Hensen's Node Research Articles

Early neurogenesis in Amniote vertebrates.

Labelling of Hensen's node in a 6-somite stage chick embryo by the quail/chick chimera method has revealed that, while moving caudalwards as the embryo elongates, the node leaves in its wake not only the notochord but also the floor plate and a longitudinal strand of dorsal endoderm. The node itself contains cells endowed with the capacity to yield midline cells (i.e. notochord and floor plate) along the whole length of the neural axis. Caudal node cells function as stem cells. They are responsible for the apical growth of the cord of cells that are at the origin of the midline structures since, if removed, neither the notochord nor the floor plate, are formed caudally to the ablation. The embryo extends however in the absence of midline cells and a neural tube develops posterior to the excision. Only dorsal molecular markers are detectable on this neural tube (e.g. Pax3 and Slug). The posterior region of the embryo in which the structures secreting Shh are missing undergo cell death within the 24 to 48 hours following its formation. Unpublished results indicate that rescue of the posterior region of the embryo can be obtained by implantation of Shh secreting cells. One of the critical roles of floor plate and notochord is therefore to inhibit the cell death programme in the axial and paraxial structures of the embryo at gastrulation and neurulation stages.

Classification scheme for genes expressed during formation and progression of the avian primitive streak.

We have systematically examined the expression patterns of thirteen genes by in situ hybridization during the formation and progression of the avian primitive streak. Based on common patterns of expression, we classify these genes into three distinct groups. Group 1 genes, subdivided into group 1A (Wnt8c, Slug, Vg1, and Nodal) and group 1B (Fgf8, Brachyury, and Cripto), were expressed first in the epiblast and then, throughout most of the length of the primitive streak. Group 2 genes, namely, cNot1, Sonic hedgehog (Shh), Hnf3 beta and Chordin, were confined to the rostral end of the primitive streak, and then, to Hensen's node. In contrast, Group 3 genes, comprising Goosecoid (GSC) and Crescent, were expressed in the hypoblast. This classification scheme provides a rational basis for categorizing genes expressed during avian gastrulation, and such systematization is likely to provide insight into the relationships among different genes and their potential roles in key events of gastrulation.

Left-right asymmetry in BMP4 signalling pathway during chick gastrulation

Determination of the left-right (L-R) axis was shown recently to implicate several genes, among which TGFβ-related molecules such as Activin βB, lefty1 and 2 and Nodal. We show here that Bmp4 and its signal transduction pathway partners BMPR IA and Smad1 are transiently expressed on the right side of Hensen's node, when L-R polarity is being established. Moreover, Smad1 is expressed asymmetrically in the nascent notochord. These observations suggest a role for a BMP4-dependent autocrine or paracrine mechanism during early L-R determination.

Temporospatial patterns of apoptosis in chick embryos during the morphogenetic period of development.

We examined the temporospatial pattern of naturally occurring apoptosis in chick embryos to five days of incubation (H.H. stages 1-25; Hamburger and Hamilton, 1951) using TUNEL labeling. The initial TUNEL-positive structure was the embryonic shield at stage 1. Apoptotic cells became ubiquitously present within embryos by stage 3, which is early in gastrulation. Until stage 6, TUNEL-positive cells were restricted to the headfold region. In embryos of stages 7-8, most cell death was localized at the most anterior neural plate. TUNEL-positive neural plate, notochord and somites appeared at stage 9. Otic and optic regions became TUNEL-positive at stage 11. The aggregation of cells from which the tail bud arises contains apoptotic cells from stage 11 onwards. At stage 16, scattered TUNEL-positive cells appeared in the branchial arches. Three streams of apoptotic neural crest cells in the cranial region became most clearly visible at stage 18. The secondary neural tube from which caudal structures develop contains apoptotic cells at stage 14. Apoptotic cells are present in the branchial arches and lateral body wall for extended periods, stages 16-25 and 25 respectively. At stages 24-25, intense positive regions of cell death were confined to the caudal regions of the arches, to limb and tail buds and to the lateral body wall, the latter in relation to body wall closure. The new findings in this study are discussed along with past studies to provide the temporospatial pattern of cell death during early chick development.

