Hensen's Node Research Articles

Notch signalling regulates the contribution of progenitor cells from the chick Hensen's node to the floor plate and notochord

Hensen's node of the chick embryo contains multipotent self-renewing progenitor cells that can contribute to either the floor plate or the notochord. Floor plate cells are a population of epithelial cells that lie at the ventral midline of the developing neural tube, whereas the notochord is a rod of axial mesoderm that lies directly beneath the floor plate. These two tissues serve as a source of a potent signalling morphogen, sonic hedgehog (Shh), which patterns the dorsoventral axis of the neural tube. We show, through both gain- and loss-of-function approaches, that Notch signalling promotes the contribution of chick axial progenitor cells to the floor plate and inhibits contribution to the notochord. Thus, we propose that Notch regulates the allocation of appropriate numbers of progenitor cells from Hensen's node of the chick embryo to the notochord and the floor plate.

Discovery, characterization and expression of a novel zebrafish gene, znfr, important for notochord formation

Genes specifically expressed in the notochord may be crucial for proper notochord development. Using the digital differential display program offered by the National Center for Biotechnology Information, we identified a novel EST sequence from a zebrafish ovary library (No. XM_701450). The full-length cDNA of this transcript was cloned by performing 3' and 5'-RACE and was further confirmed by PCR and sequencing. The resulting 614 bp gene was found to encode a novel 94 amino acid protein that did not share significant homology with any other known protein. Characterization of the genomic sequence revealed that the gene spanned 4.9 kb and was composed of four exons and three introns. RT-PCR gene expression analysis revealed that our gene of interest was expressed in ovary, kidney, brain, mature oocytes and during the early stages of embryogenesis. During embryonic development, znfr mRNA was found to be expressed in the embryonic shield, chordamesoderm and the vacuolated notochord cells by in situ hybridization. Based on this information, we hypothesize that this novel gene is an important maternal factor required for zebrafish notochord formation during early embryonic development. We have thus named this gene znfr (zebrafish notochord formation related).

Axial differentiation and early gastrulation stages of the pig embryo

Differentiation of the principal body axes in the early vertebrate embryo is based on a specific blueprint of gene expression and a series of transient axial structures such as Hensen's node and the notochord of the late gastrulation phase. Prior to gastrulation, the anterior visceral endoderm (AVE) of the mouse egg-cylinder or the anterior marginal crescent (AMC) of the rabbit embryonic disc marks the anterior pole of the embryo. For phylogenetic and functional reasons both these entities are addressed here as the mammalian anterior pregastrulation differentiation (APD). However, mouse and rabbit show distinct structural differences in APD and the molecular blueprint, making the search of general rules for axial differentiation in mammals difficult. Therefore, the pig was analysed here as a further species with a mammotypical flat embryonic disc. Using light and electron microscopy and in situ hybridisation for three key genes involved in early development ( sox17, nodal and brachyury), two axial structures of early gastrulation in the pig were identified: (1) the anterior hypoblast (AHB) characterised by increased cellular height and density and by sox17 expression, and (2) the early primitive streak characterised by a high pseudostratified epithelium with an almost continuous but unusually thick basement membrane, by localised epithelial–mesenchymal transition, and by brachyury expression in the epiblast. The stepwise appearance of these two axial structures was used to define three stages typical for mammals at the start of gastrulation. Intriguingly, the round shape and gradual posterior displacement of the APD in the pig appear to be species-specific (differing from all other mammals studied in detail to date) but correlate with ensuing specific primitive streak and extraembryonic mesoderm development. APD and, hence, the earliest axial structure presently known in the mammalian embryo may thus be functionally involved in shaping extraembryonic membranes and, possibly, the specific adult body form.

Left-right asymmetry in the chick embryo requires core planar cell polarity protein Vangl2.

Consistent left-right patterning is a fascinating and biomedically important problem. In the chick embryo, it is not known how cells determine their position (left or right) relative to the primitive streak, which is required for subsequent asymmetric gene expression cascades. We show that the subcellular localization of Vangl2, a core planar cell polarity (PCP) protein, is consistently polarized, giving cells in the blastoderm a vector pointing toward the primitive streak. Moreover, morpholino-mediated loss-of-function of Vangl2 by electroporation into chicks at very early stages randomizes the normally left-sided expression of Sonic hedgehog. Strikingly, Vangl2 morpholinos also induce a desynchronization of asymmetric gene expression within the left and right domains of Hensen's node. These data reveal the existence of polarized planar cell polarity protein localization in gastrulating chick and demonstrate that the PCP pathway is functionally required for normal asymmetry in the chick upstream of Sonic hedgehog. These data suggest a new and widely applicable class of models for the spread and coordination of left-right patterning information in the embryonic blastoderm.

