The chick oligozeugodactyly (ozd) mutant lacks sonic hedgehog function in the limb

We have analyzed a new limb mutant in the chicken that we name oligozeugodactyly (ozd). The limbs of this mutant have a longitudinal postaxial defect, lacking the posterior element in the zeugopod (ulna/fibula) and all digits except digit 1 in the leg. Classical recombination experiments show that the limb mesoderm is the defective tissue layer in ozd limb buds. Molecular analysis revealed that the ozd limbs develop in the absence of Shh expression, while all other organs express Shh and develop normally. Neither Ptc1 nor Gli1 are detectable in mutant limb buds. However, Bmp2 and dHAND are expressed in the posterior wing and leg bud mesoderm, although at lower levels than in normal embryos. Activation of Hoxd11-13 occurs normally in ozd limbs but progressively declines with time. Phase III of expression is more affected than phase II, and expression is more severely affected in the more 5' genes. Interestingly, re-expression of Hoxd13 occurs at late stages in the distal mesoderm of ozd leg buds, correlating with formation of digit 1. Fgf8 and Fgf4 expression are initiated normally in the mutant AER but their expression is progressively downregulated in the anterior AER. Recombinant Shh protein or ZPA grafts restore normal pattern to ozd limbs; however, retinoic acid fails to induce Shh in ozd limb mesoderm. We conclude that Shh function is required for limb development distal to the elbow/knee joints, similar to the Shh(-/-) mouse. Accordingly we classify the limb skeletal elements as Shh dependent or independent, with the ulna/fibula and digits other than digit 1 in the leg being Shh dependent. Finally we propose that the ozd mutation is most likely a defect in a regulatory element that controls limb-specific expression of Shh.     

Manifestation of the limb prepattern: limb development in the absence of sonic hedgehog function

The secreted protein encoded by the Sonic hedgehog (Shh) gene is localized to the posterior margin of vertebrate limb buds and is thought to be a key signal in establishing anterior-posterior limb polarity. In the Shh(-/-) mutant mouse, the development of many embryonic structures, including the limb, is severely compromised. In this study, we report the analysis of Shh(-/-) mutant limbs in detail. Each mutant embryo has four limbs with recognizable humerus/femur bones that have anterior-posterior polarity. Distal to the elbow/knee joints, skeletal elements representing the zeugopod form but lack identifiable anterior-posterior polarity. Therefore, Shh specifically becomes necessary for normal limb development at or just distal to the stylopod/zeugopod junction (elbow/knee joints) during mouse limb development. The forelimb autopod is represented by a single distal cartilage element, while the hindlimb autopod is invariably composed of a single digit with well-formed interphalangeal joints and a dorsal nail bed at the terminal phalanx. Analysis of GDF5 and Hoxd11-13 expression in the hindlimb autopod suggests that the forming digit has a digit-one identity. This finding is corroborated by the formation of only two phalangeal elements which are unique to digit one on the foot. The apical ectodermal ridge (AER) is induced in the Shh(-/-) mutant buds with relatively normal morphology. We report that the architecture of the Shh(-/-) AER is gradually disrupted over developmental time in parallel with a reduction of Fgf8 expression in the ridge. Concomitantly, abnormal cell death in the Shh(-/-) limb bud occurs in the anterior mesenchyme of both fore- and hindlimb. It is notable that the AER changes and mesodermal cell death occur earlier in the Shh(-/-) forelimb than the hindlimb bud. This provides an explanation for the hindlimb-specific competence to form autopodial structures in the mutant. Finally, unlike the wild-type mouse limb bud, the Shh(-/-) mutant posterior limb bud mesoderm does not cause digit duplications when grafted to the anterior border of chick limb buds, and therefore lacks polarizing activity. We propose that a prepattern exists in the limb field for the three axes of the emerging limb bud as well as specific limb skeletal elements. According to this model, the limb bud signaling centers, including the zone of polarizing activity (ZPA) acting through Shh, are required to elaborate upon the axial information provided by the native limb field prepattern.     

