and motor neurons compared with free NTFs. For in vivo assays, the rat sciatic nerve was transected and repaired with a silicone tube filled with collagen gel or collagen mixed with PBS encapsulated MPs (control groups) and with free or encapsulated NGF, BDNF, GDNF or FGF-2. After 20 days, a retrotracer was applied to the regenerated nerve to quantify motor and sensory axonal regeneration. NTF encapsulation in MPs improved regeneration of both motor and sensory axons, as evidenced by increased numbers of retrolabeled neurons. Hence, our results show that slow release of NTFs with PLGA MP enhance nerve regeneration

Amorphous silica nanoparticles do not induce cytotoxicity, cell transformation or genotoxicity in Balb/3T3 mouse fibroblasts

Although amorphous silica nanoparticles (aSiO2NPs) are believed to be non-toxic and are currently used in several industrial and biomedical applications including cosmetics, food additives and drug delivery systems, there is still no conclusive information on their cytotoxic, genotoxic and carcinogenic potential. For this reason, this work has investigated the effects of aSiO2NPs on Balb/3T3 mouse fibroblasts, focusing on cytotoxicity, cell transformation and genotoxicity. Results obtained using aSiO2NPs, with diameters between 15 nm and 300 nm and exposure times up to 72 h, have not shown any cytotoxic effect on Balb/3T3 cells as measured by the MTT test and the Colony Forming Efficiency (CFE) assay. Furthermore, aSiO2NPs have induced no morphological transformation in Balb/3T3 cells and have not resulted in genotoxicity, as shown by Cell Transformation Assay (CTA) and Micronucleus (MN) assay, respectively. To understand whether the absence of any toxic effect could result from a lack of internalization of the aSiO2NPs by Balb/3T3 cells, we have investigated the uptake and the intracellular distribution following exposure to 85 nm fluorescently-labelled aSiO2NPs. Using fluorescence microscopy, it was observed that fluorescent aSiO2NPs are internalized and located exclusively in the cytoplasmic region. In conclusion, we have demonstrated that although aSiO2NPs are internalized in vitro by Balb/3T3 mouse fibroblasts, they do not trigger any cytotoxic or genotoxic effect and do not induce morphological transformation, suggesting that they might be a useful component in industrial applications.JRC.I.4-Nanobioscience

Comparative expression analysis of pfdn6a and tcp1α during Xenopus development

We recently identified pfdn6a and tcp1α (also known as cct-α) as genes coregulated by the transcription factor Rx1. The proteins encoded by these genes belong to two interacting complexes (Prefoldin and "chaperonin containing t-complex polypeptide 1"), which promote the folding of actin and tubulin and have more recently been reported to be involved in a variety of additional functions including cell cycle control and transcription regulation. However, little is known about the expression and function of these two genes during vertebrate development. To assess whether pfdn6a and tcp1α display a general coordinated expression during Xenopus development, we determined, by RT-PCR and in situ hybridization, the spatio-temporal expression pattern of pfnd6a, which was not previously described, and compared it to that of tcp1α, extending the analysis to stages not previously investigated for this gene. We detected maternal transcripts of pfnd6a in the animal hemisphere at early blastula stage. During gastrulation, pfdn6a was expressed in the involuting mesoderm and subsequently in the anterior and dorsal neural plate. At tailbud and tadpole stages, pfdn6a RNA was mainly detected in the forebrain, midbrain, eye vesicle, otic vesicle, branchial arches, and developing pronephros. The pfnd6a expression pattern largely overlaps with that of tcp1α indicating a spatio-temporal transcriptional coregulation of these genes in the majority of their expression sites, which is suggestive of a possible involvement in the same developmental events