Cellular behavior in the anteroposterior axis of the regenerating forelimb of the axolotl, Ambystoma mexicanum

Cellular behavior along the anteroposterior axis of the regenerating axolotl forelimb was studied by use of triploid (3N) tissue grafted into diploid (2N) hosts and three-dimensional computer reconstructions. Asymmetrical upper forelimbs were surgically constructed with one half (anterior or posterior) 3N and the other half 2N. Limbs were amputated immediately after grafting or were permitted to heal for 5 or 30 days prior to amputation. When regenerates had attained the stage of digital outgrowth, the limbs were harvested and sectioned in the transverse axis for histological analysis. When all limbs bearing anterior grafts were considered as a group, 77% of the 3N mesodermal cells were observed in the anterior side of the regenerates and 23% were located in the posterior side of the regenerates. When all limbs bearing posterior grafts were considered as a group, 76% of the 3N mesodermal cells were found in the posterior side of the regenerate and 24% had crossed into the anterior side. Healing times of 0, 5, or 30 days prior to amputation had no effect on the experimental outcome. Three-dimensional computer reconstructions revealed that most 3N cells of mesodermal origin underwent short-distance migration from anterior to posterior or from posterior to anterior and intermixed with diploid mesodermal cells near the midpoint of the regenerated anteroposterior axis. Some 3N cells were observed at greater distances from the graft-host interface. By contrast, labeled epidermal cells from both anterior and posterior grafts exhibited long-distance migration across all surfaces of regenerated limbs. Details of a computer-assisted reconstructive method for studying the three-dimensional distribution of labeled cells in tissues are presented.     

VegT, eFGF and Xbra cause overall posteriorization while Xwnt8 causes eye-level restricted posteriorization in synergy with chordin in early Xenopus development

We examined several candidate posterior/mesodermal inducing molecules using permanent blastula-type embryos (PBEs) as an assay system. Candidate molecules were injected individually or in combination with the organizer factor chordin mRNA. Injection of chordin alone resulted in a white hemispherical neural tissue surrounded by a large circular cement gland, together with anterior neural gene expression and thus the development of the anterior-most parts of the embryo, without mesodermal tissues. When VegT, eFGF or Xbra mRNAs were injected into a different blastomere of the chordin -injected PBEs, the embryos elongated and formed eye, muscle and pigment cells, and expressed mesodermal and posterior neural genes. These embryos formed the full spectrum of the anteroposterior embryonic axis. In contrast, injection of CSKA-Xwnt8 DNA into PBEs injected with chordin resulted in eye formation and expression of En2 , a midbrain/hindbrain marker, and Xnot , a notochord marker, but neither elongation, muscle formation nor more posterior gene expression. Injection of chordin and posteriorizing molecules into the same cell did not result in elongation of the embryo. Thus, by using PBEs as the host test system we show that (i) overall anteroposterior neural development, mesoderm (muscle) formation, together with embryo elongation can occur through the synergistic effect(s) of the organizer molecule chordin , and each of the 'overall posteriorizing molecules' eFGF , VegT and Xbra ; (ii) Xwnt8-mediated posteriorization is restricted to the eye level and is independent of mesoderm formation; and (iii) proper anteroposterior patterning requires a separation of the dorsalizing and posteriorizing gene expression domains.     

The control of the anteroposterior and dorsoventral axes in embryonic chick limbs constructed of dissociated and reaggregated limb-bud mesoderm

In the 3- to 4-day embryonic avian limb bud, a unique zone of mesodermal tissue is located posteriorly at the junction of bud and body wall. Appropriately grafted to a host limb bud, it induces the formation of a supernumerary limb outgrowth from preaxial tissue and determines that its posterior side will face the graft. It is called the zone of polarizing activity (ZPA).When limb-bud mesoderm is isolated, dissociated, reaggregated centrifugally, jacketed in the mesoderm-free hull of another limb bud, and grown as a graft on a host embryo, the recombinant frequently forms a limb-like structure terminating in digits that fail to show differentiation with respect to the anteroposterior axis. When, however, a bit of ZPA tissue is implanted in the recombinant subjacent to the anterior or posterior margin of the ectoderm, the resulting outgrowth shows a characteristic anteroposterior order of digits that corresponds to the placement of the implant, regardless of its relationship with the anteroposterior axis of the ectoderm or of the host embryo.Dorsoventral differentials have been recognized only in limbs formed from reaggregated leg-bud mesoderm. The direction of the dorsoventral axis always corresponds to the original axis of the ectodermal jacket regardless of the orientation of the recombinant on the host.     

