Hensen's Node Research Articles

Expression of the avian VEGF receptor homologues Quek1 and Quek2 in blood-vascular and lymphatic endothelial and non-endothelial cells during quail embryonic development.

We have studied the expression of Quek1 and Quek2 (VEGFR-2 and VEGFR-3, respectively) in quail embryos from day 2 to day 16 by in situ hybridization with digoxigenin-labelled riboprobes on whole-mounts and paraffin sections. Parallel sections were also stained with the QH1 antibody to detect all endothelial cells and with an antibody against alpha-smooth-muscle-actin to reveal the media of blood vessels. Quek1/VEGFR-2 is a marker of blood-vascular and lymphatic endothelial cells throughout development. In 2-day-old embryos, it is expressed in the intra-embryonic vascular plexus, in cells (most probably angioblasts) located in the paraxial head mesoderm and in the somites, and caudo-laterally from Hensen's node. Thereafter, until about day 9, Quek1 is expressed in all endothelial cells. Cells positive and negative for Quek1 can later be found within the same vessel. Quek1 is additionally expressed in lymphatic endothelial cells. Occasionally, some non-endothelial cell types express Quek1. Quek2/VEGFR-3 is also a marker of endothelial cells; however, its expression pattern differs from that of Quek1. In 2-day-old embryos, Quek2 is expressed in the notochord and the intra-embryonic vascular plexus. Whereas all endothelial cells are Quek2-positive in 3-day-old embryos, expression is subsequently reduced to a subset of endothelial cells: arteries become Quek2-negative and then expression of Quek2 is limited to a few vessels that appear to be lymphatic. Endothelial cells of lymph nodes and the periaortal lymphatic vessels are Quek2-positive in later stages. A few non-endothelial cells express Quek2.

Chick noggin is expressed in the organizer and neural plate during axial development, but offers no evidence of involvement in primary axis formation.

We have cloned and examined the early developmental expression of the chick homolog of noggin, a gene originally isolated in Xenopus that can dorsalize gastrular mesoderm and induce anterior neural tissue from gastrular ectoderm when expressed experimentally. Chick noggin is expressed at relatively low levels, but at sites equivalent to those seen in amphibian development, namely Hensen's node and the endo- and mesodermal head process. There is also diffuse expression in the early CNS, centered on the ventral midline, and later hindbrain-associated expression. Since the earlier of these expression sites are consistent with endogenous organizer functions suggested by the properties of the protein in Xenopus experiments, we have used recombinant mammalian Noggin protein secreted by CHO cells in tests for developmental disturbance on the early gastrula-staged chick blastoderm. Comparable tests sensitively detect effects, on chick, of various other secreted proteins that simulate or replicate early developmental signals in Xenopus. We have been unable to observe such effects with a range of Noggin concentrations including those that dramatically dorsalize Xenopus ventral marginal zones. To illustrate effects observed in such tests with secreted proteins active on early stages, we show results with the known Xenopus ventralizer Bone Morphogenetic Protein 4 (BMP-4).

Cash4, a novel achaete-scute homolog induced by Hensen's node during generation of the posterior nervous system.

In vertebrate embryos, the precursor cells of the central nervous system (CNS) are induced by signaling from the organizer region. Here we report the isolation of a novel vertebrate achaete-scute homolog, cash4, which is expressed in the presumptive posterior nervous system in response to such signaling. cash4 is first expressed in epiblast cells flanking the late-phase organizer (Hensen's node), which retains its ability to induce cash4 during regression to the caudal end of the embryo. We show that these node-derived signals can be mimicked in vivo by the activity of fibroblast growth factor (FGF). We demonstrate that cash4 can substitute for the achaete/scute genes in the fly and that it also has proneural activity in vertebrate embryos. Together these results suggest that cash4 functions as a proneural gene downstream of node-derived signals (including FGF) to promote the formation of the neural precursors that will give rise to the posterior CNS in the chick embryo.

Localized axis determinant in the early cleavage embryo of the goldfish, Carassius auratus.

