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Vertebrate development is orchestrated by secreted signalling molecules that regulate cell behaviour and cell fate decisions during early embryogenesis. The\ud activity of key signalling molecules including members of Hedgehog, Bone Morphogenetic Proteins and Wnt families are regulated by Glypicans, a family of GPI linked polypeptides. Glypicans either promote or inhibit the action of signalling molecules and add a layer of complexity that needs to be understood in order to fully\ud decipher the processes that regulate early vertebrate development. Here we present a detailed expression profile of all six Glypicans and their modifying enzyme Notum during chick embryogenesis. Our results strongly suggest that these proteins have many as yet undiscovered roles to play during early embryogenesis. Finally, we have taken an experimental approach to investigate their role during the patterning of a key embryonic structure - the neural tube. In particular, we show that overexpression of Notum leads to the dorsalisation of this structure.

The development of vertebrate appendages, especially the limb and feather buds are orchestrated by numerous secreted signalling molecules including Sonic Hedgehog, Bone Morphogenetic Proteins, Fibroblast Growth Factors and Wnts. These proteins coordinate the growth and patterning of ectodermal and mesenchymal cells. The influence of signalling molecules is affected over large distances by their concentration (morphogen activity) but also at local levels by the presence of proteins that either attenuate or promote their activity. Glypicans are cell surface molecules that regulate the activity of the major secreted signalling molecules expressed in the limb and feather bud. Here we investigated the expression of all Glypicans during chick limb and feather development. In addition we profiled the expression of Notum, an enzyme that regulates Glypican activity. We show that five of the six Glypicans and Notum are expressed in a dynamic manner during the development of limbs and feathers. We also investigated the expression of key Glypicans and show that they are controlled by signalling molecules highlighting the presence of feedback loops. Lastly we show that Glypicans and Notum are expressed in a tissue specific manner in adult chicken tissues. Our results strongly suggest that the Glypicans and Notum have many as yet undiscovered roles to play during the development of vertebrate appendages.

The Drosophila melanogaster genome has six physically clustered NK-related homeobox genes in just 180 kb. Here we show that the NK homeobox gene cluster was an ancient feature of bilaterian animal genomes, but has been secondarily split in chordate ancestry. The NK homeobox gene clusters of amphioxus and vertebrates are each split and dispersed at two equivalent intergenic positions. From the ancestral NK gene cluster, only the Tlx–Lbx and NK3–NK4 linkages have been retained in chordates. This evolutionary pattern is in marked contrast to the Hox and ParaHox gene clusters, which are compact in amphioxus and vertebrates, but have been disrupted in Drosophila .

The COE/EBF gene family marks a subset of prospective neurons in the vertebrate central and peripheral nervous system, including neurons deriving from some ectodermal placodes. Since placodes are often considered unique to vertebrates, we have characterised an amphioxus COE/EBF gene with the aim of using it as a marker to examine the timing and location of peripheral neuron differentiation. A single COE/EBF family member, AmphiCoe, was isolated from the amphioxus Branchiostoma floridae. AmphiCoe lies basal to the vertebrate COE/EBF genes in molecular phylogenetic analysis, suggesting that the duplications that formed the vertebrate COE/EBF family were specific to the vertebrate lineage. AmphiCoe is expressed in the central nervous system and in a small number of scattered ectodermal cells on the flanks of neurulae stage embryos. These cells become at least largely recessed beneath the ectoderm. Scanning electron microscopy was used to examine embryos in which the ectoderm had been partially peeled away. This revealed that these cells have neuronal morphology, and we infer that they are the precursors of epidermal primary sensory neurons. These characters lead us to suggest that differentiation of some ectodermal cells into sensory neurons with a tendency to sink beneath the embryonic surface represents a primitive feature that has become incorporated into placodes during vertebrate evolution.

