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In JoVE (1)
Other Publications (9)
- Developmental Biology
- Proceedings of the National Academy of Sciences of the United States of America
- Journal of the American Society of Nephrology : JASN
- Matrix Biology : Journal of the International Society for Matrix Biology
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Developmental Dynamics : an Official Publication of the American Association of Anatomists
- Development (Cambridge, England)
- Development Genes and Evolution
- The Journal of Investigative Dermatology
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Articles by Alan R. Godwin in JoVE
JoVE General
In vivo Elektroporering av Morpholinos i Regenerating Adult Zebrafish stjärtfenan
1Department of Biological Sciences, Center for Zebrafish Research, University of Notre Dame, 2Department of Microbiology, Immunology, and Pathology, Colorado State University, 3Departments of Anatomy and Cell Biology and Ophthalmology, Wayne State University School of Medicine
Vi beskriver en metod för att villkorligt knockdown uttrycket av ett målprotein vid vuxen zebrafisk fena regenerering. Denna teknik innebär att mikro-injektion och elektroporera morpholinos Antisensoligonukleotid i fin vävnad, vilket gör att testa proteinets roll i olika stadier av fin förnyelse, inklusive sårläkning, blastema bildning, och regenerativ utväxt.
Other articles by Alan R. Godwin on PubMed
Differences in Expression Pattern and Function Between Zebrafish Hoxc13 Orthologs: Recruitment of Hoxc13b into an Early Embryonic Role
Vertebrate Hox genes are generally believed to initiate expression at the primitive streak or early neural plate stages. The timing and spatial restrictions of the Hox expression patterns during these stages correlate well with their demonstrated role in axial patterning. Here we demonstrate that one zebrafish hoxc13 ortholog, hoxc13a, has an expression pattern in the developing tail bud that is consistent with the gene playing a role in axial patterning. However, the second hoxc13 ortholog, hoxc13b, is maternally expressed and is detectable in every cell of early cleavage embryos through gastrulae. In addition, both transcript and protein are detectable at these stages. At 19 h post fertilization (hpf), hoxc13b expression is up-regulated in the tail bud, becoming restricted to the tail bud by 24 hpf. Importantly, by 24 hpf, hoxc13b morphants show a specific developmental delay, which can be rescued by co-injecting synthetic capped hoxc13a or hoxc13b message. These data suggest some functional divergence due to altered expression patterns of the two hoxc13 orthologs after duplication. Further characterization of the hoxc13b morphant delay reveals that it is biphasic in nature, with the first phase of the delay occurring before gastrulation, suggesting a new role for vertebrate Hox genes before their conserved role in axial patterning. The extent of the delay does not change through 20 hpf; however, an additional delay emerges at this time. Notably, this second phase of the delay correlates with hoxc13b expression pattern becoming restricted to the tail bud.
A Prolactin Family Paralog Regulates Reproductive Adaptations to a Physiological Stressor
Successful species develop strategies to optimize their reproductive performance. This optimization likely includes the evolution of genes that specifically permit reproduction in physiologically challenging conditions. The prolactin (PRL) family gene cluster is one of 25 mouse-specific gene clusters, the majority of which are associated with reproduction. A prevailing theme characterizing the PRL family is its connection with pregnancy and mechanisms controlling viviparity. PRL-like protein A (PLP-A) is one of 26 genes located within the PRL family locus. It is a nonclassical member of the PRL family (e.g., PLP-A does not use the PRL receptor) produced by trophoblast cells of the chorioallantoic placenta and acts on uterine natural killer cells. In this report, the biology of PLP-A has been investigated by generating mice with a PLP-A null mutation. Under standardized animal husbandry conditions, PLP-A possesses modest effects on reproductive performance. However, this same gene is critical for reproduction when mice are exposed to a physiological stressor. Wild-type mice exposed to hypobaric hypoxia during gestation readily adapt and maintain their pregnancies, whereas PLP-A null mutant mice fail to adapt, resulting in pregnancy failure. PLP-A contributes to hypoxia-induced adaptations critical to hemochorial placentation and thus nutrient flow to extraembryonic and embryonic tissues. The findings provide insights into species-specific reproductive adaptations.
