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During limb bud formation, axis polarities are established as evidenced by the spatially restricted expression of key regulator genes. In particular, the mutually antagonistic interaction between the GLI3 repressor and HAND2 results in distinct and non-overlapping anterior-distal Gli3 and posterior Hand2 expression domains. This is a hallmark of the establishment of antero-posterior limb axis polarity, together with spatially restricted expression of homeodomain and other transcriptional regulators. Here, we show that TBX3 is required for establishment of the posterior expression boundary of anterior genes in mouse limb buds. ChIP-seq and differential gene expression analysis of wild-type and mutant limb buds identifies TBX3-specific and shared TBX3-HAND2 target genes. High sensitivity fluorescent whole-mount in situ hybridisation shows that the posterior expression boundaries of anterior genes are positioned by TBX3-mediated repression, which excludes anterior genes such as Gli3, Alx4, Hand1 and Irx3/5 from the posterior limb bud mesenchyme. This exclusion delineates the posterior mesenchymal territory competent to establish the Shh-expressing limb bud organiser. In turn, HAND2 is required for Shh activation and cooperates with TBX3 to upregulate shared posterior identity target genes in early limb buds. [ABSTRACT FROM AUTHOR]

Transcriptional responses to the Hedgehog (HH) signaling pathway are primarily modulated by GLI repression in the mouse limb. Previous studies suggested a role for the BAF chromatin remodeling complex in mediating GLI repression. Consistent with this possibility, the core BAF complex protein SMARCC1 is present at most active limb enhancers including the majority of GLI enhancers. However, in contrast to GLI repression which reduces chromatin accessibility, SMARCC1 maintains chromatin accessibility at most enhancers, including those bound by GLI. Moreover, SMARCC1 binding at GLI-regulated enhancers occurs independently of GLI3. Consistent with previous studies, some individual GLI target genes are mis-regulated in Smarcc1 conditional knockouts, though most GLI target genes are unaffected. Moreover, SMARCC1 is not necessary for mediating constitutive GLI repression in HH mutant limb buds. We conclude that SMARCC1 does not mediate GLI3 repression, which we propose utilizes alternative chromatin remodeling complexes. [Display omitted] • The BAF component SMARCC1 maintains enhancer accessibility in limb buds. • SMARCC1 binding to enhancers occurs independently of GLI3. • Most GLI target genes are not transcriptionally regulated by SMARCC1. • SMARCC1 is not sufficient to mediate constitutive GLI repression. [ABSTRACT FROM AUTHOR]

Recent studies illustrate the importance of regulation of cellular metabolism, especially glycolysis and pathways branching from glycolysis, during vertebrate embryo development. For example, glycolysis generates cellular energy ATP. Glucose carbons are also directed to the pentose phosphate pathway, which is needed to sustain anabolic processes in the rapidly growing embryos. However, our understanding of the exact status of glycolytic metabolism as well as genes that regulate glycolytic metabolism are still incomplete. Sall4 is a zinc finger transcription factor that is highly expressed in undifferentiated cells in developing mouse embryos, such as blastocysts and the post-implantation epiblast. TCre; Sall4 conditional knockout mouse embryos exhibit various defects in the posterior part of the body, including hindlimbs. Using transcriptomics approaches, we found that many genes encoding glycolytic enzymes are upregulated in the posterior trunk, including the hindlimb-forming region, of Sall4 conditional knockout mouse embryos. In situ hybridization and qRT-PCR also confirmed upregulation of expression of several glycolytic genes in hindlimb buds. A fraction of those genes are bound by SALL4 at the promoters, gene bodies or distantly-located regions, suggesting that Sall4 directly regulates expression of several glycolytic enzyme genes in hindlimb buds. To further gain insight into the metabolic status associated with the observed changes at the transcriptional level, we performed a comprehensive analysis of metabolite levels in limb buds in wild type and Sall4 conditional knockout embryos by high-resolution mass spectrometry. We found that the levels of metabolic intermediates of glycolysis are lower, but glycolytic end-products pyruvate and lactate did not exhibit differences in Sall4 conditional knockout hindlimb buds. The increased expression of glycolytic genes would have caused accelerated glycolytic flow, resulting in low levels of intermediates. This condition may have prevented intermediates from being re-directed to other pathways, such as the pentose phosphate pathway. Indeed, the change in glycolytic metabolite levels is associated with reduced levels of ATP and metabolites of the pentose phosphate pathway. To further test whether glycolysis regulates limb patterning downstream of Sall4 , we conditionally inactivated Hk2 , which encodes a rate-limiting enzyme gene in glycolysis and is regulated by Sall4. The TCre; Hk2 conditional knockout hindlimb exhibited a short femur, and a lack of tibia and anterior digits in hindlimbs, which are defects similarly found in the TCre; Sall4 conditional knockout. The similarity of skeletal defects in Sall4 mutants and Hk2 mutants suggests that regulation of glycolysis plays a role in hindlimb patterning. These data suggest that Sall4 restricts glycolysis in limb buds and contributes to patterning and regulation of glucose carbon flow during development of limb buds. [Display omitted] • Transcription of glycolytic enzyme genes is upregulated in Sall4 conditional knockout limb buds. • SALL4 binds to many genes encoding glycolytic enzymes. • Levels of glycolytic intermediates are reduced in Sall4 conditional knockout limb buds. • Sall4 restricts glycolysis in limb buds. • Hk2 conditional knockout phenocopies hindlimb defects of Sall4 conditional knockout. [ABSTRACT FROM AUTHOR]

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]

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: Mice with a loss of function mutation in Wdpcp were described previously to display severe birth defects in the developing heart, neural tube, and limb buds. Further characterization of the skeletal phenotype of Wdpcp null mice was limited by perinatal lethality. Results: We utilized Prx1-Cre mice to generate limb bud mesenchyme specific deletion of Wdpcp. These mice recapitulated the appendicular skeletal phenotype of the Wdpcp null mice including polydactyl and limb bud signaling defects. Examination of late stages of limb development demonstrated decreased size of cartilage anlagen, delayed calcification, and abnormal growth plates. Utilizing in vitro assays, we demonstrated that loss of Wdpcp in skeletal progenitors lead to loss of hedgehog signaling responsiveness and associated proliferative response. In vitro chondrogenesis assays showed this loss of hedgehog and proliferative response was associated with decreased expression of early chondrogenic marker N-Cadherin. E14.5 forelimbs demonstrated delayed ossification and expression of osteoblast markers Runx2 and Sp7. P0 growth plates demonstrated loss of hedgehog signaling markers and expansion of the hypertrophic zones of the growth plate. In vitro osteogenesis assays demonstrated decreased osteogenic differentiation of Wdpcp null mesenchymal progenitors in response to hedgehog stimulation. Conclusions: These findings demonstrate how Wdpcp and associated regulation of the hedgehog signaling pathway plays an important role at multiple stages of skeletal development. Wdpcp is necessary for positive regulation of hedgehog signaling and associated proliferation is key to the initiation of chondrogenesis. At later stages, Wdpcp facilitates the robust hedgehog response necessary for chondrocyte hypertrophy and osteogenic differentiation. [ABSTRACT FROM AUTHOR]

