Normal skeletal pattern formation in chick limb bud with a mesenchymal hole is mediated by adjustment of cellular properties along the anterior-posterior axis in the limb bud.

The chick limb bud has plasticity to reconstruct a normal skeletal pattern after a part of mesenchymal mass is excised to make a hole in its early stage of development. To understand the details of hole closure and re-establishment of normal limb axes to reconstruct a normal limb skeleton, we focused on cellular and molecular changes during hole repair and limb restoration. We excised a cube-shaped mass of mesenchymal cells from the medial region of chick hindlimb bud (stage 23) and observed the following morphogenesis. The hole had closed by 15 â€‹h after excision, followed by restoration of the limb bud morphology, and the cartilage pattern was largely restored by 48 â€‹h. Lineage analysis of the mesenchymal cells showed that cells at the anterior and posterior margins of the hole were adjoined at the hole closure site, whereas cells at the proximal and distal margins were not. To investigate cell polarity during hole repair, we analyzed intracellular positioning of the Golgi apparatus relative to the nuclei. We found that the Golgi apparatus tended to be directed toward the hole among cells at the anterior and posterior margins but not among cells at identical positions in normal limb buds or cells at the proximal and distal hole margins. In the manipulated limb buds, the frequency of cell proliferation was maintained compared with the control side. Tbx3 expression, which was usually restricted to anterior and posterior margins of the limb bud, was temporarily expanded medially and then reverted to a normal pattern as limb reconstruction proceeded, with Tbx3 negative cells reappearing in the medial regions of the limb buds. Thus, mesenchymal hole closure and limb reconstruction are mainly mediated by cells at the anterior and posterior hole margins. These results suggest that adjustment of cellular properties along the anteroposterior axis is crucial to restore limb damage and reconstruct normal skeletal patterns.

Cell-specific alterations in Pitx1 regulatory landscape activation caused by the loss of a single enhancer.

Developmental genes are frequently controlled by multiple enhancers sharing similar specificities. As a result, deletions of such regulatory elements have often failed to reveal their full function. Here, we use the Pitx1 testbed locus to characterize in detail the regulatory and cellular identity alterations following the deletion of one of its enhancers (Pen). By combining single cell transcriptomics and an in-embryo cell tracing approach, we observe an increased fraction of Pitx1 non/low-expressing cells and a decreased fraction of Pitx1 high-expressing cells. We find that the over-representation of Pitx1 non/low-expressing cells originates from a failure of the Pitx1 locus to coordinate enhancer activities and 3D chromatin changes. This locus mis-activation induces a localized heterochrony and a concurrent loss of irregular connective tissue, eventually leading to a clubfoot phenotype. This data suggests that, in some cases, redundant enhancers may be used to locally enforce a robust activation of their host regulatory landscapes.

Spatial regulation by multiple Gremlin1 enhancers provides digit development with cis-regulatory robustness and evolutionary plasticity.

Precise cis-regulatory control of gene expression is essential for normal embryogenesis and tissue development. The BMP antagonist Gremlin1 (Grem1) is a key node in the signalling system that coordinately controls limb bud development. Here, we use mouse reverse genetics to identify the enhancers in the Grem1 genomic landscape and the underlying cis-regulatory logics that orchestrate the spatio-temporal Grem1 expression dynamics during limb bud development. We establish that transcript levels are controlled in an additive manner while spatial regulation requires synergistic interactions among multiple enhancers. Disrupting these interactions shows that altered spatial regulation rather than reduced Grem1 transcript levels prefigures digit fusions and loss. Two of the enhancers are evolutionary ancient and highly conserved from basal fishes to mammals. Analysing these enhancers from different species reveal the substantial spatial plasticity in Grem1 regulation in tetrapods and basal fishes, which provides insights into the fin-to-limb transition and evolutionary diversification of pentadactyl limbs.

Tbx4 function during hindlimb development reveals a mechanism that explains the origins of proximal limb defects.

We dissect genetically a gene regulatory network that involves the transcription factors Tbx4, Pitx1 and Isl1 acting cooperatively to establish the hindlimb bud, and identify key differences in the pathways that initiate formation of the hindlimb and forelimb. Using live image analysis of murine limb mesenchyme cells undergoing chondrogenesis in micromass culture, we distinguish a series of changes in cellular behaviours and cohesiveness that are required for chondrogenic precursors to undergo differentiation. Furthermore, we provide evidence that the proximal hindlimb defects observed in Tbx4 mutant mice result from a failure in the early differentiation step of chondroprogenitors into chondrocytes, providing an explanation for the origins of proximally biased limb defects.

