The homeobox gene irx1a is required for the propagation of the neurogenic waves in the zebrafish retina

Neurogenesis in the compound eyes of Drosophila and the camera eyes of vertebrates spreads in a wave-like fashion. In both phyla, waves of hedgehog expression are known to drive the wave of neuronal differentiation. The mechanism controlling the propagation of hedgehog expression during retinogenesis of the vertebrate eye is poorly understood. The Iroquois homeobox genes play important roles in Drosophila eye development; they are required for the up-regulation of hedgehog expression during propagation of the morphogenetic furrow. Here, we show that the zebrafish Iroquois homolog irx1a is expressed during retinogenesis and knockdown of irx1a results in a retinal phenotype strikingly similar to those of sonic hedgehog (shh) mutants. Analysis of shh-GFP transgene expression in irx1a knockdown retinas revealed that irx1a is required for the propagation of shh expression through the retina. Transplantation experiments illustrated that the effects of irx1a on shh expression are both cell-autonomous and non-cell-autonomous. Our results reveal a role for Iroquois genes in controlling hedgehog expression during vertebrate retinogenesis.     

Functional association of retinoic acid and hedgehog signaling in Xenopus primary neurogenesis.

Previous work has shown that the posteriorising agent retinoic acid can accelerate anterior neuronal differentiation in Xenopus laevis embryos (Papalopulu, N. and Kintner, C. (1996) Development 122, 3409-3418). To elucidate the role of retinoic acid in the primary neurogenesis cascade, we investigated whether retinoic acid treatment of whole embryos could change the spatial expression of a set of genes known to be involved in neurogenesis. We show that retinoic acid expands the N-tubulin, X-ngnr-1, X-MyT1, X-&Dgr;-1 and Gli3 domains and inhibits the expression of Zic2 and sonic hedgehog in the neural ectoderm, whereas a retinoid antagonist produces opposite changes. In contrast, sonic and banded hedgehog overexpression reduced the N-tubulin stripes, enlarged the neural plate at the expense of the neural crest, downregulated Gli3 and upregulated Zic2. Thus, retinoic acid and hedgehog signaling have opposite effects on the prepattern genes Gli3 and Zic2 and on other genes acting downstream in the neurogenesis cascade. In addition, retinoic acid cannot rescue the inhibitory effect of Notch(ICD), Zic2 or sonic hedgehog on primary neurogenesis. Our results suggest that retinoic acid acts very early, upstream of sonic hedgehog, and we propose a model for regulation of differentiation and proliferation in the neural plate, showing that retinoic acid might be activating primary neurogenesis by repressing sonic hedgehog expression.     

Malondialdehyde,Bcl-2, Superoxide Dismutase and Glutathione Peroxidase may Mediate the Association of Sonic Hedgehog Protein and Oxidative Stress in Autism

Sonic hedgehog signaling and brain-derived neurotrophic factor play a neuro-protective role against oxidative stress in autism. Sonic hedgehog also increases Bcl-2 expression and the activities of superoxide dismutase and glutathione peroxidase. The level or activity of Bcl-2, brain-derived neurotrophic factor, and the activities of superoxide dismutase and glutathione peroxidase are decreased in autism. Sonic hedgehog also decreases the production of malondialdehyde that its level is high in autism. Therefore, it is supposed that sonic hedgehog may be associated with oxidative stress in autism through other pathways too.     

Retinal ganglion cell-derived sonic hedgehog locally controls proliferation and the timing of RGC development in the embryonic mouse retina

The timing of cell cycle exit and temporal changes in the developmental competence of precursor cells are key components for the establishment of the normal complement of cell types in the mammalian retina. The identity of cell extrinsic cues that control these processes is largely unknown. We showed previously in mouse retina that sonic hedgehog (Shh) signalling from retinal ganglion cells (RGCs) to retinal precursor cells (RPC) is required for the establishment of normal retinal organization. Here, we show that conditional ablation of Shh expression in the peripheral mouse results in a depletion of the RPC pool, owing to precocious cell-cycle exit and neuronal differentiation. These changes were correlated with the downregulation of cyclin D1 and Hes1 gene expression. Shh inactivation also results in an increase in RGC number owing to a bias of RPC towards RGC production. In contrast to zebrafish, where Shh signalling drives cell cycle exit and RGC development, our findings indicate that in the mouse retina Shh signalling is required to maintain RPC proliferation and to control the timing of RGC development.     

