Neurogenesis is regulated by the dynamic and coordinated activity of several extracellular signalling pathways, but the basis for crosstalk between these pathways remains poorly understood. Here we investigated regulatory interactions between two pathways that are each required for neural progenitor cell maintenance in the postnatal retina; Hedgehog (Hh) and Notch signalling. Both pathways are activated in progenitor cells in the postnatal retina based on the co-expression of fluorescent pathway reporter transgenes at the single cell level. Disrupting Notch signalling, genetically or pharmacologically, induces a rapid downregulation of all three Gli proteins and inhibits Hh-induced proliferation. Ectopic Notch activation, while not sufficient to promote Hh signalling or proliferation, increases Gli2 protein. We show that Notch regulation of Gli2 in Müller glia renders these cells competent to proliferate in response to Hh. These data suggest that Notch signalling converges on Gli2 to prime postnatal retinal progenitor cells and Müller glia to proliferate in response to Hh.
The morphogen sonic hedgehog (Shh) plays a crucial role in development of the CNS, including the neural retina. Suppressor of fused (Sufu) has been recently identified as a critical regulator of Hh signaling in mammals. However, the precise roles that Sufu plays in the regulation of proliferation and cell-fate decisions in neural progenitors is unknown. Here, we have addressed these questions by conditionally deleting Sufu in mouse multipotent retinal progenitor cells (RPCs). Sufu deletion in RPCs results in transient increases in Hh activity and proliferation followed by developmentally premature cell-cycle exit. Importantly, we demonstrate a novel role for Sufu in the maintenance of multipotency in RPCs. Sufu-null RPCs downregulate transcription factors required to specify or maintain RPC identity (Rax, Vsx2) and multipotency (Pax6) but continue to express the neural progenitor marker Sox2. These cells fail to express retinal lineage-specific transcription factors, such as Math5, and adopt an amacrine or horizontal cell fate at the expense of all other classes of retinal neurons. Genetic elimination of Gli2 in Sufu-null RPCs attenuates Hh pathway activity and restores multipotency in neural progenitors. These data provide novel evidence that Sufu-mediated antagonism of Hh/Gli2 signaling is required to maintain RPC multipotency and identity.
The retina is a highly sophisticated piece of the neural machinery that begins the translation of incoming light signals into meaningful visual information. Several degenerative diseases of the retina are characterized by photoreceptor loss and eventually lead to irreversible blindness. Regenerative medicine, using tissue engineering-based constructs to deliver progenitor cells or photoreceptors along with supporting carrier matrix is a promising approach for restoration of structure and function. Fresh fibrin glue (FG) produced by the CryoSeal®FS system in combination with mouse retinal progenitor cells (RPCs) were evaluated in this study. In vitro expanded RPCs isolated from postnatal mouse retina were encapsulated into FG and cultured in the presence of the protease inhibitor, tranexamic acid. Encapsulation of RPCs into FG did not show adverse effects on cell proliferation or cell survival. RPCs exhibited fibroblast-like morphology concomitantly with attachment to the encapsulating FG surface. They expressed α7 and β3 integrin subunits that could mediate attachment to fibrin matrix via an RGD-independent mechanism. The three-dimensional environment and the attachment surface provided by FG was associated with a rapid down-regulation of the progenitor marker SOX2 and enhanced the expression of the differentiation markers cone-rod homeobox and recoverin. However, the in vitro culture conditions did not promote full differentiation into mature photoreceptors. Nevertheless, we have shown that autologous fibrin, when fabricated into a scaffold for RPCs for delivery to the retina, provides the cells with external cues that could potentially improve the differentiation events. Hence, transient encapsulation of RPCs into FG could be a valid and potential treatment strategy to promote retinal regeneration following degenerative diseases. However, further optimization is necessary to maximize the outcomes in terms of mature photoreceptors.
The tumor microenvironment is a critical modulator of carcinogenesis; however, in many tumor types, the influence of the stroma during preneoplastic stages is unknown. Here we explored the relationship between pre-tumor cells and their surrounding stroma in malignant progression of the cerebellar tumor medulloblastoma (MB). We show that activation of the vascular regulatory signalling axis mediated by Norrin (an atypical Wnt)/Frizzled4 (Fzd4) inhibits MB initiation in the Ptch+/− mouse model. Loss of Norrin/Fzd4-mediated signalling in endothelial cells, either genetically or by short-term blockade, increases the frequency of pre-tumor lesions and creates a tumor-permissive microenvironment at the earliest, preneoplastic stages of MB. This pro-tumor stroma, characterized by angiogenic remodelling, is associated with an accelerated transition from preneoplasia to malignancy. These data expose a stromal component that regulates the earliest stages of tumorigenesis in the cerebellum, and a novel role for the Norrin/Fzd4 axis as an endogenous anti-tumor signal in the preneoplastic niche.DOI: http://dx.doi.org/10.7554/eLife.16764.001
Murine cardiac and hematopoietic progenitors are derived from Mesp1 + mesoderm. Cdx function impacts both yolk sac hematopoiesis and cardiogenesis in zebrafish, suggesting that Cdx family members regulate early mesoderm cell fate decisions. We found that Cdx2 occupies a number of transcription factor loci during embryogenesis, including key regulators of both cardiac and blood development, and that Cdx function is required for normal expression of the cardiogenic transcription factors Nkx2-5 and Tbx5. Furthermore, Cdx and Brg1, an ATPase subunit of the SWI/SNF chromatin remodeling complex, cooccupy a number of loci, suggesting that Cdx family members regulate target gene expression through alterations in chromatin architecture. Consistent with this, we demonstrate loss of Brg1 occupancy and altered chromatin structure at several cardiogenic genes in Cdx-null mutants. Finally, we provide evidence for an onset of Cdx2 expression at E6.5 coinciding with egression of cardiac progenitors from the primitive streak. Together, these findings suggest that Cdx functions in multi-potential mesoderm to direct early cell fate decisions through transcriptional regulation of several novel target genes, and provide further insight into a potential epigenetic mechanism by which Cdx influences target gene expression.
