Loss of U11 small nuclear RNA in the developing mouse limb results in micromelia.

Disruption of the minor spliceosome due to mutations in RNU4ATAC is linked to primordial dwarfism in microcephalic osteodysplastic primordial dwarfism type 1, Roifman syndrome, and Lowry-Wood syndrome. Similarly, primordial dwarfism in domesticated animals is linked to positive selection in minor spliceosome components. Despite being vital for limb development and size regulation, its role remains unexplored. Here, we disrupt minor spliceosome function in the developing mouse limb by ablating one of its essential components, U11 small nuclear RNA, which resulted in micromelia. Notably, earlier loss of U11 corresponded to increased severity. We find that limb size is reduced owing to elevated minor intron retention in minor intron-containing genes that regulate cell cycle. As a result, limb progenitor cells experience delayed prometaphase-to-metaphase transition and prolonged S-phase. Moreover, we observed death of rapidly dividing, distally located progenitors. Despite cell cycle defects and cell death, the spatial expression of key limb patterning genes was maintained. Overall, we show that the minor spliceosome is required for limb development via size control potentially shared in disease and domestication.

Three-dimensional visualization of extracellular matrix networks during murine development.

The extracellular matrix (ECM) plays a crucial role in embryogenesis, serving both as a substrate to which cells attach and as an active regulator of cell behavior. However, little is known about the spatiotemporal expression patterns and 3D structure of ECM proteins during embryonic development. The lack of suitable methods to visualize the embryonic ECM is largely responsible for this gap, posing a major technical challenge for biologists and tissue engineers. Here, we describe a method of viewing the 3D organization of the ECM using a polyacrylamide-based hydrogel to provide a 3D framework within developing murine embryos. After removal of soluble proteins using sodium dodecyl sulfate, confocal microscopy was used to visualize the 3D distribution of independent ECM networks in multiple developing tissues, including the forelimb, eye, and spinal cord. Comparative analysis of E12.5 and E14.5 autopods revealed proteoglycan-rich fibrils maintain connections between the epidermis and the underlying tendon and cartilage, indicating a role for the ECM during musculoskeletal assembly and demonstrating that our method can be a powerful tool for defining the spatiotemporal distribution of the ECM during embryogenesis.

Hox11 genes are required for regional patterning and integration of muscle, tendon and bone.

Development of the musculoskeletal system requires precise integration of muscles, tendons and bones. The molecular mechanisms involved in the differentiation of each of these tissues have been the focus of significant research; however, much less is known about how these tissues are integrated into a functional unit appropriate for each body position and role. Previous reports have demonstrated crucial roles for Hox genes in patterning the axial and limb skeleton. Loss of Hox11 paralogous gene function results in dramatic malformation of limb zeugopod skeletal elements, the radius/ulna and tibia/fibula, as well as transformation of the sacral region to a lumbar phenotype. Utilizing a Hoxa11eGFP knock-in allele, we show that Hox11 genes are expressed in the connective tissue fibroblasts of the outer perichondrium, tendons and muscle connective tissue of the zeugopod region throughout all stages of development. Hox11 genes are not expressed in differentiated cartilage or bone, or in vascular or muscle cells in these regions. Loss of Hox11 genes disrupts regional muscle and tendon patterning of the limb in addition to affecting skeletal patterning. The tendon and muscle defects in Hox11 mutants are independent of skeletal patterning events as disruption of tendon and muscle patterning is observed in Hox11 compound mutants that do not have a skeletal phenotype. Thus, Hox genes are not simply regulators of skeletal morphology as previously thought, but are key factors that regulate regional patterning and integration of the musculoskeletal system.

Acoustic-induced motion of the bushcricket (Mecopoda elongata, Tettigoniidae) tympanum.

