Diana K Darnell

Interests

Research

Developmental Biology

Teaching

Gross Anatomy, Embryology, Case Based Instruction, Active learning pedagogy

Courses

Scholarly Contributions

Journals/Publications

• Antin, P. B., Yatskievych, T. A., Davey, S., & Darnell, D. K. (2014). GEISHA: an evolving gene expression resource for the chicken embryo. Nucleic acids research, 42(Database issue), D933-7.
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  GEISHA (Gallus Expression In Situ Hybridization Analysis; http://geisha.arizona.edu) is an in situ hybridization gene expression and genomic resource for the chicken embryo. This update describes modifications that enhance its utility to users. During the past 5 years, GEISHA has undertaken a significant restructuring to more closely conform to the data organization and formatting of Model Organism Databases in other species. This has involved migrating from an entry-centric format to one that is gene-centered. Database restructuring has enabled the inclusion of data pertaining to chicken genes and proteins and their orthologs in other species. This new information is presented through an updated user interface. In situ hybridization data in mouse, frog, zebrafish and fruitfly are integrated with chicken genomic and expression information. A resource has also been developed that integrates the GEISHA interface information with the Online Mendelian Inheritance in Man human disease gene database. Finally, the Chicken Gene Nomenclature Committee database and the GEISHA database have been integrated so that they draw from the same data resources.
• Darnell, D. K., & Antin, P. B. (2014). LNA-based in situ hybridization detection of mRNAs in embryos. Methods in molecular biology (Clifton, N.J.), 1211, 69-76.
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  In situ hybridization (ISH) in embryos allows the visualization of specific RNAs as a readout of gene expression during normal development or after experimental manipulations. ISH using short DNA probes containing locked nucleic acid nucleotides (LNAs) holds the additional advantage of allowing the detection of specific RNA splice variants or of closely related family members that differ in only short regions, creating new diagnostic and detection opportunities. Here we describe methods for using short (14-24 nt) DNA probes containing LNA nucleotides to detect moderately to highly expressed RNAs in whole chick embryos during the first 5 days of embryonic development. The protocol is easily adaptable for use with embryos of other vertebrate species.
• Darnell, D. K., Zhang, L. S., Hannenhalli, S., & Yaklichkin, S. Y. (2014). Developmental expression of chicken FOXN1 and putative target genes during feather development. The International journal of developmental biology, 58(1), 57-64.
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  FOXN1 is a member of the forkhead box family of transcription factors. FOXN1 is crucial for hair outgrowth and thymus differentiation in mammals. Unlike the thymus, which is found in all amniotes, hair is an epidermal appendage that arose after the last shared common ancestor between mammals and birds, and hair and feathers differ markedly in their differentiation and gene expression. Here, we show that FOXN1 is expressed in embryonic chicken feathers, nails and thymus, demonstrating an evolutionary conservation that goes beyond obvious homology. At embryonic day (ED) 12, FOXN1 is expressed in some feather buds and at ED13 expression extends along the length of the feather filament. At ED14 FOXN1 mRNA is restricted to the proximal feather filament and is not detectable in distal feather shafts. At the base of the feather, FOXN1 is expressed in the epithelium of the feather sheath and distal barb and marginal plate, whereas in the midsection FOXN1 transcripts are mainly detected in the barb plates of the feather filament. FOXN1 is also expressed in claws; however, no expression was detected in skin or scales. Despite expression of FOXN1 in developing feathers, examination of chick homologs of five putative mammalian FOXN1 target genes shows that, while these genes are expressed in feathers, there is little similarity to the FOXN1 expression pattern, suggesting that some gene regulatory networks may have diverged during evolution of epidermal appendages.
• Yaklichkin, S. Y., Darnell, D. K., Pier, M. V., Antin, P. B., & Hannenhalli, S. (2011). Accelerated evolution of 3'avian FOXE1 genes, and thyroid and feather specific expression of chicken FoxE1. BMC evolutionary biology, 11, 302.
