<p>A-L) Bright field images of WT (A–F) and cKO (G–L) embryos (EM) alone or embryos within ... more <p>A-L) Bright field images of WT (A–F) and cKO (G–L) embryos (EM) alone or embryos within their yolk sacs (YS) at the indicated stages. A, G) 8.5 <i>dpc</i> mutant embryos (inset in G) are sometimes slightly delayed compared with WT (inset in A) but display no noticeable yolk sac defects. B–C, H–I) 9.0 <i>dpc</i> mutants display relatively normal yolk sac blood vessel development. D–E, J–K) 9.5 <i>dpc</i> mutant yolk sacs have dilated vessels (asterisk in J) and poor vessel organization (compare D to J). cKO embryos (K) are smaller than WT embryos (E) from the same litter. F, L) While prominent large blood vessels are easily detected in 10.5 <i>dpc</i> WT yolk sacs (F), the yolk sacs of mutants are uniformly pale (L). M–P) A comparison of WT and mutant H&E stained yolk sac sections demonstrates that while no differences are found at 8.5 <i>dpc</i> (M, O), the 9.5 <i>dpc</i> cKO yolk sac (P) has fewer and larger vessels compared with WT (N). Q) Investigation of the same sized area at 9.5 <i>dpc</i> revealed significantly fewer vessels in mutant compared with WT yolk sacs (*** = p<0.001; error bar = standard error). R) A size distribution chart at 9.5 <i>dpc</i> reveals that mutants contain fewer of the small vessels (<100 µm) and more of the larger vessels (>100 µm) compared with WT yolk sacs.</p
<p>A–B, D–E) Whole mount immunofluorescence of 9.5 <i>dpc</i> WT (A–B) or mutan... more <p>A–B, D–E) Whole mount immunofluorescence of 9.5 <i>dpc</i> WT (A–B) or mutant (D–E) yolk sacs (YS) using the endothelial marker PECAM (green) and the vascular smooth muscle marker (αSMA) demonstrates that the large disorganized vessels in the cKO (D) are not surrounded by αSMA (E). C, F) Section immunofluorescence of WT (C) and cKO (F) 9.5 <i>dpc</i> yolk sacs demonstrates loss of αSMA in the cKO. G–L) Section immunofluorescence of VEGFA (green) demonstrates relatively uniform VEGF levels in the 9.0, 9.25 and 9.5 <i>dpc</i> WT yolk sac (G–I) while VEGF distribution in the visceral endoderm of the mutant is progressively diminished at each stage (J–L). M) A Western blot of whole yolk sacs at the indicated stages. The ratio of VEGFA to GAPDH signal intensities for the cKO relative to each stage-matched WT control is displayed under each band. N) Cleaved Caspase-3 staining was used to assess the percentage of cell death in the yolk sacs layers of WT and cKO sections at 8.5 and 9.0 <i>dpc</i>. A significant increase in apoptosis was observed in the cKO mesoderm (ME) at 9.0 <i>dpc</i>. O) Phosphohistone-H3 (PH-3) staining was similarly used to assess proliferation and a significant decrease in proliferation was found in the cKO yolk sac mesoderm at 9.0 <i>dpc</i>. *** = p<0.001, ** = p<0.01; error bars = standard error; dotted line is drawn between the visceral endoderm (VE) and mesoderm derivatives (ME) on yolk sac sections.</p
<p>A, B) A summary of the signaling events downstream of YY1 in WT and mutant yolk sacs. A)... more <p>A, B) A summary of the signaling events downstream of YY1 in WT and mutant yolk sacs. A) In the presence of YY1, normal VEGF levels produced by the visceral endoderm (VE) allow the underlying mesoderm derivatives (ME) to undergo events associated with vascular remodeling. The underlying vascular tissue is the source of a VEGF-dependant paracrine signal(s) that is required by the visceral endoderm to maintain characteristics such as epithelial polarity, large apical lysosomes and HNF4α expression. B) In the absence of YY1 in the visceral endoderm, decreased levels of paracrine VEGF result in defective angiogenesis, increased apoptosis and decreased proliferation in the adjacent mesoderm. Because of reduced VEGF signaling, the yolk sac mesoderm does not generate the paracrine signal(s) needed to maintain epithelial characteristics in the visceral endoderm, resulting in decreased HNF4α, a loss of large lysosomes and reduced CDH1 levels.</p