This review describes the Story of the Human Hand. It traces the functional needs that led to evolution of the human hand as well as its embryological development. The various in utero stages of formation of the human hand are covered along with a description of the various molecular and genetic factors that control this process. [ABSTRACT FROM AUTHOR]

The coordinated spatial and temporal regulation of gene expression in the murine hindlimb determines the identity of mesenchymal progenitors and the development of diversity of musculoskeletal tissues they form. Hindlimb development has historically been studied with lineage tracing of individual genes selected a priori , or at the bulk tissue level, which does not allow for the determination of single cell transcriptional programs yielding mature cell types and tissues. To identify the cellular trajectories of lineage specification during limb bud development, we used single cell mRNA sequencing (scRNA-seq) to profile the developing murine hindlimb between embryonic days (E)11.5-E18.5. We found cell type heterogeneity at all time points, and the expected cell types that form the mouse hindlimb. In addition, we used RNA fluorescence in situ hybridization (FISH) to examine the spatial locations of cell types and cell trajectories to understand the ancestral continuum of cell maturation. This data provides a resource for the transcriptional program of hindlimb development that will support future studies of musculoskeletal development and generate hypotheses for tissue regeneration. • The coordinated regulation of gene expression during embryonic development determines the diversity of tissue formation. • We used single cell mRNA sequencing to profile the developing murine hindlimb between embryonic days E11.5-E18.5. • Cell heterogeneity was present throughout development and specific genes were validated by RNA-FISH. • An understanding of the transcriptional program of hindlimb development can inform strategies for tissue regeneration. [ABSTRACT FROM AUTHOR]

Numerous hypotheses invoke tissue stiffness as a key parameter that regulates morphogenesis and disease progression. However, current methods are insufficient to test hypotheses that concern physical properties deep in living tissues. Here we introduce, validate, and apply a magnetic device that generates a uniform magnetic field gradient within a space that is sufficient to accommodate an organ-stage mouse embryo under live conditions. The method allows rapid, nontoxic measurement of the three-dimensional (3D) spatial distribution of viscoelastic properties within mesenchyme and epithelia. Using the device, we identify an anteriorly biased mesodermal stiffness gradient along which cells move to shape the early limb bud. The stiffness gradient corresponds to a Wnt5adependent domain of fibronectin expression, raising the possibility that durotaxis underlies cell movements. Three-dimensional stiffness mapping enables the generation of hypotheses and potentially the rigorous testing of mechanisms of development and disease. [ABSTRACT FROM AUTHOR]

Our studies conducted on reptilian limb muscle development revealed, for the first time, early forelimb muscle differentiation at the morphological and molecular level. Sand lizard skeletal muscle differentiation in the early forelimb bud was investigated by light, confocal, and transmission electron microscopy as well as western blot. The early forelimb bud, filled with mesenchymal cells, is surrounded by monolayer epithelium cells. The immunocytochemical analysis revealed the presence of Pax3- and Lbx-positive cells in the vicinity of the ventro-lateral lip (VLL) of the dermomyotome, suggesting that VLL is the source of limb muscle progenitor cells. Furthermore, Pax3- and Lbx-positive cells were observed in the dorsal and ventral myogenic pools of the forelimb bud. Skeletal muscle development in the early limb bud is asynchronous, which is manifested by the presence of myogenic cells in different stages of differentiation: multinucleated myotubes with well-developed contractile apparatus, myoblasts, and mitotically active premyoblasts. The western blot analysis revealed the presence of MyoD and Myf5 proteins in all investigated developmental stages. The MyoD western blot analysis showed two bands corresponding to monomeric (mMyoD) and dimeric (dMyoD) fractions. Two separate bands were also detected in the case of Myf5. The observed bands were related to non-phosphorylated (Myf5) and phosphorylated (pMyf5) fractions of Myf5. Our investigations on sand lizard forelimb myogenesis showed that the pattern of muscle differentiation in the early forelimb bud shares many features with rodents and chicks. [ABSTRACT FROM AUTHOR]

The early limb bud consists of mesenchymal limb progenitors derived from the lateral plate mesoderm (LPM). The LPM also gives rise to the mesodermal components of the flank and neck. However, the cells at these other levels cannot produce the variety of cell types found in the limb. Taking advantage of a direct reprogramming approach, we find a set of factors (Prdm16, Zbtb16, and Lin28a) normally expressed in the early limb bud and capable of imparting limb progenitor-like properties to mouse non-limb fibroblasts. The reprogrammed cells show similar gene expression profiles and can differentiate into similar cell types as endogenous limb progenitors. The further addition of Lin41 potentiates the proliferation of the reprogrammed cells. These results suggest that these same four factors may play pivotal roles in the specification of endogenous limb progenitors. [Display omitted] • Established a primary 3D limb culture system that maintains early limb progenitor cells • Used a reprogramming approach to identify factors imbuing a limb progenitor state • Together, Prdm16, Zbtb16, and Lin28a can convert non-limb cells to limb progenitors • Trajectory analysis identifies Lin41 as facilitating reprogramming by the other factors Atsuta et al. identified a 3D limb culture protocol that can maintain limb progenitors in vitro and screened for factors that can reprogram non-limb mesenchyme to a limb progenitor state. [ABSTRACT FROM AUTHOR]

