Self-organized formation of developing appendages from murine pluripotent stem cells.

Limb development starts with the formation of limb buds (LBs), which consist of tissues from two different germ layers; the lateral plate mesoderm-derived mesenchyme and ectoderm-derived surface epithelium. Here, we report means for induction of an LB-like mesenchymal/epithelial complex tissues from murine pluripotent stem cells (PSCs) in vitro. The LB-like tissues selectively differentiate into forelimb- or hindlimb-type mesenchymes, depending on a concentration of retinoic acid. Comparative transcriptome analysis reveals that the LB-like tissues show similar gene expression pattern to that seen in LBs. We also show that manipulating BMP signaling enables us to induce a thickened epithelial structure similar to the apical ectodermal ridge. Finally, we demonstrate that the induced tissues can contribute to endogenous digit tissue after transplantation. This PSC technology offers a first step for creating an artificial limb bud in culture and might open the door to inducing other mesenchymal/epithelial complex tissues from PSCs.

Newts can normalize duplicated proximal-distal disorder during limb regeneration.

BACKGROUND: Urodele animals can regenerate their limbs from the blastemas. The previous results of grafting proximal blastemas to distal limb levels (P to D transplantation) led to serial duplication of limb segments. However, it is unknown whether grafting to any distal levels in P to D transplantation causes serial duplication. In other words, it is unknown whether or not newt limbs can normalize such a kind of duplicated type of positional disorder in the proximal-distal axis. Therefore, we grafted the most proximal blastemas to various distal levels of the proximal-distal axis using newts (Pleurodeles waltl). The transgenic newts expressing green fluorescent protein or mCherry were used to clearly distinguish between donor and host tissues. RESULTS: Normal segmental formation without duplication occurred in P to D transplantation within the stylopod. In addition, donor blastemas lost the fates of the stylopods, and the missing portion in the stylopod by amputation was restored by the insertion of host cells. In contrast, the blastemas from the stylopod formed whole limbs after transplantation to the tail. CONCLUSIONS: These results showed that urodele limbs can normalize the duplicated type of positional disorder within the stylopod by erasing a part of the fate in the blastemas. Developmental Dynamics 247:1276-1285, 2018. © 2018 Wiley Periodicals, Inc.

Polarizing Region Tissue Grafting in the Chick Embryo Limb Bud.

The polarizing region of the developing limb bud is an important organizing center that is involved in anteroposterior (thumb to little finger) patterning and has three main functions that are now considered to depend on the secreted protein Sonic hedgehog (Shh). These are (1) specifying anteroposterior positional values by autocrine and graded paracrine signaling; (2) promoting growth in adjacent mesenchyme; (3) maintaining the distal epithelium that is essential for limb outgrowth by induction of a factor in adjacent mesenchyme. The polarizing region was identified using classical tissue grafting techniques in chicken embryos. Here we describe this procedure using tissue from transgenic Green Fluorescent Protein-expressing chicken embryos that allows the long-term fate of the polarizing region to be determined. This technique provides a highly useful and effective method to understand how the polarizing region patterns the limb and has implications for other organizing centers.

Skeleton pattern and joint formation in chorioallantoic grafts containing the distal parts of the chick wing bud.

Skeleton pattern formation was examined in chick wing bud grafts using the chorioallantoic grafting method. The distal parts of the wing bud were excised from the donor wing and transplanted onto the chorioallantoic membrane (the experimental groups). Transplants with intact limb bud material served as the control group. The skeleton pattern formation in the grafts depended on the amount of transplanted material and donor's limb bud stage. The younger the donor's stage and the bigger the piece of the transplanted material the more proximal parts grafts had, more retarded growth and abnormal skeleton in the zeugopod and autopod was. The percentage of the signs of insufficient blood supply in the experimental groups was less than that in the control group. As the amount of the transplanted limb bud material decreased and donor's limb bud aged, post-axial polydactyly changed to the pre-axial one.

Diffusible signals, not autonomous mechanisms, determine the main proximodistal limb subdivision.

