Molecular mechanism of synovial joint site specification and induction in developing vertebrate limbs.

The vertebrate appendage comprises three primary segments, the stylopod, zeugopod and autopod, each separated by joints. The molecular mechanisms governing the specification of joint sites, which define segment lengths and thereby limb architecture, remain largely unknown. Existing literature suggests that reciprocal gradients of retinoic acid (RA) and fibroblast growth factor (FGF) signaling define the expression domains of the putative segment markers Meis1, Hoxa11 and Hoxa13. Barx1 is expressed in the presumptive joint sites. Our data demonstrate that RA-FGF signaling gradients define the expression domain of Barx1 in the first presumptive joint site. When misexpressed, Barx1 induces ectopic interzone-like structures, and its loss of function partially blocks interzone development. Simultaneous perturbations of RA-FGF signaling gradients result in predictable shifts of Barx1 expression domains along the proximo-distal axis and, consequently, in the formation of repositioned joints. Our data suggest that during early limb bud development in chick, Meis1 and Hoxa11 expression domains are overlapping, whereas the Barx1 expression domain resides within the Hoxa11 expression domain. However, once the interzone is formed, the expression domains are refined and the Barx1 expression domain becomes congruent with the border of these two putative segment markers.

A dot-stripe Turing model of joint patterning in the tetrapod limb.

Iterative joints are a hallmark of the tetrapod limb, and their positioning is a key step during limb development. Although the molecular regulation of joint formation is well studied, it remains unclear what controls the location, number and orientation (i.e. the pattern) of joints within each digit. Here, we propose the dot-stripe mechanism for joint patterning, comprising two coupled Turing systems inspired by published gene expression patterns. Our model can explain normal joint morphology in wild-type limbs, hyperphalangy in cetacean flippers, mutant phenotypes with misoriented joints and suggests a reinterpretation of the polydactylous Ichthyosaur fins as a polygonal joint lattice. By formulating a generic dot-stripe model, describing joint patterns rather than molecular joint markers, we demonstrate that the insights from the model should apply regardless of the biological specifics of the underlying mechanism, thus providing a unifying framework to interrogate joint patterning in the tetrapod limb.

Co-option of the limb patterning program in cephalopod eye development.

BACKGROUND: Across the Metazoa, similar genetic programs are found in the development of analogous, independently evolved, morphological features. The functional significance of this reuse and the underlying mechanisms of co-option remain unclear. Cephalopods have evolved a highly acute visual system with a cup-shaped retina and a novel refractive lens in the anterior, important for a number of sophisticated behaviors including predation, mating, and camouflage. Almost nothing is known about the molecular-genetics of lens development in the cephalopod. RESULTS: Here we identify the co-option of the canonical bilaterian limb patterning program during cephalopod lens development, a functionally unrelated structure. We show radial expression of transcription factors SP6-9/sp1, Dlx/dll, Pbx/exd, Meis/hth, and a Prdl homolog in the squid Doryteuthis pealeii, similar to expression required in Drosophila limb development. We assess the role of Wnt signaling in the cephalopod lens, a positive regulator in the developing Drosophila limb, and find the regulatory relationship reversed, with ectopic Wnt signaling leading to lens loss. CONCLUSION: This regulatory divergence suggests that duplication of SP6-9 in cephalopods may mediate the co-option of the limb patterning program. Thus, our study suggests that this program could perform a more universal developmental function in radial patterning and highlights how canonical genetic programs are repurposed in novel structures.

Time-sequenced transcriptomes of developing distal mouse limb buds: A comparative tissue layer analysis.

BACKGROUND: The development of the amniote limb has been an important model system to study patterning mechanisms and morphogenesis. For proper growth and patterning, it requires the interaction between the distal sub-apical mesenchyme and the apical ectodermal ridge (AER) that involve the separate implementation of coordinated and tissue-specific genetic programs. RESULTS: Here, we produce and analyze the transcriptomes of both distal limb mesenchymal progenitors and the overlying ectodermal cells, following time-coursed dissections that cover from limb bud initiation to fully patterned limbs. The comparison of transcriptomes within each layer as well as between layers over time, allowed the identification of specific transcriptional signatures for each of the developmental stages. Special attention was given to the identification of genes whose transcription dynamics suggest a previously unnoticed role in the context of limb development and also to signaling pathways enriched between layers. CONCLUSION: We interpret the transcriptomic data in light of the known development pattern and we conclude that a major transcriptional transition occurs in distal limb buds between E9.5 and E10.5, coincident with the switch from an early phase continuation of the signature of trunk progenitors, related to the initial proximo distal specification, to a late intrinsic phase of development.