The teleost dorsoventral axis cannot be distinguished morphologically before gastrulation. In order to examine whether the yolk cell affects axis determination, we bisect early cleavage embryos of the goldfish, Carassius auratus. When the vegetal yolk hemisphere is removed by bisection along the equatorial plane at the 2-cell stage, the embryos develop abnormally and exhibit a symmetrical morphology. No dorsal structures, such as notochord, somites and neural tube, differentiate and no embryonic shield is formed during gastrulation. In addition, no goosecoid mRNA is expressed before gastrulation. The frequency of abnormality decreases as the age at which the vegetal yolk hemisphere is removed increases. Most embryos removed at the 32-cell stage develop normally. Their morphological phenotype is similar to that of a Xenopus ventralized embryo generated by ultraviolet irradiation on the vegetal hemisphere soon after fertilization. We also observed that, when the embryos were bisected along the first cleavage plane at the 2-cell stage, the proportion of pairs of embryos of which one embryo developed normally was 44.8%. These results indicate that the vegetal yolk hemisphere of the early cleavage embryo of the goldfish contains axis determination factor(s), which are necessary for generation of dorsal structures. Furthermore, it is suggested that these determinant(s) are distributed asymmetrically within the vegetal yolk hemisphere.

Embryonic Neural Chimeras in the Study of Vertebrate Brain and Head Development

Construction of neural chimeras between quail and chick embryos has been employed since 1969 when the unique nucleolar structure of the quail nucleus and its use to devise a cell marking technique by associating quail and chick cells in ovo were described in the "Bulletin Biologique de la France et de la Belgique." This method was first applied to the ontogeny of the neural crest, a structure whose development involves extensive cell migration, and, since 1984, to that of the central nervous system (CNS). This chapter highlights some of the most significant findings provided by this approach concerning the CNS, such as (i) demonstration of the common origin of the floor plate and notochord from a group of cells localized in the "organizer", i.e., Hensen's node, and the way in which these two structures become positioned respectively within and under the neural tube during gastrulation and neurulation in Amniotes; (ii) the neural crest origin of the skull vault and the facial and hypobranchial skeleton. This means that the mesodermal contribution to the skull is limited to the occipital and otic regions and extends only to the rostral limit of the notochord. A correlation can be drawn between the development of the telencephalon and the mesectodermally derived skull in the vertebrate phylum; (iii) demonstration that the midbrain-hindbrain junction, at the stage of the encephalic vesicles, acts as an organizing center for tectal and cerebellar structures. This function was correlated with the activity of several developmental genes, thus providing insight into their function during neurogenesis; (iv) the pattern of morphogenetic movements and cell migration taking place in defined brain-to-be areas, as well as the origin of various cell types of nervous tissues; and (v) a new avenue for studying brain localization of either behavioral traits or genetically encoded brain disorders.

Formation of reticulated endoderm, Reichert's membrane, and amniogenesis in blastocysts of captive-bred, short-tailed fruit bats, Carollia perspicillata.

As part of an effort to develop the short-tailed fruit bat (Carollia perspicillata) as a new animal model for the study of interstitial implantation and trophoblast-uterine interactions, early embryogenesis was examined histologically and ultrastructurally in captive-bred females at different intervals after the first appearance of spermatozoa in daily vaginal smears (day 1 postcoitum [p.c.]). In most of the early uterine embryos examined on days 16-18 p.c., much of the endoderm appeared as a reticulated meshwork; however, a unilocular yolk sac was formed prior to the development of any mesoderm. Early blastocysts of Carollia were also unusual in that endoderm surrounded much of the inner cell mass (ICM), Reichert's membrane continued over the dorsal side of the ICM, and basal laminalike material was observed around many of the endoderm and epiblast cells. A primordial amniotic cavity was formed between days 19 and 26 p.c. by cavitation. The first mesoderm appeared between days 23 and 26 p.c., concommitant with the development of an embryonic shield. The unusual reticulated appearance of early endoderm in Carollia, which is reminiscent of that seen in early human blastocysts, may be attributable to constraints imposed on growth of the blastocyst by the site and mode of implantation, temporary retardation of trophoblastic invasion by the basal laminae of endometrial epithelial elements, and endodermal proliferation in anticipation of rapid yolk sac expansion. Reichert's membrane appears to play an important role in this species in tethering the ICM and embryonic shield to the developing placenta prior to the formation of significant amounts of mesoderm.

