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D K Darnell

Publications and source records attributed to D K Darnell.

At least 19 recordsLinked to original sources
PubMed2001

Cell interactions underlying notochord induction and formation in the chick embryo.

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.

Animals↗
PubMed2000

The dynamic expression pattern of frzb-1 suggests multiple roles in chick development.

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.

Amino Acid Sequence↗
PubMed1999

Timing and cell interactions underlying neural induction in the chick embryo.

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.

Animals↗
PubMed1997

State of commitment of prospective neural plate and prospective mesoderm in late gastrula/early neurula stages of avian embryos.

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.

Animals↗
PubMed1997

Vertical induction of engrailed-2 and other region-specific markers in the early chick embryo.

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.

Animals↗
PubMed1995

Mesodermal patterning during avian gastrulation and neurulation: experimental induction of notochord from non-notochordal precursor cells.

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.

Animals↗
PubMed1995

Dorsoventral patterning of the avian mesencephalon/metencephalon: role of the notochord and floor plate in suppressing Engrailed-2.

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.

Animals↗
PubMed1995

Identification of inducing, responding, and suppressing regions in an experimental model of notochord formation in avian embryos.

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.

Animals↗
PubMed1992

Changes in dorsoventral but not rostrocaudal regionalization of the chick neural tube in the absence of cranial notochord, as revealed by expression of engrailed-2.

Notochord has been implicated in previous studies in both the dorsoventral and rostrocaudal patterning of the developing neural tube. This possibility has been further explored by analyzing the expression of Engrailed-2 in chick embryos developing with cranial notochord defects. Control embryos containing intact notochords expressed Engrailed-2 protein within the neural tube and in a subset of the neural crest and overlying surface ectoderm at the future mesencephalon and cranial metencephalon levels. Within the neural tube, expression was confined to cell nuclei in the roof plate and lateral walls; floor plate nuclei directly overlying the notochord typically failed to show expression. After surgical removal of Hensen's node, the source of notochord precursor cells, embryos were cultured through neurulation and assayed for expression of Engrailed-2 protein. All embryos that partially or completely lacked cranial notochord expressed Engrailed-2 in a pattern similar to that of control embryos containing intact notochords, except that when notochord and floor plate were absent, Engrailed-2 was also expressed in the most ventral part of the neural tube. These results indicate that 1) Engrailed-2 expression is suppressed in the most ventral neural tube owing to induction of the floor plate by the notochord, and 2) that the presence of an underlying notochord is not required for correct rostrocaudal expression, suggesting that multiple pathways act in the patterning of the rudiment of the central nervous system.

Animals↗
PubMed1992

Relationship between Wnt-1 and En-2 expression domains during early development of normal and ectopic met-mesencephalon.

Grafting a met-mesencephalic portion of neural tube from a 9.5-day mouse embryo into the prosencephalon of a 2-day chick embryo results in the induction of chick En-2 (ChickEn) expression in cells in contact with the graft (Martinez et al., 1991). In this paper we investigate the possibility of Wnt-1 being one of the factors involved in En-2 induction. Since Wnt-1 and En-2 expression patterns have been described as diverging during development of the met-mesencephalic region, we first compared Wnt-1 and En-2 expression in this domain by in situ hybridization in mouse embryos after embryonic day 8.5. A ring of Wnt-1-expressing cells is detected encircling the neural tube in the met-mesencephalic region at least until day 12.5. This ring consistently overlapped with the En-2 expression domain, and corresponds to the position of this latter gene's maximal expression. We subsequently studied ChickEn ectopic induction in chick embryos grafted with various portions of met-mesencephalon. When the graft originated from the level of the Wnt-1-positive ring, ChickEn induction was observed in 71% of embryos, and in these cases correlated with Wnt-1 expression in the grafted tissue. In contrast, this percentage dropped significantly when the graft was taken from more rostral or caudal parts of the mesencephalic vesicle. Taken together, these results are compatible with a prolonged role of Wnt-1 in the specification and/or development of the met-mesencephalic region, and show that Wnt-1 could be directly or indirectly involved in the regulation of En-2 expression around the Wnt-1-positive ring during this time. We also provide data on the position of the Wnt-1-positive ring relative to anatomical boundaries in the neural tube, which suggest a more general role for the Wnt-1 protein as a positional signal involved in organizing the met-mesencephalic domain.

Animals↗
PubMed1989

Metabolic and anatomic development of the chick embryo as studied by phosphorus-31 magnetic resonance spectroscopy and proton MRI.

Thirty fertilized chick eggs were studied sequentially over the 22-day maturation period of the egg by phosphorous-31 (P-31) magnetic resonance spectroscopy (MRS) and proton (H-1) magnetic resonance imaging (MRI) at 2.0 Tesla. The total MR observable P-31 volume in the egg decreased by nearly 80% from day 4 through day 22, suggesting P-31 uptake into tissues in which the P-31 signal is not visible by MR, such as phospholipid bilayers, membranes, cellular structures, and bone mineral phosphates. Of the remaining visible phosphates, the relative phospholipid peak area decreased and the relative peak areas of the phosphocreatine and nucleoside di- and triphosphate metabolites increased. Inorganic phosphate also showed an increase in peak area during maturation. The metabolic development correlated with the anatomic development of visible structures such as brain, eyes, spine and organs in the chick as seen by MRI. In-vivo MR techniques offers the potential to follow changes in prenatal development and maturation.

Adenosine Triphosphate↗
PubMed1982

Pinealectomy in ground squirrels: effect on behavioral and physiological responses to heat stress.

Richardson's ground squirrels, 1 year after pinealectomy, showed altered behavioral and physiological responses to heat stress when compared to an intact group. Pinealectomized squirrels bar-pressed more often for a cool temperature reward in a hot environment. when deprived of behavioral control of the hot environment, pinealectomized squirrels increased their oxygen consumption, had a higher body temperature, and displayed signs of greater thermal stress, including death, as compared to intact animals. When the intact group was pinealectomized and the experiments were repeated, the difference in behavioral and physiological responses to heat stress were not as great as with the 1 year pinealectomized group. A speculative explanation of the pineal gland's influence on central and peripheral control of evaporative water loss and peripheral blood circulation are offered. The pineal gland may exert a subtle influence on heat transfer mechanisms and adaptations to thermal stress.

Animals↗