Bushcrickets have a tonotopically organised hearing organ, the so-called crista acustica, in the tibia of the forelegs. This organ responds to a frequency range of about 5-80 kHz and lies behind the anterior tympanum on top of a trachea branch. We analyzed the sound-induced vibration pattern of the anterior tympanum, using a Laser-Doppler-Vibrometer Scanning microscope system, in order to identify frequency-dependent amplitude and phase of displacement. The vibration pattern evoked by a frequency sweep (4-79 kHz) showed an amplitude maximum which would correspond to the resonance frequency of an open tube system. At higher frequencies of about 30 kHz a difference in the amplitude and phase response between the distal and the proximal part of the tympanum was detected. The inner plate of the tympanum starts to wobble at this frequency. This higher mode in the motion pattern is not explained by purely acoustic characteristics of the tracheal space below the tympanum but may depend on the mechanical impedance of the tympanum plate. In accordance with a previous hypothesis, the tympanum moves over the whole tested frequency range in the dorso-ventral direction like a hinged flap with the largest displacement in its ventral part and no higher modes of vibration.

Scanning electron microscopy of legs of two species of sucking lice (Anoplura: Phthiraptera).

Pretarsal, tarsal and tibial structures of the forelegs, midlegs and hindlegs of Pediculus humanus of humans and of Haematopinus apri Goureau, 1866 (Phthiraptera), a parasite of feral hogs, were studied using light microscopy and scanning electron microscopy. Details of the tibial thumb-like process (tl) with the spine of the thumb (spn), tarsal apophysis (ta) and the coupled finger-like process (cfl) can be observed in the leg photomicrograph of both species. A frontal view of the leg in open position shows the articulation of the claw: the structures of an open-closed system, a tooth row (te), rack-system (rs) and two telescopic columns (tc) which are present near the base of the claw in both species. In H. apri, we observed a pad-like structure, the flap-like tibial lobe (fl) on the ventral surface on the tarsus, the euplantulae, with several sensilla basiconica, which is present in each leg.

Hierarchical bioinspired adhesive surfaces-a review.

The extraordinary adherence and climbing agility of geckos on rough surfaces has been attributed to the multiscale hierarchical structures on their feet. Hundreds of thousands of elastic hairs called setae, each of which split into several spatulae, create a large number of contact points that generate substantial adhesion through van der Waals interactions. The hierarchical architecture provides increased structural compliance on surfaces with roughness features ranging from micrometers to millimeters. We review synthetic adhesion surfaces that mimic the naturally occurring hierarchy with an emphasis on microfabrication strategies, material choice and the adhesive performance achieved.

Tissue patterning in the developing mouse limb.

The developing mouse fore- and hindlimbs begin as bumps on the flank of the embryo and grow out to form miniature models of the adult limb during a five day period from E9.5 to E14.5. In this paper I show a series of embryos taken at half-day intervals during limb development and outline the timetable of patterning for each of the component tissues of a limb: epidermis, connective tissues, muscle, nerves and blood vessels. Scanning electron micrographs, supplemented by histological sections, are presented to define a set of standard stages for the description of mouse limb development. I discuss my observations of the mouse limb in the light of current theories of vertebrate limb development, which are based on classic manipulation experiments in the chick as well as more recent molecular data from the mouse system. The limb skeletal pattern in a mouse is laid down in a proximodistal direction, as it is in a bird: the E11.5 forelimb reveals the first signs of a humerus and by E14.5 even the most distal phalanges of the hand are formed. At this late stage ossification sleeves are seen around the proximal limb elements as the cartilage template begins to be converted to a bony skeleton. Myogenic cells stainable with the MF20 antibody against early muscle myosin heavy chain are first seen in the mouse forelimb at E11.5, which is also when the first nerve fascicles begin to enter the limb. From E11.5 to E14.5 both muscle and nerve patterns mature to give distinct muscles at all proximodistal levels of the limb, each muscle with its own nerve branch, and a cutaneous nerve plexus that extends to the fingertips. The developing skin of the mouse limb matures from a bi-layered epidermis overlying an avascular, but otherwise nondescript, prospective dermal layer of mesenchyme at E9.5, to a 4- or 5-layered epidermis with early hair placodes and the first signs of a distinct dermal layer at E14.5. Notable differences between mouse and chick limb development lie in the relatively late formation of the apical ectodermal ridge in the mouse and its unexpectedly close relations with blood vessels, in the absence of anterior and posterior necrotic zones and, possibly, in a late migration of myogenic cells into the mouse limb bud.

Chondrogenic differentiation in cultures of embryonic rat mesenchyme.