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  The forkhead transcription factor gene E1 (FOXE1) plays an important role in regulation of thyroid development, palate formation and hair morphogenesis in mammals. However, avian FOXE1 genes have not been characterized and as such, codon evolution of FOXE1 orthologs in a broader evolutionary context of mammals and birds is not known.
• Antin, P. B., Pier, M., Sesepasara, T., Yatskievych, T. A., & Darnell, D. K. (2010). Embryonic expression of the chicken Krüppel-like (KLF) transcription factor gene family. Developmental dynamics : an official publication of the American Association of Anatomists, 239(6), 1879-87.
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  The Krüppel-like transcription factors (KLF) are zinc finger proteins that activate and suppress target gene transcription. Although KLF factors have been implicated in regulating many developmental processes, a comprehensive gene expression analysis has not been reported. Here we present the chicken KLF gene family and expression during the first five days of embryonic development. Fourteen chicken KLF genes or expressed sequences have been previously identified. Through synteny analysis and cDNA mapping, we have identified the KLF9 gene and determined that the gene presently named KLF1 is the true ortholog of KLF17 in other species. In situ hybridization expression analyses show that in general KLFs are broadly expressed in multiple cell and tissue types. Expression of KLFs 3, 7, 8, and 9, is widespread at all stages examined. KLFs 2, 4, 5, 6, 10, 11, 15, and 17 show more restricted patterns that suggest multiple functions during early stages of embryonic development.
• Darnell, D. K., Stanislaw, S., Kaur, S., & Antin, P. B. (2010). Whole mount in situ hybridization detection of mRNAs using short LNA containing DNA oligonucleotide probes. RNA (New York, N.Y.), 16(3), 632-7.
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  In situ hybridization is widely used to visualize transcribed sequences in embryos, tissues, and cells. For whole mount detection of mRNAs in embryos, hybridization with an antisense RNA probe is followed by visual or fluorescence detection of target mRNAs. A limitation of this approach is that a cDNA template of the target RNA must be obtained in order to generate the antisense RNA probe. Here we investigate the use of short (12-24 nucleotides) locked nucleic acid (LNA) containing DNA probes for whole mount in situ hybridization detection of mRNAs. Following extensive protocol optimization, we show that LNA probes can be used to localize several mRNAs of varying abundances in chicken embryos. LNA probes also detected alternatively spliced exons that are processed in a tissue specific manner. The use of LNA probes for whole mount in situ detection of mRNAs will enable in silico design and chemical synthesis and will expand the general use of in situ hybridization for studies of transcriptional regulation and alternative splicing.
• Antin, P. B., Kaur, S., Stanislaw, S., Davey, S., Konieczka, J. H., Yatskievych, T. A., & Darnell, D. K. (2007). Gallus expression in situ hybridization analysis: a chicken embryo gene expression database. Poultry science, 86(7), 1472-7.
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  With sequencing of the chicken genome largely completed, significant effort is focusing on gene annotation, including acquiring information about the patterns of gene expression. The chicken embryo is ideally suited to provide detailed temporal and spatial expression information through in situ hybridization gene expression analysis in vivo. We have developed the Gallus expression in situ hybridization analysis (GEISHA) database and user interface (http://geisha.arizona.edu) to serve as a centralized repository of in situ hybridization photos and metadata from chicken embryos. This report describes the design and implementation the GEISHA database and Web site and illustrates its usefulness for researchers in the biomedical and poultry science communities. Results from a recent comprehensive expression analysis of microRNA expression in chicken embryos are also presented.
• Darnell, D. K., Kaur, S., Stanislaw, S., Davey, S., Konieczka, J. H., Yatskievych, T. A., & Antin, P. B. (2007). GEISHA: an in situ hybridization gene expression resource for the chicken embryo. Cytogenetic and genome research, 117(1-4), 30-5.