Integration of Embryonic Stem Cells in Metanephric Kidney Organ Culture
Many stages of nephrogenesis can be studied using cultured embryonic kidneys, but there is no efficient technique available to readily knockdown or overexpress transgenes for rapid evaluation of resulting phenotypes. Embryonic stem (ES) cells have unlimited developmental potential and can be manipulated at the molecular genetic level by a variety of methods. The aim of this study was to determine if ES cells could respond to developmental signals within the mouse embryonic day 12 to embryonic day 13 (E12 to E13) kidney microenvironment and incorporate into kidney structures. ROSA26 ES cells were shown to express beta-galactosidase ubiquitously when cultured in the presence of leukemia inhibitory factor to suppress differentiation. When these cells were microinjected into E12 to E13 metanephroi and then placed in transwell organ culture, ES cell-derived, beta-galactosidase-positive cells were identified in epithelial structures resembling tubules. On rare occasions, individual ES cells were observed in structures resembling glomerular tufts. Electron microscopy showed that the ES cell-derived tubules were surrounded by basement membrane and had apical microvilli and junctional complexes. Marker analysis revealed that a subset of these epithelial tubules bound Lotus tetragonolobus and expressed alpha(1) Na(+)/K(+) ATPase. ES cells were infected before injection with a cytomegalovirus promoter-green fluorescence protein (GFP) adenovirus and GFP expression was found as early as 18 h, persisting for up to 48 h in cultured kidneys. This ES cell technology may achieve the objective of obtaining a versatile cell culture system in which molecular interventions can be used in vitro and consequences of these perturbations on the normal kidney development program in vivo can be studied.
Matrix Metalloproteinase Expression and Function During Fin Regeneration in Zebrafish: Analysis of MT1-MMP, MMP2 and TIMP2
Matrix metalloproteinases (MMPs) play key roles in the turnover of extracellular matrix (ECM) and, thereby, function as key regulators of cell-ECM interactions during development. In spite of their importance during developmental processes, relatively little has been reported about the role of these metalloproteinases during limb development and regeneration. To approach the problem of cell-ECM interactions during limb (fin) regeneration, we have utilized zebrafish as an experimental model. Based on previous MMP cloning studies from our laboratory, the current study has focused on the expression of membrane-type 1 metalloproteinase (MT1-MMP), gelatinase A (MMP-2) and endogenous tissue inhibitor 2 of metalloproteinases (TIMP-2) during fin regeneration in adult zebrafish. In situ analysis indicated co-expression of zmt1-mmp, zmmp-2, and ztimp-2 mRNA transcripts in regenerating caudal fins. In situ gelatin-zymography confirmed the presence of active metalloproteinases in regenerating fins. zmt1-mmp, zmmp-2, and ztimp-2 mRNA transcripts were expressed in the blastema and basal epithelium during caudal fin regeneration while expression of type IV collagen [zcol-IV(a5)] transcripts (a basal lamina component) was restricted to the basal epithelium. Fin outgrowth was greatly reduced in the presence of GM6001 (an inhibitor of MMP activity) indicating the importance of these enzymes during fin regeneration. Previous studies by Itoh (EMBO, 2001) indicated that expression of a vertebrate MT1-MMP construct containing only the hemopexin-transmembrane-cytoplasmic domains (MT1HPX) resulted in blockage of MT1-MMP homophilic complex formation and subsequent inhibition of pro-MMP-2 activation. Interference with homophilic complex formation was attributed to expression of the hemopexin domain at the cell surface. Building upon these earlier findings, the current study found that ectopic expression of MT1HPX in fin regenerates inhibited the regeneration process and resulted in a reduction in cell proliferation in the blastema. Taken together, these results indicate that MMPs have an important role during fin regeneration in zebrafish.
Cre-mediated Site-specific Recombination in Zebrafish Embryos
Cre-mediated site-specific recombination has become an invaluable tool for manipulation of the murine genome. The ability to conditionally activate gene expression or to generate chromosomal alterations with this same tool would greatly enhance zebrafish genetics. This study demonstrates that the HSP70 promoter can be used to inducibly control expression of an enhanced green fluorescent protein (EGFP) -Cre fusion protein. The EGFP-Cre fusion protein is capable of promoting recombination between lox sites in injected plasmids or in stably inherited transgenes as early as 2 hr post-heat shock induction. Finally, the levels of Cre expression achieved in a transgenic fish line carrying the HSP70-EGFP-cre transgene are compatible with viability and both male and female transgenic fish are fertile subsequent to induction of EGFP-Cre expression. Hence, our data suggests that Cre-mediated recombination is a viable means of manipulating gene expression in zebrafish.
