Susan C. Chapman

Improved method for chick whole-embryo culture using a filter paper carrier.

We describe a simple method of chick whole‐embryo culture, which uses a filter paper carrier to hold the early blastoderm and vitelline membranes under tension while the embryo grows on a substratum of agar‐albumen. This is a quick and efficient means of setting up cultures of chick embryos beginning at pre‐primitive streak stages to stage 10 (stages X–XIV, Eyal‐Giladi and Kochav [1976] Dev Biol 49:321–337; stages 1–10, Hamburger and Hamilton [ 1951 ] J Morphol 88:49–92). This is an improvement on the original method of New, which used a glass ring and watch glass (New [1955] Exp Morphol 3:320–331). Our modification of News method, which we call EC (Early Chick, pronounced EASY) culture, facilitates several manipulations in early chick embryos, including microsurgery, grafting, bead implantation, microinjection, and electroporation. Using the EC method, embryos at stage 8 and older can be readily cultured either dorsal‐side up (in contrast to News method) or ventral‐side up, as desired; embryos younger than stage 8 can be culture only ventral‐side up (as with News method). We also discuss some alternative methods for setting up these cultures.

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.

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. Developmental Dynamics 229:668–676, 2004.

Dysregulation of the PDGFRA gene causes inflow tract anomalies including TAPVR: integrating evidence from human genetics and model organisms

Total anomalous pulmonary venous return (TAPVR) is a congenital heart defect inherited via complex genetic and/or environmental factors. We report detailed mapping in extended TAPVR kindreds and mutation analysis in TAPVR patients that implicate the PDGFRA gene in the development of TAPVR. Gene expression studies in mouse and chick embryos for both the Pdgfra receptor and its ligand Pdgf-a show temporal and spatial patterns consistent with a role in pulmonary vein (PV) development. We used an in ovo function blocking assay in chick and a conditional knockout approach in mouse to knock down Pdgfra expression in the developing venous pole during the period of PV formation. We observed that loss of PDGFRA function in both organisms causes TAPVR with low penetrance (approximately 7%) reminiscent of that observed in our human TAPVR kindreds. Intermediate inflow tract anomalies occurred in a higher percentage of embryos (approximately 30%), suggesting that TAPVR occurs at one end of a spectrum of defects. We show that the anomalous pulmonary venous connection seen in chick and mouse is highly similar to TAPVR discovered in an abnormal early stage embryo from the Kyoto human embryo collection. Whereas the embryology of the normal venous pole and PV is becoming understood, little is known about the embryogenesis or molecular pathogenesis of TAPVR. These models of TAPVR provide important insight into the pathogenesis of PV defects. Taken together, these data from human genetics and animal models support a role for PDGF-signaling in normal PV development, and in the pathogenesis of TAPVR.

Chick embryo proliferation studies using EdU labeling

Cell proliferation studies are an important experimental tool. The most commonly used thymidine analogues, tritiated thymidine and bromodeoxyuridine (BrdU) label cells during S‐phase. Both methods have significant drawbacks: low sensitivity in the case of tritiated thymidine and a denaturation step during BrdU detection that destroys most cellular epitopes, requiring careful optimization. The antibody against BrdU is also large and tissue penetration can be difficult. EdU (5′‐ethynyl‐2′‐deoxyuridine) is closely chemically related to BrdU, with detection achieved by a copper catalyzed reaction requiring a small fluorescently conjugated azide. Cell cultures, flow cytometry and high throughput studies using EdU‐labeled cells is exceptionally fast and does not require denaturation or antibodies. We have developed a tissue‐labeling technique in chick embryos using EdU. Following EdU chemistry to detect proliferating cells, the tissue can undergo immunolabeling. We demonstrate fluorescent EdU chemistry followed by Tuj1 antibody staining resulting in multiplex fluorescent tissues. Developmental Dynamics 238:944–949, 2009.

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. J. Comp. Neurol. 487:428–440, 2005.

Identification of differentially expressed genes in early inner ear development.

To understand the etiology of congenital hearing loss, a comprehensive understanding of the molecular genetic mechanisms underlying normal ear development is required. We are identifying genes involved in otogenesis, with the longer term goal of studying their mechanisms of action, leading to inner ear induction and patterning. Using Agilent microarrays, we compared the differential expression of a test domain (which consisted of the pre-otic placodal ectoderm with the adjacent hindbrain ectoderm and the underlying mesendodermal tissues) with a rostral control domain (which included tissue that is competent, but not specified, to express inner ear markers in explant assays). We identified 1261 transcripts differentially expressed between the two domains at a 2-fold or greater change: 463 were upregulated and 798 were downregulated in the test domain. We validated the differential expression of several signaling molecules and transcription factors identified in this array using in situ hybridization. Furthermore, the expression patterns of the validated group of genes from the test domain were explored in detail to determine how the timing of their expression relates to specific events of otic induction and development. In conclusion, we identified a number of novel candidate genes for otic placode induction.

Can you hear me now? Understanding vertebrate middle ear development.

The middle ear is a composite organ formed from all three germ layers and the neural crest. It provides the link between the outside world and the inner ear, where sound is transduced and routed to the brain for processing. Extensive classical and modern studies have described the complex morphology and origin of the middle ear. Non-mammalian vertebrates have a single ossicle, the columella. Mammals have three functionally equivalent ossicles, designated the malleus, incus and stapes. In this review, I focus on the role of genes known to function in the middle ear. Genetic studies are beginning to unravel the induction and patterning of the multiple middle ear elements including the tympanum, skeletal elements, the air-filled cavity, and the insertion point into the inner ear oval window. Future studies that elucidate the integrated spatio-temporal signaling mechanisms required to pattern the middle ear organ system are needed. The longer-term translational benefits of understanding normal and abnormal ear development will have a direct impact on human health outcomes.

Lbx1 marks a subset of interneurons in chick hindbrain and spinal cord

The putative transcription factor Lbx1 is expressed in the mantle zone of the hindbrain and spinal cord caudal to rhombomere 1, in a specific domain of the alar plate. The Lbx1 domain overlaps with the expression domains for Tlx3 and partially with the domains for Pax2/Lim1. The ventral border of the Lbx1 domain coincides with the ventral border of the dorsalmost Serrate1 stripe in the ventricular zone. The latter borders the intermediate stripe of both Delta and Lunatic fringe expression. The Lbx1 domain contains differentiated interneurons that project into the lateral longitudinal fasciculus.

Computational and Experimental Approaches to Reveal the Effects of Single Nucleotide Polymorphisms with Respect to Disease Diagnostics

DNA mutations are the cause of many human diseases and they are the reason for natural differences among individuals by affecting the structure, function, interactions, and other properties of DNA and expressed proteins. The ability to predict whether a given mutation is disease-causing or harmless is of great importance for the early detection of patients with a high risk of developing a particular disease and would pave the way for personalized medicine and diagnostics. Here we review existing methods and techniques to study and predict the effects of DNA mutations from three different perspectives: in silico, in vitro and in vivo. It is emphasized that the problem is complicated and successful detection of a pathogenic mutation frequently requires a combination of several methods and a knowledge of the biological phenomena associated with the corresponding macromolecules.