Heterochronically early decline of Hox expression prior to cartilage formation in the avian hindlimb zeugopod

The fibula, a zeugopod bone in the hindlimb, exhibits various morphologies in tetrapod species. The fibula in some species has a similar length with the other zeugopod element, the tibia, while other species have obvious differences in the sizes of the two elements. In the avian hindlimb, for example, the fibula is extremely short, thin, and truncated. Basic morphology of the fibula is established during development, and cartilage primordium of the bone emerges in a certain region defined by a distinct combination of expression of Hox genes (Hox code). In order to elucidate how the different morphologies are produced from a region that is defined as the fixed Hox code, we examined spatial and temporal patterns of Hoxd11/Hoxd12 expression in the developing limb bud, which defines the region from which the fibula emerges, in comparison with the sites of precartilaginous mesenchymal condensations representing regions for cartilage formation among chick, mouse, and gecko embryos. We found that in the chick hindlimb, expression of Hoxd11/Hoxd12 decreased and disappeared from the presumptive zeugopod region before cartilage formation. This heterochronically early decline of expression of Hox genes is strongly correlated with the peculiar trait of the fibula in the avian hindlimb, since in the other species examined, expression of those genes continued after the onset of cartilage formation. This is morphological phenotype-related because the early disappearance was not seen in the chick forelimb. Our results suggest that temporal change of the Hox code governs diversification in morphology of homologous structures among related species.     

The evolutionary and developmental basis of parallel reduction in mammalian zeugopod elements

Understanding the mechanisms by which parallel evolution occurs has the potential to clarify the complex relationship between evolution and development. In this study, we examine the role of development in the repeated reduction of zeugopod elements during mammalian evolution, a functionally important phenomenon enabling locomotor specialization. By completing a morphometric study (incorporating both analyses of variation and phylogenetics) of mammalian limbs, we are able to demonstrate an evolutionary trend toward width reduction in posterior zeugopod elements of the forelimbs and hindlimbs, the ulna and fibula, respectively. We also examine the developmental basis of limb reduction in three test cases, the bat Carollia perspicillata ulna and fibula and the mouse Mus musculus fibula. The most common pattern of reduction, that of reduced element width, was achieved via the same developmental process in both bat and mouse limbs (i.e., by a slower growth rate relative to other skeletal elements), suggesting that the parallel reduction of the posterior zeugopod element within mammals could have occurred primarily by the repeated evolution of the same developmental mechanism. However, our findings also suggest that the developmental mechanisms behind the parallel evolution of other, more taxon-specific characteristics of limb reduction (i.e., element fusion) are not conserved.     

Replicated anterior zeugopod (raz): a polydactylous mouse mutant with lowered Shh signaling in the limb bud

A unique limb phenotype is described in a radiation-induced mutant mouse resulting from an inversion of a proximal segment of chromosome 5. The limb phenotype in the homozygous mutant presents with two anterior skeletal elements in the zeugopod but no posterior bone, hence the name replicated anterior zeugopod, raz. The zeugopod phenotype is accompanied by symmetrical central polydactyly of hand and foot. The chromosomal inversion includes the Shh gene and the regulatory locus, located approximately 1 Mb away, within the Lmbr1 gene. In homozygous mutants, the expression of Shh mRNA and Shh protein is severely downregulated to about 20% of wild-type limb buds, but Shh expression appears normal throughout the remainder of the embryo. Correspondingly, Gli3 expression is upregulated and posteriorly expanded in the raz/raz limb bud. We propose that the double anterior zeugopod and symmetrical central polydactyly are due to an increased and uniform concentration of the Gli3 repressor form because of lowered Shh signaling.     

Ectopic Hoxa2 induction after neural crest migration results in homeosis of jaw elements in Xenopus

Hox genes are required to pattern neural crest (NC) derived craniofacial and visceral skeletal structures. However, the temporal requirement of Hox patterning activity is not known. Here, we use an inducible system to establish Hoxa2 activity at distinct NC migratory stages in Xenopus embryos. We uncover stage-specific effects of Hoxa2 gain-of-function suggesting a multistep patterning process for hindbrain NC. Most interestingly, we show that Hoxa2 induction at postmigratory stages results in mirror image homeotic transformation of a subset of jaw elements, normally devoid of Hox expression, towards hyoid morphology. This is the reverse phenotype to that observed in the Hoxa2 knockout. These data demonstrate that the skeletal pattern of rhombomeric mandibular crest is not committed before migration and further implicate Hoxa2 as a true selector of hyoid fate. Moreover, the demonstration that the expression of Hoxa2 alone is sufficient to transform the upper jaw and its joint selectively may have implications for the evolution of jaws.     

Forced chondrocyte expression of sonic hedgehog impairs joint formation affecting proliferation and apoptosis.