Unique pelvic fin in a tetrapod-like fossil fish, and the evolution of limb patterning.

All living tetrapods have a one-to-two branching pattern in the embryonic proximal limb skeleton, with a single element at the base of the limb (the humerus or femur) that articulates distally with two parallel radials (the ulna and radius or the tibia and fibula). This pattern is also seen in the fossilized remains of stem-tetrapods, including the fishlike members of the group, in which despite the absence of digits, the proximal parts of the fin skeleton clearly resemble those of later tetrapods. However, little is known about the developmental mechanisms that establish and canalize this highly conserved pattern. We describe the well-preserved pelvic fin skeleton of Rhizodus hibberti, a Carboniferous sarcopterygian (lobe-finned) fish, and member of the tetrapod stem group. In this specimen, three parallel radials, each robust with a distinct morphology, articulate with the femur. We review this unexpected morphology in a phylogenetic and developmental context. It implies that the developmental patterning mechanisms seen in living tetrapods, now highly constrained, evolved from mechanisms flexible enough to accommodate variation in the zeugopod (even between pectoral and pelvic fins), while also allowing each element to have a unique morphology.

Hox5 interacts with Plzf to restrict Shh expression in the developing forelimb.

To date, only the five most posterior groups of Hox genes, Hox9-Hox13, have demonstrated loss-of-function roles in limb patterning. Individual paralog groups control proximodistal patterning of the limb skeletal elements. Hox9 genes also initiate the onset of Hand2 expression in the posterior forelimb compartment, and collectively, the posterior HoxA/D genes maintain posterior Sonic Hedgehog (Shh) expression. Here we show that an anterior Hox paralog group, Hox5, is required for forelimb anterior patterning. Deletion of all three Hox5 genes (Hoxa5, Hoxb5, and Hoxc5) leads to anterior forelimb defects resulting from derepression of Shh expression. The phenotype requires the loss of all three Hox5 genes, demonstrating the high level of redundancy in this Hox paralogous group. Further analyses reveal that Hox5 interacts with promyelocytic leukemia zinc finger biochemically and genetically to restrict Shh expression. These findings, along with previous reports showing that point mutations in the Shh limb enhancer lead to similar anterior limb defects, highlight the importance of Shh repression for proper patterning of the vertebrate limb.

Sufu and Kif7 in limb patterning and development.

BACKGROUND: The vertebrate digit pattern is defined by the morphogen Sonic hedgehog (Shh), which controls the activity of Gli transcription factors. Gli1, 2 and 3 are dynamically expressed during patterning. Downstream of Shh, their activity is regulated by Sufu and Kif7, core components of the Shh signaling cascade. The precise roles of these regulators during limb development have not been fully described. We analyze the role of Sufu and Kif7 in the limb and demonstrate that their loss has distinct and synergistic effects on Gli activity and digit pattern. RESULTS: Using a series of mouse mutants, we show that Sufu and Kif7 are expressed throughout limb development and their deletion has distinct effects on Gli levels and limb formation. Concomitant deletion of Sufu and Kif7 results in constitutive pathway activity and severe limb truncation. This is consistent with the recently published two-population model, which suggests that precocious activation of Shh signaling inhibits organizing center formation and limb outgrowth. CONCLUSIONS: Together, our findings demonstrate that perturbations of Sufu and Kif7 affect Gli activity and recapitulate the full spectrum of vertebrate limb defects, ranging from severe truncation to polydactyly.

Coxa Vara Deformity in Fibrous Dysplasia/McCune-Albright Syndrome: Prevalence, Natural History and Risk Factors: A Two-Center Study.