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  An important and ongoing focus of biomedical and agricultural avian research is to understand gene function, which for a significant fraction of genes remains unknown. A first step is to determine when and where genes are expressed during development and in the adult. Whole mount in situ hybridization gives precise spatial and temporal resolution of gene expression throughout an embryo, and a comprehensive analysis and centralized repository of in situ hybridization information would provide a valuable research tool. The GEISHA project (gallus expression in situ hybridization analysis) was initiated to explore the utility of using high-throughput in situ hybridization as a means for gene discovery and annotation in chicken embryos, and to provide a unified repository for in situ hybridization information. This report describes the design and implementation of a new GEISHA database and user interface (www.geisha.arizona.edu), and illustrates its utility for researchers in the biomedical and poultry science communities. Results obtained from a high throughput screen of microRNA expression in chicken embryos are also presented.
• Ason, B., Darnell, D. K., Wittbrodt, B., Berezikov, E., Kloosterman, W. P., Wittbrodt, J., Antin, P. B., & Plasterk, R. H. (2006). Differences in vertebrate microRNA expression. Proceedings of the National Academy of Sciences of the United States of America, 103(39), 14385-9.
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  MicroRNAs (miRNAs) attenuate gene expression by means of translational inhibition and mRNA degradation. They are abundant, highly conserved, and predicted to regulate a large number of transcripts. Several hundred miRNA classes are known, and many are associated with cell proliferation and differentiation. Many exhibit tissue-specific expression, which aids in evaluating their functions, and it has been assumed that their high level of sequence conservation implies a high level of expression conservation. A limited amount of data supports this, although discrepancies do exist. By comparing the expression of approximately 100 miRNAs in medaka and chicken with existing data for zebrafish and mouse, we conclude that the timing and location of miRNA expression is not strictly conserved. In some instances, differences in expression are associated with changes in miRNA copy number, genomic context, or both between species. Variation in miRNA expression is more pronounced the greater the differences in physiology, and it is enticing to speculate that changes in miRNA expression may play a role in shaping the physiological differences produced during animal development.
• Darnell, D. K., Kaur, S., Stanislaw, S., Konieczka, J. H., Konieczka, J. K., Yatskievych, T. A., & Antin, P. B. (2006). MicroRNA expression during chick embryo development. Developmental dynamics : an official publication of the American Association of Anatomists, 235(11), 3156-65.
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  MicroRNAs (miRNAs) are small, abundant, noncoding RNAs that modulate protein abundance by interfering with target mRNA translation or stability. miRNAs are detected in organisms from all domains and may regulate 30% of transcripts in vertebrates. Understanding miRNA function requires a detailed determination of expression, yet this has not been reported in an amniote species. High-throughput whole mount in situ hybridization was performed on chicken embryos to map expression of 135 miRNA genes including five miRNAs that had not been previously reported in chicken. Eighty-four miRNAs were detected before day 5 of embryogenesis, and 75 miRNAs showed differential expression. Whereas few miRNAs were expressed during formation of the primary germ layers, the number of miRNAs detected increased rapidly during organogenesis. Patterns highlighted cell-type, organ or structure-specific expression, localization within germ layers and their derivatives, and expression in multiple cell and tissue types and within sub-regions of structures and tissues. A novel group of miRNAs was highly expressed in most tissues but much reduced in one or a few organs, including the heart. This study presents the first comprehensive overview of miRNA expression in an amniote organism and provides an important foundation for investigations of miRNA gene regulation and function.
• Doyle, S. E., Scholz, M. J., Greer, K. A., Hubbard, A. D., Darnell, D. K., Antin, P. B., Klewer, S. E., & Runyan, R. B. (2006). Latrophilin-2 is a novel component of the epithelial-mesenchymal transition within the atrioventricular canal of the embryonic chicken heart. Developmental dynamics : an official publication of the American Association of Anatomists, 235(12), 3213-21.