Conservation of BMP signaling in zebrafish mesoderm patterning

To investigate the conservation of mechanisms for mesodermal patterning between zebrafish and Xenopus, we isolated two cDNA clones encoding bone morphogenetic protein (BMP)-related proteins from a zebrafish cDNA library. Based on their predicted amino acid sequences, these two clones were designated as zbmp-2 and zbmp-4. Whole-mount in situ hybridization analysis revealed that in gastrula embryo, both genes were localized in the ventral part of the embryo, consistent with the proposed function of Xenopus BMP-4 in ventral mesoderm specification. zbmp-4 expression, however, was also seen in the embryonic shield, the most dorsal mesodermal structure. To examine the ability of zbmp-2 to ventralize mesoderm, we injected synthetic mRNA into zebrafish embryos and found that overexpression of this gene eliminated dorsal structures including notochord at both morphological and molecular level. In contrast, expression of ventral marker gene eve1 was expanded to the dorsal side. These effects are analogous to the ventralization of embryos caused by ectopic xBMP-4 expression. Taken together, one may conclude that the developmental mechanisms for mesodermal patterning regulated by BMPs are evolutionarily conserved between amphibians and teleosts.

Formation of reticulated endoderm, Reichert's membrane, and amniogenesis in blastocysts of captive‐bred, short‐tailed fruit bats, Carollia perspicillata

Background and Methods As part of an effort to develop the short-tailed fruit bat (Carollia perspicillata) as a new animal model for the study of interstitial implantation and trophoblast-uterine interactions, early embryogenesis was examined histologically and ultrastructurally in captive-bred females at different intervals after the first appearance of spermatozoa in daily vaginal smears (day 1 postcoitum [p.c.]). Results In most of the early uterine embryos examined on days 16-18 p.c., much of the endoderm appeared as a reticulated meshwork; however, a unilocular yolk sac was formed prior to the development of any mesoderm. Early blastocysts of Carollia were also unusual in that endoderm surrounded much of the inner cell mass (ICM), Reichert's membrane continued over the dorsal side of the ICM, and basal laminalike material was observed around many of the endoderm and epiblast cells. A primordial amniotic cavity was formed between days 19 and 26 p.c. by cavitation. The first mesoderm appeared between days 23 and 26 p.c., concommitant with the development of an embryonic shield. Conclusion The unusual reticulated appearance of early endoderm in Carollia, which is reminiscent of that seen in early human blastocysts, may be attributable to constraints imposed on growth of the blastocyst by the site and mode of implantation, temporary retardation of trophoblastic invasion by the basal laminae of endometrial epithelial elements, and endodermal proliferation in anticipation of rapid yolk sac expansion. Reichert's membrane appears to play an important role in this species in tethering the ICM and embryonic shield to the developing placenta prior to the formation of significant amounts of mesoderm. Anat. Rec. 247:85-101 © 1997 Wiley-Liss, Inc.

Spatially distinct domains of cell behavior in the zebrafish organizer region

To determine the sequence of cell behaviors that is involved in the morphogenesis of the zebrafish organizer region, we have examined the dorsal marginal zone of vitally stained zebrafish embryos using time-lapse confocal microscopy. During the late-blastula stage, the zebrafish dorsal marginal zone segregates into several cellular domains, including a group of noninvoluting, highly endocytic marginal (NEM) cells. The NEM cell cluster, which lies in a superficial location of the dorsal marginal zone, is composed of both enveloping layer cells and one or two layers of underlying deep cells. The longitudinal position of this cellular domain accurately predicts the site of embryonic shield formation and occupies a homologous location to the organizer epithelium in Xenopus laevis. At the onset of gastrulation, deep cells underneath the superficial NEM cell domain undergo involution to form the nascent hypoblast of the embryonic shield. Deep cells within the NEM cell cluster, however, do not involute during early shield formation, but instead move in front of the blastoderm margin to form a loose mass of cells called forerunner cells. Forerunner cells coalesce into a wedge-shaped mass during late gastrulation and eventually become overlapped by the converging lateral lips of the germ ring. During early zebrafish tail elongation, most forerunner cells are incorporated into the epithelial lining of Kupffer's vesicle, a transient teleostean organ rudiment long thought to be an evolutionary vestige of the neurenteric canal. Owing to the location of NEM cells at the dorsal margin of blastula-stage embryos, as well as their early segregation from other deep cells, we hypothesized that NEM cells are specified by an early-acting dorsalizing signal. To test this possibility, we briefly treated early-blastula stage embryos with LiCl, an agent known to produce hyperdorsalized zebrafish embryos with varying degrees of expanded organizer tissue. In Li + -treated embryos, NEM cells appear either within expanded spatial domains or in ectopic locations, primarily within the marginal zone of the blastoderm. These results suggest that NEM cells represent a specific cell type that is specified by an early dorsal patterning pathway.