The tissues and regulatory pattern of limb chondrogenesis.

Initial limb chondrogenesis offers the first differentiated tissues that resemble the mature skeletal anatomy. It is a developmental progression of three tissues. The limb begins with undifferentiated mesenchyme-1, some of which differentiates into condensations-2, and this tissue then transforms into cartilage-3. Each tissue is identified by physical characteristics of cell density, shape, and extracellular matrix composition. Tissue specific regimes of gene regulation underlie the diagnostic physical and chemical properties of these three tissues. These three tissue based regimes co-exist amid a background of other gene regulatory regimes within the same tissues and time-frame of limb development. The bio-molecular indicators of gene regulation reveal six identifiable patterns. Three of these patterns describe the unique bio-molecular indicators of each of the three tissues. A fourth pattern shares bio-molecular indicators between condensation and cartilage. Finally, a fifth pattern is composed of bio-molecular indicators that are found in undifferentiated mesenchyme prior to any condensation differentiation, then these bio-molecular indicators are upregulated in condensations and downregulated in undifferentiated mesenchyme. The undifferentiated mesenchyme that remains in between the condensations and cartilage, the interdigit, contains a unique set of bio-molecular indicators that exhibit dynamic behaviour during chondrogenesis and therefore argue for its own inclusion as a tissue in its own right and for more study into this process of differentiation.

The Role of Retinoic Acid in Establishing the Early Limb Bud.

Retinoic acid (RA) was one of the first molecules in the modern era of experimental embryology to be shown capable of generating profound effects on limb development. In this review, we focus on the earliest events of limb development and specifically on the role of RA in establishing the domain of cells that will go on to form the limb itself. Although there is some consensus on the role of RA during the earliest stages of limb formation, some controversy remains on the mechanism of RA action and the requirement for RA signaling in forming the hindlimb buds.

Primary myogenesis in the sand lizard (Lacerta agilis) limb bud.

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.

The Murine Limb Bud in Culture as an In Vitro Teratogenicity Test System.

There is widespread interest today in the use of in vitro methods to study normal and abnormal development. The limb is attractive in this context since much is known about pattern formation during limb development. The murine limb bud culture technique described in this chapter was developed and refined in the 1970s. In this culture system, limb development mimics the in vivo process, although at a slower rate, where growth and cartilage differentiation lead to the formation of proximal and distal structures with an "in vivo-like" 3D shape. Uniform developmental stages are selected for assessment, exposures are controlled precisely, and the confounding influences of maternal metabolism and transport are avoided. The existence of transgenic mice with fluorescent markers for the different stages of endochondral ossification adds a further dimension to the technique by allowing striking time course observations of the developing limb. Today, limb bud cultures are used to study the roles of genes during embryogenesis and the mechanisms by which chemicals interfere with critical signalling pathways.

SOX9 is dispensable for the initiation of epigenetic remodeling and the activation of marker genes at the onset of chondrogenesis.

SOX9 controls cell lineage fate and differentiation in major biological processes. It is known as a potent transcriptional activator of differentiation-specific genes, but its earliest targets and its contribution to priming chromatin for gene activation remain unknown. Here, we address this knowledge gap using chondrogenesis as a model system. By profiling the whole transcriptome and the whole epigenome of wild-type and Sox9-deficient mouse embryo limb buds, we uncover multiple structural and regulatory genes, including Fam101a, Myh14, Sema3c and Sema3d, as specific markers of precartilaginous condensation, and we provide evidence of their direct transactivation by SOX9. Intriguingly, we find that SOX9 helps remove epigenetic signatures of transcriptional repression and establish active-promoter and active-enhancer marks at precartilage- and cartilage-specific loci, but is not absolutely required to initiate these changes and activate transcription. Altogether, these findings widen our current knowledge of SOX9 targets in early chondrogenesis and call for new studies to identify the pioneer and transactivating factors that act upstream of or along with SOX9 to prompt chromatin remodeling and specific gene activation at the onset of chondrogenesis and other processes.

A small population of resident limb bud mesenchymal cells express few MSC-associated markers, but the expression of these markers is increased immediately after cell culture.