Inhibition of Zebrafish Fin Regeneration Using in Vivo Electroporation of Morpholinos Against Fgfr1 and Msxb
Increased interest in using zebrafish as a model organism has led to a resurgence of fin regeneration studies. This has allowed for the identification of a large number of gene families, including signaling molecules and transcription factors, which are expressed during regeneration. However, in cases where no specific inhibitor is available for the gene product of interest, determination of a functional role for these genes has been difficult. Here we demonstrate that in vivo electroporation of morpholino oligonucleotides is a feasible approach for protein knock-down during fin regeneration. Morpholino oligonucleotides against fgfr1 and msxb were utilized and knock-down of both proteins resulted in reduced fin outgrowth. Importantly, Fgfr1 knock-down phenocopied outgrowth inhibition obtained with an Fgfr1 inhibitor. Furthermore, this method provided direct evidence for a functional role for msxb in caudal fin regeneration. Finally, knock-down of Fgfr1, but not Msxb, affected the blastemal expression of msxc, suggesting this technique can be used to determine epistasis in genetic pathways affecting regeneration. Thus, this convenient reverse genetic approach allows researchers to quickly (1) assess the function of genes known to be expressed during fin regeneration, (2) screen genes for functional relevance during fin regeneration, and (3) assign genes to the molecular pathways underlying fin regeneration.
A Uterine Decidual Cell Cytokine Ensures Pregnancy-dependent Adaptations to a Physiological Stressor
In the mouse, decidual cells differentiate from uterine stromal cells in response to steroid hormones and signals arising from the embryo. Decidual cells are crucially involved in creating the intrauterine environment conducive to embryonic development. Among their many functions is the production of cytokines related to prolactin (PRL), including decidual prolactin-related protein (DPRP). DPRP is a heparin-binding cytokine, which is abundantly expressed in uterine decidua. In this investigation, we have isolated the mouse Dprp gene, characterized its structure and evaluated its biological role. Dprp-null mice were made by replacing exons 2 to 6 of the Dprp gene with an in-frame enhanced green fluorescent protein (EGFP) gene and a neomycin (neo) resistance cassette. Heterozygous intercross breeding of the mutant mice yielded the expected mendelian ratio. Pregnant heterozygote females expressed EGFP within decidual tissue in locations identical to endogenous Dprp mRNA and protein expression. Homozygous Dprp-null mutant male and female mice were viable, exhibited normal postnatal growth rates, were fertile and produced normal litter sizes. A prominent phenotype was observed when pregnant Dprp-null mice were exposed to a physiological stressor. DPRP deficiency interfered with pregnancy-dependent adaptations to hypoxia resulting in pregnancy failure. Termination of pregnancy was associated with aberrations in mesometrial decidual cells, mesometrial vascular integrity, and disruptions in chorioallantoic placenta morphogenesis. The observations suggest that DPRP participates in pregnancy-dependent adaptations to a physiological stressor.
Both Hoxc13 Orthologs Are Functionally Important for Zebrafish Tail Fin Regeneration
Hox genes are re-expressed during regeneration in many species. Given their important role in body plan development, it has been assumed, but not directly shown, that they play a functional role in regeneration. In this paper we show that morpholino-mediated knockdown of either Hoxc13a or Hoxc13b during the process of zebrafish tail fin regeneration results in a significant reduction of regenerative outgrowth. Furthermore, cellular proliferation within the blastema is directly affected in both knockdowns. Hence, similar to the demonstration of unique functions of multiple Hox genes during limb formation, both Hoxc13 orthologs have distinct functions in regeneration.
The Nude Mutant Gene Foxn1 is a HOXC13 Regulatory Target During Hair Follicle and Nail Differentiation
Among the Hox genes, homeobox C13 (Hoxc13) has been shown to be essential for proper hair shaft differentiation, as Hoxc13 gene-targeted (Hoxc13(tm1Mrc)) mice completely lack external hair. Because of the remarkable overt phenotypic parallels to the Foxn1(nu) (nude) mutant mice, we sought to determine whether Hoxc13 and forkhead box N1 (Foxn1) might act in a common pathway of hair follicle (HF) differentiation. We show that the alopecia exhibited by both the Hoxc13(tm1Mrc) and Foxn1(nu) mice is because of strikingly similar defects in hair shaft differentiation and that both mutants suffer from a severe nail dystrophy. These phenotypic similarities are consistent with the extensive overlap between Hoxc13 and Foxn1 expression patterns in the HF and the nail matrix. Furthermore, DNA microarray analysis of skin from Hoxc13(tm1Mrc) mice identified Foxn1 as significantly downregulated along with numerous hair keratin genes. This Foxn1 downregulation apparently reflects the loss of direct transcriptional control by HOXC13 as indicated by our results obtained through co-transfection and chromatin immunoprecipitation (ChIP) assays. As presented in the discussion, these data support a regulatory model of keratinocyte differentiation in which HOXC13-dependent activation of Foxn1 is part of a regulatory cascade controlling the expression of terminal differentiation markers.