Separate elements cause lineage restriction and specify boundaries of Hox-1.1 expression.

The Hox genes are a class of putative developmental control genes that are thought to be involved in the specification of positional identity along the anteroposterior axis of the vertebrate embryo. It is apparent from their expression pattern that their regulation is dependent upon positional information. In a previous analysis of the Hox-1.1 promoter in transgenic mice, we identified sequences that were sufficient to establish transgene expression in a specific region of the embryo. The construct used, however, did not contain enough regulatory sequences to reproduce all aspects of Hox-1.1 expression. In particular, neither a posterior boundary nor a restriction of expression to prevertebrae was achieved. Here we show correct regulation by Hox-1.1 sequences in transgenic mice and identify the elements responsible for different levels of control. Concomitant with the subdivision of mesodermal cells into different lineages during gastrulation and organogenesis, Hox-1.1 expression is restricted to successively smaller sets of cells. Distinct elements are required at different stages of development to execute this developmental programme. One position-responsive element (130 bp nontranslated leader) was shown to be crucial for the restriction of expression not only along the anteroposterior axis of the embryo, setting the posterior border, but also along the dorsoventral axis of the neural tube and to the lineage giving rise to the prevertebrae. Thus, Hox-1.1 expression is established in a specific region of the embryo and in a specific lineage of the mesoderm by restricting the activity of the promoter by the combined effect of several regulatory elements.     

The development of three identified motor neurons in the larva of an ascidian, Halocynthia roretzi

The generation of distinct classes of motor neurons underlies the development of complex motile behavior in all animals and is well characterized in chordates. Recent molecular studies indicate that the ascidian larval central nervous system (CNS) exhibits anteroposterior regionalization similar to that seen in the vertebrate CNS. To extend the understanding about the diversity of motor neurons in the ascidian larva, we have identified the number, position, and projection of individual motor neurons in Halocynthia roretzi, using a green fluorescent protein under the control of a neuron-specific promoter. Three pairs of motor neurons, each with a distinct shape and innervation pattern, were identified along the anteroposterior axis of the neural tube: the anterior and posterior pairs extend their axons toward dorsal muscle cells, whereas the middle pair project their axons toward ventral muscle. Overexpression of a dominant-negative form of a potassium channel in these cells resulted in paralysis on the injected side, thus these cells must constitute the major population of motor neurons responsible for swimming behavior. Lim class homeobox genes have been known as candidate genes that determine subtypes of motor neurons. Therefore, the expression pattern of Hrlim, which is a Lim class homeobox gene, was examined in the motor neuron precursors. All three motor neurons expressed Hrlim at the tailbud stage, although each down-regulated Hrlim at a different time. Misexpression of Hrlim in the epidermal lineage led to ectopic expression of TuNa2, a putative voltage-gated channel gene normally expressed predominantly in the three pairs of motor neurons. Hrlim may control membrane excitability of motor neurons by regulating ion channel gene expression.     

Posterior apical ectodermal ridge removal in the chick wing bud triggers a series of events resulting in defective anterior pattern formation

The ability of the anterior apical ectodermal ridge to promote outgrowth in the chick wing bud when disconnected from posterior apical ridge was examined by rotating the posterior portion of the stage-19/20 to stage-21 wing bud around its anteroposterior axis. This permitted contact between the anterior and posterior mesoderm, without removing wing bud tissue. In a small but significant number of cases (10/54), anterior structures (digit 2) formed spatially isolated from posterior structures (digits 3 and 4). Thus, continuity with posterior ridge is not a prerequisite for anterior-ridge function in the wing bud. Nevertheless, posterior-ridge removal does result in anterior limb truncation. To investigate events leading to anterior truncation, we examined cell death patterns in the wing bud following posterior-ridge removal. We observed an abnormal area of necrosis along the posterior border of the wing bud at 6-12 h following posterior-ridge removal. This was followed by necrosis in the distal, anterior mesoderm at 48 h postoperatively and subsequent anterior truncation. Clearly, healthy posterior limb bud mesoderm is needed for anterior limb bud survival and development. We propose that anterior truncation is the direct result of anterior mesodermal cell death and that this may not be related to positional specification of anterior cells. In our view, cell death of anterior mesoderm, after posterior mesoderm removal, should not be used as evidence for a role in position specification by the polarizing zone during the limb bud stages of development. We suggest that the posterior mesoderm that maintains the anterior mesoderm need not be restricted to the mapped polarizing zone, but is more extensively distributed in the limb bud.     

