To achieve the task of fabricating functional tissues, scaffold materials that can be sufficiently vascularized to mimic functionality and complexity of native tissues are yet to be developed. Here, we report development of synthetic, biomimetic hydrogels that allow the rapid formation of a stable and mature vascular network both in vitro and in vivo. Hydrogels were fabricated with integrin binding sites and protease-sensitive substrates to mimic the natural provisional extracellular matrices, and endothelial cells cultured in these hydrogels organized into stable, intricate network of capillarylike structures. The resulting structures were further stabilized by recruitment of mesenchymal progenitor cells that differentiated into smooth muscle cell lineage and deposited collagen IV and laminin in vitro. In addition, hydrogels transplanted into mouse cornea were infiltrated with host vasculature, resulting in extensive vascularization with functional blood vessels. These results indicate that these hydrogels may be useful for applications in basic biological research, tissue engineering, and regenerative medicine.
Purpose-Classical measures of vessel morphology including diameter and density are employed to study microvasculature in endothelial membrane labeled mice. These measurements prove sufficient for some studies; however they are less well suited for quantifying changes in microcirculatory networks lacking hierarchical structure. We demonstrate automated multifractal analysis and lacunarity may be used with classical methods to quantify microvascular morphology.Methods-We present an automated extraction tool with a processing pipeline to characterize 2D representations of 3D microvasculature, using multifractal analysis and lacunarity. We apply our analysis on four tissues and the hyaloid vasculature during remodeling.Results-We found that the vessel networks analyzed have multifractal geometries and that kidney microvasculature has the largest fractal dimension and the lowest lacunarity compared to microvasculature networks in the cortex, skin, and thigh muscle. Also, we found that during hyaloid remodeling, there were differences in multifractal spectra reflecting the functional transition from a space filling vasculature which nurtures the lens to a less dense vasculature as it regresses, permitting unobstructed vision.Conclusion-Multifractal analysis and lacunarity are valuable additions to classical measures of vascular morphology and will have utility in future studies of normal, developing and pathological tissues.
Although much is known about the transcriptional regulation that coordinates retinal cell fate determination, very little is known about the developmental processes that establish the characteristic laminar architecture of the retina, in particular, the specification of neuronal positioning. The LIM class homeodomain transcription factor Lim1 (Lhx1) is expressed in postmitotic, differentiating, and mature retinal horizontal cells. We show that conditional ablation of Lim1 results in the ectopic localization of horizontal cells to inner aspects of the inner nuclear layer, among the retinal amacrine cells. The ectopic cells maintain a molecular phenotype consistent with horizontal cell identity; however, these neurons adopt a unique morphology more reminiscent of amacrine cells, including a dendritic arbor positioned within the inner plexiform layer. All other retinal cell populations appear unaltered. Our data suggest a model whereby Lim1 lies downstream of horizontal cell fate determination factors and functions cell autonomously to instruct differentiating horizontal cells to the appropriate laminar position in the developing retina. This study is the first to describe a cell type-specific genetic program that is essential for targeting a discrete retinal neuron population to the proper lamina.
It is widely accepted that the process of retinal cell fate determination is under tight transcriptional control mediated by a combinatorial code of transcription factors. However, the exact repertoire of factors necessary for the genesis of each retinal cell type remains to be fully defined. Here we show that the HMG-box transcription factor, Sox9, is expressed in multipotent mouse retinal progenitor cells throughout retinogenesis. We also find that Sox9 is downregulated in differentiating neuronal populations, yet expression in Müller glial cells persists into adulthood. Furthermore, by employing a conditional knockout approach, we show that Sox9 is essential for the differentiation and/or survival of postnatal Müller glial cells.
SUMMARY In response to retinal damage, the Müller glial cells (MGs) of the zebrafish retina have the ability to undergo a cellular reprogramming event in which they enter the cell cycle and divide asymmetrically, thereby producing multipotent retinal progenitors capable of regenerating lost retinal neurons. However, mammalian MGs do not exhibit such a proliferative and regenerative ability. Here, we identify Hippo pathway-mediated repression of the transcription cofactor YAP as a core regulatory mechanism that normally blocks mammalian MG proliferation and cellular reprogramming. MG-specific deletion of Hippo pathway components Lats1 and Lats2 , as well as transgenic expression of a Hippo non-responsive form of YAP (YAP5SA), resulted in dramatic Cyclin D1 upregulation, loss of adult MG identity, and attainment of a highly proliferative, progenitor-like cellular state. Our results reveal that mammalian MGs may have latent regenerative capacity that can be stimulated by repressing Hippo signaling.
Cryosection of an embryonic day 12.5 eye labeled for Sox9 (orange), Pax6 (blue), and DAPI (purple). J. Comp. Neurol. 510:237–250, 2008. © 2008 Wiley‐Liss, Inc.
Neuronal populations display conspicuous variability in their size among individuals, but the genetic sources of this variation are largely undefined. We demonstrate a large and highly heritable variation in neuron number within the mouse retina, affecting a critical population of interneurons, the horizontal cells. Variation in the size of this population maps to the distal end of chromosome (Chr) 13, a region homologous to human Chr 5q11.1–11.2. This region contains two genes known to modulate retinal cell number. Using conditional knock-out mice, we demonstrate that one of these genes, the LIM homeodomain gene Islet-1 ( Isl1 ), plays a role in regulating horizontal cell number. Genetic differences in Isl1 expression are high during the period of horizontal cell production, and cis -regulation of Isl1 expression within the retina is demonstrated directly. We identify a single nucleotide polymorphism in the 5′ UTR of Isl1 that creates an E-box sequence as a candidate causal variant contributing to this variation in horizontal cell number.
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