Investigating the role connective tissue fibroblasts play in the altered muscle anatomy associated with the limb abnormality, Radial Dysplasia.

Radial dysplasia (RD) is a congenital upper limb birth defect that presents with changes to the upper limb anatomy, including a shortened or absent radius, bowed ulna, thumb malformations, a radially deviated hand and a range of muscle and tendon malformations, including absent or abnormally shaped muscle bundles. Current treatments to address wrist instability caused by a shortened or absent radius frequently require an initial soft tissue distraction intervention followed by a wrist stabilisation procedure. Following these surgical interventions, however, recurrence of the wrist deviation remains a common, long-term problem following treatment. The impact of the abnormal soft connective tissue (muscle and tendon) anatomy on the clinical presentation of RD and the complications following surgery are not understood. To address this, we have examined the muscle, fascia and the fascial irregular connective tissue (ICT) fibroblasts found within soft connective tissues, from RD patients. We show that ICT fibroblasts isolated from RD patients are functionally abnormal when compared to the same cells isolated from control patients and secrete a relatively disordered extracellular matrix (ECM). Furthermore, we show that ICT fibroblast dysfunction is a unifying feature found in RD patients, even when the RD clinical presentation is caused by distinct genetic syndromes.

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.

4D formation of human embryonic forelimb musculature.

The size, shape and insertion sites of muscles enable them to carry out their precise functions in moving and supporting the skeleton. Although forelimb anatomy is well described, much less is known about the embryonic events that ensure individual muscles reach their mature form. A description of human forelimb muscle development is needed to understand the events that control normal muscle formation and to identify what events are disrupted in congenital abnormalities in which muscles fail to form normally. We provide a new, 4D anatomical characterisation of the developing human upper limb muscles between Carnegie stages 18 and 22 using optical projection tomography. We show that muscles develop in a progressive wave, from proximal to distal and from superficial to deep. We show that some muscle bundles undergo splitting events to form individual muscles, whereas others translocate to reach their correct position within the forelimb. Finally, we show that palmaris longus fails to form from early in development. Our study reveals the timings of, and suggests mechanisms for, crucial events that enable nascent muscle bundles to reach their mature form and position within the human forelimb.

Identification of a Primary Stroma and Novel Endothelial Cell Projections in the Developing Human Cornea.

Purpose: To investigate the initial events in the development of the human cornea, focusing on cell migration, and extracellular matrix synthesis and organization. To determine whether elastic fibers are present in the extracellular matrix during early human corneal development. Methods: Human corneas were collected from week 7 to week 17 of development. An elastic fiber-enhancing stain, tannic acid-uranyl acetate, was applied to all tissue. Three-dimensional serial block-face scanning electron microscopy combined with conventional transmission electron microscopy was used to analyze the corneal stroma. Results: An acellular collagenous primary stroma with an orthogonal arrangement of fibrils was identified in the central cornea from week 7 of corneal development. At week 7.5, mesenchymal cells migrated toward the central cornea and associated with the acellular collagenous matrix. Novel cell extensions from the endothelium were identified. Elastic fibers were found concentrated in the posterior peripheral corneal stroma from week 12 of corneal development. Conclusions: This study provides novel evidence of an acellular primary stroma in the early development of the embryonic human cornea. Cell extensions exist as part of a communication system and are hypothesized to assist in the migration of the mesenchymal cells and the development of the mature cornea. Elastic fibers identified in early corneal development may play an important role in establishing corneal shape.

Individual Limb Muscle Bundles Are Formed through Progressive Steps Orchestrated by Adjacent Connective Tissue Cells during Primary Myogenesis.

Although the factors regulating muscle cell differentiation are well described, we know very little about how differentiating muscle fibers are organized into individual muscle tissue bundles. Disruption of these processes leads to muscle hypoplasia or dysplasia, and replicating these events is vital in tissue engineering approaches. We describe the progressive cellular events that orchestrate the formation of individual limb muscle bundles and directly demonstrate the role of the connective tissue cells that surround muscle precursors in controlling these events. We show how disruption of gene activity within or genetic ablation of connective tissue cells impacts muscle precursors causing disruption of muscle bundle formation and subsequent muscle dysplasia and hypoplasia. We identify several markers of the populations of connective tissue cells that surround muscle precursors and provide a model for how matrix-modifying proteoglycans secreted by these cells may influence muscle bundle formation by effects on the local extracellular matrix (ECM) environment.