Skeletal progenitors are derived from resident limb bud mesenchymal cells of the vertebrate embryos. However, it remains poorly understood if they represent stem cells, progenitors, or multipotent mesenchymal stromal cells (MSC). Derived-MSC of different adult tissues under in vitro experimental conditions can differentiate into the same cellular lineages that are present in the limb. Here, comparing non-cultured versus cultured mesenchymal limb bud cells, we determined the expression of MSC-associated markers, the in vitro differentiation capacity and their gene expression profile. Results showed that in freshly isolated limb bud mesenchymal cells, the proportion of cells expressing Sca1, CD44, CD105, CD90, and CD73 is very low and a low expression of lineage-specific genes was observed. However, recently seeded limb bud mesenchymal cells acquired Sca1 and CD44 markers and the expression of the key differentiation genes Runx2 and Sox9, while Scx and Pparg genes decreased. Also, their chondrogenic differentiation capacity decreased through cellular passages while the osteogenic increased. Our findings suggest that the modification of the cell adhesion process through the in vitro method changed the limb mesenchymal cell immunophenotype leading to the expression and maintenance of common MSC-associated markers. These findings could have a significant impact on MSC study and isolation strategy because they could explain common variations observed in the MSC immunophenotype in different tissues.

Pitx1 determines characteristic hindlimb morphologies in cartilage micromass culture.

The shapes of homologous skeletal elements in the vertebrate forelimb and hindlimb are distinct, with each element exquisitely adapted to their divergent functions. Many of the signals and signalling pathways responsible for patterning the developing limb bud are common to both forelimb and hindlimb. How disparate morphologies are generated from common signalling inputs during limb development remains poorly understood. We show that, similar to what has been shown in the chick, characteristic differences in mouse forelimb and hindlimb cartilage morphology are maintained when chondrogenesis proceeds in vitro away from the endogenous limb bud environment. Chondrogenic nodules that form in high-density micromass cultures derived from forelimb and hindlimb buds are consistently different in size and shape. We described analytical tools we have developed to quantify these differences in nodule morphology and demonstrate that characteristic hindlimb nodule morphology is lost in the absence of the hindlimb-restricted limb modifier gene Pitx1. Furthermore, we show that ectopic expression of Pitx1 in the forelimb is sufficient to generate nodule patterns characteristic of the hindlimb. We also demonstrate that hindlimb cells are less adhesive to the tissue culture substrate and, within the limb environment, to the extracellular matrix and to each other. These results reveal autonomously programmed differences in forelimb and hindlimb cartilage precursors of the limb skeleton are controlled, at least in part, by Pitx1 and suggest this has an important role in generating distinct limb-type morphologies. Our results demonstrate that the micromass culture system is ideally suited to study cues governing morphogenesis of limb skeletal elements in a simple and experimentally tractable in vitro system that reflects in vivo potential.

Biophysical regulation of early limb bud morphogenesis.

The physical basis of morphogenesis is a fascinating concern that has been a longstanding interest of developmental biologists. In this review, I attempt to incorporate earlier and recent biophysical concepts and data to explain basic features of early limb bud morphogenesis. In particular, I discuss the influence of mesenchymal cohesion and physical properties that might contribute to phase separation of the bud from the lateral plate, the possibility that the early dorsoventral limb bud axis is moulded by the surface ectoderm, and endogenous electric fields that might contribute to oriented cell movements which generate the early limb bud. A combination of quantitative biophysical experimentation and modelling will likely advance this field.

A quantitative approach to understanding vertebrate limb morphogenesis at the macroscopic tissue level.

To understand organ morphogenetic mechanisms, it is essential to clarify how spatiotemporally-regulated molecular/cellular dynamics causes physical tissue deformation. In the case of vertebrate limb development, while some of the genes and oriented cell behaviors underlying morphogenesis have been revealed, tissue deformation dynamics remains incompletely understood. We here introduce our recent work on the reconstruction of tissue deformation dynamics in chick limb development from cell lineage tracing data. This analysis has revealed globally-aligned anisotropic tissue deformation along the proximo-distal axis not only in the distal region but also in the whole limb bud. This result points to a need, as a future challenge, to find oriented molecular/cellular behaviors for realizing the observed anisotropic tissue deformation in both proximal and distal regions, which will lead to systems understanding of limb morphogenesis.

Intrinsic properties of limb bud cells can be differentially reset.