Anteroposterior polarity of the early limb bud is essential for proper skeletal pattern formation. In order to establish anterior identity, hedgehog signalling needs to be repressed by GLI3 repressor activity, although the mechanism of repression is not well defined. Here we describe genetic interaction between Gli3 and Enhancer of Zeste 2 ( Ezh2 ) that encodes the histone methyltransferase subunit of Polycomb Repressive Complex 2. Loss of anterior limb identity was evident in both Gli3 and conditional Ezh2 single mutant embryos. This phenotype was enhanced in Ezh2 ; Gli3 double mutant embryos, but more closely resembled that of Ezh2 single mutants. Absent anterior skeletal elements in the Ezh2 mutant background were not rescued by either reduction of Gli activator or forced expression of Gli repressor. The data imply that Ezh2 is epistatic to Gli3 and suggest the possibility that hedghehog activation is repressed by the recruitment of polycomb repressive complex 2. [ABSTRACT FROM AUTHOR]

Background The vertebrate limb bud develops as an outgrowth of mesoderm, which forms all their elements (muscles, nerves, vessels, bone, cartilage, and tendon). Myogenic precursor cells are seen at E11.5 mouse embryo, when the first nerve fascicles begin to enter the limb. The first signs of musculature masses are seen at E12.5 in both fore and hind limb buds. Apoptosis or programmed cell death is essential in the development of the limbs. In vertebrate, the developing limb morphogenesis depends on the appropriate spatial and temporal balance between cell death and cell proliferation. Objective To perform comprehensive analysis of the proximo-distal pattern of cell death, evaluated by (TUNEL test) in cross sections of mouse limbs during prenatal development after onset of primary myogenesis. Methods Fifteen pregnant female mice (Musmusculus) were divided into three groups according to the days of pregnancy into day (14, 16 and 19), only two embryos were taken from each mouse. All the limb buds were involved in this study. Paraffin embedded histological cross-sections of the limb buds were prepared, histological staining (using H&E stain) and TUNEL test labeling were done. Assessment of the number of apoptotic cells in the limb bud mesenchyme was done by counting these cells. Results The H&E stained sections of the limb buds showed less amounts of mesenchymal tissues in older embryos (day 19). The TUNEL stain showed active apoptotic changes at proximal parts of the limb buds at gestational day 19, while the distal parts of the limbs buds showed active apoptotic changes at the early days (day 14). The evaluation of TUNEL test reaction in the proximal regions showed statistical significant increase of apoptotic cells in day 19 compared to day 14 (p = 0.001 for both). The mean number of apoptotic cells in the proximal regions were statistically significant (p = 0.001) between day 16 and day 19. While the mean number of apoptotic cells of distal regions of the limb buds was higher at day 14 compared to that of day 16 and day 19. These differences between day 14 and day 16 were statistically significant and between day 16 and day 19 while statistically non-significant between day 14 and day 19. Comparison of mean number of apoptotic cells between proximal and distal regions in all the three groups showed a statistically significant higher mean number of apoptotic cells in the distal regions compared to proximal region (p = 0.001). The mean number of the apoptotic cells in both regions (proximal and distal) of the limb buds revealed statistically significant differences between day 16 and day 19 (p = 0.001). Conclusion Apoptosis was higher in all parts of the developing limbs during day 19, and that could be associated with degenerative changes occurring at the apical ectodermal ridge. Moreover, apoptosis was higher in the distal part of the limb bud and this may be due to more differentiation of the distal parts than in the proximal part of the limb bud. [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]

Summary Limb reduction and loss are hallmarks of snake evolution. Although advanced snakes are completely limbless, basal and intermediate snakes retain pelvic girdles and small rudiments of the femur. Moreover, legs may have re-emerged in extinct snake lineages [ 1–5 ], suggesting that the mechanisms of limb development were not completely lost in snakes. Here we report that hindlimb development arrests in python embryos as a result of mutations that abolish essential transcription factor binding sites in the limb-specific enhancer of Sonic hedgehog ( SHH ). Consequently, SHH transcription is weak and transient in python hindlimb buds, leading to early termination of a genetic circuit that drives limb outgrowth. Our results suggest that degenerate evolution of the SHH limb enhancer played a role in reduction of hindlimbs during snake evolution. By contrast, HOXD digit enhancers are conserved in pythons, and HOXD gene expression in the hindlimb buds progresses to the distal phase, forming an autopodial (digit) domain. Python hindlimb buds then develop transitory pre-chondrogenic condensations of the tibia, fibula, and footplate, raising the possibility that re-emergence of hindlimbs during snake evolution did not require de novo re-evolution of lost structures but instead could have resulted from persistence of embryonic legs. Video Abstract [ABSTRACT FROM AUTHOR]

The SHH signaling pathway is essential for proper formation of the limb skeleton, as is required for the survival and expansion of distal chondrogenic progenitor cells. At the same time, SHH is important to specify digit identities along the anterior–posterior axis. Upon gain or loss of activity of the SHH pathway, bones are gained, lost or malformed, and such deregulation underlies the aetiology of various human congenital limb defects. Likewise, accumulating evidence suggests that evolutionary tampering with SHH signaling underlies the morphological diversification of the tetrapod appendicular skeleton. This review summarizes the roles of the SHH pathway in the context of limb development and evolution and incorporates recent evidence into a mechanistic view of how the positioning of digit condensations is integrated with the specification of distinct bone morphologies. [ABSTRACT FROM AUTHOR]

Gremlin1 ( grem1 ) has been previously identified as being significantly up-regulated during regeneration of Xenopus laevis limbs. Grem1 is an antagonist of bone morphogenetic proteins (BMPs) with a known role in limb development in amniotes. It forms part of a self-regulating feedback loop linking epithelial (FGF) and mesenchymal (shh) signalling centres, thereby controlling outgrowth, anterior posterior and proximal distal patterning. Spatiotemporal regulation of the same genes in developing and regenerating Xenopus limb buds supports conservation of this mechanism. Using a heat shock inducible grem1 ( G ) transgene to created temperature regulated stable lines, we have shown that despite being upregulated in regeneration, grem1 overexpression does not enhance regeneration of tadpole hindlimbs. However, both the regenerating and contralateral, developing limb of G transgenics developed skeletal defects, suggesting that overexpressing grem1 negatively affects limb patterning. When grem1 expression was targeted earlier in limb bud development, we saw dramatic bifurcations of the limbs resulting in duplication of anterior posterior (AP) pattern, forming a phenotypic continuum ranging from duplications arising at the level of the femoral head to digit bifurcations, but never involving the pelvis. Intriguingly, the original limbs have AP pattern inversion due to de-restricted Shh signalling. We discuss a possible role for Grem1 regulation of limb BMPs in regulation of branching pattern in the limbs. [ABSTRACT FROM AUTHOR]