This study aimed to evaluate the prevalence of and risk factors for coxa vara deformity in patients with fibrous dysplasia/McCune-Albright syndrome (FD/MAS). This study was conducted at the National Institutes of Health and Leiden University Medical Center. All patients with any subtype of FD/MAS, FD involving the proximal femur, one or more X-rays available and age <30 years were included. X-rays were scored for the neck-shaft angle (NSA). Varus deformity was defined as NSA <110 degrees or >10 degrees below age-specific values. Risk factors for deformity were assessed by nested case-control analysis, comparing patients and femurs with and without deformity, and by linear mixed effects model, modeling temporal NSA decrease (the natural course of the NSA) in non-operated femurs with two or more X-rays. Assessed variables included growth hormone excess, hyperthyroidism, hypophosphatemia, >25% of the femur affected, calcar destruction, radiolucency, and bilateral involvement. In total 180 patients were studied, 57% female. Mean ± SD baseline age was 13.6 ± 7.5 years; median follow-up 5.4 (interquartile range [IQR], 11.1) years. Sixty-three percent (63%) were diagnosed with MAS. A total of 94 patients were affected bilaterally; 274 FD femurs were analyzed; 99 femurs had a varus deformity (36%). In the nested case-control analysis, risk factors were as follows: presence of MAS (p < 0.001), hyperthyroidism (p < 0.001), hypophosphatemia (p < 0.001), high percentage of femur affected (p < 0.001), and calcar destruction (p < 0.001). The linear mixed effects model included 114 femurs, identified risk factors were: growth hormone excess (ß = 7.2, p = 0.013), hyperthyroidism (ß = 11.3, p < 0.001), >25% of the femur affected (ß = 13.2, p = 0.046), calcar destruction (ß = 8.3, p = 0.004), radiolucency (ß = 3.9, p = 0.009), and bilateral involvement (ß = 9.8, p = 0.010). Visual inspection of the graph of the model demonstrated most progression of deformity if NSA <120 degrees with age < 15 years. In conclusion, in tertiary care centers, the prevalence of FD/MAS coxa vara deformity was 36%. Risk factors included presence of MAS, high percentage of femur affected, calcar destruction, radiolucency, NSA <120 degrees and age < 15 years. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Integration failure of regenerated limb tissue is associated with incongruencies in positional information in the Mexican axolotl.

Introduction: Little is known about how the newly regenerated limb tissues in the Mexican axolotl seamlessly integrate with the remaining stump tissues to form a functional structure, and why this doesn't occur in some regenerative scenarios. In this study, we evaluate the phenomenological and transcriptional characteristics associated with integration failure in ectopic limb structures generated by treating anterior-located ectopic blastemas with Retinoic Acid (RA) and focusing on the "bulbus mass" tissue that forms between the ectopic limb and the host site. We additionally test the hypothesis that the posterior portion of the limb base contains anterior positional identities. Methods: The positional identity of the bulbus mass was evaluated by assaying regenerative competency, the ability to induce new pattern in the Accessory Limb Model (ALM) assay, and by using qRTPCR to quantify the relative expression of patterning genes as the bulbus mass deintegrates from the host site. We additionally use the ALM and qRTPCR to analyze the distribution of anterior and posterior positional identities along the proximal/distal limb axis of uninjured and regenerating limbs. Results: The bulbus mass regenerates limb structures with decreased complexity when amputated and is able to induce complex ectopic limb structure only when grafted into posterior-located ALMs. Expressional analysis shows significant differences in FGF8, BMP2, TBX5, Chrdl1, HoxA9, and HoxA11 expression between the bulbus mass and the host site when deintegration is occuring. Grafts of posterior skin from the distal limb regions into posterior ALMs at the base of the limb induce ectopic limb structures. Proximally-located blastemas express significantly less HoxA13 and Ptch1, and significantly more Alx4 and Grem1 than distally located blastemas. Discussion: These findings show that the bulbus mass has an anterior-limb identity and that the expression of limb patterning genes is mismatched between the bulbus mass and the host limb. Our findings additionally show that anterior positional information is more abundant at the limb base, and that anterior patterning genes are more abundantly expressed in proximally located blastemas compared to blastemas in the more distal regions of the limb. These experiments provide valuable insight into the underlying causes of integration failure and further map the distribution of positional identities in the mature limb.