Maternally controlled (beta)-catenin-mediated signaling is required for organizer formation in the zebrafish.

We have identified and characterized a zebrafish recessive maternal effect mutant, ichabod, that results in severe anterior and dorsal defects during early development. The ichabod mutation is almost completely penetrant, but exhibits variable expressivity. All mutant embryos fail to form a normal embryonic shield; most fail to form a head and notochord and have excessive development of ventral tail fin tissue and blood. Abnormal dorsal patterning can first be observed at 3.5 hpf by the lack of nuclear accumulation of (beta)-catenin in the dorsal yolk syncytial layer, which also fails to express bozozok/dharma/nieuwkoid and znr2/ndr1/squint. At the onset of gastrulation, deficiencies in expression of dorsal markers and expansion of expression of markers of ventral tissues indicate a dramatic alteration of dorsoventral identity. Injection of (beta)-catenin RNA markedly dorsalized ichabod embryos and often completely rescued the phenotype, but no measurable dorsalization was obtained with RNAs encoding upstream Wnt pathway components. In contrast, dorsalization was obtained when RNAs encoding either Bozozok/Dharma/Nieuwkoid or Znr2/Ndr1/Squint were injected. Moreover, injection of (beta)-catenin RNA into ichabod embryos resulted in activation of expression of these two genes, which could also activate each other. RNA injection experiments strongly suggest that the component affected by the ichabod mutation acts on a step affecting (beta)-catenin nuclear localization that is independent of regulation of (beta)-catenin stability. This work demonstrates that a maternal gene controlling localization of (beta)-catenin in dorsal nuclei is necessary for dorsal yolk syncytial layer gene activity and formation of the organizer in the zebrafish.

Anterior Neural Induction by Nodes from Rabbits and Mice

The organizer of vertebrate embryos represents the major regulatory center for the formation of the embryonic axis during gastrulation. The early blastopore lip of amphibia and Hensen's node of the chick at the full-length primitive streak stage possess both a head- and a trunk-inducing potential. In mice, a head-inducing activity was identified in the extraembryonic, anterior visceral endoderm (AVE) by tissue ablation and genetic experiments. Evidence for a similar activity in the AVE from the rabbit was obtained by transplanting below the avian epiblast. However, it was still unclear whether the AVE is the exclusive origin of anterior neural induction or if this activity is recapitulated by the node and/or its derivatives. We report here that nodes from both rabbit and mouse embryos can induce a complete neural axis including forebrain structures upon grafting to chick hosts. Thus, in rabbits and mice not only the AVE, but also the node, possesses a potential for the induction of anterior neural tissue.

Axis-inducing activities and cell fates of the zebrafish organizer.

We have investigated axis-inducing activities and cellular fates of the zebrafish organizer using a new method of transplantation that allows the transfer of both deep and superficial organizer tissues. Previous studies have demonstrated that the zebrafish embryonic shield possesses classically defined dorsal organizer activity. When we remove the morphologically defined embryonic shield, embryos recover and are completely normal by 24 hours post-fertilization. We find that removal of the morphological shield does not remove all goosecoid- and floating head-expressing cells, suggesting that the morphological shield does not comprise the entire organizer region. Complete removal of the embryonic shield and adjacent marginal tissue, however, leads to a loss of both prechordal plate and notochord. In addition, these embryos are cyclopean, show a significant loss of floor plate and primary motorneurons and display disrupted somite patterning. Motivated by apparent discrepancies in the literature we sought to test the axis-inducing activity of the embryonic shield. A previous study suggested that the shield is capable of only partial axis induction, specifically being unable to induce the most anterior neural tissues. Contrary to this study, we find shields can induce complete secondary axes when transplanted into host ventral germ-ring. In induced secondary axes donor tissue contributes to notochord, prechordal plate and floor plate. When explanted shields are divided into deep and superficial fragments and separately transplanted we find that deep tissue is able to induce the formation of ectopic axes with heads but lacking posterior tissues. We conclude that the deep tissue included in our transplants is important for proper head formation.