An intrinsic timing mechanism specifies the positional values of the zeugopod (i.e. radius/ulna) and then autopod (i.e. wrist/digits) segments during limb development. Here, we have addressed whether this timing mechanism ensures that patterning events occur only once by grafting GFP-expressing autopod progenitor cells to the earlier host signalling environment of zeugopod progenitor cells. We show by detecting Hoxa13 expression that early and late autopod progenitors fated for the wrist and phalanges, respectively, both contribute to the entire host autopod, indicating that the autopod positional value is irreversibly determined. We provide evidence that Hoxa13 provides an autopod-specific positional value that correctly allocates cells into the autopod, most likely through the control of cell-surface properties as shown by cell-cell sorting analyses. However, we demonstrate that only the earlier autopod cells can adopt the host proliferation rate to permit normal morphogenesis. Therefore, our findings reveal that the ability of embryonic cells to differentially reset their intrinsic behaviours confers robustness to limb morphogenesis. We speculate that this plasticity could be maintained beyond embryogenesis in limbs with regenerative capacity.

mTORC1 Signaling Promotes Limb Bud Cell Growth and Chondrogenesis.

mTORC1 signaling has been shown to promote limb skeletal growth through stimulation of protein synthesis in chondrocytes. However, potential roles of mTORC1 in prechondrogenic mesenchyme have not been explored. In this study, we first deleted Raptor, a unique and essential component of mTORC1, in prechondrogenic limb mesenchymal cells. Deletion of Raptor reduced the size of limb bud cells, resulting in overall diminution of the limb bud without affecting skeletal patterning. We then examined the potential role of mTORC1 in chondrogenic differentiation in vitro. Both pharmacological and genetic disruption of mTORC1 significantly suppressed the number and size of cartilage nodules in micromass cultures of limb bud mesenchymal cells. Similarly, inhibition of mTORC1 signaling in chondrogenic ATDC5 cells greatly impaired cartilage nodule formation, and decreased the expression of the master transcriptional factor Sox9, along with the cartilage matrix genes Acan and Col2a1. Thus, we have identified an important role for mTORC1 signaling in promoting limb mesenchymal cell growth and chondrogenesis during embryonic development. J. Cell. Biochem. 118: 748-753, 2017. © 2016 Wiley Periodicals, Inc.

Chondrogenesis of embryonic limb bud cells in micromass culture progresses rapidly to hypertrophy and is modulated by hydrostatic pressure.

Chondrogenesis in vivo is precisely controlled in time and space. The entire limb skeleton forms from cells at the core of the early limb bud that condense and undergo chondrogenic differentiation. Whether they form stable cartilage at the articular surface of the joint or transient cartilage that progresses to hypertrophy as endochondral bone, replacing the cartilage template of the skeletal rudiment, is spatially controlled over several days in the embryo. Here, we follow the differentiation of cells taken from the early limb bud (embryonic day 11.5), grown in high-density micromass culture and show that a self-organising pattern of evenly spaced cartilage nodules occurs spontaneously in growth medium. Although chondrogenesis is enhanced by addition of BMP6 to the medium, the spatial pattern of nodule formation is disrupted. We show rapid progression of the entire nodule to hypertrophy in culture and therefore loss of the local signals required to direct formation of stable cartilage. Dynamic hydrostatic pressure, which we have previously predicted to be a feature of the forming embryonic joint region, had a stabilising effect on chondrogenesis, reducing expression of hypertrophic marker genes. This demonstrates the use of micromass culture as a relatively simple assay to compare the effect of both biophysical and molecular signals on spatial and temporal control of chondrogenesis that could be used to examine the response of different types of progenitor cell, both adult- and embryo-derived.

Specification of osteoblast cell fate by canonical Wnt signaling requires Bmp2.

Enhanced BMP or canonical Wnt (cWnt) signaling are therapeutic strategies employed to enhance bone formation and fracture repair, but the mechanisms each pathway utilizes to specify cell fate of bone-forming osteoblasts remain poorly understood. Among all BMPs expressed in bone, we find that singular deficiency of Bmp2 blocks the ability of cWnt signaling to specify osteoblasts from limb bud or bone marrow progenitors. When exposed to cWnts, Bmp2-deficient cells fail to progress through the Runx2/Osx1 checkpoint and thus do not upregulate multiple genes controlling mineral metabolism in osteoblasts. Cells lacking Bmp2 after induction of Osx1 differentiate normally in response to cWnts, suggesting that pre-Osx1+ osteoprogenitors are an essential source and a target of BMP2. Our analysis furthermore reveals Grainyhead-like 3 (Grhl3) as a transcription factor in the osteoblast gene regulatory network induced during bone development and bone repair, which acts upstream of Osx1 in a BMP2-dependent manner. The Runx2/Osx1 transition therefore receives crucial regulatory inputs from BMP2 that are not compensated for by cWnt signaling, and this is mediated at least in part by induction and activation of Grhl3.