Developmental abnormalities in the cornea of a mouse model for Marfan syndrome.

Elastic fibres provide tissues with elasticity and flexibility. In the healthy human cornea, elastic fibres are limited to the posterior region of the peripheral stroma, but their specific functional role remains elusive. Here, we examine the physical and structural characteristics of the cornea during development in the mgΔloxPneo dominant-negative mouse model for Marfan syndrome, in which the physiological extracellular matrix of its elastic-fibre rich tissues is disrupted by the presence of a dysfunctional fibrillin-1 glycoprotein. Optical coherence tomography demonstrated a reduced corneal thickness in the mutant compared to wild type mice from embryonic day 16.5 until adulthood. X-ray scattering and electron microscopy revealed a disruption to both the elastic fibre and collagen fibril ultrastructure in the knockout mice, as well as abnormally low levels of the proteoglycan decorin. It is suggested that these alterations might be a result of increased transforming growth factor beta signalling. To conclude, this study has demonstrated corneal structure and ultrastructure to be altered when fibrillin-1 is disrupted and has provided insights into the role of fibrillin-1 in developing a functional cornea.

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.

Three-dimensional imaging of the extracellular matrix and cell interactions in the developing prenatal mouse cornea.

As the outer lens in the eye, the cornea needs to be strong and transparent. These properties are governed by the arrangement of the constituent collagen fibrils, but the mechanisms of how this develops in mammals is unknown. Using novel 3-dimensional scanning and conventional transmission electron microscopy, we investigated the developing mouse cornea, focusing on the invading cells, the extracellular matrix and the collagen types deposited at different stages. Unlike the well-studied chick, the mouse cornea had no acellular primary stroma. Collagen fibrils initially deposited at E13 from the presumptive corneal stromal cells, become organised into fibril bundles orthogonally arranged between cells. Extensive cell projections branched to adjacent stromal cells and interacted with the basal lamina and collagen fibrils. Types I, II and V collagen were expressed from E12 posterior to the surface ectoderm, and became widespread from E14. Type IX collagen localised to the corneal epithelium at E14. Type VII collagen, the main constituent of anchoring filaments, was localised posterior to the basal lamina. We conclude that the cells that develop the mouse cornea do not require a primary stroma for cell migration. The cells have an elaborate communication system which we hypothesise helps cells to align collagen fibrils.

A comparative study of the elastic fibre system within the mouse and human cornea.

The cornea relies on its organised extracellular matrix for maintaining transparency and biomechanical strength. Studies have identified an elastic fibre system within the human posterior cornea, thought to allow for slight deformations in response to internal pressure fluctuations within the eye. However, the type of elastic fibres that exist within the cornea and their roles remain elusive. The aim of this study was to compare the distribution and organisation of the elastic fibres within the posterior peripheral mouse and human cornea, and elucidate how these fibres integrate with the trabecular meshwork, whilst characterising the distribution of their main likely components (fibrillin-1, elastin and type VI collagen) in different parts of the cornea and adjacent sclera. We identified key differences in the elastic fibre system between the human and mouse cornea. True elastic fibres (containing elastin) were identified within the human posterior peripheral cornea. Elastic fibres appeared to present as an extensive network throughout the mouse corneal stroma, but as fibrillin-rich microfibril bundles rather than true elastic fibres. However, tropoelastin staining indicated the possibility that true elastic fibres had yet to develop in the young mice studied. Differences were also apparent within the anatomy of the trabecular meshwork. The human trabecular meshwork appeared to insert between the corneal stroma and Descemet's membrane, with elastic fibres continuing into the stroma from the trabecular meshwork anterior to Descemet's membrane. Within the mouse cornea, no clear insertion point of the trabecular meshwork was seen, instead the elastic fibres within the trabecular meshwork continued into Descemet's membrane, with the trabecular meshwork joining posterior to Descemet's membrane.