α2B-Adrenoceptor Deficiency Leads to Postnatal Respiratory Failure in Mice

α₂-Adrenoceptors belong to the family of adrenergic receptors, which regulate the neuronal release of norepinephrine as part of a negative feedback loop. Among the α₂-adrenoceptors, the α_2B-subtype may also influence developmental signaling pathways involved in angiogenesis of the placenta. Thus, the aim of the present study was to determine whether α_2B-adrenoceptors are also involved in other developmental processes beyond placental angiogenesis. Ablation of α_2B-adrenoceptors led to lethality of mutant mice during the first hours after birth. Despite normal breathing and drinking behavior, mutant mice developed cyanosis, which could be traced back to a defect in lung morphology with significantly reduced alveolar volume and thickened interalveolar septi. In α_2B-deficient lungs and in isolated alveolar type II cells, expression of sonic hedgehog (SHH) was significantly increased, resulting in mesenchymal proliferation. In vitro α_2B-adrenoreceptor stimulation suppressed expression of sonic hedgehog and the cell cycle genes cyclin D1 and Ki67. In vivo inhibition of enhanced SHH signaling by the smoothened antagonist cyclopamine partially rescued perinatal lethality, lung morphology, and altered gene expression in mutant mice. Thus, α_2B-adrenoceptors in lung epithelia play an important role in suppressing sonic hedgehog-mediated proliferation of mesenchymal cells and thus prevent respiratory failure.     

Hedgehog signaling in normal urothelial cells and in urothelial carcinoma cell lines

Constitutive activation of hedgehog signaling, often caused by PTCH1 inactivation and leading to inappropriate activation of GLI target genes, is crucial for the development of several human tumors including basal cell carcinoma of the skin and medulloblastoma. The PTCH1 gene at 9q22 is also considered as a candidate tumor suppressor in transitional cell carcinoma (TCC), of which >50% show LOH in this region. However, only rare mutations have been found in PTCH1. We have therefore investigated GLI-dependent promoter activity and expression of hedgehog pathway components in TCC cell lines and proliferating normal urothelial cells. Normal urothelial cells cultured in serum-free medium, but not TCC lines exhibited low, but significant promoter activity under standard growth conditions. Accordingly, GLI1-3 and PTCH1 mRNAs were expressed at moderate levels, and sonic hedgehog (SHH) mRNA expression was low to undetectable. In co-transfection experiments GLI1 increased promoter activity significantly in one TCC line and further in normal urothelial cells, but less strongly in other TCC lines. Expression patterns of GLI factor mRNAs did not correlate with inducibility. No significant effects of SHH or cyclopamine on proliferation were observed, ruling out autocrine effects. However, SHH induced GLI-dependent promoter activity in normal urothelial cells. Taken together, our data suggest that the hedgehog pathway is weakly active in normal adult urothelial cells and of limited importance in TCC.     

Function for Hedgehog genes in zebrafish retinal development

The hedgehog (hh) genes encode secreted signaling proteins that have important developmental functions in vertebrates and invertebrates. In Drosophila, expression of hh coordinates retinal development by propagating a wave of photoreceptor differentiation across the eye primordium. Here we report that two vertebrate hh genes, sonic hedgehog (shh) and tiggy-winkle hedgehog (twhh), may perform similar functions in the developing zebrafish. Both shh and twhh are expressed in the embryonic zebrafish retinal pigmented epithelium (RPE), initially in a discrete ventral patch which then expands outward in advance of an expanding wave of photoreceptor recruitment in the subjacent neural retina. A gene encoding a receptor for the hedgehog protein, ptc-2, is expressed by retinal neuroepithelial cells. Injection of a cocktail of antisense (alphashh/alphatwhh) oligonucleotides reduces expression of both hh genes in the RPE and slows or arrests the progression of rod and cone photoreceptor differentiation. Zebrafish strains known to have mutations in Hh signaling pathway genes similarly exhibit retardation of photoreceptor differentiation. We propose that hedgehog genes may play a role in propagating photoreceptor differentiation across the developing eye of the zebrafish.     

An in vivo comparative study of sonic, desert and Indian hedgehog reveals that hedgehog pathway activity regulates epidermal stem cell homeostasis

Despite the well-characterised role of sonic hedgehog (Shh) in promoting interfollicular basal cell proliferation and hair follicle downgrowth, the role of hedgehog signalling during epidermal stem cell fate remains largely uncharacterised. In order to determine whether the three vertebrate hedgehog molecules play a role in regulating epidermal renewal we overexpressed sonic (Shh), desert (Dhh) and Indian (Ihh) hedgehog in the basal cells of mouse skin under the control of the human keratin 14 promoter. We observed no overt epidermal morphogenesis phenotype in response to Ihh overexpression, however Dhh overexpression resulted in a range of embryonic and adult skin manifestations indistinguishable from Shh overexpression. Two distinct novel phenotypes were observed amongst Shh and Dhh transgenics, one exhibiting epidermal progenitor cell hyperplasia with the other displaying a complete loss of epidermal tissue renewal indicating deregulation of stem cell activity. These data suggest that correct temporal regulation of hedgehog activity is a key factor in ensuring epidermal stem cell maintenance. In addition, we observed Shh and Dhh transgenic skin from both phenotypes developed lesions reminiscent of human basal cell carcinoma (BCC), indicating that BCCs can be generated despite the loss of much of the proliferative (basal) compartment. These data suggest the intriguing possibility that BCC can arise outside the stem cell population. Thus the elucidation of Shh (and Dhh) target gene activation in the skin will likely identify those genes responsible for increasing the proliferative potential of epidermal basal cells and the mechanisms involved in regulating epidermal stem cell fate.     