Differential Activation of the Clustered Homeobox GenesCNOT2andCNOT1during Notogenesis in the Chick

CNOT2,a newly identified homeobox gene, is physically linked to theCNOT1gene in the chicken genome. The two chicken genes represent two different subgroups of theNotgene family, the first includingCNOT1and theXenopusgenesXNot1andXNot2,and the secondCNOT2and the zebrafishfloating headgene. The overall expression pattern ofCNOT2in Hensen's node, notochord, neural plate, tailbud, and epiphysis resembled theCNOT1pattern. However, several significant differences occurred:CNOT2expression was much stronger and more widespread in the pregastrulation embryo, it showed an additional, transient domain on the anterior intestinal portal, and lacked expression on the early anterior neural folds and the anterodistal limb bud. We studied CNOT expression by transplanting parts of the primitive streak into growing embryos or by explanting them into tissue culture.CNOTgene expression from young nodes was maintainedin vivo,but requiredin vitrothe addition of retinoic acid. The generation of differentiated notochord structures could only be obtained, if either older node grafts were usedin vitroor young node grafts were transplanted close to the primary axisin vivo.We conclude thatCNOTexpression in the anterior streak is not enough for notochord differentiation, but further influences are necessary. ANot-related gene has previously been isolated fromDrosophila melanogasterand its expression was detected in the posterior brain and the neuroblasts (Dessain and McGinnis, 1993.Adv. Dev. Biochem.2, 1–55). The correspondence betweenNotgene-expressing cells in the nervous system ofDrosophilaand the early neuroectoderm in the chick and its implication for a phylogenetic relationship between neuroectoderm and the notochord is discussed.

A Cluster of Noninvoluting Endocytic Cells at the Margin of the Zebrafish Blastoderm Marks the Site of Embryonic Shield Formation

In zebrafish embryos, the nascent embryonic shield first appears as a thickening in the germ ring of the mid-epiboly blastoderm. This site defines the dorsal side of the developing embryo. In this paper, we report that the site of embryonic axis formation is marked earlier at the late-blastula stage by the appearance of a cluster of cells with unique endocytic activities. This cluster of cells is composed of enveloping layer epithelial cells and one to two layers of underlying deep cells. Unlike other marginal blastomeres, cells in this cluster do not participate in involution as the blastoderm undergoes epiboly. These noninvoluting endocytic marginal (NEM) cells can be selectively labeled by applying membrane impermeant fluorescent probes to pre-epiboly and mid-epiboly embryos. During embryonic shield formation, deep cells in the NEM cell cluster rearrange and are displaced forward to the leading edge of the blastoderm. As deep NEM cells move into this location, they become a group of cells known as “forerunner cells.” Between 60%- and 80%-epiboly, the forerunner cells coalesce into a coherent cell cluster that forms a wedge-shaped cap at the leading edge of the blastoderm. During embryonic axis formation, deep cells migrate and converge toward the embryonic midline, which is defined by the center of the forerunner cell cluster. At approximately 90% epiboly, the forerunner cell cluster becomes overlapped by the constricting germ ring. At tailbud stage, forerunner cells form the dorsal roof of Kupffer's vesicle, which is located ventral to the nascent chordoneural hinge. On the basis of previous grafting studies and known dorsal gene expression patterns, we discuss possible roles that the NEM/forerunner cell cluster may play in teleost axis formation.

Unusual aspects of inner cell mass formation, endoderm differentiation, Reichert's membrane development, and amniogenesis in the lesser bulldog bat, Noctilio albiventris.

The early embryogenesis of the lesser bulldog bat, Noctilio albiventris (family Noctilionidae), was examined histologically in 59 pregnant females collected from a reproductively synchronized population in Colombia. Early blastocysts of Noctilio are unusual in lacking a typical inner cell mass. Instead, cells inside of the trophoblast are dispersed for a period as a monolayer. A typical inner cell mass (ICM) only forms and becomes properly oriented after the initiation of implantation. Several features of Reichert's membrane in this species are also noteworthy: it develops between the ICM and trophoblast and between the parietal endoderm and trophoblast; it becomes linked to a meshwork of basal laminalike material that extends into the ICM; and it appears to be continuous, or fused, with prominent basal laminae that develop within the cytotrophoblastic villi that radiate throughout the preplacenta. Amniogenesis occurs by cavitation and converts the ICM into a hollow epiblastic vesicle. Gastrulation commences before this vesicle exhibits obvious differentiation into an embryonic shield and amniotic ectoderm. Because development and proper orientation of a typical ICM in Noctilio occur after the initiation of implantation, these may involve the migration of cells on the interior of the blastocyst and/or an unusual method of early endoderm differentiation. The possibility exists that epiblast, endoderm, and cytotrophoblast may all contribute to the secretion of Reichert's membrane in this bat. Although the early embryogenesis of Noctilio exhibits many similarities to that in phyllostomid bats, substantial differences also exist between these closely related species.