Initiation of neural induction by FGF signalling before gastrulation.

During neural induction, the 'organizer' of the vertebrate embryo instructs neighbouring ectodermal cells to become nervous system rather than epidermis. This process is generally thought to occur around the mid-gastrula stage of embryogenesis. Here we report the isolation of ERNI, an early response gene to signals from the organizer (Hensen's node). Using ERNI as a marker, we present evidence that neural induction begins before gastrulation--much earlier in development than previously thought. We show that the organizer and some of its precursor cells produce a fibroblast growth factor signal, which can initiate, and is required for, neural induction.

Involvement of fibroblast growth factor (FGF)18-FGF8 signaling in specification of left-right asymmetry and brain and limb development of the chick embryo

To elucidate roles of fibroblast growth factors (FGF)18 during vertebrate development, we examined expression patterns of Fgf18 in chick embryos and observed effects of FGF18 protein on the Hensen's node, isthmus, and limb buds. Fgf18 is expressed on the right side of the node before the expression of Fgf8 starts. FGF18 protein can induce expression of Fgf8 on the left side of the node, indicating involvement of both FGFs in specification of left-right asymmetry. In the developing brain, Fgf18 is expressed in the isthmus, following the Fgf8 expression. Since Fgf18 is induced ectopically during formation of the second midbrain by FGF8 protein, both FGFs also elaborate midbrain development. In the limb bud, Fgf18 is expressed in the mesenchyme and ectopic application of FGF18 protein inhibits bone growth in the limb. FGF18 is thus likely an endogenous ligand of FGF receptor 3, whose mutation causes bone dysplasia in humans. These results demonstrate that the FGF18-FGF8 signaling is involved in various organizing activities and the signaling hierarchies between FGF18 and FGF8 seem to change during patterning of different structures.

An early requirement for FGF signalling in the acquisition of neural cell fate in the chick embryo.

In Xenopus embryos, fibroblast growth factors (FGFs) and secreted inhibitors of bone morphogenetic protein (BMP)-mediated signalling have been implicated in neural induction. The precise roles, if any, that these factors play in neural induction in amniotes remains to be established. To monitor the initial steps of neural induction in the chick embryo, we developed an in vitro assay of neural differentiation in epiblast cells. Using this assay, we found evidence that neural cell fate is specified in utero, before the generation of the primitive streak or Hensen's node. Early epiblast cells expressed both Bmp4 and Bmp7, but the expression of both genes was downregulated as cells acquired neural fate. During prestreak and gastrula stages, exposure of epiblast cells to BMP4 activity in vitro was sufficient to block the acquisition of neural fate and to promote the generation of epidermal cells. Fgf3 was also found to be expressed in the early epiblast, and ongoing FGF signalling in epiblast cells was required for acquisition of neural fate and for the suppression of Bmp4 and Bmp7 expression. The onset of neural differentiation in the chick embryo occurs in utero, before the generation of Hensen's node. Fgf3, Bmp4 and Bmp7 are each expressed in prospective neural cells, and FGF signalling appears to be required for the repression of Bmp expression and for the acquisition of neural fate. Subsequent exposure of epiblast cells to BMPs, however, can prevent the generation of neural tissue and induce cells of epidermal character.

Sequential programs of retinoic acid synthesis in the myocardial and epicardial layers of the developing avian heart.