Proliferation and apoptosis are two fundamental processes that occur during limb development, and in particular in joint formation. To study the role of hedgehog proteins in limbs, we have misexpressed Sonic Hedgehog specifically in chondrocytes. We found that the appendicular skeleton was severely misshapen while pelvic and shoulder girdles developed normally. In particular, we detected fusion of the elbow/knee joint, no definite carpal/tarsal, metacarpal/metatarsal bones and absence of distinct phalanges, fused in a continuous cartilaginous rod. Molecular markers of joints, such as Gdf5 and sFrp2 were absent at presumptive joint sites and Tenascin C, a molecule associated with joint formation and expressed in permanent cartilage, was expressed in a wider region in transgenic animals as compared to the wild type. The ratio of proliferating to non-proliferating chondrocytes was about two times higher in transgenic developing cartilage as compared to the wild type. Accordingly, the proapoptotic gene Bax was barely detectable in the growth plate of transgenic mice and Tunel assay showed the absence of apoptosis in presumptive joints at E15.5. Taken together, these results suggest that misexpression of Sonic Hedgehog causes apoptosis and proliferation defects leading to the lack of joint cavity and fusion of selected limb skeletal elements.     

JAWS coordinates chondrogenesis and synovial joint positioning

Properly positioned synovial joints are crucial to coordinated skeletal movement. Despite their importance for skeletal development and function, the molecular mechanisms that underlie joint positioning are not well understood. We show that mice carrying an insertional mutation in a previously uncharacterized gene, which we have named Jaws (joints abnormal with splitting), die perinatally with striking skeletal defects, including ectopic interphalangeal joints. These ectopic joints develop along the longitudinal axis and persist at birth, suggesting that JAWS is uniquely required for the orientation and consequent positioning of interphalangeal joints within the endochondral skeleton. Jaws mutant mice also exhibit severe chondrodysplasia characterized by delayed and disorganized maturation of growth plate chondrocytes, together with impaired chondroitin sulfation and abnormal metabolism of the chondroitin sulfate proteoglycan aggrecan. Our findings identify JAWS as a key regulator of chondrogenesis and synovial joint positioning required for the restriction of joint formation to discrete stereotyped locations in the embryonic skeleton.     

Shape of articular surface of crocodilian (Archosauria) elbow joints and its relevance to sauropsids

The determination of area and shape of articular surfaces on the limb bones of extinct archosaurs is difficult because of postmortem decomposition of the fibrous tissue and articular cartilages that provide the complex three‐dimensional joint surfaces in vivo. This study aims at describing the shape of the articular cartilages in the elbow joints of six crocodilian specimens; comparing its structure with that of four birds, three testudines, and five squamates; and comparing the shapes of the surfaces of the calcified and the articular cartilages in the elbow joints of an Alligator specimen. The shapes of the articular cartilages of crocodilian elbow joint are shown to resemble those of birds. The humerus possesses an olecranon fossa positioned approximately at the midportion of the distal epiphysis and bordering the margin of the extensor side of the articular surface. The ulna possesses a prominent intercotylar process at approximately the middle of its articular surface, and splits the surface into the radial and ulnar cotylae. This divides the articular cartilage into an articular surface on the flexor portion, and the olecranon on the extensor portion. The intercotylar process fits into the olecranon fossa to restrict elbow joint extension. Dinosaurs and pterosaurs, phylogenetically bracketed by Crocodylia and Aves (birds), may have possessed a similar olecranon fossa and intercotylar process on their articular cartilages. Although these shapes are rarely recognizable on the bones, their impressions on the surfaces of the calcified cartilages provide an important indication of the extensor margin of the articular surfaces. This, in turn, helps to determine the maximum angle of extension of the elbow joint in archosaurs. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Mycobacterium chelonae osteoarthritis in a Kemp's ridley sea turtle (Lepidochelys kempii)

A stranded Kemp's ridley sea turtle (Lepidochelys kempii) was rescued and treated at the National Aquarium in Baltimore (Maryland, USA) for inappetence and epidermal appendicular and plastral lesions. After 4 mo of care, the turtle developed a swollen left elbow joint. Within 1 mo of initial swelling, osteolytic lesions developed in the proximal radius and ulna. The elbow joint was surgically debrided, flushed, and cultured. The incision dehisced 10 days after surgery. Mycobacterium chelonae was cultured from the left elbow joint and from a skin nodule of the dorsum of the right front flipper. The turtle was euthanized due to apparent systemic infection with M. chelonae. Mycobacterium chelonae was isolated from cultures taken at necropsy of the lung, liver, spleen, kidney, and pericardium. Osteoarthritic infections with M. chelonae have not been reported in reptiles. Additionally, primary osteoarthritic diseases of synovial joints are uncommon in reptilian species. Due to the paucity of reports of mycobacterial diseases in sea turtles, the continued documentation of these cases will increase knowledge and understanding in caring for these endangered animals.     

