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Gary C Schoenwolf

Publications and source records attributed to Gary C Schoenwolf.

At least 19 recordsLinked to original sources
PubMed2006

Restricted expression of Fgf16 within the developing chick inner ear.

Fibroblast growth factor (FGF) signaling is required for otic placode induction and patterning of the developing inner ear. We have cloned the chick ortholog of Fgf16 and analyzed its expression pattern in the early chick embryo. Expression is restricted to the otic placode and developing inner ear through all the stages examined. By the closed otocyst stage, expression has resolved to anterior and posterior domains that partially overlap with those of bone morphogenetic protein 4 (Bmp4), a marker of the developing sensory patches, the cristae of the anterior and posterior semicircular canals. Platelet-derived growth factor alpha (PDGFA), another growth factor with restricted otic expression, also overlaps with Fgf16 expression. The restricted expression pattern of Fgf16 suggests a role for FGF signaling in the patterning of the sensory cristae, together with BMP signaling.

Amino Acid Sequence↗
PubMed2006

Optimized cationic lipid-based gene delivery reagents for use in developing vertebrate embryos.

We have used cationic lipid-based transfection reagents for ectopic gene expression experiments in developing vertebrate embryos. Lipofectamine, Lipofectamine 2000, and Lipofectamine enhanced with a disulfide linked pegylated lipid (mPEG-SS-DOPE) were initially tested and optimized in cell culture. Two reagent formulations, 1:4 (DNA:Lipofectamine 2000) Lipofectamine 2000, and 7.5% pegylated Lipofectamine, produced the highest levels of gene expression in vitro. Those formulations, containing the enhance green fluorescent protein reporter gene, were microinjected into intact vertebrate embryos -- systemically through the vasculature and locally into selected tissues -- to assess in vivo transfection efficiency. Whereas both formulations are capable of transfecting cells in developing embryos in vivo, greater transfection efficiencies in a broader range of tissue types were obtained with the pegylated Lipofectamine formulation. We conclude that in developing vertebrate embryos, optimized cationic lipid-based reagents are capable of producing significant levels of ectopic gene expression and can be used as alternatives to electroporation and viral-mediated gene delivery.

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PubMed2005

A three-dimensional atlas of pituitary gland development in the zebrafish.

The pituitary gland is unique to Chordates, with significant variation within this group, offering an excellent opportunity to increase insight into phylogenetic relationships within this phylum. The structure of the pituitary in adult Teleosts (class: Osteichthyes) is quite different from that in other chordates and is also variable among members of the class. Therefore, a complete description of the structure and development of the pituitary in members of this class is a critical component to our overall understanding of this gland. An obvious teleost model organism is the zebrafish, Danio rerio, as a significant amount of work has been done on the molecular control of pituitary development in this fish. However, very little work has been published on the morphological development of the pituitary in the zebrafish; the present study aims to fill this void. The pituitary develops from cells on the rostrodorsal portion of the head and reaches its final position, ventral to the hypothalamus, as the cephalic flexure occurs and the jaws and mouth form. The pituitary placode is juxtaposed to cells that will form the olfactory vesicles, the stomodeum, and the hatching gland. The volume of the pituitary is greatest at 24 hours post fertilization (hpf). From 24 to 120 hpf, the pituitary decreases in height and width as it undergoes convergent extension, increasing in length with the axis. The adenohypophysis is a morphologically distinct structure by 24 hpf, whereas the neurohypophysis remains indistinct until 72 hpf. The findings of this study correlate well with the available molecular data.

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PubMed2005

FGF8 initiates inner ear induction in chick and mouse.

In both chick and mouse, the otic placode, the rudiment of the inner ear, is induced by at least two signals, one from the cephalic paraxial mesoderm and the other from the neural ectoderm. In chick, the mesodermal signal, FGF19, induces neural ectoderm to express additional signals, including WNT8c and FGF3, resulting in induction of the otic placode. In mouse, mesodermal Fgf10 acting redundantly with neural Fgf3 is required for induction of the placode. To determine how the mesodermal inducers of the otic placode are localized, we took advantage of the unique strengths of the two model organisms. We show that endoderm is necessary for otic induction in the chick and that Fgf8, expressed in the chick endoderm subjacent to Fgf19, is both sufficient and necessary for the expression of Fgf19 in the mesoderm. In the mouse, Fgf8 is also expressed in endoderm as well as in other germ layers in the periotic placode region. We show that otic induction fails in embryos null for Fgf3 and hypomorphic for Fgf8 and expression of mesodermal Fgf10 is reduced. Thus, Fgf8 plays a critical upstream role in an FGF signaling cascade required for otic induction in chick and mouse.