Endogenous patterns of retinoic acid (RA) signaling in avian cardiac morphogenesis were characterized by localized expression of a key RA-synthetic enzyme, RALDH2, which displayed a biphasic pattern during heart development. RALDH2 immunoreactivity was initially apparent posterior to Hensen's node of stage 5-6 embryos and subsequently in somites and unsegmented paraxial and lateral plate mesoderm overlapping atrial precursors in the cardiogenic plate of stage 9- embryos. Initial RALDH2 synthesis in the posterior myocardium coincided with activation of the AMHC1 gene, a RA-responsive marker of inflow heart segments. A wave of RALDH2 synthesis then swept the myocardium in a posterior-to-anterior direction, reaching the outflow tract by stage 13, then fading from the myocardial layer. The second phase of RALDH2 expression, initiated at stage 18 in the proepicardial organ, persisted in migratory epicardial cells that completely enveloped the heart by stage 24. Early restriction of RALDH2 expression to the posterior cardiogenic plate, overlapping RA-inducible gene activation, provides evidence for commitment of posterior avian heart segments by localized production of RA, whereas subsequent RALDH2 expression exclusively in the migratory epicardium suggests a role for the morphogen in ventricular expansion and morphogenesis of underlying myocardial tissues.

Identification of chick frizzled-10 expressed in the developing limb and the central nervous system

We have identified chick frizzled ( Fz)- 10, encoding a Wnt receptor, and examined the expression pattern during embryogenesis. Fz-10 is expressed in the region posterior to the Hensen's node at stage 6. Fz-10 expression is detected in the dorsal domain of the neural tube and the central nervous system of the developing embryo. In the developing limb, Fz-10 expression starts at stage 18 in the posterior–dorsal region of the distal mesenchyme, and gradually expands to the anterior–distal region. Fz-10 is also expressed in the feather bud and branchial arch. Implantation of Sonic hedgehog ( Shh)-expressing cells into the anterior margin of the limb bud resulted in the induction of Fz-10 expression in anterior-dorsal mesenchyme.

Regulation of left-right patterning in mice by growth/differentiation factor-1

The transforming growth factor-beta (TGF-beta) superfamily encompasses a large group of structurally related polypeptides that are capable of regulating cell growth and differentiation in a wide range of embryonic and adult tissues. Growth/differentiation factor-1 (Gdf-1, encoded by Gdf1) is a TGF-beta family member of unknown function that was originally isolated from an early mouse embryo cDNA library and is expressed specifically in the nervous systemin late-stage embryos and adult mice. Here we show that at early stages of mouse development, Gdfl is expressed initially throughout the embryo proper and then most prominently in the primitive node, ventral neural tube, and intermediate and lateral plate mesoderm. To examine its biological function, we generated a mouse line carrying a targeted mutation in Gdf1. Gdf1-/- mice exhibited a spectrum of defects related to left-right axis formation, including visceral situs inversus, right pulmonary isomerism and a range of cardiac anomalies. In most Gdf1-/- embryos, the expression of Ebaf (formerly lefty-1) in the left side of the floor plate and Leftb (formerly lefty-2), nodal and Pitx2 in the left lateral plate mesoderm was absent, suggesting that Gdf1 acts upstream of these genes either directly or indirectly to activate their expression. Our findings suggest that Gdf1 acts early in the pathway of gene activation that leads to the establishment of left-right asymmetry.

Zebrafish Dkk1 Functions in Forebrain Specification and Axial Mesendoderm Formation