A single-cell census of mouse limb development identifies complex spatiotemporal dynamics of skeleton formation.

Limb development has long served as a model system for coordinated spatial patterning of progenitor cells. Here, we identify a population of naive limb progenitors and show that they differentiate progressively to form the skeleton in a complex, non-consecutive, three-dimensional pattern. Single-cell RNA sequencing of the developing mouse forelimb identified three progenitor states: naive, proximal, and autopodial, as well as Msx1 as a marker for the naive progenitors. In vivo lineage tracing confirmed this role and localized the naive progenitors to the outer margin of the limb, along the anterior-posterior axis. Sequential pulse-chase experiments showed that the progressive transition of Msx1+ naive progenitors into proximal and autopodial progenitors coincides with their differentiation to Sox9+ chondroprogenitors, which occurs along all the forming skeletal segments. Indeed, tracking the spatiotemporal sequence of differentiation showed that the skeleton forms progressively in a complex pattern. These findings suggest an alternative model for limb skeleton development.

Brd4 is required for chondrocyte differentiation and endochondral ossification.

Differentiation of multi-potent mesenchymal stromal cells (MSCs) is directed by the activities of lineage-specific transcription factors and co-factors. A subset of these proteins controls the accessibility of chromatin by recruiting histone acetyl transferases or deacetylases that regulate acetylation of the N-termini of H3 and H4 histone proteins. Bromodomain (BRD) proteins recognize these acetylation marks and recruit the RNA pol II containing transcriptional machinery. Our previous studies have shown that Brd4 is required for osteoblast differentiation in vitro. Here, we investigated the role of Brd4 on endochondral ossification in C57BL/6 mice and chondrogenic differentiation in cell culture models. Conditional loss of Brd4 in the mesenchyme (Brd4 cKO, Brd4fl/fl: Prrx1-Cre) yields smaller mice that exhibit alteration in endochondral ossification. Importantly, abnormal growth plate morphology and delayed long bone formation is observed in juvenile Brd4 cKO mice. One week old Brd4 cKO mice have reduced proliferative and hypertrophic zones within the physis and exhibit a delay in the formation of the secondary ossification center. At the cellular level, Brd4 function is required for chondrogenic differentiation and maturation of both ATDC5 cells and immature mouse articular chondrocytes. Mechanistically, Brd4 loss suppresses Sox9 levels and reduces expression of Sox9 and Runx2 responsive endochondral genes (e.g., Col2a1, Acan, Mmp13 and Sp7/Osx). Collectively, our results indicate that Brd4 is a key epigenetic regulator required for normal chondrogenesis and endochondral ossification.

Loss of Sprouty Produces a Ciliopathic Skeletal Phenotype in Mice Through Upregulation of Hedgehog Signaling.

The Sprouty family is a highly conserved group of intracellular modulators of receptor tyrosine kinase (RTK)-signaling pathways, which have been recently linked to primary cilia. Disruptions in the structure and function of primary cilia cause inherited disorders called ciliopathies. We aimed to evaluate Sprouty2 and Sprouty4 gene-dependent alterations of ciliary structure and to focus on the determination of its association with Hedgehog signaling defects in chondrocytes. Analysis of the transgenic mice phenotype with Sprouty2 and Sprouty4 deficiency revealed several defects, including improper endochondral bone formation and digit patterning, or craniofacial and dental abnormalities. Moreover, reduced bone thickness and trabecular bone mass, skull deformities, or chondroma-like lesions were revealed. All these pathologies might be attributed to ciliopathies. Elongation of the ciliary axonemes in embryonic and postnatal growth plate chondrocytes was observed in Sprouty2-/- and Sprouty2+/- /Sprouty4-/- mutants compared with corresponding littermate controls. Also, cilia-dependent Hedgehog signaling was upregulated in Sprouty2/4 mutant animals. Ptch1 and Ihh expression were upregulated in the autopodium and the proximal tibia of Sprouty2-/- /Sprouty4-/- mutants. Increased levels of the GLI3 repressor (GLI3R) form were detected in Sprouty2/4 mutant primary fibroblast embryonic cell cultures and tissues. These findings demonstrate that mouse lines deficient in Sprouty proteins manifest phenotypic features resembling ciliopathic phenotypes in multiple aspects and may serve as valuable models to study the association between overactivation of RTK and dysfunction of primary cilia during skeletogenesis. © 2021 American Society for Bone and Mineral Research (ASBMR).