Vertebrate limbs develop three main proximodistal (PD) segments (upper arm, forearm, and hand) in a proximal-to-distal sequence. Despite extensive research into limb development, whether PD specification occurs through nonautonomous or autonomous mechanisms is not resolved. Heterotopic transplantation of intact and recombinant chicken limb buds identifies signals in the embryo trunk that proximalize distal limb cells to generate a complete PD axis. In these transplants, retinoic acid induces proximalization, which is counteracted by fibroblast growth factors from the distal limb bud; these related actions suggest that the first limb-bud PD regionalization results from the balance between proximal and distal signals. The plasticity of limb progenitor cell identity in response to diffusible signals provides a unifying view of PD patterning during vertebrate limb development and regeneration.

Regulative patterning in limb bud transplants is induced by distalizing activity of apical ectodermal ridge signals on host limb cells.

We have used the chick limb as a model to gain insight into the longstanding question of regulative vs. mosaic development. To test the influence of signals on limb proximodistal development, distal limb bud tips of several stages were grafted to regions of the embryo known to provide different signaling environments. Of interest, thin grafts (100-micron thick) formed elements more proximal in character when grafted to the proximal limb region than when grafted to other regions. The extra elements were derived from host tissue, presumably distalized and recruited by the graft's apical ectodermal ridge signals. The results of classic and recent experiments have been reinterpreted in light of our conclusions.

A reevaluation of X-irradiation-induced phocomelia and proximodistal limb patterning.

Phocomelia is a devastating, rare congenital limb malformation in which the long bones are shorter than normal, with the upper portion of the limb being most severely affected. In extreme cases, the hands or fingers are attached directly to the shoulder and the most proximal elements (those closest to the shoulder) are entirely missing. This disorder, previously known in both autosomal recessive and sporadic forms, showed a marked increase in incidence in the early 1960s due to the tragic toxicological effects of the drug thalidomide, which had been prescribed as a mild sedative. This human birth defect is mimicked in developing chick limb buds exposed to X-irradiation. Both X-irradiation and thalidomide-induced phocomelia have been interpreted as patterning defects in the context of the progress zone model, which states that a cell's proximodistal identity is determined by the length of time spent in a distal limb region termed the 'progress zone'. Indeed, studies of X-irradiation-induced phocomelia have served as one of the two major experimental lines of evidence supporting the validity of the progress zone model. Here, using a combination of molecular analysis and lineage tracing in chick, we show that X-irradiation-induced phocomelia is fundamentally not a patterning defect, but rather results from a time-dependent loss of skeletal progenitors. Because skeletal condensation proceeds from the shoulder to fingers (in a proximal to distal direction), the proximal elements are differentially affected in limb buds exposed to radiation at early stages. This conclusion changes the framework for considering the effect of thalidomide and other forms of phocomelia, suggesting the possibility that the aetiology lies not in a defect in the patterning process, but rather in progenitor cell survival and differentiation. Moreover, molecular evidence that proximodistal patterning is unaffected after X-irradiation does not support the predictions of the progress zone model.

Modification of the zone of polarizing activity signal by trypsin.

Patterning of the developing vertebrate limb along the anterior-posterior axis is controlled by the zone of polarizing activity (ZPA) via the expression of Sonic hedgehog (Shh) and along the proximal-distal axis by the apical ectodermal ridge (AER) through the production of fibroblast growth factors (FGFs). ZPA grafting, as well as ectopic application of SHH to the anterior chick limb bud, demonstrate that digit patterning is largely influenced by these secreted factors. Although signal transduction pathways have been well characterized for SHH and for FGFs, little is known of how these signals are regulated extracellularly in the limb. The present study shows that alteration of the extracellular environment through trypsin treatment can have profound effects on digit patterning. These effects appear to be mediated by the induction of Shh in host tissues and by ectopic AER formation, implicating the extracellular matrix in regulating the signaling activities of key patterning genes in the limb.

Characteristics of initiation and early events for muscle development in the Xenopus limb bud.