We know little about the genomic events that led to the advent of a multicellular grade of organization in animals, one of the most dramatic transitions in evolution. Metazoan multicellularity is correlated with the evolution of embryogenesis, which presumably was underpinned by a gene regulatory network reliant on the differential activation of signaling pathways and transcription factors. Many transcription factor genes that play critical roles in bilaterian development largely appear to have evolved before the divergence of cnidarian and bilaterian lineages. In contrast, sponges seem to have a more limited suite of transcription factors, suggesting that the developmental regulatory gene repertoire changed markedly during early metazoan evolution. Using whole-genome information from the sponge Amphimedon queenslandica, a range of eumetazoans, and the choanoflagellate Monosiga brevicollis, we investigate the genesis and expansion of homeobox, Sox, T-box, and Fox transcription factor genes. Comparative analyses reveal that novel transcription factor domains (such as Paired, POU, and T-box) arose very early in metazoan evolution, prior to the separation of extant metazoan phyla but after the divergence of choanoflagellate and metazoan lineages. Phylogenetic analyses indicate that transcription factor classes then gradually expanded at the base of Metazoa before the bilaterian radiation, with each class following a different evolutionary trajectory. Based on the limited number of transcription factors in the Amphimedon genome, we infer that the genome of the metazoan last common ancestor included fewer gene members in each class than are present in extant eumetazoans. Transcription factor orthologues present in sponge, cnidarian, and bilaterian genomes may represent part of the core metazoan regulatory network underlying the origin of animal development and multicellularity.

Despite significant advances in tissue engineering such as the use of scaffolds, bioreactors and pluripotent stem cells, effective cardiac tissue engineering for therapeutic purposes has remained a largely intractable challenge. For this area to capitalise on such advances, a novel approach may be to unravel the physiological mechanisms underlying the development of tissues that exhibit rhythmic contraction yet do not originate from the cardiac lineage. Considerable attention has been focused on the physiology of the avian lymph heart, a discrete organ with skeletal muscle origins yet which displays pacemaker properties normally only found in the heart. A functional lymph heart is essential for avian survival and growth in ovo. The histological nature of the lymph heart is similar to skeletal muscle although molecular and bioelectrical characterisation during development to assess mechanisms that contribute towards lymph heart contractile rhythmicity have not been undertaken. A better understanding of these processes may provide exploitable insights for therapeutic rhythmically contractile tissue engineering approaches in this area of significant unmet clinical need. Here, using molecular and electrophysiological approaches, we describe the molecular development of the lymph heart to understand how this skeletal muscle becomes fully functional during discrete in ovo stages of development. Our results show that the lymph heart does not follow the normal transitional programme of myogenesis as documented in most skeletal muscle, but instead develops through a concurrent programme of precursor expansion, commitment to myogenesis and functional differentiation which offers a mechanistic explanation for its rapid development. Extracellular electrophysiological field potential recordings revealed that the peak-to-peak amplitude of electrically evoked local field potentials elicited from isolated lymph heart were significantly reduced by treatment with carbachol; an effect that could be fully reversed by atropine. Moreover, nifedipine and cyclopiazonic acid both significantly reduced peak-to-peak local field potential amplitude. Optical recordings of lymph heart showed that the organ's rhythmicity can be blocked by the HCN channel blocker, ZD7288; an effect also associated with a significant reduction in peak-to-peak local field potential amplitude. Additionally, we also show that isoforms of HCN channels are expressed in avian lymph heart. These results demonstrate that cholinergic signalling and L-type Ca2+ channels are important in excitation and contraction coupling, while HCN channels contribute to maintenance of lymph heart rhythmicity.

Stem cells that can be directed to differentiate into specific cell types offer the prospect of a renewable source of replacement cells to treat diseases. This study evaluates the reprogramming of 2 readily available stem cell populations into skeletal muscle. We show for the first time that freshly isolated muscle fibers reprogram bone marrow or white fat stem cells far more efficiently than muscle cell lines. In addition, we show that the ability of muscle fibers to reprogram stem cells can be almost doubled through the use of chromatin remodeling reagents such as trichostatin A. This novel approach permits the generation of myogenic cells that could be used to treat a range of muscle-wasting diseases.

The forelimbs of higher vertebrates are composed of two portions: the appendicular region (stylopod, zeugopod and autopod) and the less prominent proximal girdle elements (scapula and clavicle) that brace the limb to the main trunk axis. We show that the formation of the muscles of the proximal limb occurs through two distinct mechanisms. The more superficial girdle muscles (pectoral and latissimus dorsi) develop by the "In-Out" mechanism whereby migration of myogenic cells from the somites into the limb bud is followed by their extension from the proximal limb bud out onto the thorax. In contrast, the deeper girdle muscles (e.g. rhomboideus profundus and serratus anterior) are induced by the forelimb field which promotes myotomal extension directly from the somites. Tbx5 inactivation demonstrated its requirement for the development of all forelimb elements which include the skeletal elements, proximal and distal muscles as well as the sternum in mammals and the cleithrum of fish. Intriguingly, the formation of the diaphragm musculature is also dependent on the Tbx5 programme. These observations challenge our classical views of the boundary between limb and trunk tissues. We suggest that significant structures located in the body should be considered as components of the forelimb. (C) 2011 Elsevier Inc. All rights reserved.

