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Hedgehog (HH) signaling is a conserved pathway that drives developmental growth and is essential for the formation of most organs. The expression of HH target genes is regulated by a dual switch mechanism where GLI proteins function as bifunctional transcriptional activators (in the presence of HH signaling) and transcriptional repressors (in the absence of HH signaling). This results in a tight control of GLI target gene expression during rapidly changing levels of pathway activity. It has long been presumed that GLI proteins also repress target genes prior to the initial expression of HH in a given tissue. This idea forms the basis for the limb bud pre‐patterning model for regulating digit number. Recent findings indicate that GLI repressor proteins are indeed present prior to HH signaling but contrary to this model, GLI proteins are inert as they do not regulate transcriptional responses or enhancer chromatin modifications at this time. These findings suggest that GLI transcriptional repressor activity is not a default state as assumed, but is itself regulated in an unknown fashion. We discuss these findings and their implications for understanding pre‐patterning, digit regulation, and HH‐driven disease. [ABSTRACT FROM AUTHOR]

Background: Limb buds develop as bilateral outgrowths of the lateral plate mesoderm and are patterned along three axes. Current models of proximal to distal patterning of early amniote limb buds suggest that two signals, a distal organizing signal from the apical epithelial ridge (AER, Fgfs) and an opposing proximal (retinoic acid [RA]) act early on pattern this axis. Results: Transcriptional analysis of stage 51 Xenopus laevis hindlimb buds sectioned along the proximal‐distal axis showed that the distal region is distinct from the rest of the limb. Expression of capn8.3, a novel calpain, was located in cells immediately flanking the AER. The Wnt antagonist Dkk1 was AER‐specific in Xenopus limbs. Two transcription factors, sall1 and zic5, were expressed in distal mesenchyme. Zic5 has no described association with limb development. We also describe expression of two proximal genes, gata5 and tnn, not previously associated with limb development. Differentially expressed genes were associated with Fgf, Wnt, and RA signaling as well as differential cell adhesion and proliferation. Conclusions: We identify new candidate genes for early proximodistal limb patterning. Our analysis of RA‐regulated genes supports a role for transient RA gradients in early limb bud in proximal‐to‐distal patterning in this anamniote model organism. Key Findings: Transcriptome analysis revealed seven patterns of differential expression across stage 51 Xenopus hindlimb buds, with the distal region most transcriptionally distinct.Two distal (capn8.3, zic5) and two proximal (gata5 and tnn) genes with no previously described role in limb development were identified.Genes linked to Wnt, Fgf and retinoic acid (RA) signaling, cell adhesion and proliferation are differentially expressed across the proximodistal axis.All genes associated with RA signaling are proximal, and genes previously shown to be down‐regulated by RA are distal.Our findings support the presence of patterning gradients of RA across the early limb proximodistal axis in Xenopus. [ABSTRACT FROM AUTHOR]

Cartilage differentiates in rat limb buds cultivated in a chemically defined protein‐free medium in the same manner as in the richer serum‐supplemented medium. We aimed to investigate the remaining differentiation potential of pre‐cultivated limb buds by subsequent transplantation in vivo. Rat front (FLBs) and hind‐limb buds (HLBs) were isolated from Fischer rat dams at the 14th gestation day (GD 14) and cultivated at the air‐liquid interface in Eagle's Minimum Essential Medium (MEM) alone; with 5 μM of 5‐azacytidine (5azaC) or with rat serum (1:1). Overall growth was measured seven times during the culture by an ocular micrometre. After 14 days, explants were transplanted under the kidney capsule of adult males. Growth of limb buds was significantly lower in all limb buds cultivated in MEM than in those cultivated with serum. In MEM with 5azaC, growth of LBs was significantly lower only on day 3 of culture. Afterwards, it was higher throughout the culture period, although a statistically significant difference was assessed only for HLBs. In transplants, mixed structures developed with the differentiated transmembranous bone, cartilage with enchondral ossification, bone‐marrow, sebaceous gland, and hair that have never been found in vitro. Nerves differentiated only in transplants precultivated in the serum‐supplemented medium. We conclude that pre‐cultivation of LBs in a chemically defined protein‐free medium does not restrict osteogenesis and formation of epidermal appendages but is restrictive for neural tissue. These results are important for understanding limb development and regenerative medicine strategies. [ABSTRACT FROM AUTHOR]