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PubMed2005

Ubiquitous GFP expression in transgenic chickens using a lentiviral vector.

We report the first ubiquitous green fluorescent protein expression in chicks using a lentiviral vector approach, with eGFP under the control of the phosphoglycerol kinase promoter. Several demonstrations of germline transmission in chicks have been reported previously, using markers that produce tissue-specific, but not ubiquitous, expression. Using embryos sired by a heterozygous male, we demonstrate germline transmission in the embryonic tissue that expresses eGFP uniformly, and that can be used in tissue transplants and processed by in situ hybridization and immunocytochemistry. Transgenic tissue is identifiable by both fluorescence microscopy and immunolabeling, resulting in a permanent marker identifying transgenic cells following processing of the tissue. Stable integration of the transgene has allowed breeding of homozygous males and females that will be used to produce transgenic embryos in 100% of eggs laid upon reaching sexual maturity. These results demonstrate that a transgenic approach in the chick model system is viable and useful even though a relatively long generation time is required. The transgenic chick model will benefit studies on embryonic development, as well as providing the pharmaceutical industry with an economical bioreactor.

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PubMed2005

Vasculogenesis drives pulmonary vascular growth in the developing chick embryo.

Formation of the pulmonary vasculature has been described as occurring by outgrowth of existing vessels (angiogenesis), de novo formation of new vessels (vasculogenesis), or a combination of both processes. Uncertainty about the contribution of angiogenesis and vasculogenesis to pulmonary vascular formation is partly due to methodologic approaches. Evidence in favor of angiogenesis stems from studies that used vascular-filling methods. Such methods identify only directly continuous lumina. Evidence for vasculogenesis has been provided by the use of molecular markers of blood vessel endothelium. Use of both methods has not been combined in the same species, however. We hypothesized, based on published evidence from quail and mouse, that chick pulmonary vascular formation occurs by vasculogenesis. To test that hypothesis, we used vascular filling, serial section, and immunohistochemical methods to analyze the developing lungs of chick embryos from Hamburger and Hamilton stages 20 to 43. Vascular filling suggested that the lumen of the pulmonary arteries sprouted from the sixth pharyngeal arch arteries. However, serial sections and immunohistochemical localization of fetal liver kinase-1 protein, the receptor for vascular endothelial growth factor, showed that the pulmonary arterial tree formed from endothelial cell precursors and coalescence of isolated blood vessels in the mediastinal splanchnic mesenchyme centrally to the developing lung tissue distally. Pulmonary veins grew from the left atrium to the developing lungs. Pulmonary blood vessel formation occurred continuously throughout the embryonic period studied. Our results show that vasculogenesis is the main process by which the pulmonary vasculature forms in the developing chick embryo.

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PubMed2004

Mouse FGF15 is the ortholog of human and chick FGF19, but is not uniquely required for otic induction.

The inner ear develops from an ectodermal placode that is specified by inductive signals from the adjacent neurectoderm and underlying mesoderm. In chick, fibroblast growth factor (Fgf)-19 is expressed in mesoderm underlying the presumptive otic placode, and human FGF19 induces expression of otic markers in a tissue explant containing neural plate and surface ectoderm. We show here that mouse Fgf15 is the sequence homolog of chick and human Fgf19/FGF19. In addition, we show that FGF15, like FGF19, is sufficient to induce expression of otic markers in a chick explant assay, suggesting that these FGFs are orthologs. Mouse embryos lacking Fgf15, however, do not have otic abnormalities at E9.5-E10.5, suggesting that Fgf15 is not uniquely required for otic induction or early patterning of the otocyst. To compare FGF15 and FGF19 signaling components and assess where signals potentially redundant with FGF15 might function, we determined the expression patterns of Fgf15 and Fgf19. Unlike Fgf19, Fgf15 is not expressed in mesoderm underlying the presumptive otic placode, but is expressed in the adjacent neurectoderm. Fgfr4, which encodes the likely receptor for both FGF19 and FGF15, is expressed in the neurectoderm of both species, and is also expressed in the mesoderm only in chick. These results suggest the hypotheses that during otic induction, FGF19 signals in either an autocrine fashion to the mesoderm or a paracrine fashion to the neurectoderm, whereas FGF15 signals in an autocrine fashion to the neurectoderm. Thus, the FGFs that signal to the neurectoderm are the best potential candidates for redundancy with FGF15 during mouse otic development.