<p>Immunofluorescence analysis of sectioned WT (A–D, I–K) and cKO tissue (E–H, L–N) at the ... more <p>Immunofluorescence analysis of sectioned WT (A–D, I–K) and cKO tissue (E–H, L–N) at the stages indicated. A–C, I) YY1 (green) is ubiquitous in WT embryonic and extraembryonic tissues. A–B, D, K) HNF4α (red, orange when co-expressed with YY1) labels the visceral endoderm (A–B, D) and the developing liver bud (K). E–G, L) In cKO embryos, YY1 is downregulated in the extraembryonic visceral endoderm (VE) at 7.5 <i>dpc</i> (E) and is completely lost in the embryonic visceral endoderm by 8.75 <i>dpc</i> (F), when YY1 is also depleted in the definitive endoderm (DE) of the foregut. By 9.25 <i>dpc</i> YY1 is lost in most cells of the liver bud (L). E–F, H, N) Although HNF4α is present in the YY1-deficient visceral endoderm until 8.75 <i>dpc</i> (E, F) it is greatly reduced in both the visceral endoderm and in the nascent liver bud by 9.5 <i>dpc</i>. J, M) Despite the loss of YY1 in the nascent liver bud, the liver bud differentiation marker PROX1 is maintained in the cKO liver bud (M) at levels comparable to that observed in WT (J). The dotted line in C–D and G–H represent the division between the visceral endoderm and mesoderm derivative of the yolk sac, while in I–N the dashed line outlines the liver bud (LB).</p
Holothuria glaberrima exhibits the capacity to regenerate body parts after injury or loss. This a... more Holothuria glaberrima exhibits the capacity to regenerate body parts after injury or loss. This ability is mediated by an initial blastemal formation which requires proliferation and reorganization...
In recent years, transcriptomic databases have become one of the main sources for protein discove... more In recent years, transcriptomic databases have become one of the main sources for protein discovery. In our studies of nervous system and digestive tract regeneration in echinoderms, we have identified several transcripts that have attracted our attention. One of these molecules corresponds to a previously unidentified transcript (Orpin) from the sea cucumber Holothuria glaberrima that appeared to be upregulated during intestinal regeneration. We have now identified a second highly similar sequence and analyzed the predicted proteins using bioinformatics tools. Both sequences have EF-hand motifs characteristic of calcium-binding proteins (CaBPs) and N-terminal signal peptides. Sequence comparison analyses such as multiple sequence alignments and phylogenetic analyses only showed significant similarity to sequences from other echinoderms or from hemichordates. Semi-quantitative RT-PCR analyses revealed that transcripts from these sequences are expressed in various tissues including m...
Mouse embryos lacking the polycomb group gene member Yin-Yang1 (YY1) die during the peri-implanta... more Mouse embryos lacking the polycomb group gene member Yin-Yang1 (YY1) die during the peri-implantation stage. To assess the post-gastrulation role of YY1, a conditional knock-out (cKO) strategy was used to delete YY1 from the visceral endoderm of the yolk sac and the definitive endoderm of the embryo. cKO embryos display profound yolk sac defects at 9.5 days post coitum (dpc), including disrupted angiogenesis in mesoderm derivatives and altered epithelial characteristics in the visceral endoderm. Significant changes in both cell death and proliferation were confined to the YY1-expressing yolk sac mesoderm indicating that loss of YY1 in the visceral endoderm causes defects in the adjacent yolk sac mesoderm. Production of Vascular Endothelial Growth Factor A (VEGFA) by the visceral endoderm is essential for normal growth and development of the yolk sac vasculature. Reduced levels of VEGFA are observed in the cKO yolk sac, suggesting a cause for the angiogenesis defects. Ex vivo culture with exogenous VEGF not only rescued angiogenesis and apoptosis in the cKO yolk sac mesoderm, but also restored the epithelial defects observed in the cKO visceral endoderm. Intriguingly, blocking the activity of the mesoderm-localized VEGF receptor, FLK1, recapitulates both the mesoderm and visceral endoderm defects observed in the cKO yolk sac. Taken together, these results demonstrate that YY1 is responsible for maintaining VEGF in the developing visceral endoderm and that a VEGF-responsive paracrine signal, originating in the yolk sac mesoderm, is required to promote normal visceral endoderm development.