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  Endothelial cells in the atrioventricular canal of the heart undergo an epithelial-mesenchymal transition (EMT) to form heart valves. We surveyed an on-line database (http://www.geisha.arizona.edu/) for clones expressed during gastrulation to identify novel EMT components. One gene, latrophilin-2, was identified as expressed in the heart and appeared to be functional in EMT. This molecule was chosen for further examination. In situ localization showed it to be expressed in both the myocardium and endothelium. Several antisense DNA probes and an siRNA for latrophilin-2 produced a loss of EMT in collagen gel cultures. Latrophilin-2 is a putative G-protein-coupled receptor and we previously identified a pertussis toxin-sensitive G-protein signal transduction pathway. Microarray experiments were performed to examine whether these molecules were related. After treatment with antisense DNA against latrophilin-2, expression of 1,385 genes and ESTs was altered. This represented approximately 12.5% of the microarray elements. In contrast, pertussis toxin altered only 103 (0.9%) elements of the array. There appears to be little overlap between the two signal transduction pathways. Latrophilin-2 is thus a novel component of EMT and provides a new avenue for investigation of this cellular process.
• Healy, K. H., Schoenwolf, G. C., & Darnell, D. K. (2001). Cell interactions underlying notochord induction and formation in the chick embryo. Developmental dynamics : an official publication of the American Association of Anatomists, 222(2), 165-77.
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  The development of the notochord in the chick is traditionally associated with Hensen's node (the avian equivalent of the organizer). However, recent evidence has shown that two areas outside the node (called the inducer and responder) are capable of interacting after ablation of Hensen's node to form a notochord. It was not clear from these studies what effect (if any) signals from these areas had on normal notochord formation. A third area, the postnodal region, may also contribute to notochord formation, although this has also been questioned. Using transection and grafting experiments, we have evaluated the timing and cellular interactions involved in notochord induction and formation in the chick embryo. Our results indicate that the rostral primitive streak, including the node, is not required for formation of the notochord in rostral blastoderm isolates transected at stages 3a/b. In addition, neither the postnodal region nor the inducer is required for the induction and formation of the most rostral notochordal cells. However, inclusion of the inducer results in considerable elongation of the notochord in this experimental paradigm. Our results also demonstrate that the responder per se is not required for notochord formation, provided that at least the inducer and postnodal region are present, although in the absence of the responder, formation of the notochord occurs far less frequently. We also show that the node is not specified to form notochord until stage 4 and concomitant with this, the inducer loses its ability to induce notochord from the responder. The coincident timing of these changes in the node and inducer suggests that notochord specification and the activity of the inducer are regulated through a negative feedback loop. We propose a model relating our results to the induction of head and trunk organizer activity in the node.
• Baranski, M., Berdougo, E., Sandler, J. S., Darnell, D. K., & Burrus, L. W. (2000). The dynamic expression pattern of frzb-1 suggests multiple roles in chick development. Developmental biology, 217(1), 25-41.
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  The Wnt family of secreted proteins has been shown to have multiple roles in embryonic development. Wnt signals are thought to be propagated by binding to the cysteine-rich extracellular domain (CRD) of Frizzled, a seven-transmembrane-domain cell surface receptor. Secreted Frizzled-related proteins (generally denoted Frzb or Sfrp) possess a domain with a high degree of sequence identity and structural similarity with the CRD of Frizzled. Current data indicate that the cysteine-rich domain of secreted Frzb proteins can bind Wnt proteins, suggesting the possibility that Frzbs compete with membrane-bound Frizzled for Wnt binding and consequently act as competitive inhibitors of Wnt signaling. In order to gain a better understanding of the potential roles of Frzb-1 in chick development, we utilized the polymerase chain reaction to isolate a partial cDNA of the chick orthologue of frzb-1, cfrzb-1, and compared its expression pattern to that of Wnt-1, Wnt-3a, Wnt-5a, Wnt-7a, and Wnt-8c. Whole-mount in situ hybridizations have revealed three major phases of expression for cfrzb-1 in the developing chick. The earliest expression of cfrzb-1 is in cells fated to become neural ectoderm in streak-stage embryos. Expression of cfrzb-1 in the neural ectoderm continues up through stage 8. After stage 8, cfrzb-1 expression is gradually attenuated in the closing neural tube of the trunk and is concomitantly up-regulated in neural crest cells. Finally, cfrzb-1 appears in the condensing mesenchyme of the bones in both the limb and the trunk in stage 25+ embryos. Comparative analysis of the cfrzb-1 and the Wnt gene expression patterns suggests possible interactions between cFrzb-1 and all of the Wnt family members examined.