The structural origin of the vertebrates' paired limbs is still an unsolved problem. Historically, two hypotheses have been raised to explain the origin of vertebrate limbs: the Archipterygium Hypothesis and the Fin Fold Hypothesis. Current knowledge provides support for both ideas. In the recent years, it has been also suggested that (1) all appendages correspond to body axis duplications and (2) they are originated by the ventralization of the developmental program present in the median fins. The tail bud is also a relevant structure in the attempt to understand the origin of the vertebrates' limbs. Due to their similarities in gene expression and general organization, both structures should be studied more closely to understand their potential evolutionary link. Interestingly, in non-vertebrate chordates such as Amphioxus, it is possible to find a tail fin that during development expresses several genes that are conserved with other vertebrates' limbs and tails. This shared gene expression could be considered as an evidence of potential co-option of the same genetic tool kit from the tail to the extremities. This observation is congruent with the hypothesis of Axis paramorphism, which previously suggested similarities between the tail and limb buds. [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]

The rapid growth of nanotechnological products for biomedical applications has exacerbated the need for suitable biological tests to evaluate the potential toxic effects of nanomaterials. The possible consequences of exposure during embryo and fetal development are of particular concern. The limb bud culture is anex vivo3D model in which growth, cell differentiation, and tissue organization occur and both molecular and functional endpoints can be quantitatively assessed. We employed this model to assess biochemical and morphological changes induced during organogenesis by two classes of nanostructured materials: quantum dot nanocrystals and organic polyglycerol sulfate dendrimers (dPGS). We show that quantum dots carrying mercaptopropionic acid (QD-MPA) on the surface, commonly used in biological studies, inhibit the development of limb buds from CD1 wildtype andCol2a1;Col10a1;Col1a1triple transgenic fluorescent reporter mice, as revealed by changes in several morphological and biochemical markers. QD-MPA interfere with chondrogenesis and osteogenesis and disrupt the expression of COL10A1 and COL1A1, key markers of differentiation. In contrast, equivalent (3–100 nM) concentrations of dPGS do not adversely affect limb development. Neither QD-MPA nor dPGS-Cy5 alters the expression of several markers of cell proliferation or apoptosis. Collectively, these results suggest that murine limb buds in culture constitute a convenient, inexpensive and reliable developmental model for the assessment of the nanotoxicological effects of nanocrystals and polymers. In these 3D cultures, any effect that is observed can be directly ascribed to the nanostructuresper seor a degradation component released from the complex nanostructure. [ABSTRACT FROM PUBLISHER]

Objective: There is a wide application of titanium dioxide (TiO2) nanoparticles (NPs) in industry. These particles are used in various products, and they also has biological effects on cells and organs through direct contact. Materials and Methods: In this experimental research, the effect of TiO2 on chondrogenesis of forelimb buds of mice embryos was assessed in in vivo condition. Concentrations of 30, 150 and 500 mg/kg body weight (BW) TiO2 NPs (20 nm size) dissolved in distilled water were injected intraperitoneally to Naval Medical Research Institute (NMRI) mice on day 11.5 of gestation. On day 15, limb buds were amputated from the embryos and skeletogeneis of limb buds were studied. Results: TiO2 NPs caused the significant changes in chondrocytes in the following developmental stages: resting, proliferating, hypertrophy, degenerating, perichondrium and mesenchymal cells. Decreased number of mesenchymal cells and increased level of chondrocytes were observed after the injection of different concentrations of TiO2, which proves the unpredictable effects of TiO2 on limb buds. Conclusion: Results of the present study showed TiO2 NPs accelerated the chondrogenesis of limb buds, but further studies are recommended to predict TiO2 toxicity effects on organogenesis. [ABSTRACT FROM AUTHOR]

LIM homeobox transcription factor Lhx2 is known to be an important regulator of neuronal development, homeostasis of hair follicle stem cells, and self-renewal of hematopoietic stem cells; however, its function in skeletal muscle development is poorly understood. In this study, we found that overexpression of Lhx2 completely inhibits the myotube-forming capacity of C2C12 cells and primary myoblasts. The muscle dedifferentiation factors Msx1 and Msx2 were strongly induced by the Lhx2 overexpression. Short interfering RNA-mediated knockdown of Lhx2 in the developing limb buds of mouse embryos resulted in a reduction in Msx1 and Msx2 mRNA levels, suggesting that they are downstream target genes of Lhx2. We found two Lhx2 consensus-binding sites in the −2097 to −1189 genomic region of Msx1 and two additional sites in the −536 to +73 genomic region of Msx2 . These sequences were shown by luciferase reporter assay to be essential for Lhx2-mediated transcriptional activation. Moreover, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that Lhx2 is present in chromatin DNA complexes bound to the enhancer regions of the Msx1 and Msx2 genes. These data demonstrate that Msx1 and Msx2 are direct transcriptional targets of Lhx2. In addition, overexpression of Lhx2 significantly enhanced the mRNA levels of bone morphogenetic protein 4 and transforming growth factor beta family genes. We propose that Lhx2 is involved in the early stage of skeletal muscle development by inducing multiple differentiation inhibitory factors. [ABSTRACT FROM AUTHOR]

Gestational diabetes has an adverse impact on fetal musculoskeletal development, but the mechanism involved is still not completely understood. In this study, we investigated the effects of high glucose on the developing somites and their derivate using the chick embryo as a model. We demonstrated that under high glucose, the number of generated somites was reduced and their morphology altered in 2-day old chick embryos. In addition, high glucose repressed the development of the limb buds in 5.5-day old chick embryos. We also demonstrated that high glucose abridged the development of the sclerotome and the cartilage in the developing limb bud. The sonic hedgehog (Shh) gene has been reported to play a crucial role in the development and differentiation of sclerotome. Hence, we examined how Shh expression in the sclerotome was affected under high glucose. We found that high glucose treatment significantly inhibited Shh expression. The high glucose also impaired myotome formation at trunk level – as revealed by immunofluorescent staining with MF20 antibodies. In the neural tube, we established that Wnt3a expression was also significantly repressed. In summary, our study demonstrates that high glucose concentrations impair somite and limb bud development in chick embryos, and suggests that Shh and Wnt genes may play a role in the underlying mechanism. [ABSTRACT FROM AUTHOR]