The ubiquitin proteasome system (UPS) is the main proteolytic system of cells. Recent evidence su... more The ubiquitin proteasome system (UPS) is the main proteolytic system of cells. Recent evidence suggests that the UPS plays a regulatory role in regeneration processes. Here, we explore the possibility that the UPS is involved during intestinal regeneration of the sea cucumber Holothuria glaberrima. These organisms can regenerate most of their digestive tract following a process of evisceration. Initially, we identified components of H. glaberrima UPS, including sequences for Rpn10, β3, and ubiquitin-RPL40. Predicted proteins from the mRNA sequences showed high degree of conservation that ranged from 60% (Rpn10) to 98% (Ub-RPL40). Microarrays and RT-PCR experiments showed that these genes were upregulated during intestinal regeneration. In addition, we demonstrated expression of alpha 20S proteasome subunits and ubiquitinated proteins during intestinal regeneration and detected them in the epithelium and connective tissue of the regenerating intestine. Finally, the intestinal regener...
Among deuterostomes, the regenerative potential is maximally expressed in echinoderms, animals th... more Among deuterostomes, the regenerative potential is maximally expressed in echinoderms, animals that can quickly replace most injured organs. In particular, sea cucumbers are excellent models for studying organ regeneration since they regenerate their digestive tract after evisceration. However, echinoderms have been sidelined in modern regeneration studies partially because of the lack of genome-wide profiling approaches afforded by modern genomic tools.For the last decade, our laboratory has been using the sea cucumber Holothuria glaberrima to dissect the cellular and molecular events that allow for such amazing regenerative processes. We have already established an EST database obtained from cDNA libraries of normal and regenerating intestine at two different regeneration stages. This database now has over 7000 sequences. In the present work we used a custom-made microchip from Agilent with 60-mer probes for these ESTs, to determine the gene expression profile during intestinal re...
Not Available Bibtex entry for this abstract Preferred format for this abstract (see Preferences)... more Not Available Bibtex entry for this abstract Preferred format for this abstract (see Preferences) Find Similar Abstracts: Use: Authors Title Return: Query Results Return items starting with number Query Form Database: Astronomy Physics arXiv e-prints
<p>A-L) Bright field images of WT (A–F) and cKO (G–L) embryos (EM) alone or embryos within ... more <p>A-L) Bright field images of WT (A–F) and cKO (G–L) embryos (EM) alone or embryos within their yolk sacs (YS) at the indicated stages. A, G) 8.5 <i>dpc</i> mutant embryos (inset in G) are sometimes slightly delayed compared with WT (inset in A) but display no noticeable yolk sac defects. B–C, H–I) 9.0 <i>dpc</i> mutants display relatively normal yolk sac blood vessel development. D–E, J–K) 9.5 <i>dpc</i> mutant yolk sacs have dilated vessels (asterisk in J) and poor vessel organization (compare D to J). cKO embryos (K) are smaller than WT embryos (E) from the same litter. F, L) While prominent large blood vessels are easily detected in 10.5 <i>dpc</i> WT yolk sacs (F), the yolk sacs of mutants are uniformly pale (L). M–P) A comparison of WT and mutant H&E stained yolk sac sections demonstrates that while no differences are found at 8.5 <i>dpc</i> (M, O), the 9.5 <i>dpc</i> cKO yolk sac (P) has fewer and larger vessels compared with WT (N). Q) Investigation of the same sized area at 9.5 <i>dpc</i> revealed significantly fewer vessels in mutant compared with WT yolk sacs (*** = p<0.001; error bar = standard error). R) A size distribution chart at 9.5 <i>dpc</i> reveals that mutants contain fewer of the small vessels (<100 µm) and more of the larger vessels (>100 µm) compared with WT yolk sacs.</p