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PubMed2004

Expression analysis of chick Wnt and frizzled genes and selected inhibitors in early chick patterning.

Wnt signaling is an important component in patterning the early embryo and specifically the neural plate. Studies in Xenopus, mouse, and zebrafish have shown that signaling by members of the Wnt family of secreted signaling factors, their Frizzled receptors and several inhibitors (sFRP1, sFRP2, sFRP3/Frzb1, Crescent/Frzb2, Dkk1, and Cerberus) are involved. However, very little is known about the expression of genes in the Wnt signaling pathway during early anterior neural patterning in chick. We have performed an expression analysis at neural plate stages of several Wnts, Frizzled genes, and Wnt signaling pathway inhibitors using in situ hybridization. The gene expression patterns of these markers are extremely dynamic. We have identified two candidate molecules for anterior patterning of the neural plate, Wnt1 and Wnt8b, which are expressed in the rostral ectoderm at these stages. Further functional studies on the roles of these markers are underway.

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PubMed2004

Rapid triple-labeling method combining in situ hybridization and double immunocytochemistry.

A new, rapid method is described for combining in situ hybridization and immunocytochemistry to define cell populations and to map three-dimensional movements of groups of labeled cells within developing chick embryos. The method allows fluorescently labeled cells to be followed in living embryos and subsequently detected as a permanent reaction product for detailed three-dimensional analysis by immunocytochemistry in histological serial sections. Cell identity can be ascertained using a specific riboprobe and in situ hybridization. With this approach, the movements of two groups of cells can be mapped simultaneously (using two different fluorescent trackers and, subsequently, two different chromogens for immunocytochemistry) to analyze relative movements within an embryo, and when combined with in situ hybridization with a specific riboprobe for cell identity, allows fate mapping studies to be conducted using molecular criteria, rather than solely at morphological/positional criteria. The improved method enables the investigator to extract substantially more information from individual embryos, maximizing the results obtained from labor-intensive fate mapping studies.

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PubMed2004

DD achieves Zen.

Explore the source record for details and available documents.

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PubMed2003

Anterior identity is established in chick epiblast by hypoblast and anterior definitive endoderm.

Previous studies of head induction in the chick have failed to demonstrate a clear role for the hypoblast and anterior definitive endoderm (ADE) in patterning the overlying ectoderm, whereas data from both mouse and rabbit suggest patterning roles for anterior visceral endoderm (AVE) and ADE. Based on similarity of gene expression patterns, fate and a dual role in 'protecting' the prospective forebrain from caudalising influences of the organiser, the chick hypoblast has been suggested to be the homologue of the mouse anterior visceral endoderm. In support of this, when transplanted to chick embryos, the rabbit AVE induces anterior markers in the chick epiblast. To reevaluate the role of the hypoblast/ADE (lower layer) in patterning the chick ectoderm, we used rostral blastoderm isolates (RBIs) as an assay, that is, rostral regions of blastoderms transected at levels rostral to the node. RBIs are, therefore, free from the influences of Hensen's node and ingressing axial mesoderm - tissues that are able to induce Ganf, the earliest specific marker of anterior neural plate. We demonstrate, using such RBIs (or RBIs dissected to remove the lower layer with or without tissue replacement), that the hypoblast/ADE (lower layer) is required and sufficient for patterning anterior positional identity in the overlying ectoderm, leading to expression of Ganf in neuroectoderm. Our results suggest that patterning of anterior positional identity and specification of neural identity are separable events operating to pattern the rostral end of the early chick embryo. Based on this new evidence we propose a revised model for establishing anteroposterior polarity, neural specification and head patterning in the early chick that is consonant with that occurring in other vertebrates.