Unusual aspects of inner cell mass formation, endoderm differentiation, Reichert's membrane development, and amniogenesis in the lesser bulldog bat, Noctilio albiventris

Background and Methods The early embryogenesis of the lesser bulldog bat, Noctilio albiventris (family Noctilionidae), was examined histologically in 59 pregnant females collected from a reproductively synchronized population in Colombia. Results Early blastocysts of Noctilio are unusual in lacking a typical inner cell mass. Instead, cells inside of the trophoblast are dispersed for a period as a monolayer. A typical inner cell mass (ICM) only forms and becomes properly oriented after the initiation of implantation. Several features of Reichert's membrane in this species are also noteworthy: it develops between the ICM and trophoblast and between the parietal endoderm and trophoblast; it becomes linked to a meshwork of basal laminalike material that extends into the ICM; and it appears to be continuous, or fused, with prominent basal laminae that develop within the cytotrophoblastic villi that radiate throughout the preplacenta. Amniogenesis occurs by cavitation and converts the ICM into a hollow epiblastic vesicle. Gastrulation commences before this vesicle exhibits obvious differentiation into an embryonic shield and amniotic ectoderm. Conclusions Because development and proper orientation of a typical ICM in Noctilio occur after the initiation of implantation, these may involve the migration of cells on the interior of the blastocyst and/or an unusual method of early endoderm differentiation. The possibility exists that epiblast, endoderm, and cytotrophoblast may all contribute to the secretion of Reichert's membrane in this bat. Although the early embryogenesis of Noctilio exhibits many similarities to that in phyllostomid bats, substantial differences also exist between these closely related species. © 1996 Wiley-Liss, Inc.

Boundary-fitted parametrization of unstructured grids on arbitrary surfaces

This paper presents a numerical algorithm for handling arbitrary surfaces which are described through an unstructured set of grid points. Surface unstructured grids can be produced using any CAD software and the present method allows its transformation into high-quality structured grids which are appropriate for flow and heat-transfer calculations, by means of finite-difference techniques. The connectivity and the coordinates of the initial surface nodes are the data for the calculation. The structured grid is defined by introducing a convenient set of two parametric coordinates that satisfy the surface Laplace equation. These equations are solved in the unstructured surface grid using the finite element technique, and provide the parametric values over the primitive unstructured grid nodes. The curvilinear coordinate lines, which coincide with the structured grid lines, are defined by searching for the solution contours inside the surface patch. The quality of the so-calculated grid is further improved via a structured-to-structured grid transformation method which improves grid characteristics, such as orthogonality, smoothness and user-defined grid-lines clustering. The proposed method has been successfully applied in several surface grid generation problems of industrial interest, some of them being illustrated herein.

Cloning and Early Dorsal Axial Expression of Flik, a Chick Follistatin-Related Gene: Evidence for Involvement in Dorsalization/Neural Induction

We have cloned and sequenced a chick gene, Flik (follistatin-like) that appears to be the homolog of the mammalian TSC36. The ORF encodes a secreted protein of approx 38 kDa, containing a single cysteine-rich domain that shows a strong relationship with the second of the four from which Follistatin is constructed. The remainder of the Flik protein shows no strong family affinities. We describe here the normal expression pattern of the gene during primitive streak and neurula stages. We also give revised data for early neurectodermal expression of chick follistatin (Connollyet al.,1995,Developmental Genetics17(1), 65–77) and follow the new ectopic expression of both genes during the induction of a second neural axis, after grafting of Hensen's node into a peripheral position in a host blastoderm. Both genes mark the organizer (node and/or mesodermal head process) and early neural plate, and could thus be involved in intercellular signaling during mesodermal dorsalization and neural induction. Flik expression appears earlier than that of follistatin however, and unlike that of follistatin, it is maintained strongly in the dorsal midline with intensity smoothly declining into presumptive lateral regions. We show that both genes are upregulated in host tissue in the neighborhood of node grafts, but whereas follistatin is transcribed after 8–10 hr in host epiblast that has formed new neural plate, Flik is expressed within 4 hr in this region, sometimes detectable before the first structural changes (columnarization) of neuralization. Thus, although ectodermal Flik expression is later confined within neural plate, and mesodermal expression concentrated in dorsal axial tissue, its early distribution is consistent with the idea that the encoded protein may first be involved in generating a graded system that positions the boundaries of both neural and dorsal axial mesodermal territories. The results are discussed in relation to this hypothesis and to other recent findings regarding control of vertebrate dorsoventral patterning.