The analysis of vertebrate limb bud development provides insight of general relevance into the signaling networks that underlie the controlled proliferative expansion of large populations of mesenchymal progenitors, cell fate determination and initiation of differentiation. In particular, extensive genetic analysis of mouse and experimental manipulation of chicken limb bud development has revealed the self-regulatory feedback signaling systems that interlink the main morphoregulatory signaling pathways including BMPs and their antagonists. It this review, we showcase the key role of BMPs and their antagonists during limb bud development. This review provides an understanding of the key morphoregulatory interactions that underlie the highly dynamic changes in BMP activity and signal transduction as limb bud development progresses from initiation and setting-up the signaling centers to determination and formation of the chondrogenic primordia for the limb skeletal elements. [ABSTRACT FROM AUTHOR]

Highlights: [•] Transcriptome analysis of Xenopus limb buds predicted differential expression of three sprouty (spry1a, 2 and 4) genes. [•] Distal expression of all three preceded restriction of spry1a and 4 to anterior distal mesenchyme and spry2 to the AER. [•] Spry genes exhibited de novo expression during limb regeneration but were not localized along the anterior posterior axis. [Copyright &y& Elsevier]

Abstract: Species of frogs that develop directly have removed the tadpole from their ontogeny and form adult structures precociously. To see whether cell cycle regulators could be involved in this altered embryogenesis, we examined the expression of ccnd1, ccnd2, and mycn in embryos of the direct developing frog, Eleutherodactylus coqui. Notable differences compared to embryos of Xenopus laevis, a species with a tadpole, included prominent expression of ccnd2 in the midbrain and ccnd1 in the mandibular neural crest. The former may contribute to the precocious appearance of the adult-type visual system and the latter to the adult-type jaw. Large domains of ccnd2 and mycn presage the early appearance of limb buds, and ccnd1 and mycn are implicated in digit development. [Copyright &y& Elsevier]

High density micromass culture of limb bud mesenchymal stem cells isolated from mouse embryos represents a well-established model to study chondro- and osteogenesis. In spite of wide usage of the limb bud model, the mechanisms underlying cartilage nodule growth remain unclear. To determine whether cartilage nodules grow solely by induction of surrounding cells or proliferation of cells within the nodules, we performed BrdU/Collagen II (Col II) double-labelling and 3D reconstruction of growing cartilage nodules. We demonstrated that Col II-positive replicating chondrocytes are present throughout the nodules with the majority of replicating cells localized on the top (cell-medium interface) and periphery/sides of nodules. Kinetic analysis of cellular proliferation within the nodules demonstrated the time-dependent reduction in number of Col II-positive replicating cells. The sequential expression of Col I, Col II, Col X, parathyroid hormone related peptide receptor 1 ( Pthr1), bone sialoprotein ( Bsp) and osteocalcin ( Ocn) mRNAs was similar to that characterizing chondrocyte differentiation and maturation in vivo. We conclude that the limb bud model recapitulates events seen during endochondral bone formation: cellular aggregation, proliferation, differentiation and maturation to hypertrophy. We also conclude that not only induction of peri-nodular mesenchymal cells but also proliferation of chondrocytes within cartilage nodules contribute to cartilage nodule growth. [ABSTRACT FROM AUTHOR]

The article presents information regarding a study to evaluate the requirement of Smad4 protein for inducing digit ray primordia in order to initiate aggregation and differentiation of chondrogenic progenitors in mouse limb buds. It mentions that the Smad4 is a mediator of canonical signal transduction. It mentions that the Smad4 is required for initiating Sox9-positive digit ray primordia formation.

Abstract: During development, the mammalian embryo must integrate signals to control growth and proliferation. A failure in the ability to respond to mitogenic stimuli can cause embryonic growth restriction. We have identified a mouse mutant, l11Jus15, from a mutagenesis screen that exhibits growth defects and late-gestation lethality. Here we demonstrate that this phenotype results from a mutation in the Mediator complex gene Med31, which causes degradation of Med31 protein. The Med31 mutant phenotype is not similar to other Mediator complex mouse mutants, and target genes of other Mediator proteins are expressed normally in Med31 mutants, suggesting that Med31 has distinct target genes required for mammalian development. Med31 mutant embryos have fewer proliferating cells than controls, especially in regions that expand rapidly during development such as the forelimb buds. Likewise, embryonic fibroblast cells cultured from mutant embryos have a severe proliferation defect, as well as reduced levels of the cell cycle protein Cdc2. Med31 mutants have normal limb bud patterning but defective or delayed chondrogenesis due to a lack of Sox9 and Col2a1 expression. As the Mediator complex is a transcriptional co-activator, our results suggest that Med31 functions to promote the transcription of genes required for embryonic growth and cell proliferation. [Copyright &y& Elsevier]

Abstract: While it is well accepted that mammalian embryos may develop abnormally in response to harmful environmental stresses, the molecular changes that embryonic cells undergo are not always clear. In the present study we examined the induction of mitogen-activated protein kinase (MAPK) pathways by cadmium chloride (CdCl2) in micromass cultures of limb bud cells from day 11 BALB/c mouse embryos. CdCl2 exposure was found to cause a marked reduction in both cell survival and differentiation in these cultures. To study stress signalling pathways, cells were lysed and were examined for the phosphorylation of three MAPKs. It was shown that 4μM CdCl2 treatment of limb bud cells caused the phosphorylation of c-Jun N-terminal kinase (JNK), but not p38 or extracellular signal-regulated kinase (ERK), over a 120min period. The results indicate a specific activation of the JNK pathway in response to CdCl2-induced toxicity in mouse limb bud cells. [Copyright &y& Elsevier]

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]

Abstract: While the various internal organs of vertebrates display many obvious left–right asymmetries in their location and/or morphology, external features exhibit a high degree of bilateral symmetry. How this external bilateral symmetry is established during development is largely unknown. In this review, we explore several mechanisms, in place during development, that regulate the final size of the limb. These mechanisms rely on the presence of positive signaling feedback loops during limb bud growth. Through the activity of these signaling loops and their eventual breakdown when the limb bud has reached a certain size, bilateral symmetry can be achieved. [Copyright &y& Elsevier]