Background: Interdigits (IDs) determine digit identity in chick limbs. They are located between the digital rays and act as secondary signaling centers downstream of sonic hedgehog to provide positional information for determining digit identity in the phalanx‐forming region (PFR). We examined the dynamic developmental mechanism by which PFR cells obtain positional information from IDs to determine the identity of individual digits in the chick hindlimb. Results: We identified the specific region of the IDs responsible for determining digit identity and showed that PFR cells actively receive positional information only from the posteriorly, and not the anteriorly, located IDs. We also demonstrated that digits 1, 2, and 3 are interchangeable with each other, but not with digit 4. Finally, we found that both ID4 and digital ray 4 are necessary for determining digit 4 identity. Conclusions: The digital rays are naïve during the initial stages of their development, at which time digit identity is not determined. To determine digit identity, each PFR cell shows a unidirectional response to obtain positional information specifically from the IDs located posterior to the PFR, regardless of the signal strength from the anteriorly located IDs. Key Findings: We identified the specific region of the interdigits responsible for determining digit identity and showed that phalanx‐forming region (PFR) cells actively receive positional information only from the posteriorly, and not the anteriorly, located interdigits.We demonstrated that digits 1, 2, and 3 are interchangeable with each other, but not with digit 4 in the chick hindlimb.Interdigit 4 and digital ray 4 are necessary for determining digit 4 identity.The digital rays are na=C3=AFve during the initial stages of their development, at which time digit identity is not determined. [ABSTRACT FROM AUTHOR]

Background: Xenopus embryos and tadpoles are versatile models for embryological, cell biological, and regenerative studies. Genomic and transcriptomic approaches have been increasingly employed in these frogs. Most of these genome‐wide analyses have profiled tissues in bulk, but there are many scenarios where isolation of single cells may be advantageous, including isolation of a preferred cell type, or generation of a single‐cell suspension for applications such as scRNA‐Seq. Results: Here we present a protocol for the disaggregation of complex tail and limb bud tissue, and use cell type‐specific fluorescence in transgenic X. tropicalis appendages to isolate specific cell populations using fluorescence activated cell sorting (FACS). Our protocol addresses a specific challenge in Xenopus embryos and tadpoles: the storage of maternal yolk platelets in each cell, which can introduce light scatter and thereby false positives into FACS analysis. Conclusions: Here we gate against both nontransgenic and ubiquitously transgenic animals to reduce both false positives and false negatives. We use the Xtr.Tg(pax6:GFP;cryga:RFP;actc1:RFP)Papal transgenic line as a test case to demonstrate that nucleic acid preparations made from sorted cells are high quality and specific. We anticipate this method will be adaptable to study various cell types that have transgenic reporter lines to better profile cell types of interest. Key Findings: A protocol for tissue disaggregation in tadpole tails and limb buds.Setting gates with cells from non‐transgenic animals eliminates light scattering and false positive cells.Sorted cells do not have significantly different gene expression relative to unsorted cells of the same type.Sorted cells have similar chromatin accessibility profiles to unsorted cells of the same type. [ABSTRACT FROM AUTHOR]

Paired limbs were acquired in the ancestor of tetrapods and their morphology has been highly diversified in amniotes in relation to the adaptive radiation to the terrestrial environment. These morphological changes may have been induced by modification of the developmental program of the skeletal or muscular system. To complete limb modification, it is also important to change the neuronal framework, because the functions of the limbs rely on neural circuits that involve coordinated movement. Previous studies have shown that class 3 semaphorins (Sema3 semaphorins), which act as repulsive axonal guidance cues, play a crucial role in the formation of the peripheral nerves in mice. Here, we studied the expression pattern of Sema3A orthologues in embryos of developing amniotes, including mouse, chick, soft-shelled turtle, and ocelot gecko. Sema3A transcripts were expressed in restricted mesenchymal parts of the developing limb primordium in all animals studied, and developing spinal nerves appeared to extend through Sema3A-negative regions. These results suggest that a Sema3A-dependent guidance system plays a key role in neuronal circuit formation in amniote limbs. We also found that Sema3A partially overlapped with the distribution of cartilage precursor cells. Based on these results, we propose a model in which axon guidance and skeletogenesis are linked by Sema3A; such mechanisms may underlie functional neuron rearrangement during limb diversification. [ABSTRACT FROM AUTHOR]