Expression of the cCdx-B homeobox gene in chick embryo suggests its participation in rostrocaudal axial patterning.

cCdx-B (formerly cHox-cad 2) is a chick homeobox-containing gene related to the Drosophila caudal. Compared with other caudal homologues, its similarity is highest with the murine Cdx-4. In the present study, we characterize the localization of cCdx-B transcripts to the caudal region of the embryo by using reverse transcription-polymerase chain reaction (RT-PCR) and, in detail, by using in situ hybridization. Chick embryos from gastrulation to early organogenesis were hybridized with digoxigenin-labeled riboprobes, and the pattern of expression of cCdx-B mRNA was analyzed in wholemount embryos and in tissue sections. In the early gastrula, transcripts were localized in a gradient through the caudal half of the embryo, in the epiblast and the mesoderm cells, but not including Hensen's node. During neurulation, cCdx-B transcripts were found more rostrally, with high levels localized in Hensen's node and the posterior neural plate. Expression was also high in paraxial mesoderm, with a rostral limit in the most recently formed somite. There was no expression in definitive endoderm. During late neurulation and tail bud formation, cCdx-B mRNA expression regressed posteriorly and was finally confined to the tail bud region. This pattern of expression of cCdx-B, regulated in time and space, is different from that of the other known chick caudal homologue, cCdx-A. Both genes may play a coordinated role in the posterior axial patterning of the chick embryo, whereas cCdx-B may specify further the identity of the tail region.

Expression of Zebrafishconnexin43.4in the Notochord and Tail Bud of Wild-Type and Mutantno tailEmbryos

Communication via gap junctions provides a mechanism for the cell–cell transfer and coordination of developmental signals. The spatial restriction of gap junctions may also serve to organize cells into domains of coordinated behavior. To investigate the role of gap junctions during embryogenesis, we have characterized the expression of a member of the gap junction gene family, zebrafishconnexin43.4,a homolog ofconnexin45in chicken and mammals. Expression ofconnexin43.4was induced in the early gastrula, coincident with the first definitive assignments of axial cell fate and the onset of the cell movements comprising convergence and extension in zebrafish.In situhybridization and immunohistochemistry revealed that during gastrulationconnexin43.4mRNA and protein were progressively enriched in the germ ring and in the notochord primordia on the dorsal side of the embryo. Later in developmentconnexin43.4expression was detected in the notochord, the paraxial mesoderm, and the tail bud but was not observed after the differentiation of these tissues. Inno tailmutant embryos which are defective in tail formation and proper morphogenesis of the notochord,connexin43.4expression was absent during gastrulation from the caudal embryonic shield and notochord primordia. During somite stages inno tailembryos,connexin43.4expression remained absent in the notochordal precursor cells and was lost in the tail bud. Thus, theno tailgene product, a transcription factor, was required for the expression ofconnexin43.4in both the notochord and tail bud during morphogenesis. By microinjection of mRNA coding for a connexin43.4/green fluorescent protein fusion in the 1-cell zebrafish embryo, we showed that connexin43.4 is capable of assembling into structures reminiscent of gap junctions. The progressively restricted, developmental expression of the zebrafishconnexin43.4gene suggests that this gap junctional protein participates in the coordination of gastrulation and the formation of the notochord and tail.