FoxC, FoxF, FoxL1 and FoxQ1 genes have been shown to be clustered in some animal genomes, with mesendodermal expression hypothesised as a selective force maintaining cluster integrity. Hypotheses are, however, constrained by a lack of data from the Lophotrochozoa. Here we characterise members of the FoxC, FoxF, FoxL1 and FoxQ1 families from the annelid Capitella teleta and the molluscs Lottia gigantea and Patella vulgata. We cloned FoxC, FoxF, FoxL1 and FoxQ1 genes from C. teleta, and FoxC, FoxF and FoxL1 genes from P. vulgata, and established their expression during development. We also examined their genomic organisation in C. teleta and L. gigantea, and investigated local syntenic relationships. Our results show mesodermal and anterior gut expression is a common feature of these genes in lophotrochozoans. In L. gigantea FoxC, FoxF and FoxL1 are closely linked, while in C. teleta Ct-foxC and Ct-foxL1 are closely linked, with Ct-foxF and Ct-foxQ1 on different scaffolds. Adjacent to these genes there is limited evidence of local synteny. This demonstrates conservation of genomic organisation and expression of these genes can be traced in all three bilaterian Superphyla. These data are evaluated against competing theories for the long-term maintenance of gene clusters.

Satellite cells represent the stem cell population of adult skeletal muscle. The molecular mechanisms that control the proliferation of satellite cells are not well understood. In this study, we show that in response to injury, myofibres activate Wnt ligand transcription and activate a reporter cell line that is sensitive to the canonical Wnt-signalling pathway. Activated satellite cells on isolated cultured myofibres show robust expression of activated-β-catenin (Act-β-Cat), a key downstream transcriptional coactivator of canonical Wnt signalling. We provide evidence that the Wnt family of secreted glycoproteins act on satellite cells in a ligand-specific manner. Overexpression of Wnt1, Wnt3a or Wnt5a protein causes a dramatic increase in satellite-cell proliferation. By contrast, exposure of satellite cells to Wnt4 or Wnt6 diminishes this process. Moreover, we show that the prolonged satellite-cell quiescence induced by inhibitory Wnt is reversible and exposing inhibited satellite cells to stimulatory Wnt signalling restores their proliferation rate. Stimulatory Wnt proteins induce premature satellite cell BrdU incorporation as well as nuclear translocation of Act-β-Cat. Finally, we provide evidence that the Act-β-Cat translocation observed in single fibres during in vitro culture also occurs in cases of acute and chronic skeletal muscle regeneration in rodents and humans. We propose that Wnt proteins may be key factors that regulate the rate of satellite-cell proliferation on adult muscle fibres during the wound-healing response.

The FoxQ1 genes form a distinct group within the Fox (also known as forkhead) gene family. We have isolated a gene from the amphioxus Branchiostoma floridae that encodes a forkhead domain with high identity to FoxQ1 genes in other chordates. Molecular phylogenetic analysis places AmphiFoxQ1 in a robust grouping with vertebrate FoxQ1 genes and with Ciona intestinalis Ci-FoxQ1. This group is separate from that containing AmphiFoxQ2, which instead groups with other invertebrate Fox genes. The expression of AmphiFoxQ1 was analysed by whole mount in situ hybridisation. The results show that AmphiFoxQ1 expression is confined to the developing endoderm, and specifically marks the endostyle and associated peripharyngeal bands of amphioxus larvae. Ci-FoxQ1 is also expressed in the endostyle, highlighting this as a conserved site of FoxQ1 gene expression in basal chordates.

We describe a polymerase chain reaction which amplifies part of the Eco RI repeat unit of the fowl W chromosome. The resulting 447 bp fragment enables DNA from female birds to be identified. The composition of this DNA is confirmed by a nested polymerase chain reaction which specifically amplifies a known internal 263 bp region in this fragment. Using this technique it is possible to follow the fate of female cells in male germline chimaeras. The polymerase chain reaction fragment can be traced in cells of the embryonic and hatchling gonad and in adult sperm implying that cells containing the W chromosome are capable of being processed through the avian testis.

