Highlights•Characterization of the events that prefigure the formation of individual muscle bundles•Direct demonstration of the role of connective tissue cells in muscle morphogenesis•Identification of markers of limb irregular connective tissue (ICT)•Demonstration of molecularly distinct ICT subdomains in the limbSummaryAlthough 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.Graphical abstract
We dissect genetically a gene regulatory network, including the transcription factors Tbx4, Pitx1 and Isl1 that act 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 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 in the Tbx4 mutant result from a failure in the early differentiation step of chondroprogenitors into chondrocytes, providing a novel explanation for the origins of proximally-biased limb defects.
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.
This thesis aimed to understand the structural changes that occur during the development of the mammalian cornea. The imaging techniques used included novel three-dimensional serial block-face scanning electron microscopy, transmission electron microscopy, optical coherence tomography, X-ray diffraction and immunofluorescence. These techniques were utilised to investigate the human, mouse and the fibrillin-1 knockout mouse cornea.
The mouse cornea had no collagenous primary stroma to direct mesenchymal cell migration. Stromal cell projections associated with adjacent corneal stromal cells and the corneal epithelium, and appeared to direct collagen alignment. The mouse stroma expressed types I, II and V collagen, and later type IX collagen in the epithelium. Proteoglycans were observed before collagen deposition in the mouse stroma, associated with stromal cells and collagen fibrils.
A collagenous primary stroma was identified in the human embryonic cornea prior to mesenchymal cell migration. The corneal endothelium contained novel cell extensions that associated with the mesenchymal cells and the acellular collagenous matrix; these results suggested that the endothelium assists mesenchymal cell migration.
The human adult cornea contained true elastic fibres in the peripheral posterior cornea with fibrillin-rich microfibrils in the central posterior cornea. The elastic fibres in the mouse contained only fibrillin-rich microfibrils. In the human, elastic fibres were detected from week 12 of development and had a distribution similar to the mature human cornea. This included elastic fibre
sheets directly anterior to the endothelium and individual elastic fibres in the posterior peripheral stroma.
The fibrillin-1 knockout mouse cornea had reduced stromal thickness and a disorganised extracellular matrix. It is thought that elevated transforming growth factor-beta disrupted the corneal architecture.
This thesis has contributed novel findings of the events that develop the mammalian cornea. The results identified fundamental differences and similarities between the mouse and human models and have suggested new mechanisms in the developmental process.
Purpose : To characterise and compare the elastic fibre system of mouse and human posterior cornea.
Methods : 4 adult human corneas were obtained from a UK eye bank and 20 corneas taken from 3 month old C5BL/6 mice which had been euthanized. The samples were examined using Serial block face scanning electron microscopy (SBF-SEM) and transmission electron microscopy (TEM). The corneas were fixed and processed for SBF-SEM through a series of staining solutions prior to embedding and polymerization in epoxy resin blocks. The sample blocks were trimmed and transferred to a Zeiss Sigma VP FEG scanning electron microscope equipped with a Gatan 3View system, where data sets of up to 1000 images were acquired of the block surface every 50nm through automated sectioning. Selected serial image sequences were extracted and 3D reconstructions were generated using Amira 6.4 software. For electron microscopy ultrathin sections were prepared and examined in a JEOL 1010 TEM. The corneas were also examined using immunofluorescence labelling of elastin and fibrillin-1.
Results : Immunofluorescence revealed an extensive fibrillin-1-rich microfibril system running throughout the mouse corneal stroma. Human corneas were also positive for fibrillin-1 within the posterior cornea, but in addition exhibited positive elastin staining confined to the posterior peripheral cornea. TEM confirmed the presence of true elastic fibres containing an amorphous elastin core in peripheral human corneas, which were absent within the mouse microfibrils. Clear structural differences at the convergence of the trabecular meshwork (TM) into the elastic fibre system were also observed between mouse and human cornea using SBF-SEM. In mouse cornea the TM merged directly with Descemet’s membrane whereas in human cornea the TM inserted into the elastic fibre system anterior to Descemet’s.
Conclusions : Clear differences exist between the elastic fibre system and TM anatomy of mouse and human cornea. These differences suggest the fibre system of mouse and human have different biomechanical properties and function.
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.
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.
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.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)