Rotation of organizer tissue contributes to left‐right asymmetry in avian embryos

Establishment of left‐right sidedness is a crucial early event in vertebrate morphogenesis. It is currently believed that left‐right asymmetry patterns result from genetic regulatory networks via extracellular morphogens and/or ciliary activity in all vertebrates. However the role of biomechanical motion near the organizer region, has been overlooked. Electroporation of histone 2B‐YFP nuclear marker into avian nodal epiblastic cells and time‐lapse imaging (HH 4‐6) revealed the movements of labeled cells during the interval when morphological and gene expression asymmetries are first observed. The recordings indicated that relative motion occurs between the organizer (Hensen's node) and the surrounding epiblastic epithelium in avians. Specifically, an asymmetric counterclockwise rotation of epiblastic nodal tissue was observed following electroporation on the embryonic left side of the node. However, on the embryonic right, a two‐pronged migratory path was observed. The rotational motion occurs during the interval when initial expression of Shh and Fgf8 is symmetrical, and continues until establishment of Shh and Fgf8 left‐right asymmetry. Ongoing work is aimed at influencing the biomechanical tissue motion and analysis of resulting gene expression patterns. Supported by an AHA Fellowship 0620105Z, the NIH P20 RR016475 from the INBRE Program, NCRR; NIHLB R01 068855; and the Mathers Foundation.

Fate map and morphogenesis of presumptive neural crest and dorsal neural tube

In contrast to the classical assumption that neural crest cells are induced in chick as the neural folds elevate, recent data suggest that they are already specified during gastrulation. This prompted us to map the origin of the neural crest and dorsal neural tube in the early avian embryo. Using a combination of focal dye injections and time-lapse imaging, we find that neural crest and dorsal neural tube precursors are present in a broad, crescent-shaped region of the gastrula. Surprisingly, static fate maps together with dynamic confocal imaging reveal that the neural plate border is considerably broader and extends more caudally than expected. Interestingly, we find that the position of the presumptive neural crest broadly correlates with the BMP4 expression domain from gastrula to neurula stages. Some degree of rostrocaudal patterning, albeit incomplete, is already evident in the gastrula. Time-lapse imaging studies show that the neural crest and dorsal neural tube precursors undergo choreographed movements that follow a spatiotemporal progression and include convergence and extension, reorientation, cell intermixing, and motility deep within the embryo. Through these rearrangement and reorganization movements, the neural crest and dorsal neural tube precursors become regionally segregated, coming to occupy predictable rostrocaudal positions along the embryonic axis. This regionalization occurs progressively and appears to be complete in the neurula by stage 7 at levels rostral to Hensen's node.

Developmental stages until hatching of the Lake Victoria cichlid Haplochromis piceatus (Teleostei: Cichlidae)

Because little is known about embryonic developmental stages in any haplochromine cichlid, we describe here a series of normal stages of the Lake Victoria cichlid Haplochromis piceatus. We collected 273 embryos and scored them for 47 morphological characters. The result was an illustrated series of 12 stages from embryonic shield until hatching based on live, fixed, and histological material. We defined each stage according to a single "key" character that applied to all embryos of that stage. Other characters forming part of the stage descriptions were not necessarily present in all embryos of a stage. We compare our findings with published studies of other freshwater teleosts and find wide variation in staging systems. Our data will form a baseline for further research on cichlid development.

The heart forming region of early chick embryo is an alternative source of embryonic stem cells

In early chick embryo, the precardiac cells reside within distinct groups of mesodermal cells known as presumptive heart forming regions (HFRs). HFRs are located on the lateral sides of the Hensens node. In an effort to study fate of HFRs in isolation, HFRs were excised from early gastrulating chick embryos and cultured in vitro. A very small proportion of HFRs from 18 h incubated embryos differentiated into beating cardiomyocytes whereas about 43% of HFRs from embryos incubated for longer durations (20, 23 and 28 h) showed beating activity. The potential of HFRs, from 18 h incubated embryos, to differentiate into cardiomyocytes increased significantly in presence of Hensens node. About one third of the HFR cells underwent spontaneous differentiation into adipocytes in culture. Simultaneously, some of the cells derived from HFRs exhibited alkaline phosphatase (AP) activity indicating presence of stem cells in the culture. HFR cells were positive for vimentin indicating their mesenchymal origin. FGF supplement increased the proportion of AP-positive cells in a dose dependent manner. The present study demonstrates that HFRs can serve as a source of mesenchymal stem cells which can be gainfully employed for various purposes. The results also suggest that even though the in vitro cultured HFRs from 18 h incubated HH stage 4 chick embryo retain the potential to undergo cardiac differentiation, certain instructive signals from Hensens node may reinforce the fate.