In Xenopus laevis, limb buds start to develop at a later point of the larval stage, prior to metamorphosis. This onset of limb development in Xenopus is totally different from that in amniotes such as birds and mammals, in which limb buds emerge at an early stage of embryogenesis, in parallel with other organogenesis. We investigated limb myogenesis in Xenopus, focusing on myogenic gene expression, myogenic ability of limb bud cells in the early stage, and the origin of myogenic precursor cells in the limb bud. The Xenopus early limb bud contains myoD/cardiac actin-positive and pax3/pax7-negative cells. Interestingly, results of transplantation experiments have revealed that this early limb bud contains myogenic precursor cells. In order to know the contribution of myogenic cells in somites to myogenic precursor cells in the early limb bud, we used a Cre-LoxP system for tracing over a long period. The results of fate tracing for myogenic cells in somites of the Xenopus embryo suggested that early-specified myogenic cells in somites do not contribute to limb muscle in Xenopus. Taken together, the results suggest that limb muscle development in Xenopus has characteristics of initiation and early events distinct from those of other vertebrate clades.

[Elbow joint formation in grafts of the limbs lacking the anterior or posterior necrotic zones]. / Alkunes sanario sklaida persodintose galuniu uzuomazgose, stokojanciose priekines arba uzpakalines nekrozes zonu.

UNLABELLED: The purpose of this experimental work is to examine the joint formation in limb bud grafts lacking the anterior or posterior necrotic zones where programmed cell death (apoptosis) occurs. METHODS: The embyonic chick limbs were transplanted onto the chorioallantoic membrane. The limbs of experimental group were transplanted without the prospective anterior or posterior necrotic zones. Transplants containing all limb bud material served as the control group. RESULTS: Most experimental grafts showed wide or complete fussions of the bones forming the elbow joint in the early developmental stages. The regression of the elbow joint in the control grafts was gradually progressive: from the narrow cartilaginous bridges between articular surfaces in early stages to extensive fusion of bone primodia in late developmental stages. CONCLUSIONS: The elimination of the anterior or posterior necrotic zones suppressed apoptosis in the "opaque patch" and caused the fussion of the bones in elbow joint.

Temporal restriction of migratory and lineage potential in rhombomere 1 and 2 neural crest.

Migratory cranial neural crest cells differentiate into a wide range of cell types, such as ectomesenchymal tissue (bone and connective tissues) ventrally in the branchial arches and neural tissue (neurons and glia) dorsally. We investigated spatial and temporal changes of migration and differentiation potential in neural crest populations derived from caudal midbrain and rhombomeres 1 and 2 by back-transplanting cells destined for the first branchial arch and trigeminal ganglion from HH8-HH19 quail into HH7-HH11 chicks. Branchial arch cells differentiated down ectomesenchymal lineages but largely lost both the ability to localize to the trigeminal position and neurogenic differentiation capacity by HH12-HH13, even before the arch is visible, and lost long distance migratory ability around HH17. In contrast, neural crest-derived cells from trigeminal ganglia lost ectomesechymal differentiation potential by HH17. Despite this, they retain the ability to migrate into the branchial arches until at least HH19. However, many of the neural crest-derived trigeminal ganglia cells in the branchial arch localized to the non-neural crest core of the arch from HH13 and older donors. These results suggest that long distance migration ability, finer scale localization, and lineage restriction may not be coordinately regulated in the cranial neural crest population.

Neuroectodermal origin of brain pericytes and vascular smooth muscle cells.

The origin of vascular pericytes (PCs) and smooth muscle cells (vSMCs) in the brain has hitherto remained an open question. In the present study, we used the quail-chick chimerization technique to elucidate the lineage of cranial PCs/vSMCs. We transplanted complete halves of brain anlagen, or dorsal (presumptive neural crest [NC]) or ventral cranial neural tube. Additional experiments included transplantations of neuroectoderm into limb mesenchyme, and of head mesoderm or limb mesenchyme into paraxial head mesoderm. After interspecific transplantation of quail brain rudiment, graft-derived vSMCs were found in the vessel walls of the grafted brain. Notably, transplanted ventral neural tube also gave rise to vSMCs. After grafting of quail head mesoderm, quail endothelial cells were found in the host brain, but no vSMCs of donor origin. Grafting of quail whole or ventral neural tube into the limb bud led to endowment of graft and host vessels with graft-derived vSMCs. Quail limb bud mesenchyme contributed to vSMCs in the ectopic neural graft, but, when transplanted into paraxial head mesenchyme, it did not form intraneural vSMCs. After orthotopic transplantation of cranial NC, graft-derived vSMCs were not only found in meninges and brain of the operated side, but also on the contralateral side. Our results show that 1) avian cranial neuroectoderm is able to differentiate into vSMCs of the brain; 2) this potential is not restricted to the prospective NC; and 3) neither cranial mesoderm nor cranially transplanted limb bud mesoderm can give rise to brain vSMC.