We identified a zebrafish homologue of Dickkopf-1 (Dkk1), which was previously identified in Xenopus as a Wnt inhibitor with potent head-inducing activity. Zebrafish dkk1 is expressed in the dorsal marginal blastoderm and also in the dorsal yolk syncytial layer after mid-blastula transition. At later blastula stages, the expression expands to the entire blastoderm margin. During gastrulation, dkk1-expressing cells are confined to the embryonic shield and later to the anterior axial mesendoderm, prospective prechordal plate. Embryos, in which dkk1 was ectopically expressed, exhibited enlarged forebrain, eyes, and axial mesendoderm such as prechordal plate and notochord. dkk1 expression in the dorso-anterior mesendoderm during gastrulation was prominently reduced in zebrafish mutants bozozok (boz), squint (sqt), and one-eyed pinhead (oep), which all display abnormalities in the formation and function of the Spemann organizer and axial mesendoderm. dkk1 expression was normal in these embryos during the blastula period, indicating that zygotic functions of these genes are required for maintenance but not establishment of dkk1 expression. Overexpression of dkk1 suppressed defects in the development of forebrain, eyes, and notochord in boz mutants. Overexpression of dkk1 promoted anterior neuroectoderm development in the embryos injected with antivin RNA, which lack most of the mesoderm and endoderm, suggesting that Dkk1 can affect regionalization of neuroectoderm independently of dorso-anterior mesendoderm. These data indicate that Dkk1, expressed in dorsal mesendoderm, functions in the formation of both the anterior nervous system and the axial mesendoderm in zebrafish.

Defining subregions of Hensen's node essential for caudalward movement, midline development and cell survival.

Hensen's node, also called the chordoneural hinge in the tail bud, is a group of cells that constitutes the organizer of the avian embryo and that expresses the gene HNF-3(&bgr;). During gastrulation and neurulation, it undergoes a rostral-to-caudal movement as the embryo elongates. Labeling of Hensen's node by the quail-chick chimera system has shown that, while moving caudally, Hensen's node leaves in its wake not only the notochord but also the floor plate and a longitudinal strand of dorsal endodermal cells. In this work, we demonstrate that the node can be divided into functionally distinct subregions. Caudalward migration of the node depends on the presence of the most posterior region, which is closely apposed to the anterior portion of the primitive streak as defined by expression of the T-box gene Ch-Tbx6L. We call this region the axial-paraxial hinge because it corresponds to the junction of the presumptive midline axial structures (notochord and floor plate) and the paraxial mesoderm. We propose that the axial-paraxial hinge is the equivalent of the neuroenteric canal of other vertebrates such as Xenopus. Blocking the caudal movement of Hensen's node at the 5- to 6-somite stage by removing the axial-paraxial hinge deprives the embryo of midline structures caudal to the brachial level, but does not prevent formation of the neural tube and mesoderm located posteriorly. However, the whole embryonic region generated posterior to the level of Hensen's node arrest undergoes widespread apoptosis within the next 24 hours. Hensen's node-derived structures (notochord and floor plate) thus appear to produce maintenance factor(s) that ensures the survival and further development of adjacent tissues.

Analysis of the origin and development of hatching gland cells by transplantation of the embryonic shield in the fish, Oryzias latipes

Hatching gland cells of the medaka, Oryzias latipes, have been observed to differentiate from the anterior end of the hypoblast, which seems to first involute at the onset of gastrulation. These results suggest that the hatching gland cells of medaka originate from the embryonic shield, the putative organizer of this fish. The present study investigated whether hatching gland cells really originate from the embryonic shield in the medaka. Transplantation experiments with embryonic shield and in situ hybridization detection of hatching enzyme gene expression as a sign of terminal differentiation of the gland cells were carried out. The analysis was performed according to the following processes. First, identification and functional characterization of the embryonic shield region were made by determining the expression of medaka goosecoid gene and its organizer activity. Second, it was confirmed that the embryonic shield had an organizer activity, inducing a secondary embryo, and that the developmental patterns of hatching gland cells in primary and secondary embryos were identical. Finally, the hatching gland cells as identified by hatching enzyme gene expression were found to coincide with the dye-labeled progeny cells of the transplanted embryonic shield. In conclusion, it was determined that hatching gland cells were derived from the embryonic shield that functioned as the organizer in medaka.