Resource-Scalable Joint Source-Channel MAP and MMSE Estimation of Multiple Descriptions

<para xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> A joint source-channel multiple description (JSC-MD) framework for signal estimation and communication in resource-constrained lossy networks is presented. To keep the encoder complexity at a minimum, a signal is coded by a multiple description quantizer (MDQ) with neither entropy nor channel coding. The code diversity of MDQ and the path diversity of the network are exploited by decoders to combat transmission errors. A key design objective is resource scalability: powerful nodes in the network can perform JSC-MD estimation under the criteria of maximum <emphasis emphasistype="boldital">a posteriori</emphasis> probability (MAP) or minimum mean-square error (MMSE), while primitive nodes resort to simpler MD decoding, all working with the same MDQ code. The application of JSC-MD to distributed estimation of hidden Markov models in a sensor network is demonstrated. The proposed JSC-MD MAP estimator is an algorithm of the longest path in a weighted directed acyclic graph, while the JSC-MD MMSE decoder is an extension of the well-known forward-backward algorithm to multiple descriptions. Both algorithms simultaneously exploit the source memory, the redundancy of the fixed-rate MDQ and the inter-description correlations. They outperform the existing hard-decision MDQ decoders by large margins (up to 8 dB). For Gaussian Markov sources, the complexity of JSC-MD distributed MAP sequence estimation can be made as low as that of typical single description Viterbi-type algorithms. </para>

Evolution of Otx paralogue usages in early patterning of the vertebrate head

To assess evolutional changes in the expression pattern of Otx paralogues, expression analyses were undertaken in fugu, bichir, skate and lamprey. Together with those in model vertebrates, the comparison suggested that a gnathostome ancestor would have utilized all of Otx1, Otx2 and Otx5 paralogues in organizer and anterior mesendoderm for head development. In this animal, Otx1 and Otx2 would have also functioned in specification of the anterior neuroectoderm at presomite stage and subsequent development of forebrain/midbrain at somite stage, while Otx5 expression would have already been specialized in epiphysis and eyes. Otx1 and Otx2 functions in anterior neuroectoderm and brain of the gnathostome ancestor would have been differentially maintained by Otx1 in a basal actinopterygian and by Otx2 in a basal sarcopterygian. Otx5 expression in head organizer and anterior mesendoderm seems to have been lost in the teleost lineage after divergence of bichir, and also from the amniotes after divergence of amphibians as independent events. Otx1 expression was lost from the organizer in the tetrapod lineage. In contrast, in a teleost ancestor prior to whole genome duplication, Otx1 and Otx2 would have both been expressed in the dorsal margin of blastoderm, embryonic shield, anterior mesendoderm, anterior neuroectoderm and forebrain/midbrain, at respective stages of head development. Subsequent whole genome duplication and the following genome changes would have caused different Otx paralogue usages in each teleost lineage. Lampreys also have three Otx paralogues; their sequences are highly diverged from gnathostome cognates, but their expression pattern is well related to those of skate Otx cognates.

Multiple evolutionarily conserved enhancers control expression of Eya1

Eya1 is a homolog of eyes absent in Drosophila, and essential for various organ formations in vertebrates. Mouse and chick Eya1 shows dynamic expression pattern in early development. We identified ten independent Eya1 enhancers by screening evolutionarily conserved sequences. They exhibited enhancer activities in Hensen's node, neural tube, migrating neural crest cells, otic vesicle, olfactory placode, cranial ganglia, and somites at HH6-17 of chick embryo. The sum of the enhancer activities of the enhancers covers the endogenous expression domains of Eya1 common to chick and mouse. Enhancer activities were also observed in species-specific expression domains such as trigeminal ganglia and brain. Mutational study of one of the enhancers revealed that the enhancer is composed of positive and negative cis-regulatory elements. Thus, we successfully identified a comprehensive group of enhancers around Eya1 locus, which are probably involved in the control of the complex expression pattern of Eya1 in vivo.

A ryanodine receptor-dependent Ca(i)(2+) asymmetry at Hensen's node mediates avian lateral identity.