The morphology and fine structure of day 12 rat embryonic mesenchyme from forelimb bud, mandibular arches, and frontonasal prominence is described as the cells undergo chondrogenesis in high density, micromass culture. The cultures began as a multilayered "pavement" of flattened mesenchymal cells, 3-4 deep, with moderate intercellular space but little identifiable electron-dense extracellular matrix. Pre-cartilage condensations, which consisted of aggregates of cells which had rounded up, displaying little or no intercellular space, formed within the first 24 h in limb mesenchyme and after an additional 24 h in mandibular and frontonasal cultures. Gap junctions occur between these cells, indicating a phase of direct cell-cell communication. Chondrogenesis within these aggregates began within the next 24 h in limb cultures but was delayed an additional 24-48 h in the frontonasal and especially in mandibular cultures. The aggregates in both limb and mandibular mesenchyme form discrete nodules bordered by a perichondrium consisting of 2-3 layers of flattened cells. Evidence from late stage mandibular cultures suggests that chondroblasts are added to the nodules from the perichondrium, as occurs in vivo. By contrast, the frontonasal cartilage is initially unbordered and forms anastomosing trabecular arrays. Some of these arrays fuse into larger structures with time, but others become surrounded by a single, flattened perichondrium, resulting in the stacking of these trabeculae as chondrification proceeds. The sequence of cartilage matrix production, as revealed in long-term facial cultures, appears to occur in three stages, an early phase in which the extracellular matrix consists primarily of proteoglycans, followed by a phase of homogeneous collagen-proteoglycan matrix and a mature, territorial matrix. In all three cultures the cartilage ultimately produced resembles mature rat hyaline cartilage with chondroblasts surrounded by a territorial matrix of type II collagen and proteoglycan granules.

Electron microscopic investigations on the growing tip of nerve fibres in the developing distal forelimb of the mouse.

The distal course of the sensory nerve fibres on the palmar side of the hand of 12-day-old mouse embryos was followed by serial ultrathin cross-sections of conventionally fixed tissue. Small nerves fibres are to be found in the space between the layer of the cutaneous blood vessels and the epidermis. A special arrangement of the tissue which is traversed by the nerve fibres is not visible. In most cases the nerve fibres are in intimate contact with fibroblasts. More distally the nerve fibres consist only of one axon encircled by a Schwann cell. The sheath of the Schwann cell decreases gradually, and finally disappears completely. The distal tips of the naked axons are a few microns ahead. In some cases they are considerably enlarged and contain only a fine fuzzy material without special cell organelles. Other axons may end in a more finger-like fashion, and contain also in the naked parts many cell organelles. Also the naked axons contact fibroblasts over long ranges. Collagen was not found aligned to the distal nerve fibres.

A study on the regenerative potential of partially excised mouse embryonic fore-limb bud.

The ability of day E10 mouse fore-limb bud to regulate following the removal of a portion of limb tissue was investigated. A longitudinal strip of tissue, two to three somites in width and extending from the base of the limb bud to its distal tip, was excised. The embryos were then maintained in a roller culture system for periods of 6 h, 12 h or 24 h post-operatively prior to fixation and subsequent examination. The embryos were examined with scanning electron microscopy (SEM) and light microscopy. SEM revealed that about two thirds of the operated limbs grossly restored their overall morphology. The sequence of morphological changes involved in the restoration process is described. The ability of the restored limb bud to develop an apical ectodermal ridge (AER) is shown in histological sections.

Post natal development of the canine elbow joint: a light and electron microscopical study.

The elbows of 13 puppy cadavers were dissected, samples were taken for light and electron microscopy, and the thickness of the articular cartilage of the distal humerus and proximal ulna was measured. Throughout post natal development differences were found in the arrangement of the growth plate and articular chondrocytes. At birth, the articular surface had remnants of a fibrous limiting membrane that was continuous with the perichondrium, a finding not previously recorded in dogs. Orientation of the collagen fibrils within the matrix of the articular cartilage was initially lacking but became established by three weeks. In the humerus cartilage canals were present up to 12 weeks old. The articular cartilage of the humeral condyle varied in thickness across the joint surface, being thicker on the medial than on the lateral side; it was also thicker at the apex of the medial coronoid process. These regions of thick cartilage correspond with the sites where cartilage defects arise in elbow osteochondrosis. No histological evidence was found that the medial cornoid process of the ulna is a separate centre of ossification.