Transient establishment of anteroposterior polarity in the zebrafish pectoral fin bud in the absence of sonic hedgehog activity.

Sonic hedgehog (Shh) is expressed in the posterior vertebrate limb bud mesenchyme and directs anteroposterior patterning and growth during limb development. Here we report an analysis of the pectoral fin phenotype of zebrafish sonic you mutants, which disrupt the shh gene. We show that Shh is required for the establishment of some aspects of anteroposterior polarity, while other aspects of anteroposterior polarity are established independently of Shh, and only later come to depend on Shh for their maintenance. We also demonstrate that Shh is required for the activation of posterior HoxD genes by retinoic acid. Finally, we show that Shh is required for normal development of the apical ectodermal fold, for growth of the fin bud, and for formation of the fin endoskeleton.     

Dopaminergic Neuronal Conversion from Adult Rat Skeletal Muscle-Derived Stem Cells In Vitro

Muscle-derived stem cells reside in the skeletal muscle tissues and are known for their multipotency to differentiate toward the mesodermal lineage. Recent studies have demonstrated their capacity of neuroectodermal differentiation, including neurons and astrocytes. In this study, we investigated the possibility of dopaminergic neuronal conversion from adult rat skeletal muscle-derived stem cells. Using a neurosphere protocol, muscle-derived stem cells form neurosphere-like cell clusters after cultivation as a suspension, displaying an obvious expression of nestin and a remarkable down-regulation of myogenic associated factors desmin, MyoD, Myf5 and myogenin. Subsequently, these neurosphere-like cell clusters were further directed to dopaminergic differentiation through two major induction steps, patterning to midbrain progenitors with sonic hedgehog and fibroblast growth factor 8, followed by the differentiation to dopaminergic neurons with neurotrophic factors (glial cell line-derived neurotrophic factor) and chemicals (ascorbic acid, forskolin). After the differentiation, these cells expressed tyrosine hydroxylase, dopamine transporter, dopamine D1 receptor and synapse-associated protein synapsin I. Several genes, Nurr1, Lmx1b, and En1, which are critically related with the development of dopaminergic neurons, were also significantly up-regulated. The present results indicate that adult skeletal muscle-derived stem cells could provide a promising cell source for autologous transplantation for neurodegenerative diseases in the future, especially the Parkinson's disease.     

The role of UV induced lesions in skin carcinogenesis: an overview of oncogene and tumor suppressor gene modifications in xeroderma pigmentosum skin tumors

Xeroderma pigmentosum (XP), a rare hereditary syndrome, is characterized by a hypersensitivity to solar irradiation due to a defect in nucleotide excision repair resulting in a predisposition to squamous and basal cell carcinomas as well as malignant melanomas appearing at a very early age. The mutator phenotype of XP cells is evident by the higher levels of UV specific modifications found in key regulatory genes in XP skin tumors compared to those in the same tumor types from the normal population. Thus, XP provides a unique model for the study of unrepaired DNA lesions, mutations and skin carcinogenesis. The high level of ras oncogene activation, Ink4a-Arf and p53 tumor suppressor gene modifications as well as alterations of the different partners of the mitogenic sonic hedgehog signaling pathway (patched, smoothened and sonic hedgehog), characterized in XP skin tumors have clearly demonstrated the major role of the UV component of sunlight in the development of skin tumors. The majority of the mutations are C to T or tandem CC to TT UV signature transitions, occurring at bipyrimidine sequences, the specific targets of UV induced lesions. These characteristics are also found in the same genes modified in sporadic skin cancers but with lower frequencies confirming the validity of studying the XP model. The knowledge gained by studying XP tumors has given us a greater perception of the contribution of genetic predisposition to cancer as well as the consequences of the many alterations which modulate the activities of different genes affecting crucial pathways vital for maintaining cell homeostasis.     

Epidermal growth factor promotes a neural phenotype in thymic epithelial cells and enhances neuropoietic cytokine expression

Neural crest-derived cells populate the thymus, and their coexistence with epithelial cells is required for proper organ development and T cell education function. We show here that epidermal growth factor (EGF), a major epithelial cell growth-enhancing agent, has a morphogenetic action to promote the expression of a neuronal phenotype (e.g., neurofilament expression) in cultured thymic epithelial cells that are characterized by a cytokeratin-positive epithelial cell background. The proliferation of such neurodifferentiated cells is also enhanced by EGF. Furthermore, the growth factor enhances cells that express the genes encoding the preprotachykinin A-generated neuropeptides and bipotential neuropoietic and lymphopoietic cytokines ciliary neurotrophic factor and interleukin-6. These cytokines also enhance the neuronal phenotype of thymic epithelial cells. Therefore, EGF appears to be a composite autocrine/paracrine neuromodulator in thymic stroma. This suggests that EGF may regulate thymus-dependent immune functions by promoting neuronal gene expression in neural crest- derived cells.