Characterization of Notch-class gene expression in segmentation stem cells and segment founder cells in Helobdella robusta (Lophotrochozoa; Annelida; Clitellata; Hirudinida; Glossiphoniidae)

To understand the evolution of segmentation, we must compare segmentation in all three major groups of eusegmented animals: vertebrates, arthropods, and annelids. The leech Helobdella robusta is an experimentally tractable annelid representative, which makes segments in anteroposterior progression from a posterior growth zone consisting of 10 identified stem cells. In vertebrates and some arthropods, Notch signaling is required for normal segmentation and functions via regulation of hes-class genes. We have previously characterized the expression of an hes-class gene (Hro-hes) during segmentation in Helobdella, and here, we characterize the expression of an H. robusta notch homolog (Hro-notch) during this process. We find that Hro-notch is transcribed in the segmental founder cells (blast cells) and their stem-cell precursors (teloblasts), as well as in other nonsegmental tissues. The mesodermal and ectodermal lineages show clear differences in the levels of Hro-notch expression. Finally, Hro-notch is shown to be inherited by newly born segmental founder cells as well as transcribed by them before their first cell division.     

Mesodermal cell migration during Xenopus gastrulation

The adhesive glycoprotein fibronectin (FN), which is a component of the network of extracellular matrix fibrils on the inner surface of the blastocoel roof (BCR), has been proposed to play a major role in directing mesodermal cell migration during amphibian gastrulation. In the first part of this paper, the adhesion of Xenopus mesodermal cells to FN in vitro is examined. Cells from several mesoderm regions, which differ in developmental fate and morphogenetic activity, are able to bind specifically to the RGD cell-binding site of FN. Dorsal mesodermal cell adhesion to FN varies along the anterior-posterior (a-p) axis: adhesion is strongest in the anterior head mesoderm, and gradually decreases posteriorly. This a-p gradient of mesodermal adhesiveness to FN does not change during mesodermal involution, and is reflected in the morphology of mesodermal explants on FN. An a-p strip of mesoderm develops a spreading, leading anterior margin and a compact, retracting posterior end, thus moving slowly and directionally over the FN substrate at about 0.8 micron/min. Although dissociated cells from all levels of the dorsal mesodermal axis adhere to FN, only the anterior, leading prospective head mesoderm cells migrate as single cells on a FN substrate in vitro. Locomotion by means of lamelliform protrusions occurs at an average rate of about 1.5 micron/min. Cells of the more posterior axial mesoderm merely shift position at random without substantial net translocation, and preinvolution mesoderm cells are completely stationary. On the BCR, the in vivo substrate for mesodermal cell migration, dissociated prospective head mesoderm cells spread and migrate as on FN in vitro, at 2.2 microns/min. In the presence of an RGD peptide which inhibits cell-FN interaction, cells remain globular and do not spread. They are still motile, but change direction frequently, which leads to less efficient net translocation. Apparently, interaction with the RGD cell-binding site of FN and concomitant spreading of head mesoderm cells is required for the stabilization of cell locomotion. In contrast to the directional migration of the mesoderm cell population toward the animal pole in the embryo, the pathways of dissociated cells on the BCR are randomly oriented. Coherent explants of migratory mesoderm do not move at all on the BCR, although they translocate on FN in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)     

Anteroposterior patterning and organogenesis of Xenopus laevis require a correct dose of germ cell nuclear factor (xGCNF).

