ABSTRACTThe establishment of a functional circulatory system is required for post-implantation de... more ABSTRACTThe establishment of a functional circulatory system is required for post-implantation development during murine embryogenesis. Previous studies in loss of function mouse models have shown that FOXO1, a Forkhead family transcription factor, is required for yolk sac vascular remodeling and survival beyond embryonic day (E) 11. Here, we demonstrate that loss ofFoxO1in E8.25 endothelial cells results in increasedSprouty2andSprouty4transcripts, reduced expression of arterial genes, and decreasedFlk1/Vegfr2mRNA levels without affecting overall endothelial cell identity, survival, or proliferation. Using aDll4-BAC-nlacZreporter line, we found that one of the earliest expressed arterial genes,Delta like 4(Dll4), is significantly reduced in the yolk sac ofFoxO1mutants without being substantially affected in the embryo proper. We show that in the yolk sac, FOXO1 not only binds directly to a subset of previously identifiedSprouty2gene regulatory elements (GREs), as well as newly ident...
Macrophages are well characterized as immune cells. However, in recent years, a multitude of non-... more Macrophages are well characterized as immune cells. However, in recent years, a multitude of non-immune functions have emerged many of which play essential roles in a variety of developmental processes (Wynn, Chawla and J. W. Pollard, 2013; DeFalco et al., 2014). In adult animals, macrophages are derived from circulating monocytes originating in the bone marrow, but much of the tissue-resident population arise from erythro-myeloid progenitors (EMPs) in the extra-embryonic yolk sac, appearing around the same time as primitive erythroblasts (Schulz et al., 2012; Kierdorf et al., 2013; McGrath, Frame and Palis, 2015; Gomez Perdiguero et al., 2015; Mass et al., 2016). Of particular interest to our group, macrophages have been shown to act as pro-angiogenic regulators during development (Owen and Mohamadzadeh, 2013; DeFalco et al., 2014; Hsu et al., 2015), but there is still much to learn about these early cells. The goal of the present study was to isolate and expand progenitors of yolk...
Programmed capillary regression and remodeling are essential developmental processes. However, th... more Programmed capillary regression and remodeling are essential developmental processes. However, the cellular and molecular mechanisms that regulate vessel regression are only the beginning to be understood. Here, using in vivo, dynamic, confocal imaging of mouse transgenic reporters as well as static confocal and electron microscopy, we studied the embryonic development and postnatal regression of the transient mouse pupillary membrane (PM) vasculature. This approach allowed us to directly observe the precise temporal sequence of cellular events preceding and during the elimination of the PM from the mouse eye. Imaging of Tcf/Lef-H2B::GFP Wnt-reporter mice uncovered that, unlike the hyaloid vasculature of the posterior eye, a PM endothelial cell (EC) Wnt/β-catenin response is unlikely to be part of the regression mechanism. Live imaging of EC and macrophage dynamics revealed highly active Csf1r-GFP+ macrophages making direct contact with the Flk1-myr::mCherry+ vessel surface and with...
INTRODUCTIONThe field of tissue engineering aims to recapitulate healthy human organs and 3-D tis... more INTRODUCTIONThe field of tissue engineering aims to recapitulate healthy human organs and 3-D tissue structures in vitro and then transplant these constructs in vivo where they can be effectively integrated within the recipient patient and become perfused by the host circulation. To improve the design of materials for artificial tissue scaffolds, it would be ideal to have a high-throughput imaging system that allows one to directly monitor transplanted tissue constructs in live animals over an extended period of time. By combining such an assay with transgenic, cell-specific fluorescent reporters, one could monitor such parameters as tissue construct perfusion, donor cell survival, and donor-host cell interaction/integration. Here, we describe a protocol for a modified version of the classical corneal micropocket angiogenesis assay, employing it as a live imaging “window” to monitor angiogenic poly(ethylene glycol) (PEG)-based hydrogel tissue constructs.
Although much is known about the transcriptional regulation that coordinates retinal cell fate de... more 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 ofLim1results 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 wherebyLim1lies downstream of horizontal cell fate determina...