Transcriptional Trajectories in Mouse Limb Buds Reveal the Transition from Anterior-Posterior to Proximal-Distal Patterning at Early Limb Bud Stage.

Limb patterning relies in large part on the function of the Hox family of developmental genes. While the differential expression of Hox genes shifts from the anterior-posterior (A-P) to the proximal-distal (P-D) axis around embryonic day 11 (E11), whether this shift coincides with a more global change of A-P to P-D patterning program remains unclear. By performing and analyzing the transcriptome of the developing limb bud from E10.5 to E12.5, at single-cell resolution, we have uncovered transcriptional trajectories that revealed a general switch from A-P to P-D genetic program between E10.5 and E11.5. Interestingly, all the transcriptional trajectories at E10.5 end with cells expressing either proximal or distal markers suggesting a progressive acquisition of P-D identity. Moreover, we identified three categories of genes expressed in the distal limb mesenchyme characterized by distinct temporal expression dynamics. Among these are Hoxa13 and Hoxd13 (Hox13 hereafter), which start to be expressed around E10.5, and importantly the binding of the HOX13 factors was observed within or in the neighborhood of several of the distal limb genes. Our data are consistent with previous evidence suggesting that the transition from the early/proximal to the late/distal transcriptome of the limb mesenchyme largely relies on HOX13 function. Based on these results and the evidence that HOX13 factors restrict Hoxa11 expression to the proximal limb, in progenitor cells of the zeugopod, we propose that HOX13 act as a key determinant of P-D patterning.

Morphology and development of a novel murine skeletal dysplasia.

BACKGROUND: Limb bones develop and grow by endochondral ossification, which is regulated by specific cell and molecular pathways. Changes in one or more of these pathways can have severe effects on normal skeletal development, leading to skeletal dysplasias. Many skeletal dysplasias are known to result from mis-expression of major genes involved in skeletal development, but the etiology of many skeletal dysplasias remains unknown. We investigated the morphology and development of a mouse line with an uncharacterized mutation exhibiting a skeletal dysplasia-like phenotype (Nabo). METHODS: We used µCT scanning and histology to comprehensively characterize the phenotype and its development, and to determine the developmental stage when this phenotype first appears. RESULTS: Nabo mice have shorter limb elements compared to wildtype mice, while clavicles and dermal bones of the skull are not affected. Nabo embryos at embryonic stage E14 show shorter limb cartilage condensations. The tibial growth plate in Nabo mice is wider than in wildtype, particularly in the proliferative zone, however proliferative chondrocytes show less activity than wildtype mice. Cell proliferation assays and immunohistochemistry against the chondrogenic marker Sox9 suggest relatively lower, spatially-restricted, chondrocyte proliferation activity in Nabo. Bone volume and trabecular thickness in Nabo tibiae are also decreased compared to wildtype. DISCUSSION: Our data suggest that the Nabo mutation affects endochondral ossification only, with the strongest effects manifesting in more proximal limb structures. The phenotype appears before embryonic stage E14, suggesting that outgrowth and patterning processes may be affected. Nabo mice present a combination of skeletal dysplasia-like characteristics not present in any known skeletal dysplasia. Further genomic and molecular analysis will help to identify the genetic basis and precise developmental pathways involved in this unique skeletal dysplasia.

Mechanistic insights into skeletal development gained from genetic disorders.