The germ cell nuclear factor of Xenopus laevis (xGCNF; NR6A1) is a nuclear orphan receptor that is predominantly expressed during neurula and late tailbud stages. As a strategy to analyze the role of xGCNF in embryogenesis, we have induced a gain of function by overexpression of full-length (fl) GCNF and a functional inhibition by a dominant-negative (dn) GCNF. Early events of embryogenesis including gastrulation and neurulation were not affected and the expression of several early mesodermal markers was normal. Yet specific defects were observed upon organogenesis. Ectopic posterior overexpression of the full-length xGCNF caused posterior defects and disturbed somite formation. In contrast, expression of dnGCNF interfered with differentiation of the neural tube and affected the differentiation of anterior structures, including the cement gland and the eyes. Embryos affected by dnGCNF were rescued by coexpression of flGCNF. After expression of dnGCNF, mRNA encoding the the retinoic acid receptor xRAR gamma 2 was selectively suppressed anteriorly. From the distinct phenotypes obtained, we conclude that GCNF has an essential function in anteroposterior differentiation during organogenesis.     

The orderly allocation of mesodermal cells to the extraembryonic structures and the anteroposterior axis during gastrulation of the mouse embryo.

The prospective fate of cells in the primitive streak was examined at early, mid and late stages of mouse gastrula development to determine the order of allocation of primitive streak cells to the mesoderm of the extraembryonic membranes and to the fetal tissues. At the early-streak stage, primitive streak cells contribute predominantly to tissues of the extraembryonic mesoderm as previously found. However, a surprising observation is that the erythropoietic precursors of the yolk sac emerge earlier than the bulk of the vitelline endothelium, which is formed continuously throughout gastrula development. This may suggest that the erythropoietic and the endothelial cell lineages may arise independently of one another. Furthermore, the extraembryonic mesoderm that is localized to the anterior and chorionic side of the yolk sac is recruited ahead of that destined for the posterior and amnionic side. For the mesodermal derivatives in the embryo, those destined for the rostral structures such as heart and forebrain mesoderm ingress through the primitive streak early during a narrow window of development. They are then followed by those for the rest of the cranial mesoderm and lastly the paraxial and lateral mesoderm of the trunk. Results of this study, which represent snapshots of the types of precursor cells in the primitive streak, have provided a better delineation of the timing of allocation of the various mesodermal lineages to specific compartments in the extraembryonic membranes and different locations in the embryonic anteroposterior axis.     

An expression pattern screen for genes involved in the induction of the posterior nervous system of zebrafish

The posterior nervous system, including the hindbrain and the spinal cord, has been shown to be formed by the transformation of neural plate of anterior character by signals derived from non-axial mesoderm. Although secreted factors, such as fibroblast growth factors (FGFs), Wnts, retinoic acid (RA) and Nodal, have been proposed to be the posteriorizing factors, the mechanism how neural tissue of posterior character is induced and subsequently specified along the anteroposterior axis remains elusive. To identify intercellular signaling molecules responsible for posteriorization of the neural plate as well as to find molecules induced intracellularly by the posteriorizing signal in the caudal neural plate, we screened by in situ hybridization for genes specifically expressed in posterior tissues, including the posterior neural plate and non-axial mesoderm when posteriorization of the neural plate takes place. From a subtracted library differentiating anterior versus posterior neural plate, 420 cDNA clones were tested, out of which 76 cDNA fragments showed expression restricted to the posterior tissue. These clones turned out to represent 32 different genes, including one novel secreted factor and one transmembrane protein. Seven genes were induced by non-axial mesodermal implants and bFGF beads, suggesting that these are among the early-response genes of the posteriorizing signal. Thus, our approach employing cDNA subtraction and subsequent expression pattern screening allows us to clone candidate genes involved in a novel signaling pathway contributing to the formation of the posterior nervous system.     