Opposing RA and FGF signals control proximodistal vertebrate limb development through regulation of Meis genes.

Vertebrate limbs develop in a temporal proximodistal sequence, with proximal regions specified and generated earlier than distal ones. Whereas considerable information is available on the mechanisms promoting limb growth, those involved in determining the proximodistal identity of limb parts remain largely unknown. We show here that retinoic acid (RA) is an upstream activator of the proximal determinant genes Meis1 and Meis2. RA promotes proximalization of limb cells and endogenous RA signaling is required to maintain the proximal Meis domain in the limb. RA synthesis and signaling range, which initially span the entire lateral plate mesoderm, become restricted to proximal limb domains by the apical ectodermal ridge (AER) activity following limb initiation. We identify fibroblast growth factor (FGF) as the main molecule responsible for this AER activity and propose a model integrating the role of FGF in limb cell proliferation, with a specific function in promoting distalization through inhibition of RA production and signaling.

Distribution of polarizing activity and potential for limb formation in mouse and chick embryos and possible relationships to polydactyly.

A central feature of the tetrapod body plan is that two pairs of limbs develop at specific positions along the head-to-tail axis. However, the potential to form limbs in chick embryos is more widespread. This could have implications for understanding the basis of limb abnormalities. Here we extend the analysis to mouse embryos and examine systematically the potential of tissues in different regions outside the limbs to contribute to limb structures. We show that the ability of ectoderm to form an apical ridge in response to FGF4 in both mouse and chick embryos exists throughout the flank as does ability of mesenchyme to provide a polarizing region signal. In addition, neck tissue has weak polarizing activity. We show, in chick embryos, that polarizing activity of tissues correlates with the ability either to express Shh or to induce Shh expression. We also show that cells from chick tail can give rise to limb structures. Taken together these observations suggest that naturally occurring polydactyly could involve recruitment of cells from regions adjacent to the limb buds. We show that cells from neck, flank and tail can migrate into limb buds in response to FGF4, which mimics extension of the apical ectodermal ridge. Furthermore, when we apply simultaneously a polarizing signal and a limb induction signal to early chick flank, this leads to limb duplications.

The "waiting period" of sensory and motor axons in early chick hindlimb: its role in axon pathfinding and neuronal maturation.

During embryonic development motor axons in the chick hindlimb grow out slightly before sensory axons and wait in the plexus region at the base of the limb for approximately 24 hr before invading the limb itself (Tosney and Landmesser, 1985a). We have investigated the role of this waiting period by asking, Is the arrest of growth cones in the plexus region a general property of both sensory and motor axons? Why do axons wait? Does eliminating the waiting period affect the further development of motor and sensory neurons? Here we show that sensory axons, like motor axons, pause in the plexus region and that neither sensory nor motor axons require cues from the other population to wait in or exit from the plexus region. By transplanting older or younger donor limbs to host embryos, we show that host axons innervate donor limbs on a schedule consistent with the age of the grafted limbs. Thus, axons wait in the plexus region for maturational changes to occur in the limb rather than in the neurons themselves. Both sensory and motor axons innervate their appropriate peripheral targets when the waiting period is eliminated by grafting older donor limbs. Therefore, axons do not require a prolonged period in the plexus region to sort out and project appropriately. Eliminating the waiting period does, however, accelerate the onset of naturally occurring cell death, but it does not enhance the development of central projections or the biochemical maturation of sensory neurons.

The recombinant limb as a model for the study of limb patterning, and its application to muscle development.