Investigation of foreign substances in food.

Scanning electron microscopy (SEM) together with energy-dispersive spectroscopy (EDS) and Fourier transform infrared spectroscopy (FTIR) were used to investigate foreign substances from seven categories of foreign substances in food. (1) Naturally occurring foreign substances--Using FTIR, a foreign substance was identified as a natural resin probably from the product. (2) Foreign substances introduced during food processing. Scanning electron microscopy-EDS was used to identify a foreign material found on surf clams as calcium phosphate from a product/ingredient interaction. Using SEM-EDS, a crystalline material in a meat product was identified as calcium salts of chloride and phosphate. Fourier transform infrared spectroscopy was used to identify foreign material that clogged an aerosol valve as chipboard. Using SEM-EDS, the metal in the heel of a glass bottle was identified as copper sulfide-containing metal inclusion. (3) Insects, reptiles, and rodents--Scanning electron microscopy was used to determine that a mouse found in food was not processed with the food, but entered the container after it left the factory. (4) Glass fragments--Glass from various sources can be distinguished from one another using SEM-EDS either by the level of the major elements in glass or by the presence of elements in one glass, but not in another. (5) Glass-like particles--Using SEM-EDS, glass-like particles found on beets were determined to be a fatty acid. (6) Metal foreign objects--Using SEM-EDS, metals from a variety of sources can be easily distinguished. For example, a tin-soldered container can be distinguished from a lead-soldered can. Using SEM-EDS, the metal fiber found on the bottom of a two-piece can likely enter the can during the final stage of the manufacture of the drawn and ironed food can. (7) Drug capsule identification--Fourier transform infrared spectroscopy was used to determine that a pill found in food was ibuprofen.

Comparison of results of scanning electron microscopy and magnetic resonance imaging before and after administration of a radiographic contrast agent in the tendon of the deep digital flexor muscle obtained from horse cadavers.

OBJECTIVE: To analyze the tendon of the deep digital flexor (TDDF) muscle of the forelimb in horses by use of a contrast radiographic agent (gadopentate dimeglumine [Gd-DTPA/Dimeg]) and magnetic resonance imaging (MRI) and to determine the concentration of water protons in the tendons by use of MRI. SAMPLE POPULATION: 8 TDDF harvested from the forelimbs of 6 horse cadavers. PROCEDURE: Examinations were performed on the same portion of each tendon. Tendons were examined by use of two techniques: MRI before and after treatment with Gd-DTPA/Dimeg as well as scanning electron microscopy. RESULTS: Tendons did not have detectable signal intensity on MRI before treatment with Gd-DTPA/Dimeg; however, intravascular injection of Gd-DTPA/Dimeg allowed evaluation of the internal structure of the tendons Scanning electron microscopy images correlated well with images obtained by use of MRI before and after administration of Gd-DTPA/Dimeg. Localized spectra revealed the concentration of water protons in the TDDF. CONCLUSIONS AND CLINICAL RELEVANCE: The techniques used in this study provided information about internal organization of the TDDF in horses. Analysis of results revealed that the best technique involved vascular injection of contrast medium. Results of MRI correlated well with results for scanning electron microscopy. After administration of Gd-DTPA/Dimeg, MRI provided additional information about tendon morphologic characteristics. This technique may be of value for examination of tendons in lame horses.

Structural diversity of nuptial pads in Phyllomedusinae (Amphibia: Anura: Hylidae).