Background: Pannexin 3 (PANX3) is a channel-forming protein capable of stimulating osteogenesis in vitro. Here, we studied the in vivo roles of PANX3 in the chicken embryo using the RCAS retroviral system to over-express and knockdown expression during endochondral bone formation. Results: In the limbs, PANX3 RNA was first detected in the cartilage condensations and became restricted to the prehypertrophic cartilage of the epiphyses, diaphysis, and perichondrium. The increase in PANX3 was not sufficient to alter osteogenesis; however, knockdown with a virus containing an interference RNA construct caused a 20% reduction in bone volume. The control virus containing an shEGFP cassette did not affect development. Interestingly, the phenotype was restricted to later stages rather than to proliferation of the skeletogenic mesenchyme, formation of the cartilage condensation, or creation of the hypertrophic zones. In addition, there was also no change in readouts of Hedgehog, WNT, fibroblast growth factor, or bone morphogenetic protein signaling using either quantitative real-time polymerase chain reaction or radioactive in situ hybridization. Conclusions: Based on the normal expression domains of PANX3 and the relatively late manifestation of the phenotype, it is possible that PANX3 hemichannels may be required to facilitate the transition of hypertrophic chondrocytes to osteoblasts, thereby achieving final bone size. Developmental Dynamics 245:913-924, 2016. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Xenopus laevis, the South African clawed frog, is a well-established model organism for the study of developmental biology and regeneration due to its many advantages for both classical and molecular studies of patterning and morphogenesis. While contemporary studies of limb development tend to focus on models developed from the study of chicken and mouse embryos, there are also many classical studies of limb development in frogs. These include both fate and specification maps, that, due to their age, are perhaps not as widely known or cited as they should be. This has led to some inevitable misinterpretations- for example, it is often said that Xenopus limb buds have no apical ectodermal ridge, a morphological signalling centre located at the distal dorsal/ventral epithelial boundary and known to regulate limb bud outgrowth. These studies are valuable both from an evolutionary perspective, because amphibians diverged early from the amniote lineage, and from a developmental perspective, as amphibian limbs are capable of regeneration. Here, we describe Xenopus limb morphogenesis with reference to both classical and molecular studies, to create a clearer picture of what we know, and what is still mysterious, about this process. Developmental Dynamics 245:233-243, 2016. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background: While it is known that cadmium-exposed embryonic cells have increased activation of c-Jun N-terminal kinase (JNK), the role of this stress signaling pathway in the embryotoxic response is not clear. Thus, the effects of modification of the transcription factor c-Jun, one of the downstream targets of JNK, on cadmium-induced embryotoxicity were investigated in primary cultures of mouse embryo limb bud cells. Methods: Cultures of limb bud cells harvested on day 11 of gestation were pretreated with antisense oligonucleotides (ASO) to c-Jun to reduce its expression, and then incubated with cadmium in the form of cadmium chloride. Toxicity was measured through assessments of cell proliferation and differentiation, while the effectiveness of the ASO in reducing c-Jun was assessed through Western blotting using phosphorylation-specific antibodies. Results: When cells were treated with ASO c-Jun, the total amounts of c-Jun and also cadmiuminduced c-Jun activation were diminished. Cadmium-induced cytotoxicity, indicated by reduced cell numbers and differentiation, was found to decrease when cells were exposed to the antisense oligonucleotides to c-Jun. In addition, limb cell numbers and differentiation were also enhanced by exposure to ASO in the absence of cadmium. Conclusion: The JNK pathway, and particularly the downstream effector c-Jun, appears to play an important role in regulating cell survival and differentiation in mouse embryo limb bud cells both in the presence and absence of the toxic metal cadmium. [ABSTRACT FROM AUTHOR]

The vertebrate limb with its complex anatomy develops from a small bud of undifferentiated mesoderm cells encased in ectoderm. The bud has its own intrinsic polarity and can develop autonomously into a limb without reference to the rest of the embryo. In this review, recent advances are integrated with classical embryology, carried out mainly in chick embryos, to present an overview of how the embryo makes a limb bud. We will focus on how mesoderm cells in precise locations in the embryo become determined to form a limb and express the key transcription factors Tbx4 (leg/hindlimb) or Tbx5 (wing/forelimb). These Tbx transcription factors have equivalent functions in the control of bud formation by initiating a signalling cascade involving Wnts and fibroblast growth factors ( FGFs) and by regulating recruitment of mesenchymal cells from the coelomic epithelium into the bud. The mesoderm that will form limb buds and the polarity of the buds is determined with respect to both antero-posterior and dorso-ventral axes of the body. The position in which a bud develops along the antero-posterior axis of the body will also determine its identity - wing/forelimb or leg/hindlimb. Hox gene activity, under the influence of retinoic acid signalling, is directly linked with the initiation of Tbx5 gene expression in the region along the antero-posterior axis of the body that will form wings/forelimbs and determines antero-posterior polarity of the buds. In contrast, Tbx4 expression in the regions that will form legs/hindlimbs is regulated by the homeoprotein Pitx1 and there is no evidence that Hox genes determine antero-posterior polarity of the buds. Bone morphogenetic protein ( BMP) signalling determines the region along the dorso-ventral axis of the body in which both wings/forelimbs and legs/hindlimbs develop and dorso-ventral polarity of the buds. The polarity of the buds leads to the establishment of signalling regions - the dorsal and ventral ectoderm, producing Wnts and BMPs, respectively, the apical ectodermal ridge producing fibroblast growth factors and the polarizing region, Sonic hedgehog (Shh). These signals are the same in both wings/forelimbs and legs/hindlimbs and control growth and pattern formation by providing the mesoderm cells of the limb bud as it develops with positional information. The precise anatomy of the limb depends on the mesoderm cells in the developing bud interpreting positional information according to their identity - determined by Pitx1 in hindlimbs - and genotype. The competence to form a limb extends along the entire antero-posterior axis of the trunk - with Hox gene activity inhibiting the formation of forelimbs in the interlimb region - and also along the dorso-ventral axis. [ABSTRACT FROM AUTHOR]