In mouse, the establishment of left-right (LR) asymmetry requires intracellular calcium (Ca(i)(2+)) enrichment on the left of the node. The use of Ca(i)(2+) asymmetry by other vertebrates, and its origins and relationship to other laterality effectors are largely unknown. Additionally, the architecture of Hensen's node raises doubts as to whether Ca(i)(2+) asymmetry is a broadly conserved mechanism to achieve laterality. We report here that the avian embryo uses a left-side enriched Ca(i)(2+) asymmetry across Hensen's node to govern its lateral identity. Elevated Ca(i)(2+) was first detected along the anterior node at early HH4, and its emergence and left-side enrichment by HH5 required both ryanodine receptor (RyR) activity and extracellular calcium, implicating calcium-induced calcium release (CICR) as the novel source of the Ca(i)(2+). Targeted manipulation of node Ca(i)(2+) randomized heart laterality and affected nodal expression. Bifurcation of the Ca(i)(2+) field by the emerging prechordal plate may permit the independent regulation of LR Ca(i)(2+) levels. To the left of the node, RyR/CICR and H(+)V-ATPase activity sustained elevated Ca(i)(2+). On the right, Ca(i)(2+) levels were actively repressed through the activities of H(+)K(+) ATPase and serotonin-dependent signaling, thus identifying a novel mechanism for the known effects of serotonin on laterality. Vitamin A-deficient quail have a high incidence of situs inversus hearts and had a reversed calcium asymmetry. Thus, Ca(i)(2+) asymmetry across the node represents a more broadly conserved mechanism for laterality among amniotes than had been previously believed.

Btg1 and Btg2 gene expression during early chick development

Btg/Tob genes encode for a new family of proteins with antiproliferative functions, which are also able to stimulate cell differentiation. Btg1 and Btg2 are the most closely related members in terms of gene sequence. We analyzed their expression patterns in avian embryos by in situ hybridization, from embryonic day 1 to 3. Btg1 was distinctively expressed in the Hensen's node, the notochord, the cardiogenic mesoderm, the lens vesicle, and in the apical ectodermal ridge and mesenchyme of the limb buds. On the other hand, Btg2 expression domains included the neural plate border, presomitic mesoderm, trigeminal placode, and mesonephros. Both genes were commonly expressed in the myotome, epibranchial placodes, and dorsal neural tube. The results suggest that Btg1 and Btg2 are involved in multiple developmental processes. Overlapping expression of Btg1 and Btg2 may imply redundant functions, but unique expression patterns suggest also differential regulation and function.

The dual knots of doubly primitive knots

For certain $(1,1)$-knots in lens spaces with a longitudinal surgery yielding the 3-sphere, we determine a non-negative integer derived from its $(1,1)$-splitting. The value will be an invariant for such knots. Roughly, it corresponds to a `minimal' self-intersection number when one consider projections of a knot on a Heegaard torus. As an application, we give a necessary and sufficient condition for such knots to be hyperbolic.

Expression of avian prickle genes during early development and organogenesis

Chicken homologues of prickle-1 (pk-1) and prickle-2 (pk-2) were isolated to gain insight into the extent of planar cell polarity signaling during avian embryogenesis. Bioinformatics analyses demonstrated homology and showed that pk-1 and pk-2 exhibited conserved synteny with ADAMTS20 and ADAMTS9, GON-related zinc metalloproteases. Expression of pk-1 and pk-2 was established during embryogenesis and early organogenesis, using in situ hybridization and sections of chicken embryos. At early stages, pk-1 was expressed in Hensen's node, primitive streak, ventral neural tube, and foregut. In older embryos, pk-1 transcripts were detected in dorsolateral epithelial somites, dorsomedial lip of dermomyotomes, and differentiating myotomes. Furthermore, pk-1 expression was seen in lateral body folds, limb buds, and ventral metencephalon. pk-2 was expressed in Hensen's node and neural ectoderm at early stages. In older embryos, pk-2 expression was restricted to ventromedial epithelial somites, except in the most recently formed somite pair, and limb bud mesenchyme.