We studied the morphological variation of the nuptial pads using light microscopy and scanning electron microscopy (SEM) in 26 species of phyllomedusines (Anura: Hylidae), representing the five currently recognized genera. All phyllomedusines have single nuptial pads with dark colored epidermal projections (EPs). Spine-shaped EPs occur in Cruziohyla calcarifer, Phrynomedusa appendiculata and in one species of Phasmahyla. The other species have roundish EPs. The density of the EPs on the pad is variable. Species in the Phyllomedusa hypochondrialis Group have EPs with a density that varies between 764 ± 58/mm(2) and 923 ± 160/mm(2). In all other studied species (including the Phyllomedusa burmeisteri and Phyllomedusa perinesos groups, Phyllomedusa camba, Phyllomedusa boliviana, Phyllomedusa sauvagii, Phyllomedusa bicolor, and Phyllomedusa tomopterna) the density of EPs varies between 108 ± 20/mm(2) and 552 ± 97/mm(2). Pores were observed with SEM in C. calcarifer, Agalychnis lemur, Agalychnis moreletii, but its presence is confirmed through histological sections on several other species. Its visibility using SEM seems to be related with the level of separation between adjacent EPs. The pores in the four studied species of Agalychnis are shown with SEM and histological sections to have a characteristic epidermal rim, that is absent in the otherphyllomedusines. Unlike most previous reports on breeding glands, those of phyllomedusines are alcian blue positive, indicating the presence of acidic mucosubstances on its secretions.

Organization of connective tissue patterns by dermal fibroblasts in the regenerating axolotl limb.

A set of tendons, aponeurotic sheets and retinaculae, which transduce muscle action from proximal limb levels to flexion and extension of the digits, is found in limbs of many vertebrates. This set of structures, here termed the digit tendon complex, is described for the axolotl forelimb. We show that the complex forms autonomously in muscleless axolotl limb regenerates produced from a cuff of unirradiated dermis surrounding an irradiated limb stump, and persists for up to a year after amputation. The pattern of other connective tissue structures, including the skeleton, is also normal. Fibroblast condensations that may represent sets of these cells normally associated with muscles in the extensor and flexor compartments of the carpal region also form in muscleless limbs. The results are discussed in terms of the importance of the dermis in pattern regulation, selforganization of connective tissues in general and autonomous development of the digit tendon complex in particular.

Fine structure of the forelimb regenerate of the African clawed toad, Xenopus laevis.

Forelimb regenerates from postmetamorphic Xenopus froglets were examined at various stages postamputation by light microscopy and scanning and transmission electron microscopy. The outside surface of the wound epithelium was found to exhibit progressive changes following amputation with a distinct difference in appearance between stump epidermis and wound epithelium at all stages examined. The internal structure of the wound epithelium is characterized by loosely arranged cells with numerous cell junctions and abundant intracellular filaments. The wound epithelium is separated at an early stage from the underlying cells by a thick band of extracellular matrix. Cells accumulating beneath the wound epithelium were morphologically similar to blastemal cells from completely regenerating limbs in other species but no evidence of myogenesis or abortive myogenesis was seen. Blastemal cells from the central portion of the regenerate were observed at varius stages of chrondrogenesis with those immediately beneath the wound epithelium least advanced in this respect. Those located more laterally appear not to be directly involved in chondrogenesis. Although the usual explanation for lack of complete regeneration in this species is inadequate innervation of the regenerate, the fine structure of the regenerating spikes noted here is markedly different than that of denervated, amputated newt limbs.

A study of digit fusion in the mouse embryo.

During the embryonic development of the mouse limb separation of the digit is followed by their union. This is a true, though temporary, epithelial fusion, a fused layer of epidermal cells remaining intact until separation takes place after birth. The periderm cells in the line of fusion are displaced to the dorsal or ventral surface of the foot. On the dorsal surface these displaced cells form a prominent interdigital ridge of elongated, intertwined cells which remains until the periderm is shed. During the fusion of the eyelids, and also of the pinnae to the scalp, a similar ridge of periderm cells is formed.

Axon and neuron numbers after forelimb amputation in neonatal rats.

It seems a paradox that more primary sensory neurons are lost but recovery is better after peripheral nerve injury in neonates as compared to adult mammals. A possible explanation is that surviving neurons sprout in the neonate. To test this, forelimbs in neonatal rats were amputated, which caused the death of many primary sensory neurons. The number of neurons in the dorsal root ganglia, and the number of myelinated and unmyelinated fibers in the dorsal and ventral roots were determined on the amputated and contralateral normal sides. On the amputated side, soma loss in the ganglia was 30%, and the fiber numbers were decreased by 16% in the dorsal root and increased by 20% in the ventral root. These data are compatible with the hypothesis that there is axonal branching or sprouting from surviving sensory neurons. In addition, morphometric analyses showed a changed myelin-axon relationship for central processes of sensory cells whose distal processes have been cut.