<p>A–B, D–E) Whole mount immunofluorescence of 9.5 <i>dpc</i> WT (A–B) or mutan... more <p>A–B, D–E) Whole mount immunofluorescence of 9.5 <i>dpc</i> WT (A–B) or mutant (D–E) yolk sacs (YS) using the endothelial marker PECAM (green) and the vascular smooth muscle marker (αSMA) demonstrates that the large disorganized vessels in the cKO (D) are not surrounded by αSMA (E). C, F) Section immunofluorescence of WT (C) and cKO (F) 9.5 <i>dpc</i> yolk sacs demonstrates loss of αSMA in the cKO. G–L) Section immunofluorescence of VEGFA (green) demonstrates relatively uniform VEGF levels in the 9.0, 9.25 and 9.5 <i>dpc</i> WT yolk sac (G–I) while VEGF distribution in the visceral endoderm of the mutant is progressively diminished at each stage (J–L). M) A Western blot of whole yolk sacs at the indicated stages. The ratio of VEGFA to GAPDH signal intensities for the cKO relative to each stage-matched WT control is displayed under each band. N) Cleaved Caspase-3 staining was used to assess the percentage of cell death in the yolk sacs layers of WT and cKO sections at 8.5 and 9.0 <i>dpc</i>. A significant increase in apoptosis was observed in the cKO mesoderm (ME) at 9.0 <i>dpc</i>. O) Phosphohistone-H3 (PH-3) staining was similarly used to assess proliferation and a significant decrease in proliferation was found in the cKO yolk sac mesoderm at 9.0 <i>dpc</i>. *** = p<0.001, ** = p<0.01; error bars = standard error; dotted line is drawn between the visceral endoderm (VE) and mesoderm derivatives (ME) on yolk sac sections.</p
<p>A, B) A summary of the signaling events downstream of YY1 in WT and mutant yolk sacs. A)... more <p>A, B) A summary of the signaling events downstream of YY1 in WT and mutant yolk sacs. A) In the presence of YY1, normal VEGF levels produced by the visceral endoderm (VE) allow the underlying mesoderm derivatives (ME) to undergo events associated with vascular remodeling. The underlying vascular tissue is the source of a VEGF-dependant paracrine signal(s) that is required by the visceral endoderm to maintain characteristics such as epithelial polarity, large apical lysosomes and HNF4α expression. B) In the absence of YY1 in the visceral endoderm, decreased levels of paracrine VEGF result in defective angiogenesis, increased apoptosis and decreased proliferation in the adjacent mesoderm. Because of reduced VEGF signaling, the yolk sac mesoderm does not generate the paracrine signal(s) needed to maintain epithelial characteristics in the visceral endoderm, resulting in decreased HNF4α, a loss of large lysosomes and reduced CDH1 levels.</p
<p>Immunofluorescence analysis of sectioned WT (A–D, I–K) and cKO tissue (E–H, L–N) at the ... more <p>Immunofluorescence analysis of sectioned WT (A–D, I–K) and cKO tissue (E–H, L–N) at the stages indicated. A–C, I) YY1 (green) is ubiquitous in WT embryonic and extraembryonic tissues. A–B, D, K) HNF4α (red, orange when co-expressed with YY1) labels the visceral endoderm (A–B, D) and the developing liver bud (K). E–G, L) In cKO embryos, YY1 is downregulated in the extraembryonic visceral endoderm (VE) at 7.5 <i>dpc</i> (E) and is completely lost in the embryonic visceral endoderm by 8.75 <i>dpc</i> (F), when YY1 is also depleted in the definitive endoderm (DE) of the foregut. By 9.25 <i>dpc</i> YY1 is lost in most cells of the liver bud (L). E–F, H, N) Although HNF4α is present in the YY1-deficient visceral endoderm until 8.75 <i>dpc</i> (E, F) it is greatly reduced in both the visceral endoderm and in the nascent liver bud by 9.5 <i>dpc</i>. J, M) Despite the loss of YY1 in the nascent liver bud, the liver bud differentiation marker PROX1 is maintained in the cKO liver bud (M) at levels comparable to that observed in WT (J). The dotted line in C–D and G–H represent the division between the visceral endoderm and mesoderm derivative of the yolk sac, while in I–N the dashed line outlines the liver bud (LB).</p
Holothuria glaberrima exhibits the capacity to regenerate body parts after injury or loss. This a... more Holothuria glaberrima exhibits the capacity to regenerate body parts after injury or loss. This ability is mediated by an initial blastemal formation which requires proliferation and reorganization...