Heparan sulfate (HS) interacts with numerous growth factors, morphogens, receptors, and extracellular matrix proteins. Disruption of HS synthetic enzymes causes perturbation of growth factor signaling and malformation in vertebrate and invertebrate development. Our previous studies show that the O-sulfation patterns of HS are essential for the specific binding of growth factors to HS chains, and that depletion of O-sulfotransferases results in remarkable developmental defects in Drosophila, zebrafish, chick, and mouse. Here, we show that inhibition of chick HS-6- O-sulfotransferases (HS6ST-1 and HS6ST-2) in the prospective limb region by RNA interference (RNAi) resulted in the truncation of limb buds and reduced Fgf-8 and Fgf-10 expressions in the apical ectodermal ridge and in the underlying mesenchyme, respectively. HS6ST-2 RNAi resulted in a higher frequency of limb truncation and a more marked change in both Fgf-8 and Fgf-10 expressions than that achieved with HS6ST-1 RNAi. HS6ST-1 RNAi and HS6ST-2 RNAi caused a significant but distinct reduction in the levels of different 6- O-sulfation in HS, possibly as a result of their different substrate specificities. Our data support a model where proper levels and patterns of 6- O-sulfation of HS play essential roles in chick limb bud development. [ABSTRACT FROM AUTHOR]

Gene transduction technologies are essential tools for understanding of gene functions or gene cascades underlying embryogenesis. In this review, we introduce a gene transduction method using microbubble and ultrasound (hereafter referred to as sonoporation). Sonoporation is carried out with relatively simple procedures and easily transduces genes into mesenchymal cells without significant damage to target tissues. Therefore, sonoporation is effective for gene transduction to study the molecular mechanisms of morphogenesis. [ABSTRACT FROM AUTHOR]

Limb skeletal elements develop from a cartilage template, which is formed by the process termed chondrogenesis. This process is crucial in determining the shape and size of definitive bones in vertebrates. During chondrogenesis, aggregated mesenchymal cells undergo a highly organized process of proliferation and maturation along with secretion of extracellular matrix followed by programmed cell death and replacement by bone. The molecular mechanisms underlying this sophisticated process have been extensively studied. It has been demonstrated that several transcription factors such as Sox genes and Runx genes are indispensable for the major steps in chondrogenesis. Additionally, a number of signaling molecules including Bmps, Fgfs and Ihh/ PTHrP are known to regulate chondrogenesis through highly coordinated interactions. This review is meant to provide an overview of the current knowledge of chondrogenesis with particular emphasis on the cellular and molecular aspects. [ABSTRACT FROM AUTHOR]

Mammalians have a low potency for limb regeneration compared to that of amphibians. One explanation for the low potency is the deficiency of cells for regenerating amputated limbs in mammals. Amphibians can form a blastema with dedifferentiated cells, but mammals have few such cells. In this paper, we report limb formation, especially bone/cartilage formation in amputated limbs, because bone/cartilage formation is a basic step in limb pattern regeneration. After the amputation of limbs of a neonatal mouse, hypertrophy of the stump bone was observed at the amputation site, which was preceded by cell proliferation and cartilage formation. However, no new elements of bone/cartilage were formed. Thus, we grafted limb buds of mouse embryo into amputated limbs of neonatal mice. When the intact limb bud of a transgenic green fluorescent protein (GFP) mouse was grafted to the limb stump after amputation at the digit joint level, the grafted limb bud grew and differentiated into bone, cartilage and soft tissues, and it formed a segmented pattern that was constituted by bone and cartilage. The skeletal pattern was more complicated when limb buds at advanced stages were used. To examine if the grafted limb bud autonomously develops a limb or interacts with stump tissue to form a limb, the limb bud was dissociated into single cells and reaggregated before grafting. The reaggregated limb bud cells formed similar digit-like bone/cartilage structures. The reaggregated grafts also formed segmented cartilage. When the reaggregates of bone marrow mesenchymal cells were grafted into the stump, these cells formed cartilage, as do limb bud cells. Finally, to examine the potency of new bone formation in the stump tissue without exogenously supplied cells, we grafted gelatin gel containing BMP-7. BMP induced formation of several new bone elements, which was preceded by cartilage formation. The results suggest that the environmental tissues of the stump allow the formation of cartilage and bone at least partially, and that limb formation will be possible by supplying competent cells endogenously or exogenously in the future. [ABSTRACT FROM AUTHOR]