A somitic contribution to the apical ectodermal ridge is essential for fin formation.

The transition from fins to limbs was an important terrestrial adaptation, but how this crucial evolutionary shift arose developmentally is unknown. Current models focus on the distinct roles of the apical ectodermal ridge (AER) and the signaling molecules that it secretes during limb and fin outgrowth. In contrast to the limb AER, the AER of the fin rapidly transitions into the apical fold and in the process shuts off AER-derived signals that stimulate proliferation of the precursors of the appendicular skeleton. The differing fates of the AER during fish and tetrapod development have led to the speculation that fin-fold formation was one of the evolutionary hurdles to the AER-dependent expansion of the fin mesenchyme required to generate the increased appendicular structure evident within limbs. Consequently, a heterochronic shift in the AER-to-apical-fold transition has been postulated to be crucial for limb evolution. The ability to test this model has been hampered by a lack of understanding of the mechanisms controlling apical fold induction. Here we show that invasion by cells of a newly identified somite-derived lineage into the AER in zebrafish regulates apical fold induction. Ablation of these cells inhibits apical fold formation, prolongs AER activity and increases the amount of fin bud mesenchyme, suggesting that these cells could provide the timing mechanism proposed in Thorogood's clock model of the fin-to-limb transition. We further demonstrate that apical-fold inducing cells are progressively lost during gnathostome evolution;the absence of such cells within the tetrapod limb suggests that their loss may have been a necessary prelude to the attainment of limb-like structures in Devonian sarcopterygian fish.

Spatiotemporal distribution of proliferation, proapoptotic and antiapoptotic factors in the early human limb development.

Involvement of proliferation and apoptosis in the human limb development was analyzed electronmicroscopically and immunohistochemically in histological sections of 8 human embryos, 4(th) -10(th) week old, using apoptotic (caspase-3, AIF, BAX), anti-apoptotic (Bcl-2) and proliferation (Ki-67) markers, and TUNEL method. The data were analyzed by Mann-Whitney test, Kruskal-Wallis and Dunn's post hoc test. Initially, developing human limbs consisted of mesenchymal core and surface ectoderm with apical ectodermal ridge (AER). During progression of development, strong proliferation activity gradually decreased in the mesenchyme (from 78% to 68%) and in the epithelium (from 62% to 42%), while in the differentiating finger cartilages proliferation was constantly low (26-7%). Apoptotic caspase-3 and AIF-positive cells characterized mesenchyme and AER at earliest stages, while during digit separation they appeared in interdigital mesenchyme as well. Strong Bcl-2 expression was observed in AER, subridge mesenchyme and phalanges, while BAX expression charaterized limb areas undergoing apoptosis. Ultrastructurally, proliferating cells showed mitotic figures, while apoptotic cells were characterized by nuclear fragmentation. Macrophages were observed around the apoptotic cells. We suggest that intense proliferation enables growth and elongation of human limb primordia, and differential growth of digits. Both caspase-3 and AIF-dependant pathways of cell death control the extent of AER and numer of cells in the subridge mesenchyme at earliest developmental stages, as well as process of digit separation at later stages of limb development. Spatio-temporal co-expresson of Bcl-2 and BAX indicates their role in suppression of apoptosis and selective stimulation of growth during human limb morphogenesis.

A shared role for sonic hedgehog signalling in patterning chondrichthyan gill arch appendages and tetrapod limbs.

Chondrichthyans (sharks, skates, rays and holocephalans) possess paired appendages that project laterally from their gill arches, known as branchial rays. This led Carl Gegenbaur to propose that paired fins (and hence tetrapod limbs) originally evolved via transformation of gill arches. Tetrapod limbs are patterned by asonic hedgehog(Shh)-expressing signalling centre known as the zone of polarising activity, which establishes the anteroposterior axis of the limb bud and maintains proliferative expansion of limb endoskeletal progenitors. Here, we use loss-of-function, label-retention and fate-mapping approaches in the little skate to demonstrate that Shh secretion from a signalling centre in the developing gill arches establishes gill arch anteroposterior polarity and maintains the proliferative expansion of branchial ray endoskeletal progenitor cells. These findings highlight striking parallels in the axial patterning mechanisms employed by chondrichthyan branchial rays and paired fins/limbs, and provide mechanistic insight into the anatomical foundation of Gegenbaur's gill arch hypothesis.