Edited by John M. DenuThe packaging of genomic DNA into nucleosomes creates a barrier to transcription that can be relieved through ATP-dependent chromatin remodeling via complexes such as the switch-sucrose non-fermentable (SWI-SNF) chromatin remodeling complex. The SWI-SNF complex remodels chromatin via conformational or positional changes of nucleosomes, thereby altering the access of transcriptional machinery to target genes. The SWI-SNF complex has limited ability to bind to sequencespecific elements, and, therefore, its recruitment to target loci is believed to require interaction with DNA-associated transcription factors. The Cdx family of homeodomain transcript ion factors (Cdx1, Cdx2, and Cdx4) are essential for a number of developmental programs in the mouse. Cdx1 and Cdx2 also regulate intestinal homeostasis throughout life. Although a number of Cdx target genes have been identified, the basis by which Cdx members impact their transcription is poorly understood. We have found that Cdx members interact with the SWI-SNF complex and make direct contact with Brg1, a catalytic member of SWI-SNF. Both Cdx2 and Brg1 co-occupy a number of Cdx target genes, and both factors are necessary for transcriptional regulation of such targets. Finally, Cdx2 and Brg1 occupancy occurs coincident with chromatin remodeling at some of these loci. Taken together, our findings suggest that Cdx transcription factors regulate target gene expression, in part, through recruitment of Brg1-associated SWI-SNF chromatin remodeling activity.The Cdx genes are vertebrate orthologs of Drosophila caudal and encode homeodomain transcriptional factors. In the mouse, the three members of this family (Cdx1, Cdx2, and Cdx4) are co-expressed in the caudal embryo in all three germ layers commencing at mid-gastrulation and play overlapping roles in a number of developmental programs, including axial elongation, endoderm specification, and anterior-posterior vertebral patterning (1-6). Both Cdx1 and Cdx2 are also expressed in the intestinal epithelium throughout life, where they play critical roles in intestinal homeostasis (7-11) and can function as tumor suppressors (12-15). Although Cdx members play critical roles in governing gene expression during development and in the adult intestine, little is known about the mechanisms by which Cdx members regulate target gene transcription.The packaging of DNA into nucleosomes creates a barrier to transcription by obstructing access of the basal transcription machinery as well as modulating access of other transcriptional regulators to their DNA binding motifs (16 -19). Alteration of the chromatin structure by ATP-dependent remodeling complexes can alleviate these constraints, and such complexes play important roles in the transcriptional regulation of many eukaryotic genes. Such complexes include the switch-sucrose non-fermentable (SWI-SNF) 2 chromatin-remodeling complex (16, 20 -22), which remodels the chromatin structure via conformational or positional changes of nucleosomes (23,24). Because the SWI-...
Our data show that combining Hh and mitogen signaling is sufficient to promote the expansion of RPCs in vitro, but it is insufficient to maintain competence of these cells for retinal differentiation.
The Cdx transcription factors play essential roles in primitive hematopoiesis in the zebrafish where they exert their effects, in part, through regulation of hox genes. Defects in hematopoiesis have also been reported in Cdx mutant murine embryonic stem cell models, however, to date no mouse model reflecting the zebrafish Cdx mutant hematopoietic phenotype has been described. This is likely due, in part, to functional redundancy among Cdx members and the early lethality of Cdx2 null mutants. To circumvent these limitations, we used Cre-mediated conditional deletion to assess the impact of concomitant loss of Cdx1 and Cdx2 on murine primitive hematopoiesis. We found that Cdx1/Cdx2 double mutants exhibited defects in primitive hematopoiesis and yolk sac vasculature concomitant with reduced expression of several genes encoding hematopoietic transcription factors including Scl/Tal1. Chromatin immunoprecipitation analysis revealed that Scl was occupied by Cdx2 in vivo, and Cdx mutant hematopoietic yolk sac differentiation defects could be rescued by expression of exogenous Scl. These findings demonstrate critical roles for Cdx members in murine primitive hematopoiesis upstream of Scl.
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