IGF-I and Insulin in the Acquisition of Limb-Forming Ability by the Embryonic Lateral Plate

Acquisition of limb-forming ability by discrete regions of the lateral plate of the chick embryo is dependent on a medial–lateral inductive signaling cascade moving sequentially from the area of Hensen's node to the somitic mesoderm, the intermediate mesoderm, and then to the prospective limb-forming regions of the lateral plate. IGF-I and insulin are expressed by medial tissues as they are influencing the prospective limb-forming regions of the lateral plate. Here we report that IGF-I and insulin, but not FGF-2 or FGF-4, induce the formation of limb bud-like structuresin vitrofrom prospective limb regions before they have acquired the ability to form limbs independent of medial tissues, and also induce the formation of limb bud-like structures from the prospective flank. The limb bud-like structures induced by IGF-I and insulin possess a thickened cap of ectoderm along their distal tips that resembles the apical ectodermal ridge (AER) and this thickened distal apical ectoderm expresses the AER-characteristic homeobox-containing geneMsx-2.Like in normal limb buds, a population of highly proliferating cells which express the homeobox-containing geneMsx-1are localized in the mesoderm directly subjacent to the thickened AER-like structures induced by IGF-I and insulin. However, the limb bud-like structures induced by IGF-I and insulin do not expresssonic hedgehog,which encodes a secreted signaling molecule that has been implicated in regulating the anteroposterior patterning of the developing limb bud. IGF-I- and insulin-treated prospective limb explants give rise to rudimentary limbs containing identifiable skeletal elements when grafted into the coelom or to somites of host embryos. Overall, these results suggest that IGF-I and insulin may be endogenous signals produced by medial tissues that are involved in conferring limb-forming ability to the lateral plate and may promote the initial outgrowth of limb buds and possibly induce the AER. However, other signals are necessary to promote the expression of genes such assonic hedgehogthat regulate the patterning of the developing limb.

Avian Serum Response Factor Expression Restricted Primarily to Muscle Cell Lineages Is Required for α-Actin Gene Transcription

Serum response factor (SRF) gene expression in avian embryonic muscle lineages plays a central role in activating α-actin gene activity. In early stage HH 6 avian embryos, SRF mRNA expression showed strong localization to the neural groove, primitive streak, lateral plate mesoderm, and Hensen's node, while distinct SRF expression was seen later in the neural folds and the somites by HH stage 8. SRF transcripts appeared in the precardiac splanchnic mesoderm in stage HH 9 embryos and was detected at higher levels in the myocardium, somites, and lateral mesoderm of HH 11 embryos. SRF antibody staining demonstrated significant SRF protein accumulation in the myocardium of the developing heart and the myotomal portion of somites. During primary myogenesis in culture, SRF transcripts and nuclear SRF protein content increased about 40-fold, as primary myoblasts withdrew from the cell cycle, reaching their highest levels prior to the upregulation of the skeletal α-actin gene. A dominant-negative SRF mutant, SRFpm1, which inhibited DNA binding, but not dimerization of monomeric SRF subunits, blocked transcriptional activation of a skeletal α-actin promoter–luciferase reporter gene during myogenesis. Transcriptional blockade was reversed by co-transfections of a wild-type SRF expression vector, but was not rescued by the expression of other myogenic factors, such as MyoD and Mef-2C. Thus, SRF displayed an embryonic expression pattern restricted primarily to striated muscle cell lineages, in which increased mass of nuclear SRF was obligatory for α-actin gene transcription.

The Fate Diversity of Mesodermal Cells within the Heart Field during Chicken Early Embryogenesis

In gastrulation stage embryos of birds and mammals, the heart field is established as mesodermal crescents flanking the area rostrolateral to Hensen's node. Subsequent fusion of the bilateral heart primordia gives rise to a single tubular heart consisting of two epithelial layers: an outer myocardium and an inner endocardium. To date, it is uncertain whether these two distinct cell types of the heart arise from common or separate progenitor populations of mesodermal cells within the heart field. By retroviral single cell marking and tracking, we examined the diversity of cell populations present in the heart field of stage 4 chicken embryos. Here we demonstrate that individual mesodermal cells in the heart field gave rise to a clone consisting only of one cell type, either endocardial or myocardial cells; i.e., 95.1% of the mesoderm-derived clones were localized in the myocardium, while 4.9% of them were found in endocardium. No clones containing both of these two cell types were detected. The results suggest that the heart field mesoderm at stage 4 consists of at least two distinct subpopulations, containing more premyocardial cells than preendocardial cells. If there exists a common precursor of both myocardial and endocardial cells, the lineage diversification must occur at or prior to the arrival of mesodermal cells to the heart field.