• Darnell, D. K., & Schoenwolf, G. C. (2000). Culture of avian embryos. Methods in molecular biology (Clifton, N.J.), 135, 31-8.
• Darnell, D. K., & Schoenwolf, G. C. (2000). The chick embryo as a model system for analyzing mechanisms of development. Methods in molecular biology (Clifton, N.J.), 135, 25-9.
• Darnell, D. K., & Schoenwolf, G. C. (2000). Transplantation chimeras. Use in analyzing mechanisms of avian development. Methods in molecular biology (Clifton, N.J.), 135, 367-71.
• Darnell, D. K., Garcia-Martinez, V., Lopez-Sanchez, C., Yuan, S., & Schoenwolf, G. C. (2000). Dynamic labeling techniques for fate mapping, testing cell commitment, and following living cells in avian embryos. Methods in molecular biology (Clifton, N.J.), 135, 305-21.
• Darnell, D. K., Stark, M. R., & Schoenwolf, G. C. (1999). Timing and cell interactions underlying neural induction in the chick embryo. Development (Cambridge, England), 126(11), 2505-14.
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  Previous studies on neural induction have identified regionally localized inducing activities, signaling molecules, potential competence factors and various other features of this important, early differentiation event. In this paper, we have developed an improved model system for analyzing neural induction and patterning using transverse blastoderm isolates obtained from gastrulating chick embryos. We use this model to establish the timing of neural specification and the spatial distribution of perinodal cells having organizer activity. We show that a tissue that acts either as an organizer or as an inducer of an organizer is spatially co-localized with the prospective neuroectoderm immediately rostral to the primitive streak in the early gastrula. As the primitive streak elongates, this tissue with organizing activity and the prospective neuroectoderm rostral to the streak separate. Furthermore, we show that up to and through the mid-primitive streak stage (i.e., stage 3c/3+), the prospective neuroectoderm cannot self-differentiate (i.e. , express neural markers and acquire neural plate morphology) in isolation from tissue with organizer activity. Signals from the organizer and from other more caudal regions of the primitive streak act on the rostral prospective neuroectoderm and the latter gains potency (i.e., is specified) by the fully elongated primitive streak stage (i.e., stage 3d). Transverse blastoderm isolates containing non-specified, prospective neuroectoderm provide an improved model system for analyzing early signaling events involved in neuraxis initiation and patterning.
• Darnell, D. K., & Schoenwolf, G. C. (1997). Vertical induction of engrailed-2 and other region-specific markers in the early chick embryo. Developmental dynamics : an official publication of the American Association of Anatomists, 209(1), 45-58.
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  We investigated the role of vertical signals in the regulation of Engrailed-2, a regionally restricted (mesencephalon/metencephalon) neuroectodermal marker, using epiblast grafted from prospective neuroectoderm or prospective trunk mesoderm at mid-stage 3 in the gastrulating chick embryo. Grafts that were isolated from the rostral (prospective neuroectodermal) epiblast and placed rostral to or at the future mesencephalon/metencephalon level, between the endoderm and epiblast of stage 3d to stage 8 host embryos, expressed Engrailed-2 after 24 hr in culture, whereas these same grafts failed to express this marker when placed at a more caudal level. Grafts from caudal = (prospective trunk mesodermal) epiblast, which would ordinarily not express Engrailed-2, also expressed this marker when placed at the mesencephalon/metencephalon level, and failed to express it when grafted more caudally. The expression of four other markers, L5, Fgf8, Wnt-1, and paraxis, were also evaluated. Collectively, our results show that regionally restricted vertical signals are capable of inducing neuroectoderm from naive tissue, and of patterning epiblast to express some but not all mesencephalon/metencephalon isthmus markers. Experiments using grafts taken from older embryos indicated that the competence of prospective neuroectoderm to become regionally patterned by vertical signals is gradually lost between stage 3c and stage 7. Similarly, prospective mesoderm from the caudal epiblast becomes unable to respond to vertical, neural-inductive signals at these stages. These observations support a role for vertical signals in the induction and patterning of the neuroectoderm at gastrula and early neurula stages.