The recombinant limb is a model system that has proved fruitful for analyzing epithelial-mesenchymal interactions and understanding the functional properties of the components of the limb bud. Here we present an overview of some of the insights obtained through the use of this technique. Among these are the understanding that fore or hind limb identity is inherent to the limb bud mesoderm, that the apical ectodermal ridge (AER) is a permissive signaling center and that the limb bud ectoderm plays a central role in the control of dorsoventral polarity. Recombinant limb studies have also allowed the identification of the affected tissue component in several limb mutants. More recently this model has been applied to the study of regulation of gene expressions related to patterning. In this report we use recombinant limbs to analyze pattering of the Pax3 expressing limb muscle cell lineage in the early stages of limb development. In recombinant limbs made without the zone of polarizing activity (ZPA), myoblasts appear intermingled with other mesodermal cells at the beginning of the recombinant limb development. Rapidly thereafter, the muscle precursors segregate and organize around the central forming chondrogenic core of the recombinant. Although this segregation is reminiscent of that occurring during normal development, the myoblasts in the recombinant fail to proliferate appropriately and also fail to migrate distally. Consequently, the muscle pattern in the recombinant limb is defective indicating that normal patterning cues are absent. However, recombinant limbs polarized with a ZPA exhibited a larger mass of muscle cells and a more normal morphogenesis, supporting a role for this signaling center in limb muscle development. Finally, we have ruled out host somite contributions to recombinant limbs by grafting chick recombinant limbs to quail hosts. This initial report demonstrates the value of the recombinant limb model system for dissecting the environmental cues required for normal muscle limb patterning.

Involvement of T-box genes Tbx2-Tbx5 in vertebrate limb specification and development.

We have recently shown in mice that four members of the T-box family of transcription factors (Tbx2-Tbx5) are expressed in developing limb buds, and that expression of two of these genes, Tbx4 and Tbx5, is primarily restricted to the developing hindlimbs and forelimbs, respectively. In this report, we investigate the role of these genes in limb specification and development, using the chick as a model system. We induced the formation of ectopic limbs in the flank of chick embryos to examine the relationship between the identity of the limb-specific T-box genes being expressed and the identity of limb structures that subsequently develop. We found that, whereas bud regions expressing Tbx4 developed characteristic leg structures, regions expressing Tbx5 developed characteristic wing features. In addition, heterotopic grafts of limb mesenchyme (wing bud into leg bud, and vice versa), which are known to retain the identity of the donor tissue after transplantation, retained autonomous expression of the appropriate, limb-specific T-box gene, with no evidence of regulation by the host bud. Thus there is a direct relationship between the identity of the structures that develop in normal, ectopic and recombinant limbs, and the identity of the T-box gene(s) being expressed. To investigate the regulation of T-box gene expression during limb development, we employed several other embryological manipulations. By surgically removing the apical ectodermal ridge (AER) from either wing or leg buds, we found that, in contrast to all other genes implicated in the patterning of developing appendages, maintenance of T-box gene expression is not dependent on the continued provision of signals from the AER or the zone of polarizing activity (ZPA). By generating an ectopic ZPA, by grafting a sonic hedgehog (SHH)-expressing cell pellet under the anterior AER, we found that Tbx2 expression can lie downstream of SHH. Finally, by grafting a SHH-expressing cell pellet to the anterior margin of a bud from which the AER had been removed, we found that Tbx2 may be a direct, short-range target of SHH. Our findings suggest that these genes are intimately involved in limb development and the specification of limb identity, and a new model for the evolution of vertebrate appendages is proposed.

Retinoid signaling is required for the establishment of a ZPA and for the expression of Hoxb-8, a mediator of ZPA formation.

We show that retinoid receptor antagonists applied to the presumptive wing region block the formation of a zone of polarizing activity (ZPA). This suggests a direct relationship between retinoid signaling and the establishment of the ZPA. We provide evidence that the Hox gene, Hoxb-8, is a direct target of retinoid signaling since exogenously applied RA rapidly induces this gene in the absence of protein synthesis and, moreover, retinoid receptor antagonists down-regulate Hoxb-8 expression. In addition, we find that, in the lateral plate mesoderm, the domains of Hoxb-8 expression and of polarizing activity are coextensive. Taken together, these findings support the hypothesis that retinoids are required for the establishment of a ZPA, and that retinoids act, at least in part, through Hoxb-8, a gene associated with ZPA formation (Charité et al., 1994).