Developmental rate and allocation of transgenic cells in rabbit chimeric embryos

The objective of this study was to compare developmental capacity of rabbit chimeric embryos and the allocation of the EGFP gene expression to the embryoblast (ICM) or embryonic shield. We produced chimeric embryos (TR< >N) by synchronous transfer of two or three blastomeres at the 16-cell stage from transgenic (TR) into normal host embryos (N) at the same stage. In the control group, two to three non-transgenic blastomeres were used to produce chimeric embryos. The TR embryos were produced by microinjection of EGFP into both pronuclei of fertilized rabbit eggs. The developmental rate and allocation of EGFP-positive cells of the reconstructed chimeric embryos was controlled at blastocyst (96 h PC) and embryonic shield (day 6) stage. All chimeric embryos (120/120, 100%) developed up to blastocyst stage. Using fluorescent microscope, we detected green signal (EGFP expression). In 90 chimeric (TR< >N) embryos (75%). Average total number of cells in chimeric embryos at blastocyst stage was 175+/-13.10, of which 58+/-2.76 cells were found in the ICM area. The number of EGFP-positive cells in the ICM area was 24+/-5.02 (35%). After the transfer of 50 chimeric rabbit embryos at the 16-cell stage, 20 embryos (40%) were flushed from five recipients on day 6 of pregnancy, of which five embryos (25%) were EGFP positive at the embryonic shield stage. Our results demonstrate that transgenic blastomeres in synchronous chimeric embryos reconstructed from TR embryos have an ability to develop and colonize ICM and embryonic shield area.

Lens spaces obtainable by surgery on doubly primitive knots

In this paper, we consider which lens spaces are obtainable by Dehn surgery described by Berge on doubly primitive knots. It is given an algorithm to decide whether a given lens space is obtainable by such surgery. Also included is a complete characterization of such surgery yielding lens spaces with Klein bottles.

Quantitative analysis of neural plate thickness and cell density during gastrulation in the chick embryo

We quantitatively analyzed the developing prospective neural and non-neural ectoderm during chicken gastrulation on semithin transverse sections. At stage PS8 (primitive streak stage 8 of Lopez-Sanchez et al. [C. Lopez-Sanchez, L. Puelles, V. Garcia-Martinez, L. Rodriguez-Gallardo, Morphological and molecular analysis of the early developing chick requires an expanded series of primitive streak stages, J. Morphol. 264 (2005) 105–116.], equivalent to stage HH4), the thickest area of the ectoderm agrees in extent with the fate-mapped neural plate we had reported previously. The thickness of the median ectoderm is constantly higher up to a distance of 250 μm from Hensen's node, and thickness decreases along a mediolateral gradient with a further drop at the prospective lateral border of the neural plate. A higher cell density of the developing ectoderm also coincided with the prospective neural plate. We observed that cell death does not play an important role in the spatial definition of the neural plate.

Experimental study of the dependence of embryonic development of Trachurus trachurus eggs on temperature

Abstract Cunha, M. E., Vendrell, C., and Gonçalves, P. 2008. Experimental study of the dependence of embryonic development of Trachurus trachurus eggs on temperature. – ICES Journal of Marine Science, 65: 17–24. To determine the effect of temperature on the development rates of artificially fertilized eggs of Trachurus trachurus, experiments were carried out at temperatures ranging from 10.5°C to 19°C. Egg development through to hatching only took place at 11.7–19°C. At lower temperature, eggs did not develop beyond the stage where the outline of the embryo was clearly discernible and a defined median line of the embryonic shield (stage 4 in this study) was apparent. Development time took from 46 h at 19°C to 126 h at 12°C. A generalized linear model of the stage-dependent development time (age) as a function of incubation temperature was developed. The data are also compared with those reported in the literature and related to sea temperature on the spawning grounds.

Rab23 GTPase is expressed asymmetrically in Hensen's node and plays a role in the dorsoventral patterning of the chick neural tube

The mouse Rab23 protein, a Ras-like GTPase, inhibits signaling through the Sonic hedgehog pathway and thus exerts a role in the dorsoventral patterning of the spinal cord. Rab23 mouse mutant embryos lack dorsal spinal cord cell types. We cloned the chicken Rab23 gene and studied its expression in the developing nervous system. Chick Rab23 mRNA is initially expressed in the entire neural tube but retracts to the dorsal alar plate. Unlike in mouse, we find Rab23 in chick already expressed asymmetrically during gastrulation. Ectopic expression of Rab23 in ventral midbrain induced dorsal genes (Pax3, Pax7) ectopically and reduced ventral genes (Nkx2.2 and Nkx6) without influencing cell proliferation or neurogenesis. Thus, in the developing brain of chick embryos Rab23 acts in the same manner as described for the caudal spinal cord in mouse. These data indicate that Rab23 plays an important role in patterning the dorso-ventral axis by dorsalizing the neural tube.