Abstract: Cell–cell communication is critical for regulating embryonic organ growth and differentiation. The Bone Morphogenetic Protein (BMP) family of transforming growth factor β (TGFβ) molecules represents one class of such cell–cell signaling molecules that regulate the morphogenesis of several organs. Due to high redundancy between the myriad BMP ligands and receptors in certain tissues, it has been challenging to address the role of BMP signaling using targeting of single Bmp genes in mouse models. Here, we present a detailed study of the developmental expression profiles of three BMP ligands (Bmp2, Bmp4, Bmp7) and three BMP receptors (Bmpr1a, Bmpr1b, and BmprII), as well as their molecular antagonist (noggin), in the early embryo during the initial steps of murine organogenesis. In particular, we focus on the expression of Bmp family members in the first organs and tissues that take shape during embryogenesis, such as the heart, vascular system, lungs, liver, stomach, nervous system, somites and limbs. Using in situ hybridization, we identify domains where ligand(s) and receptor(s) are either singly or co-expressed in specific tissues. In addition, we identify a previously unnoticed asymmetric expression of Bmp4 in the gut mesogastrium, which initiates just prior to gut turning and the establishment of organ asymmetry in the gastrointestinal tract. Our studies will aid in the future design and/or interpretation of targeted deletion of individual Bmp or Bmpr genes, since this study identifies organs and tissues where redundant BMP signaling pathways are likely to occur. [Copyright &y& Elsevier]

Abstract: Limb bud outgrowth in chicken embryos is initiated during the third day of development by Fibroblast Growth Factor 8 (FGF8) produced by the newly formed apical ectodermal ridge (AER). One of the earliest effects of this induction is a change in the properties of the limb field mesoderm leading to bulging of the limb buds from the body wall. Heintzelman et al. [Heintzelman, K.F., Phillips, H.M., Davis, G.S., 1978. Liquid-tissue behavior and differential cohesiveness during chick limb budding. J. Embryol. Exp. Morphol. 47, 1–15.] suggested that budding of the limbs is caused by a higher liquid-like cohesivity of limb bud tissue compared with flank. We sought additional evidence relevant to this hypothesis by performing direct measurements of the effective surface tension, a measure of relative tissue cohesivity, of 4-day embryonic chicken wing and leg bud mesenchymal tissue, and adjacent flank mesoderm. As predicted, the two types of limb tissues were 1.5- to 2-fold more cohesive than the flank tissue. These differences paralleled cell number and volume density differences: 4-day limb buds had 2- to 2.5-fold as many cells per unit area of tissue as surrounding flank, a difference also seen at 3 days, when limb budding begins. Exposure of flank tissue to exogenous FGF8 for 24 h increased its cell number and raised its cohesivity to limb-like values. Four-day flank tissue exhibited a novel and unique active rebound response to compression, which was suppressed by the drug latrunculin and therefore dependent on an intact actin cytoskeleton. Correspondingly, flank at this stage expressed high levels of α-smooth muscle actin (SMA) mRNA and protein and a dense network of microfilaments. Treatment of flank with FGF8 eliminated the rebound response. We term material properties of tissues, such as cohesivity and mechanical excitability, the “physical phenotype”, and propose that changes thereof are driving forces of morphogenesis. Our results indicate that two independent aspects of the physical phenotype of flank mesoderm can be converted to a limb-like state in response to treatment with FGF8. The higher tissue cohesivity induced by this effect will cause the incipient limb bud to phase separate from the surrounding flank, while the active mechanical response of the flank could help ensure that the limb bud bulges out from, rather than becoming engulfed by, this less cohesive tissue. [Copyright &y& Elsevier]

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]

Abstract: The Caenorhabditis elegans heterochronic gene lin-28 regulates developmental timing in the nematode trunk. We report the dynamic expression patterns of Lin-28 homologues in mouse and chick embryos. Whole mount in situ hybridization revealed specific and intriguing expression patterns of Lin-28 in the developing mouse and chick limb bud. Mouse Lin-28 expression was detected in both the forelimb and hindlimb at E9.5, but disappeared from the forelimb at E10.5, and finally from the forelimb and hindlimb at E11.5. Chicken Lin-28, which was first detected in the limb primordium at stage 15/16, was also downregulated as the stage proceeded. The amino acid sequences of mouse and chicken Lin-28 genes are highly conserved and the similar expression patterns of Lin-28 during limb development in mouse and chicken suggest that this heterochronic gene is also conserved during vertebrate limb development. [Copyright &y& Elsevier]

Abstract: The chick limb bud has been used as a model system for studying pattern formation and tissue development for more than 50 years. However, the lineal relationships among the different cell types and the migrational boundaries of individual cells within the limb mesenchyme have not been explored. We have used a retroviral lineage analysis system to track the fate of single limb bud mesenchymal cells at different times in early limb development. We find that progenitor cells labeled at stage 19–22 can give rise to multiple cell types including clones containing cells of all five of the major lateral plate mesoderm-derived tissues (cartilage, perichondrium, tendon, muscle connective tissue, and dermis). There is a bias, however, such that clones are more likely to contain the cell types of spatially adjacent tissues such as cartilage/perichondrium and tendon/muscle connective tissue. It has been recently proposed that distinct proximodistal segments are established early in limb development; however our analysis suggests that there is not a strict barrier to cellular migration along the proximodistal axis in the early stage 19–22 limb buds. Finally, our data indicate the presence of a dorsal/ventral boundary established by stage 16 that is inhibitory to cellular mixing. This boundary is demarcated by the expression of the LIM-homeodomain factor lmx1b. [Copyright &y& Elsevier]

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]

Abstract: Skeletogenesis is a complex process that involves the interaction of many tissues and cell types and that engages many intercellular signaling systems. In this review we focus on the Fibroblast Growth Factor (FGF) signaling system, which is involved in all stages of skeletogenesis, from the earliest stages of limb bud development to the growth and remodeling of bone. Examination of phenotypes of Fgf18 and Fgfr3 null mutations and conditional alleles for Fgfr1 and Fgfr2, targeted with Dermo1 Cre , Col2 Cre and Col1 Cre , is beginning to unravel the contribution of these signaling molecules to different stages of limb and skeletal development. [Copyright &y& Elsevier]