Patterns of correlation and covariation of anthropoid distal forelimb segments correspond to Hoxd expression territories

Anthropoids in general and hominoids in particular exhibit differential adaptations in forearm and digital skeletal proportions to a diverse array of locomotor modes. Hox genes act as selector genes with spatially regulated expression patterns during development. Their expression in the forelimb appears to define modules that specify differential skeletal growth. Here we explore forelimb skeletal proportions in a large sample of anthropoids from a background provided by Hoxd expression patterns in late-stage murine embryonic forelimbs. Interspecific correlation and principal components analyses of primate forelimb data indicate that morphological variation in anthropoids reflects well-defined developmental modules downstream of Hoxd expression. The phalanges of digit one appear to represent a single growth module, whereas the metacarpals and manual phalanges of the posterior digits correspond to a second, independent, expression territory that extends proximally into the distal zeugopod. In particular, hominoids show very high correlations among the posterior digits and the independence of digit one. In addition, the distal radius is generally highly correlated with the posterior digits and not digit one. Relying on established functional differences among Hox paralogs, we present a model that parsimoniously explains hominoid forearm and digital proportions as a consequence of downstream effects of Hox. We, therefore, suggest that Hox-defined developmental modules have served as evolutionary modules during manual evolution in anthropoids.     

An Investigation of the Locomotor Function of Therian Forearm Pronation Provides Renewed Support for an Arboreal,Chameleon-like Evolutionary Stage

Although investigations of forelimb characteristics are central to therian evolutionary studies, the functional origins of forearm pronation are neglected. However, recent research based on bipedal manipulations strongly suggests that proximal radioulnar joint mobility is highly conserved in tetrapods. This new information calls for a replication of previously published physical simulations of forearm bone movements, to investigate whether active therian pronation/supination evolved from the plesiomorphic mechanism via which locomotor-induced torsion is passively alleviated during forelimb retraction. Preliminary results using representative extant and extinct tetrapod forelimb elements are supportive, and also offer insight into why another overlooked forearm trait, osteological full pronation (mechanically aligned elbow and wrist/finger joints), evolved only in therians and chameleons. During forelimb retraction in tetrapods with unfused radii/ulnae, the radius unexpectedly remains fixed in place as a functional complex with the firmly planted manus/carpus, which the ulnar complex (ulna/humerus) displaces relative to. Therefore, the highly conserved functional morphology of the tetrapod forearm indicates that enhanced therian manual dexterity, which emphasizes isolated radial movements bipedally, was preceded by the locomotor evolution of ulnar supination relative to the radius quadrupedally. This counterintuitive information indicates that the traditional hypothesis, that therian pronation/supination evolved arboreally to amplify radial mobility, requires modification. The authors propose that proximal long-axis rotations of the therian ulnar complex co-evolved with osteological full pronation during a period of arboreal, chameleon-like locomotion, to continue allowing torsion at a reinforced proximal radioulnar joint. These adaptations were later or simultaneously co-opted for object manipulation using active radioulnar pronation/supination.     

Perlecan immunolocalizes to perichondrial vessels and canals in human fetal cartilaginous primordia in early vascular and matrix remodeling events associated with diarthrodial joint development.

The aim of this study was to ascertain how perlecan was localized in human fetal cartilaginous joint rudiment tissues. Perlecan was immunolocalized in human fetal (12-14-week-old) toe, finger, knee, elbow, shoulder, and hip joint rudiments using a monoclonal antibody to domain-1 of perlecan (MAb A76). Perlecan had a widespread distribution in the cartilaginous joint rudiments and growth plates and was also prominent in a network of convoluted hairpin loop-type vessels at the presumptive articulating surfaces of joints. Perlecan was also present in small perichondrial venules and arterioles along the shaft of the developing long bones, small blood vessels in the synovial lining and joint capsules, and in distinctive arrangements of cartilage canals in the knee, elbow, shoulder, and hip joint rudiments. Perlecan was notably absent from CD-31-positive metaphyseal vessels in the hip, knee, shoulder, and fingers. These vessels may have a role in the nutrition of the expanding cell populations in these developing joint tissues and in the establishment of the secondary centers of ossification in the long bones, which is essential for endochondral ossification.     

Stress and the etiology of osteoarthritis

Although degenerative joint disease is an old and exceedingly common problem, clinical investigators have not reached a consensus regarding the etiology of this disease. Comparative osteological analysis of the knee, hip, shoulder, and elbow joints of 798 individuals from four human skeletal populations (Black and White Americans, Pueblo Indians, and Alaskan Eskimos) indicates that age of onset, frequency, and location of degenerative changes are directly related to the nature and degree of environmentally associated stress, as reflected by the variable life styles of the populations sampled. Eskimos have the earliest onset and most severe involvement for all four joints studied, the right side is usually more affected than the left, and Blacks are more frequently involved than Whites in the knee, shoulder, and elbow. Functional stress, when constant and severe in nature, becomes the primary focus of degenerative disease, but other background contributing agents such as age, sex, and hormonal influence must not be ignored.