A complex cascade of highly regulated processes of cell fate determination, differentiation, proliferation and transdifferentiation dictate the patterning, morphogenesis and growth of the vertebrate skeleton, perturbation of which results in malformation. In humans over 450 different dysplasias involving the skeletal system constitute a significant fraction of documented Mendelian disorders. The combination of clinical, phenotypic characterization of rare human skeletal dysmorphologies, the discovery of causative mutations and functional validation in animal models has contributed enormously to the understanding of molecular control of skeletal development. These studies revealed a myriad of genes and pathways, such as WNT, Hedgehog (HH), planar cell polarity and primary cilia, as key regulators for skeletal patterning, growth and homeostasis. The generation of mouse models recapitulating human congenital skeletal dysplasia has provided mechanistic insights into the diverse pathologies caused by single gene mutations, integrated action of developmental pathways such as WNT and HH and the role of stress responses. Technological developments in whole genome and exome sequencing have accelerated the discovery of disease-causing mutations and are changing approaches for diagnosis. The discovery that non-coding variants and disorganization of the 3D genome are associated with limb patterning disorders has revealed an additional level of complexity in the regulatory framework of skeletal development and disease mechanisms. This chapter focuses on a selection of human skeletal pathologies which illustrate how new findings about the coding and noncoding genome, combined with functional modeling, are contributing to deeper understanding of skeletal development, mechanisms of disease, with therapeutic potential for chondrodysplasias.

Disruption of Wnt production in Shh lineage causes bone malformation in mice, mimicking human Malik-Percin-type syndactyly.

Here, we show that Shh-Cre-mediated deletion of Wntless, the Wnt cargo protein, in mouse posterior limb mesenchyme causes bone syndactyly of the 3rd and 4th digits, resembling the human Malik-Percin type. The Shh descendants gradiently distributed from digit 5 to posterior half of digit 3 in wild-type limbs, however, they abnormally increased in posterior digit 3 in WntlessShh-Cre . WntlessShh-Cre limbs displayed altered expression of hedgehog pathway genes and impaired noncanonical Wnt signaling activity. We further showed that the anterior limb mesenchymal cells in the WlsShh-Cre served as a source of Wnt5a to reorientate the adjacent Wls-lacking Shh lineage cells to move anteriorly and subsequently led to syndactyly, suggesting that aberrant mesenchymal cell movement/condensation may underlie the pathogenesis of syndactyly.

Reduced ß-catenin expression affects patterning of bone primordia, but not bone maturation.

Wnt/ß-catenin signaling is involved in patterning of bone primordia, but also plays an important role in the differentiation of chondrocytes and osteoblasts. During these processes the level of ß-catenin must be tightly regulated. Excess ß-catenin leads to conditions with increased bone mass, whereas loss of ß-catenin is associated with osteoporosis or, in extreme cases, the absence of limbs. In this study, we examined skeletogenesis in mice, which retain only 25% of ß-catenin. These embryos showed severe morphological abnormalities of which the lack of hindlimbs and misshaped front paws were the most striking. Surprisingly however, calcification of bone primordia occurred normally. Moreover, the Wnt-dependent regulatory network of transcription factors driving the differentiation of cartilage and bone, as well as the expression of extracellular matrix components, were preserved. These findings show that 25% ß-catenin is insufficient for the correct patterning of bone primordia, but sufficient for their mineralization. Our approach helps to identify bone morphogenetic processes that can proceed normally even at low ß-catenin levels, in contrast to those that require high ß-catenin dosages. This information could be exploited to improve the treatment of bone diseases by fine-tuning the individual ß-catenin dosage requirements.

The Roles of T-Box Genes in Vertebrate Limb Development.

Members of the T-box gene family have diverse roles during embryogenesis and many play critical roles in the developing limb. This is exemplified by the fact that, in humans, mutations in T-box genes are associated with several congenital syndromes that include limb defects as part of their characteristic spectrum of abnormalities. T-box genes encode for evolutionary conserved transcription factors that include both transcriptional activators and repressors. The hallmark of T-box gene members is the presence of the eponymous DNA-binding T-box domain. There are 17 mammalian T-box genes, which based on the sequence homology of the T-box domain, are grouped into five subfamilies, namely, T, Tbx1, Tbx2, Tbx6, and Tbr1. At least nine T-box genes are expressed during limb development with distinct and dynamic expression patterns. All four members of Tbx2 subfamily (Tbx2, Tbx3, Tbx4, Tbx5) and three members of Tbx1 (Tbx1, Tbx15, Tbx18), Brachyury (T) and Eomes (Tbr2) are expressed in the developing limb.