• Garcia-Martinez, V., Darnell, D. K., Lopez-Sanchez, C., Sosic, D., Olson, E. N., & Schoenwolf, G. C. (1997). State of commitment of prospective neural plate and prospective mesoderm in late gastrula/early neurula stages of avian embryos. Developmental biology, 181(1), 102-15.
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  We examined the ability of epiblast regions of known prospective fate from the late gastrula/early neurula stage of avian embryos to self-differentiate when placed heterotopically, testing their state of commitment. Three sites were examined: paranodal prospective neural plate ectoderm, containing cells fated to form a portion of the lateral wall of the neural tube at essentially all rostrocaudal levels of the neuraxis; prospective mesoderm from the caudolateral epiblast, containing cells fated to ingress through the primitive streak and to form lateral plate mesoderm; and prospective mesoderm from one level of the primitive streak, containing cells fated to continue ingressing and form paraxial mesoderm. Grafts from all sites exhibited plasticity. Grafts from the prospective neural plate ectoderm could readily substitute for regions of prospective mesoderm, when transplanted to either the epiblast or primitive streak, undergoing an epithelial-mesenchymal transition and, where appropriate, expressing paraxis, a gene expressed in paraxial mesoderm. Similarly, grafts containing prospective mesoderm from the epiblast could readily substitute for regions of the prospective neural plate ectoderm, undergoing convergent-extension movements characteristic of neuroectodermal cells and expressing appropriate genes such as Engrailed-2 and Hoxb-1. Grafts containing prospective mesoderm from the primitive streak could also incorporate into the neural plate and undergo convergence-extension movements of neurulation, although their principal contribution was to mesodermal and endodermal structures. Collectively, our results demonstrate that at the late gastrula/early neurula stage, germ layer-specific properties are not irrevocably fixed for prospective ectodermal and mesodermal regions of the blastoderm. Moreover, the signals responsible for the induction of these two tissue types must still be present and available at these late stages.
• Garcia-Martinez, V., López-Sanchez, C., Darnell, D. K., & Schoenwolf, G. C. (1996). Experimental analysis of the mechanisms implicated in the induction and commitment of precardiogenic mesodermal cells during avian gastrulation. The International journal of developmental biology, Suppl 1, 215S-216S.
• López-Sanchez, C., Sanchez-Quintana, D., Darnell, D. K., Schoenwolf, G. C., & Garcia-Martinez, V. (1996). In situ hybridization and immunocytochemical analyses of the expression of cardiac-specific genes in an experimental model, which prevents the fusion and subsequent formation of the single tubular heart in avian embryos. The International journal of developmental biology, Suppl 1, 259S-260S.
• Darnell, D. K., & Schoenwolf, G. C. (1995). Dorsoventral patterning of the avian mesencephalon/metencephalon: role of the notochord and floor plate in suppressing Engrailed-2. Journal of neurobiology, 26(1), 62-74.
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  Transcription factors that are spatially and temporally restricted within the embryo may be used for dorsoventral and rostrocaudal positional information during development. The Engrailed-2 (En-2) gene is expressed across the mesencephalon/metencephalon (mes/met) boundary in the cerebellar primordium with strong dorsolateral expression and limited expression in the floor plate. In a previous experiment we demonstrated that, after removal of Hensen's node, embryos lacked a notochord in the head and the pattern of En-2 expression was normal rostrocaudally, but it was expanded into the ventral midline of the neural tube. This suggested that the notochord suppresses En-2 in the ventral neural tube during normal development. To test further the ability of the notochord (and floor plate) to suppress En-2, we transplanted ventral midline tissues from HH 5-9 quail embryos beneath the rostral neural plate of HH 4-6 chick embryos. After 24 hours in culture, 90% of the embryos with quail notochord or floor plate near the mes/met of the host lacked En-2 expression adjacent to the graft, and suppression was distance dependent. Enzymatically isolated notochords also suppressed En-2 (71%), but the results from isolated floor plates were inconclusive. Other grafts served as controls and included tissues from the trunk ventral midline, mes/met level dorsolateral neural plate, and trunk dorsolateral neural plate/somite. Collectively, the results suggest that during normal development the notochord and possibly the floor plate are important regulators of normal En-2 expression.