The present study examined the consequences of eliminating horizontal cells from the outer retina... more The present study examined the consequences of eliminating horizontal cells from the outer retina during embryogenesis upon the organization and assembly of the outer plexiform layer (OPL). Retinal horizontal cells exhibit a migration defect inLim1-conditional knock-out (Lim1-CKO) mice and become mispositioned in the inner retina before birth, redirecting their dendrites into the inner plexiform layer. The resultant (mature) OPL, developing in the absence of horizontal cells, shows a retraction of rod spherules into the outer nuclear layer and a sprouting of rod bipolar cell dendrites to reach ectopic ribbon-protein puncta. Cone pedicles and the dendrites of type 7 cone bipolar cells retain their characteristic stratification and colocalization within the collapsed OPL, although both are atrophic and the spatial distribution of the pedicles is disrupted. Developmental analysis ofLim1-CKO retina reveals that components of the rod and cone pathways initially co-assemble within their n...
Proceedings of the National Academy of Sciences, 2011
Neuronal populations display conspicuous variability in their size among individuals, but the gen... more 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...
A group of retinal interneurons known as horizontal cells has recently been shown to exhibit a va... more A group of retinal interneurons known as horizontal cells has recently been shown to exhibit a variety of unique biological properties, as compared with other nerve cells, that challenge many long-standing assumptions in the fields of neural development and cancer biology. These features include their unusual migratory behavior, their unique morphological plasticity, and their propensity to divide at a relatively late stage during development. Here, we review these novel features, discuss their relevance for other cell types,outline open questions in our understanding of horizontal cell development and consider their implications.
ABSTRACT On page 2344, J. L. West, M. E. Dickinson, and co-workers demonstrate an image-guided me... more ABSTRACT On page 2344, J. L. West, M. E. Dickinson, and co-workers demonstrate an image-guided method for engineering complex biomimetic materials. Using laser scanning microscopes and twophoton photolithography, they use this method to create patterned poly(ethylene glycol) diacrylate hydrogels that recreate 3D structural and biochemical features of endogenous vascular microenvironments, as shown here for the coronary vasculature. These biomaterials can effectively guide cellular organization in order to recapitulate endogenous vascular structures, marking an important step forward for tissue engineering.
ABSTRACTThe establishment of a functional circulatory system is required for post-implantation de... more ABSTRACTThe establishment of a functional circulatory system is required for post-implantation development during murine embryogenesis. Previous studies in loss of function mouse models have shown that FOXO1, a Forkhead family transcription factor, is required for yolk sac vascular remodeling and survival beyond embryonic day (E) 11. Here, we demonstrate that loss ofFoxO1in E8.25 endothelial cells results in increasedSprouty2andSprouty4transcripts, reduced expression of arterial genes, and decreasedFlk1/Vegfr2mRNA levels without affecting overall endothelial cell identity, survival, or proliferation. Using aDll4-BAC-nlacZreporter line, we found that one of the earliest expressed arterial genes,Delta like 4(Dll4), is significantly reduced in the yolk sac ofFoxO1mutants without being substantially affected in the embryo proper. We show that in the yolk sac, FOXO1 not only binds directly to a subset of previously identifiedSprouty2gene regulatory elements (GREs), as well as newly ident...
Macrophages are well characterized as immune cells. However, in recent years, a multitude of non-... more Macrophages are well characterized as immune cells. However, in recent years, a multitude of non-immune functions have emerged many of which play essential roles in a variety of developmental processes (Wynn, Chawla and J. W. Pollard, 2013; DeFalco et al., 2014). In adult animals, macrophages are derived from circulating monocytes originating in the bone marrow, but much of the tissue-resident population arise from erythro-myeloid progenitors (EMPs) in the extra-embryonic yolk sac, appearing around the same time as primitive erythroblasts (Schulz et al., 2012; Kierdorf et al., 2013; McGrath, Frame and Palis, 2015; Gomez Perdiguero et al., 2015; Mass et al., 2016). Of particular interest to our group, macrophages have been shown to act as pro-angiogenic regulators during development (Owen and Mohamadzadeh, 2013; DeFalco et al., 2014; Hsu et al., 2015), but there is still much to learn about these early cells. The goal of the present study was to isolate and expand progenitors of yolk...