Over the past decade genomic approaches have begun to revolutionise the study of animal diversity. In particular, genome sequencing programmes have spread beyond the traditional model species to encompass an increasing diversity of animals from many different phyla, as well as unicellular eukaryotes that are closely related to the animals. Whole genome sequences allow researchers to establish, with reasonable confidence, the full complement of any particular family of genes in a genome. Comparison of gene complements from appropriate genomes can reveal the evolutionary history of gene families, indicating when both gene diversification and gene loss have occurred. More than that, however, assembled genomes allow the genomic environment in which individual genes are found to be analysed and compared between species. This can reveal how gene diversification occurred. Here, we focus on the Fox genes, drawing from multiple animal genomes to develop an evolutionary framework explaining the timing and mechanism of origin of the diversity of animal Fox genes. Ancient linkages between genes are a prominent feature of the Fox genes, depicting a history of gene clusters, some of which may be relevant to understanding Fox gene function.

The Wnt family of secreted signalling molecules control a wide range of developmental processes in all metazoans. The intracellular response to Wnt signalling depends on the choice of signalling cascade activated in the responding cell. Cells can activate either the canonical pathway that modulates gene expression to control cellular differentiation and proliferation, or the non-canonical pathway that controls cell polarity and movement. Recent work has identified the protein Naked Cuticle to act as an intracellular switch to promote the non-canonical pathway at the expense of the canonical pathway. We have cloned chick Naked Cuticle-1 (cNkd-1) and show that it is expressed in a dynamic manner during early embryogenesis. We show that it is expressed in the somites and in particular regions where cells are undergoing movement. Lastly, we show that the expression of cNkd-1 is regulated by Wnt expression originating from the neural tube. This study provides evidence that non-canonical Wnt signalling plays a part in somite development.

As the sister group to vertebrates, amphioxus is consistently used as a model of genome evolution for understanding the invertebrate/vertebrate transition. The amphioxus genome has not undergone massive duplications like those in the vertebrates or disruptive rearrangements like in the genome of Ciona, a urochordate, making it an ideal evolutionary model. Transposable elements have been linked to many genomic evolutionary changes including increased genome size, modified gene expression, massive gene rearrangements, and possibly intron evolution. Despite their importance in genome evolution, few previous examples of transposable elements have been identified in amphioxus. We report five novel Miniature Inverted-repeat Transposable Elements (MITEs) identified by an analysis of amphioxus DNA sequence, which we have named LanceleTn-1, LanceleTn-2, LanceleTn-3a, LanceleTn-3b and LanceleTn-4. Several of the LanceleTn elements were identified in the amphioxus ParaHox cluster, and we suggest these have had important implications for the evolution of this highly conserved gene cluster. The estimated high copy numbers of these elements implies that MITEs are probably the most abundant type of mobile element in amphioxus, and are thus likely to have been of fundamental importance in shaping the evolution of the amphioxus genome.

1. Embryos of the domestic fowl have been partially sterilised by injecting the drug busulphan into 24-h incubated eggs. 2. Some of these embryos were injected with primordial germ cells (PGCs) after 55 h of incubation to attempt to repopulate the gonads. 3. Primordial germ cells transfected with a defective retrovirus containing the reporter gene lac Z were shown to settle in these sterilised gonads. 4. Quantitative histology of 6-d embryos showed that busulphan produced 75% sterilisation but that PGCs could repopulate these gonads. 5. The technique of producing such germ line chimaeras is of value in studying cell kinetics, gonad differentiation and the production of transgenics.

The drug busulphan is known to be cytotoxic to migrating primordial germ cells (PGCs). A technique is described in which doses of 0, 25, 50 and 250 micrograms busulphan in 40 microliters sesame oil were injected into the yolk of White Leghorn eggs incubated for 0, 24, 48 and 72 h. The percentage survival values of these embryos showed that the older the embryo at the time of injection, the greater the survival. Increasing the dose of busulphan decreased the survival. The percentage of embryos showing abnormalities increased with higher doses of busulphan. The number of germ cells in histological sections from gonads of 16-day embryos was estimated and in embryos treated with 50 micrograms and 250 micrograms busulphan the number of germ cells was significantly less than in the controls. Eggs were injected with 50 micrograms busulphan at 24-30 h, and at 50-55 h the embryos received an intravascular injection of a germinal crescent cell suspension containing PGCs from Rhode Island Red embryos. Twenty hatchlings from these experiments were raised to sexual maturity. All these birds were fertile and half of the breeding groups producing offspring from the transferred germ cells at a rate of about 35% of the total. The technique would improve the efficiency of producing transgenic gametes.