Cooperation of polarized cell intercalations drives convergence and extension of presomitic mesoderm during zebrafish gastrulation

During vertebrate gastrulation, convergence and extension (C&E) movements narrow and lengthen the embryonic tissues, respectively. In zebrafish, regional differences of C&E movements have been observed; however, the underlying cell behaviors are poorly understood. Using time-lapse analyses and computational modeling, we demonstrate that C&E of the medial presomitic mesoderm is achieved by cooperation of planar and radial cell intercalations. Radial intercalations preferentially separate anterior and posterior neighbors to promote extension. In knypek;trilobite noncanonical Wnt mutants, the frequencies of cell intercalations are altered and the anteroposterior bias of radial intercalations is lost. This provides evidence for noncanonical Wnt signaling polarizing cell movements between different mesodermal cell layers. We further show using fluorescent fusion proteins that during dorsal mesoderm C&E, the noncanonical Wnt component Prickle localizes at the anterior cell edge, whereas Dishevelled is enriched posteriorly. Asymmetrical localization of Prickle and Dishevelled to the opposite cell edges in zebrafish gastrula parallels their distribution in fly, and suggests that noncanonical Wnt signaling defines distinct anterior and posterior cell properties to bias cell intercalations.     

Combinatorial gene regulation by Bmp and Wnt in zebrafish posterior mesoderm formation

Combinatorial signaling is an important mechanism that allows the embryo to utilize overlapping signaling pathways to specify different territories. In zebrafish, the Wnt and Bmp pathways interact to regulate the formation of the posterior body. In order to understand how this works mechanistically, we have identified tbx6 as a posterior mesodermal gene activated by both of these signaling pathways. We isolated a genomic fragment from the tbx6 gene that recapitulates the endogenous tbx6 expression, and used this to ask how the Bmp and Wnt signaling pathways combine to regulate gene expression. We find that the tbx6 promoter utilizes distinct domains to integrate the signaling inputs from each pathway, including multiple Tcf/LEF sites and a novel Bmp-response element. Surprisingly, we found that overexpression of either signaling pathway can activate the tbx6 promoter and the endogenous gene, whereas inputs from both pathways are required for the normal pattern of expression. These results demonstrate that both Bmp and Wnt are present at submaximal levels, which allows the pathways to function combinatorially. We present a model in which overlapping Wnt and Bmp signals in the ventrolateral region activate the expression of tbx6 and other posterior mesodermal genes, leading to the formation of posterior structures.     

Analysis of Wnt8 for neural posteriorizing factor by identifying Frizzled 8c and Frizzled 9 as functional receptors for Wnt8

The dorsal ectoderm of vertebrate gastrula is first specified into anterior fate by an activation signal and posteriorized by a graded transforming signal, leading to the formation of forebrain, midbrain, hindbrain and spinal cord along the anteroposterior (A-P) axis. Transplanted non-axial mesoderm rather than axial mesoderm has an ability to transform prospective anterior neural tissue into more posterior fates in zebrafish. Wnt8 is a secreted factor that is expressed in non-axial mesoderm. To investigate whether Wnt8 is the neural posteriorizing factor that acts upon neuroectoderm, we first assigned Frizzled 8c and Frizzled 9 to be functional receptors for Wnt8. We then, transplanted non-axial mesoderm into the embryos in which Wnt8 signaling is cell-autonomously blocked by the dominant-negative form of Wnt8 receptors. Non-axial mesodermal transplants in embryos in which Wnt8 signaling is cell-autonomously blocked induced the posterior neural markers as efficiently as in wild-type embryos, suggesting that Wnt8 signaling is not required in neuroectoderm for posteriorization by non-axial mesoderm. Furthermore, Wnt8 signaling, detected by nuclear localization of beta-catenin, was not activated in the posterior neuroectoderm but confined in marginal non-axial mesoderm. Finally, ubiquitous over-expression of Wnt8 does not expand neural ectoderm of posterior character in the absence of mesoderm or Nodal-dependent co-factors. We thus conclude that other factors from non-axial mesoderm may be required for patterning neuroectoderm along the A-P axis.     

Coupling segmentation to axis formation

A characteristic feature of the vertebrate body is its segmentation along the anteroposterior axis, as illustrated by the repetition of vertebrae that form the vertebral column. The vertebrae and their associated muscles derive from metameric structures of mesodermal origin, the somites. The segmentation of the body is established by somitogenesis, during which somites form sequentially in a rhythmic fashion from the presomitic mesoderm. This review highlights recent findings that show how dynamic gradients of morphogens and retinoic acid, coupled to a molecular oscillator, drive the formation of somites and link somitogenesis to the elongation of the anteroposterior axis.