• Yuan, S., Darnell, D. K., & Schoenwolf, G. C. (1995). Identification of inducing, responding, and suppressing regions in an experimental model of notochord formation in avian embryos. Developmental biology, 172(2), 567-84.
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  The notochord normally arises from committed cells in the rostral tip of the primitive streak. After removal of these cells from the avian gastrula, embryos with notochords nevertheless develop in the majority of cases. A region required for the formation of this reconstituted notochord lies lateral to the primitive streak. In the present study we have determined that this region acts as an inducer for more lateral cells in the epiblast, which actually give rise to the reconstituted notochord. The strongest inducing region lies between 0-250 micrometer lateral to the streak and 500-750 micrometer caudal to the rostral end of the streak and chiefly contains cells normally fated to form lateral plate and somitic mesoderm. The responding region is located 250-500 micrometer lateral to the streak and 0-750 micrometer caudal to the rostral end of the streak. This area chiefly contains cells normally fated to form neural ectoderm, although cells normally fated to form lateral plate and somitic mesoderm are also within this area. The inducing and responding areas interact to form reconstituted notochord either when the primitive streak, including its rostral end (Hensen's node), is removed from the cultured blastoderm or when the inducer and responder are grafted together into an ectopic site. Grafting Hensen's node into isolates containing both inducer and responder blocks formation of reconstituted notochord, suggesting that Hensen's node suppresses formation of lateral notochords during normal development. These findings increase our understanding of the early interactions between mesoderm and ectoderm and provide a novel model system that is well defined and accessible for studying inductive events in higher vertebrates.
• Yuan, S., Darnell, D. K., & Schoenwolf, G. C. (1995). Mesodermal patterning during avian gastrulation and neurulation: experimental induction of notochord from non-notochordal precursor cells. Developmental genetics, 17(1), 38-54.
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  The cells that are normally fated to form notochord occupy a region at the rostral tip of the primitive streak at late gastrula/early neurula stages of avian and mammalian development. If these cells are surgically removed from avian embryos in culture, a notochord will nonetheless form in the majority of cases. The origin of this reconstituted notochord previously had not been investigated and was the objective of this study. Chick embryos at late gastrulal early neurula stages were cultured, and the rostral tip of the primitive streak including Hensen's node was removed and replaced with non-node cells from quail epiblast to ensure that the cells normally fated to be notochord would be absent and that healing of the blastoderm would occur. Embryos were allowed to develop for 24 hr, and the presence and origin (host or graft) of the notochord were assessed using antibodies against notochord or quail cells. Two notochords typically developed; both were almost exclusively of host origin. The primitive streak, and in some cases adjacent tissues, was removed from another group of embryos in an attempt to estimate the mediolateral position and extent of the cells required to form reconstituted notochord. Additional experimental embryos with and without grafts were transected at various rostrocaudal levels in an attempt to estimate the rostrocaudal extent of the cells required to form reconstituted notochord. Finally, various levels of the primitive streak either were placed in a neutral environment (the germ cell crescent) or were grafted in place of the node. Collective results from all experiments indicate that the areas lateral to the rostral portion of the primitive streak, estimated to have a rostrocaudal span of less than 500 microns and a mediolateral extent of less than 250 microns, are critical for formation of the reconstituted notochord. Fate mapping and histological examination of this region identify 4 possible precursor cell populations. Further studies are underway to determine which of the 4 possible precursor cell types forms or induces the reconstituted notochord, and which tissue interactions underlie this change in cell fate.

Reviews

• Darnell, D. K., & Gilbert, S. (2017. Neuroembryology.
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  PMID: 27906497