Programmed capillary regression and remodeling are essential developmental processes. However, th... more Programmed capillary regression and remodeling are essential developmental processes. However, the cellular and molecular mechanisms that regulate vessel regression are only the beginning to be understood. Here, using in vivo, dynamic, confocal imaging of mouse transgenic reporters as well as static confocal and electron microscopy, we studied the embryonic development and postnatal regression of the transient mouse pupillary membrane (PM) vasculature. This approach allowed us to directly observe the precise temporal sequence of cellular events preceding and during the elimination of the PM from the mouse eye. Imaging of Tcf/Lef-H2B::GFP Wnt-reporter mice uncovered that, unlike the hyaloid vasculature of the posterior eye, a PM endothelial cell (EC) Wnt/β-catenin response is unlikely to be part of the regression mechanism. Live imaging of EC and macrophage dynamics revealed highly active Csf1r-GFP+ macrophages making direct contact with the Flk1-myr::mCherry+ vessel surface and with...
INTRODUCTIONThe field of tissue engineering aims to recapitulate healthy human organs and 3-D tis... more INTRODUCTIONThe field of tissue engineering aims to recapitulate healthy human organs and 3-D tissue structures in vitro and then transplant these constructs in vivo where they can be effectively integrated within the recipient patient and become perfused by the host circulation. To improve the design of materials for artificial tissue scaffolds, it would be ideal to have a high-throughput imaging system that allows one to directly monitor transplanted tissue constructs in live animals over an extended period of time. By combining such an assay with transgenic, cell-specific fluorescent reporters, one could monitor such parameters as tissue construct perfusion, donor cell survival, and donor-host cell interaction/integration. Here, we describe a protocol for a modified version of the classical corneal micropocket angiogenesis assay, employing it as a live imaging “window” to monitor angiogenic poly(ethylene glycol) (PEG)-based hydrogel tissue constructs.
Although much is known about the transcriptional regulation that coordinates retinal cell fate de... more 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 ofLim1results 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 wherebyLim1lies downstream of horizontal cell fate determina...
The present study examined the consequences of eliminating horizontal cells from the outer retina... more The present study examined the consequences of eliminating horizontal cells from the outer retina during embryogenesis upon the organization and assembly of the outer plexiform layer (OPL). Retinal horizontal cells exhibit a migration defect inLim1-conditional knock-out (Lim1-CKO) mice and become mispositioned in the inner retina before birth, redirecting their dendrites into the inner plexiform layer. The resultant (mature) OPL, developing in the absence of horizontal cells, shows a retraction of rod spherules into the outer nuclear layer and a sprouting of rod bipolar cell dendrites to reach ectopic ribbon-protein puncta. Cone pedicles and the dendrites of type 7 cone bipolar cells retain their characteristic stratification and colocalization within the collapsed OPL, although both are atrophic and the spatial distribution of the pedicles is disrupted. Developmental analysis ofLim1-CKO retina reveals that components of the rod and cone pathways initially co-assemble within their n...
Proceedings of the National Academy of Sciences, 2011
Neuronal populations display conspicuous variability in their size among individuals, but the gen... more 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...
A group of retinal interneurons known as horizontal cells has recently been shown to exhibit a va... more A group of retinal interneurons known as horizontal cells has recently been shown to exhibit a variety of unique biological properties, as compared with other nerve cells, that challenge many long-standing assumptions in the fields of neural development and cancer biology. These features include their unusual migratory behavior, their unique morphological plasticity, and their propensity to divide at a relatively late stage during development. Here, we review these novel features, discuss their relevance for other cell types,outline open questions in our understanding of horizontal cell development and consider their implications.
ABSTRACT On page 2344, J. L. West, M. E. Dickinson, and co-workers demonstrate an image-guided me... more ABSTRACT On page 2344, J. L. West, M. E. Dickinson, and co-workers demonstrate an image-guided method for engineering complex biomimetic materials. Using laser scanning microscopes and twophoton photolithography, they use this method to create patterned poly(ethylene glycol) diacrylate hydrogels that recreate 3D structural and biochemical features of endogenous vascular microenvironments, as shown here for the coronary vasculature. These biomaterials can effectively guide cellular organization in order to recapitulate endogenous vascular structures, marking an important step forward for tissue engineering.
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Papers by Ross Poche