Abstract: Scube genes encode a small group of secreted plasma membrane-associated proteins characterised by a N-terminal signal peptide sequence, multiple EGF domains, a N-linked glycosylated spacer region and a C-terminal CUB region. Here we describe expression of the mouse Scube3 gene during early embryonic development. Transcripts were initially localised to neurectoderm of the developing embryo, in the ventral rhombencephalon and caudal neuropore. However, as development progressed, strong expression was detected in ectodermal, endodermal and mesodermal tissues. In particular, the neural tube, branchial arches and fronto-nasal region, the dermomyotome of differentiating somites and the limb buds. Scube3 also demonstrated a highly restricted and specific expression domain in the developing tooth and hair follicle. At later stages, expression was also localised to cartilaginous primordia of the skeleton and regions of intramembranous bone formation in the developing craniofacial region. In addition, Scube3 transcripts were also found in the developing kidney. [Copyright &y& Elsevier]

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]

Abstract: Formation of paired limbs in vertebrate embryos has long been a particularly useful paradigm for the study of pattern formation. Here, we show that Blimp-1, a SET domain and zinc finger-containing transcriptional factor, plays an important role in the development of the pectoral fins of the zebrafish structures that are homologous to forelimbs of amniotes. The blimp-1 gene is expressed dynamically in the mesenchyme as well as the ectodermal cells of the early fin bud, and later, in the cells of the apical ectodermal ridge (AER) of the outgrowing fin. Consistent with this expression profile, loss of Blimp-1 activity severely impairs fin outgrowth and patterning. We present evidence that blimp-1 functions downstream of tbx5 and fgf24 and therefore is not required for the initial specification of the fin bud primordia. Subsequently, however, its function is necessary for the induction of fgf10 and sonic hedgehog in the mesenchyme. In addition, Blimp-1 activity is absolutely critical for the proper induction of gene expression in the ectoderm and establishment of the AER. Taken together, these results identify an additional layer of control in the genetic pathway that operates in the developing limb and provides novel insights into regulatory mechanisms that organize its pattern. [Copyright &y& Elsevier]

Abstract: Proper outgrowth of the limb bud requires a positive feedback loop between Sonic hedgehog (Shh) in the zone of polarizing activity (ZPA) and Fgfs in the overlying apical ectodermal ridge. The Bmp antagonist Gremlin is expressed in a domain anterior to the ZPA and is thought to act as a signaling intermediate between Shh and Fgf. It is currently unclear whether Shh acts directly or indirectly to initiate and maintain Gremlin. In this study, we confirm that Bmp activity is necessary and sufficient for induction of Gremlin. Beads soaked in the Bmp antagonist Noggin downregulate Gremlin, while beads soaked in Bmp2 cause its upregulation. Furthermore, Bmp2 is also capable of upregulating Gremlin in oligozeugodactyly mutant limbs that lack Shh activity, demonstrating that Gremlin expression does not depend on the combined exposure to both these factors. In spite of the ability of Bmp2 to induce Gremlin, beads soaked in high concentrations of Bmp2 downregulate Gremlin around the bead without apparent induction of cell death, whereas another target gene Msx2 is upregulated around the bead. Consistent with this concentration-dependent effect, we find that low concentrations of Bmp2 upregulate Gremlin while high concentrations of Bmp2 downregulate Gremlin in limb mesenchyme cultures. These data implicate Bmp activity as a required intermediate in the Shh–Fgf4 signaling loop. Though we show that Bmp activity is sufficient to upregulate Gremlin, Gremlin expression is excluded from a posterior domain of the limb, and expansion of this domain as limb outgrowth proceeds is important in terminating the Shh–Fgf4 signaling loop. We find that the posterior limb is refractory to Gremlin induction in response to Bmp2, suggesting that termination of the Shh–Fgf4 signaling loop results from inability of Bmp activity to induce Gremlin in the posterior. In contrast, in the oligozeugodactyly limb, we find that beads soaked in Bmp2 can induce Gremlin in the posterior, demonstrating that Shh activity is required for exclusion of Gremlin in the posterior. Finally, by blocking Shh activity with cyclopamine, we find evidence that continued Shh activity is also required to maintain refractoriness to Gremlin expression in response to Bmp activity. [Copyright &y& Elsevier]

Abstract: Sonic hedgehog (Shh) signaling controls numerous aspects of vertebrate development, including proliferation of precursors in different organs. Identification of molecules that link the Shh pathway to cell cycle machinery is therefore of major importance for an understanding of the mechanisms underlying Shh-dependent proliferation. Here, we show that an actor in the control of entry into mitosis, the phosphatase CDC25B, is transcriptionally upregulated by the Shh/Gli pathway. Unlike other G2/M regulators, CDC25B is highly expressed in domains of Shh activity, including the ventral neural tube and the posterior limb bud. Loss- and gain-of-function experiments reveal that Shh contributes to CDC25B transcriptional activation in the neural tube both of chick and mouse embryos. Moreover, CDC25B transcripts are absent from the posterior limb bud of Shh−/− mice, while anterior grafts of Shh-expressing cells in the chicken limb bud induce ectopic CDC25B expression. Arresting the cell cycle does not reduce the level of CDC25B expression in the neural tube strongly suggesting that the upregulation of CDC25B is not an indirect consequence of the Shh-dependent proliferation. These data reveal an unexpected developmental link between the Shh pathway and a participant in G2/M control. [Copyright &y& Elsevier]

Abstract: Tetrapod limbs, forelimbs and hindlimbs, emerge as limb buds during development from appropriate positions along the rostro-caudal axis of the main body. In this study, tissue interactions by which rostro-caudal level-specific limb initiation is established were analyzed. The limb bud originates from the lateral plate located laterally to the paraxial mesoderm, and we obtained evidence that level-specific tissue interactions between the paraxial mesoderm and the lateral plate mesoderm are important for the determination of the limb-type-specific gene expression and limb outgrowth. When the wing-level paraxial mesoderm was transplanted into the presumptive leg region, the wing-level paraxial mesoderm upregulated the expression of Tbx5, a wing marker gene, and downregulated the expression of Tbx4 and Pitx1, leg marker genes, in the leg-level lateral plate. The wing-level paraxial mesoderm relocated into the leg level also inhibited outgrowth of the hindlimb bud and downregulated Fgf10 and Fgf8 expression, demonstrating that the wing-level paraxial mesoderm cannot substitute for the function of the leg-level paraxial mesoderm in initiation and outgrowth of the hindlimb. The paraxial mesoderm taken from the neck- and flank-level regions also had effects on Tbx5/Tbx4 expression with different efficiencies. These findings suggest that the paraxial mesoderm has level-specific abilities along the rostro-caudal axis in the limb-type-specific mechanism for limb initiation. [Copyright &y& Elsevier]