In recent years, transcriptomic databases have become one of the main sources for protein discove... more In recent years, transcriptomic databases have become one of the main sources for protein discovery. In our studies of nervous system and digestive tract regeneration in echinoderms, we have identified several transcripts that have attracted our attention. One of these molecules corresponds to a previously unidentified transcript (Orpin) from the sea cucumber Holothuria glaberrima that appeared to be upregulated during intestinal regeneration. We have now identified a second highly similar sequence and analyzed the predicted proteins using bioinformatics tools. Both sequences have EF-hand motifs characteristic of calcium-binding proteins (CaBPs) and N-terminal signal peptides. Sequence comparison analyses such as multiple sequence alignments and phylogenetic analyses only showed significant similarity to sequences from other echinoderms or from hemichordates. Semi-quantitative RT-PCR analyses revealed that transcripts from these sequences are expressed in various tissues including m...
Mouse embryos lacking the polycomb group gene member Yin-Yang1 (YY1) die during the peri-implanta... more Mouse embryos lacking the polycomb group gene member Yin-Yang1 (YY1) die during the peri-implantation stage. To assess the post-gastrulation role of YY1, a conditional knock-out (cKO) strategy was used to delete YY1 from the visceral endoderm of the yolk sac and the definitive endoderm of the embryo. cKO embryos display profound yolk sac defects at 9.5 days post coitum (dpc), including disrupted angiogenesis in mesoderm derivatives and altered epithelial characteristics in the visceral endoderm. Significant changes in both cell death and proliferation were confined to the YY1-expressing yolk sac mesoderm indicating that loss of YY1 in the visceral endoderm causes defects in the adjacent yolk sac mesoderm. Production of Vascular Endothelial Growth Factor A (VEGFA) by the visceral endoderm is essential for normal growth and development of the yolk sac vasculature. Reduced levels of VEGFA are observed in the cKO yolk sac, suggesting a cause for the angiogenesis defects. Ex vivo culture with exogenous VEGF not only rescued angiogenesis and apoptosis in the cKO yolk sac mesoderm, but also restored the epithelial defects observed in the cKO visceral endoderm. Intriguingly, blocking the activity of the mesoderm-localized VEGF receptor, FLK1, recapitulates both the mesoderm and visceral endoderm defects observed in the cKO yolk sac. Taken together, these results demonstrate that YY1 is responsible for maintaining VEGF in the developing visceral endoderm and that a VEGF-responsive paracrine signal, originating in the yolk sac mesoderm, is required to promote normal visceral endoderm development.
The ubiquitin proteasome system (UPS) is the main proteolytic system of cells. Recent evidence su... more The ubiquitin proteasome system (UPS) is the main proteolytic system of cells. Recent evidence suggests that the UPS plays a regulatory role in regeneration processes. Here, we explore the possibility that the UPS is involved during intestinal regeneration of the sea cucumber Holothuria glaberrima. These organisms can regenerate most of their digestive tract following a process of evisceration. Initially, we identified components of H. glaberrima UPS, including sequences for Rpn10, β3, and ubiquitin-RPL40. Predicted proteins from the mRNA sequences showed high degree of conservation that ranged from 60% (Rpn10) to 98% (Ub-RPL40). Microarrays and RT-PCR experiments showed that these genes were upregulated during intestinal regeneration. In addition, we demonstrated expression of alpha 20S proteasome subunits and ubiquitinated proteins during intestinal regeneration and detected them in the epithelium and connective tissue of the regenerating intestine. Finally, the intestinal regener...
Among deuterostomes, the regenerative potential is maximally expressed in echinoderms, animals th... more Among deuterostomes, the regenerative potential is maximally expressed in echinoderms, animals that can quickly replace most injured organs. In particular, sea cucumbers are excellent models for studying organ regeneration since they regenerate their digestive tract after evisceration. However, echinoderms have been sidelined in modern regeneration studies partially because of the lack of genome-wide profiling approaches afforded by modern genomic tools.For the last decade, our laboratory has been using the sea cucumber Holothuria glaberrima to dissect the cellular and molecular events that allow for such amazing regenerative processes. We have already established an EST database obtained from cDNA libraries of normal and regenerating intestine at two different regeneration stages. This database now has over 7000 sequences. In the present work we used a custom-made microchip from Agilent with 60-mer probes for these ESTs, to determine the gene expression profile during intestinal re...
Not Available Bibtex entry for this abstract Preferred format for this abstract (see Preferences)... more Not Available Bibtex entry for this abstract Preferred format for this abstract (see Preferences) Find Similar Abstracts: Use: Authors Title Return: Query Results Return items starting with number Query Form Database: Astronomy Physics arXiv e-prints
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