Mesenchymal cells in developing limb buds of tetrapods manifest position-specific cell affinity, which is important in morphogenetic processes such as cell migration and cell condensation. The receptor tyrosine kinase EphA4 contributes to dynamic changes in position-specific cell affinity, playing an important role in normal limb development. The aim of the present study was to characterize the pattern and regulation ofEphA4gene expression during quail embryogenesis. The distribution of EphA4 mRNA in embryos was determined by whole-mountin situhybridization. Expression of theEphA4gene was not detected in the body wall before the protrusion of limb buds. EphA4 mRNA was first detected in the emerging limb buds at stage 18. As the limb buds became more distinct, EphA4 transcripts became confined to the distal tip and the base of these structures and extended into the flank.EphA4gene expression was later detected in regions of cell condensation before undergoing gradual downregulation coincident with bone formation. Removal of part of the wing bud including the zone of polarizing activity from embryos at stage 21 resulted in loss ofEphA4gene expression. Signaling from the zone of polarizing activity appears important forEphA4gene expression during limb development in quail embryos. [ABSTRACT FROM AUTHOR]

Bone morphogenetic protein 2 (BMP-2) plays a decisive role during bone regeneration and repair as well as during various stages of embryonal development. A cDNA encoding mature human BMP-2 could be efficiently expressed in Escherichia coli, and after renaturation a dimeric BMP-2 protein of Mr 26000 was prepared with a purity greater 98%. The recombinant BMP-2 was functionally active as demonstrated by the induction of alkaline phosphatase activity in the C3H10T1/2 fibroblast cell line (EC50 of 70 nM) and proteoglycan synthesis in embryonic chicken limb bud cells (EC50 of 15-20 nM). A peptide 1 - 17 representing the N-terminal basic part of BMP-2 as well as heparin increased the specific activity of the protein about fivefold in the limb bud assay. These observations suggested that the N-termini reduce the specific activity of BMP-2, probably by interacting with heparinic sites in the extracellular matrix. This conclusion was supported by a variant EHBMP-2, where the N-terminal residues 1 - 12 of BMP-2 had been substituted by a dummy sequence of equal length and which showed an EC50 value of around 1 nM which was affected neither by heparin nor by peptide 1 - 17. A physical interaction between BMP-2 and heparin could be seen in biosensor experiments, where BMP-2 bound to immobilized heparin with a dissociation constant, Kd, of approximately 20 nM, whereas the heparin-binding of variant EHBMP-2 was negligible. These results identify the basic N-terminal domains of dimeric BMP-2 as heparin-binding sites that are not obligatory for receptor activation but modulate its biological activity. [ABSTRACT FROM AUTHOR]

We asked whether specific mesenchymal/epithelial (M/E) induction generates olfactory receptor neurons (ORNs), vomeronasal neurons (VRNs), and gonadotropin-releasing hormone (GnRH) neurons, the major neuron classes associated with the olfactory epithelium (OE). To assess specificity of M/E-mediated neurogenesis, we compared the influence of frontonasal mesenchyme on frontonasal epithelium, which becomes the OE, with that of the forelimb bud. Despite differences in position, morphogenetic and cytogenic capacity, both mesenchymal tissues support neurogenesis, expression of several signaling molecules and neurogenic transcription factors in the frontonasal epithelium. Only frontonasal mesenchyme, however, supports OE-specific patterning and activity of a subset of signals and factors associated with OE differentiation. Moreover, only appropriate pairing of frontonasal epithelial and mesenchymal partners yields ORNs, VRNs, and GnRH neurons. Accordingly, the position and molecular identity of specialized frontonasal epithelia and mesenchyme early in gestation and subsequent inductive interactions specify the genesis and differentiation of peripheral chemosensory and neuroendocrine neurons., Molecular and Cellular Biology, Author's Original