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PubMed2003

Localization of cartilage linking protein 1 during primary neurulation in the chick embryo.

Primary neurulation is a form-shaping event during the early development of the vertebrate embryo in which the neural plate is rolled up into the neural tube, the rudiment of the central nervous system. In an effort to identify genes specifically expressed in tissues lateral to the chick neural plate--tissues known to generate extrinsic forces for primary neurulation--we designed a subtractive scheme and identified a positive clone as the gene encoding chick cartilage linking protein 1 (CRTL1). CRTL1 (also known as link protein) is a small glycoprotein of the extracellular matrix that was originally identified for its role in stabilizing aggregates of aggrecan and hyaluronan in cartilage. In addition to being expressed in cartilage, CRTL1 is also immunolocalized in several noncartilaginous tissues as assessed with the 4B6 monoclonal antibody. Using the 4B6 antibody and the G9 riboprobe derived from our subtraction, we report the detailed distribution of CRTL1 protein and crtl1 transcripts during primary neurulation in chick embryos. This report emphasizes and briefly discusses important differences between the RNA expression pattern and the domains of accumulation of the protein. CRTL1 prominently accumulates in the basal lamina of the epidermal ectoderm just lateral to the neural plate. Based on the crucial role of the interface between this tissue and the neuroepithelium in the formation of the neural folds, and because of the biophysical role of hyaluronan in tissue morphogenesis, we propose that crtl1 represents is an excellent candidate neurulation gene, worthy of further study.

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PubMed2003

Hensen's node gives rise to the ventral midline of the foregut: implications for organizing head and heart development.

Patterning of the ventral head has been attributed to various cell populations, including endoderm, mesoderm, and neural crest. Here, we provide evidence that head and heart development may be influenced by a ventral midline endodermal cell population. We show that the ventral midline endoderm of the foregut is generated directly from the extreme rostral portion of Hensen's node, the avian equivalent of the Spemann organizer. The endodermal cells extend caudally in the ventral midline from the prechordal plate during development of the foregut pocket. Thus, the prechordal plate appears as a mesendodermal pivot between the notochord and the ventral foregut midline. The elongating ventral midline endoderm delimits the right and left sides of the ventral foregut endoderm. Cells derived from the midline endoderm are incorporated into the endocardium and myocardium during closure of the foregut pocket and fusion of the bilateral heart primordia. Bilateral ablation of the endoderm flanking the midline at the level of the anterior intestinal portal leads to randomization of heart looping, suggesting that this endoderm is partitioned into right and left domains by the midline endoderm, thus performing a function similar to that of the notochord in maintaining left-right asymmetry. Because of its derivation from the dorsal organizer, its extent from the forebrain through the midline of the developing face and pharynx, and its participation in formation of a single midline heart tube, we propose that the ventral midline endoderm is ideally situated to function as a ventral organizer of the head and heart.

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PubMed2003

Expression of mouse fibroblast growth factor and fibroblast growth factor receptor genes during early inner ear development.

The inner ear, which mediates hearing and equilibrium, develops from an ectodermal placode located adjacent to the developing hindbrain. Induction of the placode and its subsequent morphogenesis and differentiation into the inner ear epithelium and its sensory neurons, involves signalling interactions within and between otic and non-otic tissues. Several members of the fibroblast growth factor (FGF) family play important roles at various stages of otic development; however, there are additional family members that have not been evaluated. In this study, we surveyed the expression patterns of 18 mouse Fgf and 3 Fgf receptor (Fgfr) genes during early otic development. Two members of the Fgf family, Fgf4 and Fgf16, and all three tested members of the Fgfr family, Fgfr2c, Fgfr3c, and Fgfr4, were expressed in tissues relevant to inner ear development. Fgf4 transcripts were expressed in the preplacodal and placodal ectoderm, suggesting potential roles in placode induction and/or maintenance. Fgf16 was expressed in the posterior otic cup and vesicle, suggesting roles in otic cell fate decisions and/or axis formation.

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