Flik, a Chick Follistatin-Related Gene, Functions in Gastrular Dorsalisation/Neural Induction and in Subsequent Maintenance of Midline Sonic Hedgehog Signalling

We have targetted the chick gene Flik with antisense oligodeoxynucleotide treatment at gastrular stages, when it is expressed in organiser-derived structures of the midline (K. Patel et al., 1996, Dev. Biol. 178, 327–342). A specific syndrome of deficient axial patterning and holoprosencephaly is produced. Most aspects of this syndrome can be understood as due to attenuation of dorsalising and neural-inducing signals during gastrulation, followed by failure to maintain the later signals from chordamesoderm/neural midline that pattern the mesodermal and neural cross sections during subsequent stages. Anatomical effects are first apparent at early neurula stages and correspond with what might be expected from a reduced counteraction of the ventralising Bone morphogenetic protein (BMP) pathway at the earlier stages, coupled with inadequate Sonic hedgehog (Shh) signalling subsequently. Delay in the clearing of BMP-4 RNA expression from the presumptive neural region at gastrulation is indeed seen, though chordin RNA expression within organiser derivatives remains normal. Subsequently, specific attenuation of chordamesoderm and neural midline Shh expression is observed. Brief preincubation of stage 4 chick blastoderms in supernatant from Xenopus oocytes that have been injected with Flik RNA prolongs and enhances the competence of their peripheral epiblast to respond to neural inductive signals from grafted Hensen's nodes. This effect specifically mimics that recently observed using μg/ml solutions of recombinant Follistatin (D. J. Connolly et al., 1999, Int. J. Dev. Biol., in press), further suggesting that Flik protein might act in vivo by somehow modulating activity of signalling pathways through BMP or other TGFβ-related ligands. We discuss the significance of the observations in relation to recent ideas about neural induction, about possible redundancy in gene action, and about subsequent patterning of the axial cross section, suggesting that a Flik function in autocrine/paracrine maintenance of later midline Shh signalling represents a role of the gene separate from that in primary dorsalisation/neural induction.

Molecular Interactions Continuously Define the Organizer during the Cell Movements of Gastrulation

The organizer is a unique region in the gastrulating embryo that induces and patterns the body axis. It arises before gastrulation under the influence of the Nieuwkoop center. We show that during gastrulation, cell movements bring cells into and out of the chick organizer, Hensen's node. During these movements, cells acquire and lose organizer properties according to their position. A “node inducing center,” which emits Vg1 and Wnt8C, is located in the middle of the primitive streak. Its activity is inhibited by ADMP produced by the node and by BMPs at the periphery. These interactions define the organizer as a position in the embryo, whose cellular makeup is constantly changing, and explain the phenomenon of organizer regeneration.

Differential expression of trkC mRNA in the chicken embryo from gastrulation to development of secondary brain vesicles

In situ hybridization revealed that mRNA for the neurotrophin receptor trkC first appears in the chicken embryo at stage 4 anterior to Hensen's node. At stages 6 and 8, trkC mRNA was restricted to the neural plate. By stage 11, trkC mRNA was absent from much of the prosencephalon, the entire mesencephalon and rhombomeres 1 and 4. At stage 15, trkC mRNA expression was limited to rhombomeres 3 and 5, and, by stage 18, there was no apparent expression of trkC mRNA in the hindbrain.

Antagonistic Signaling by Caronte, a Novel Cerberus-Related Gene, Establishes Left–Right Asymmetric Gene Expression

Left–right asymmetry is initiated during chick embryogenesis in small domains near Hensen's node. Subsequently, broad asymmetric gene expression domains are established in the lateral plate mesoderm, ultimately determining the directionality of morphogenetic events. The transfer of asymmetric information from the node to the lateral plate is mediated by Caronte (Car), a novel member of the Cerberus/Dan gene family, which induces targets by antagonizing symmetrically expressed BMP signals. In addition, BMP antagonism by Car induces asymmetric expression of Lefty in the midline, preventing spread of left-sided signals to the contralateral side.