In contrast to the pattern of limb emergence in mammals, chicks, and the newt N. viridescens, embryos such as Xenopus laevis and Eleutherodactylus coqui initiate pelvic limb buds before they develop pectoral ones. We studied the expression of Pitx1 in X. laevis and E. coqui to determine if this paired-like homeodomain transcription factor directs differentiation specifically of the hindlimb, or if it directs the second pair of limbs to form, namely the forelimbs. We also undertook to determine if embryonic expression patterns were recapitulated during the regeneration of an amputated limb bud. Pitx1 is expressed in hindlimbs in both X. laevis and E. coqui, and expression is similar in both developing and regenerating limb buds. Expression in hindlimbs is restricted to regions of proliferating mesenchyme. [ABSTRACT FROM AUTHOR]

Abstract: The tetrapod limb, which has served as a paradigm for the study of development and morphological evolution, is becoming a paradigm for developmental evolution as well. In its origin and diversification, the tetrapod limb has undergone a great deal of remodeling. These morphological changes and other evolutionary phenomena have produced variation in mechanisms of tetrapod limb development. Here, we review that variation in the four major clades of limbed tetrapods. Comparisons in a phylogenetic context reveal details of development and evolution that otherwise may have been unclear. Such details include apparent differences in the mechanisms of dorsal–ventral patterning and limb identity specification between mouse and chick and mechanistic novelties in amniotes, anurans, and urodeles. As we gain a better understanding of the details of limb development, further differences among taxa will be revealed. The use of appropriate comparative techniques in a phylogenetic context thus sheds light on evolutionary transitions in limb morphology and the generality of developmental models across species and is therefore important to both evolutionary and developmental biologists. [Copyright &y& Elsevier]

Abstract: Spontaneous and engineered mutations in the Notch ligand Jagged2 produced the Syndactylism phenotype (Jiang, R.L., Lan, Y., Chapman, H.D., Shawber, C., Norton, C.R., Serreze, D.V., Weinmaster, G., Gridley, T., 1998. Defects in limb, craniofacial, and thymic Development in Jagged2 mutant mice. Genes Dev. 12, 1046–1057; Sidow, A., Bulotsky, M.S., Kerrebrock, A.W., Bronson, R.T., Daly, M.J., Reeve, M.P., Hawkins, T.L., Birren, B.W., Jaenisch, R., Lander, E.S., 1997. Serrate2 is disrupted in the mouse limb-development mutant syndactylism. Nature 389, 722–725). Given that additional ligands may be expressed in the developing limb bud, it was possible that loss of Jagged2 disabled only part of Notch function in the limb. In addition, it is not clear from the expression pattern of Jagged2 in the apical ectodermal ridge (AER) whether the ectodermal or mesenchymal compartment of the limb bud receives the Jagged2 signal. To elucidate the requirement for the Notch pathway in limb development, we have analyzed single and compound Notch receptor mutants as well as γ-secretase-deficient limbs. Floxed alleles were removed either from the developing limb bud ectoderm (using Msx2-Cre) or from the mesenchyme (using Prx1-Cre). Our results confirm that Jagged2 loss describes the contribution of the entire Notch pathway to the mouse limb development and revealed that both Notch1 and 2 are required in the ectoderm to receive the Jagged2 signal. Interestingly, our allelic series allowed us to determine that Notch receives this signal at an early stage in the developmental process and that memory of this event is retained by the mesenchyme, where Notch signaling appears to be dispensable. Thus, Notch signaling plays a non-autonomous role in digit septation. [Copyright &y& Elsevier]

Abstract: Groucho-related genes (Grgs) encode transcriptional co-repressors of Lef/Tcf and Hes proteins, which are mediators of Wnt and Notch signalling, respectively. Thus, they are important players in the developmental processes controlled by Wnt and Notch signalling, including lateral inhibition, segmentation and dorso-ventral patterning. We have cloned the avian homologues of Grg genes and examined their expression pattern by whole-mount in situ hybridisation between Hamburger–Hamilton (HH) stages 3 and 24. At HH stage 3, Grg gene expression is detected in the primitive streak and Hensen''s node. Later, Grg genes are expressed at high levels in the developing head fold and by HH stage 11, throughout the anterior CNS and in the ventricular zone of the neural tube. In addition, Grg2, Grg4 and Grg5 are expressed in the notochord. In the paraxial mesoderm, Grg genes are activated as soon as somites form. As somites mature, Grg1 and Grg5/AES are expressed predominantly in the medial myotome and dermomyotome, whereas Grg2, Grg3 and Grg4 are expressed throughout the myotome. In HH stage 20 limbs, Grg1, Grg3 and Grg4 transcripts are more abundant in the posterior limb bud, whereas Grg2 and Grg5/AES are expressed throughout. By HH stage 24, Grg1, Grg2 and Grg3 become localized to the dorsal and ventral limb muscle masses, whereas Grg4 and Grg5/AES occupy a more central and ventro-proximal domain, respectively. Overall, our expression data are consistent with a role for Grg genes in Lef/Tcf and Wnt signalling during somitogenesis and with a role in Hes and Notch signalling in neurogenesis. [Copyright &y& Elsevier]

Sirenomelia or mermaid-like phenotype is one of the principal human congenital malformations that can be traced back to the stage of gastrulation. Sirenomelia is characterized by the fusion of the two hindlimbs into a single one. In the mouse, sirens have been observed in crosses between specific strains and as the consequence of mutations that increase retinoic acid levels. We report that the loss of bone morphogenetic protein 7 (Bmp7) in combination with a half dose or complete loss of twisted gastrulation (Tsg) causes sirenomelia in the mouse. Tsg is a Bmp- and chordin-binding protein that has multiple effects on Bmp metabolism in the extracellular space; Bmp7 is one of many Bmps and is shown here to bind to Tsg. In Xenopus, co-injection of Tsg and Bmp7 morpholino oligonucleotides (MO) has a synergistic effect, greatly inhibiting formation of ventral mesoderm and ventral fin tissue. In the mouse, molecular marker studies indicate that the sirenomelia phenotype is associated with a defect in the formation of ventroposterior mesoderm. These experiments demonstrate that dorsoventral patterning of the mouse posterior mesoderm is regulated by Bmp signaling, as is the case in other vertebrates. Sirens result from a fusion of the hindlimb buds caused by a defect in the formation of ventral mesoderm. [ABSTRACT FROM AUTHOR]