Christopher Wylie

Overexpression of cadherins and underexpression of β-catenin inhibit dorsal mesoderm induction in early Xenopus embryos

The cadherin-catenin complex has an important role in cell-cell adhesion and may also function in signaling pathways. We report that overexpression of three cadherin types in Xenopus embryos causes them to develop with reduced dorsal axial structures. The same phenotype is produced in embryos that have been depleted of maternal beta-catenin protein by an antisense oligodeoxynucleotide complementary to beta-catenin mRNA. They show an inhibition in the expression of dorsal mesodermal markers MyoD and goosecoid, but not of ventral and general mesodermal markers. They lack notochords, somites, and neural tubes and are defective in dorsal mesodermal signaling in Nieuwkoop assays. The phenotype can be rescued by the injection of beta-catenin mRNA and not by the injection of Xwnt-8 mRNA. These results show that beta-catenin has an important role in dorsal mesoderm induction. They directly demonstrate the activity of a maternal mRNA in axis specification.

The Role of Maternal VegT in Establishing the Primary Germ Layers in Xenopus Embryos

VegT is a T-box transcription factor whose mRNA is synthesized during oogenesis and localized in the vegetal hemisphere of the egg and early embryo. We show that maternally expressed VegT controls the pattern of primary germ layer specification in Xenopus embryos. Reduction of the maternal store completely alters the fates of different regions of the blastula so that animal cell fate is changed from epidermis and nervous system to epidermis only, equatorial cell fate is changed from mesoderm to ectoderm, and vegetal cell fate is changed from endoderm to mesoderm and ectoderm. Vegetal cells lose their capacity both to form endoderm and to release mesoderm-inducing signals. These results show that a single maternally expressed gene controls the patterning of the Xenopus blastula.

Xklp15 a chromosomal xenopus kinesin-like protein essential for spindle organization and chromosome positioning

Xklp1 is a novel Xenopus kinesin-like protein with a motor domain at the amino terminus, nuclear localization sequences in the stalk, and a putative zinc finger-like sequence in the tail. It is nuclear during interphase and chromosomal during mitosis. During late anaphase, a fraction of the protein relocalizes to the spindle interzone and accumulates in the midbody during telophase. Depletion of Xklp1 protein by antisense oligo knockout in oocytes leads to defective mitosis during the first cell cycles following fertilization. The bipolarity of spindles assembled in vitro in the presence of anti-Xklp1 antibodies is unstable, and the chromosomes fail to congress on the metaphase plate.

The role of cadherins during primordial germ cell migration and early gonad formation in the mouse

Primordial germ cells (PGCs) are the founder cells of the gametes. In mammals, PGCs migrate from the hindgut to the genital ridges, where they coalesce with each other and with somatic cells to form the primary sex cords. We show here that, in both sexes, PGCs express P- and E-cadherins during and after migration, and N-cadherin at post-migratory stages. E-Cadherin is not expressed by PGCs whilst in the hindgut, but is upregulated as they leave. Blocking antibodies against E-, but not P-cadherin cause defective PGC-PGC coalescence, and in some cases, ectopic PGCs.

A Kinesin-like Protein Is Required for Germ Plasm Aggregation in Xenopus

In embryos of X. laevis, and many other species, early development requires targeted movements of molecules and molecular aggregates within the oocyte or egg cytoplasm. One well-known example in Xenopus is the aggregation of germ plasm, a group of cytoplasmic islands that become distributed during the first few cell cycles to cells that will give rise to the germ line. Nothing is known about the cytoskeletal motor proteins that may drive these movements. We show here that a recently identified Xenopus kinesin-like protein, Xklp1, is required for the aggregation of germ plasm in early Xenopus embryos, thus assigning this protein a role in a developmentally important cytoplasmic localization.

Impaired growth of denervated muscle contributes to contracture formation following neonatal brachial plexus injury.

BACKGROUND The etiology of shoulder and elbow contractures following neonatal brachial plexus injury is incompletely understood. With use of a mouse model, the current study tests the novel hypothesis that reduced growth of denervated muscle contributes to contractures following neonatal brachial plexus injury. METHODS Unilateral brachial plexus injuries were created in neonatal mice by supraclavicular C5-C6 nerve root excision. Shoulder and elbow range of motion was measured four weeks after injury. Fibrosis, cross-sectional area, and functional length of the biceps, brachialis, and subscapularis muscles were measured over four weeks following injury. Muscle satellite cells were cultured from denervated and control biceps muscles to assess myogenic capability. In a comparison group, shoulder motion and subscapularis length were assessed following surgical excision of external rotator muscles. RESULTS Shoulder internal rotation and elbow flexion contractures developed on the involved side within four weeks following brachial plexus injury. Excision of the biceps and brachialis muscles relieved the elbow flexion contractures. The biceps muscles were histologically fibrotic, whereas fatty infiltration predominated in the brachialis and rotator cuff muscles. The biceps and brachialis muscles displayed reduced cross-sectional and longitudinal growth compared with the contralateral muscles. The upper subscapularis muscle similarly displayed reduced longitudinal growth, with the subscapularis shortening correlating with internal rotation contracture. However, excision of the external rotators without brachial plexus injury caused no contractures or subscapularis shortening. Myogenically capable satellite cells were present in denervated biceps muscles despite impaired muscle growth in vivo. CONCLUSIONS Injury of the upper trunk of the brachial plexus leads to impaired growth of the biceps and brachialis muscles, which are responsible for elbow flexion contractures, and impaired growth of the subscapularis muscle, which correlates with internal rotation contracture of the shoulder. Shoulder muscle imbalance alone causes neither subscapularis shortening nor internal rotation contracture. Impaired muscle growth cannot be explained solely by absence of functioning satellite cells.

Shh Signaling from the Nucleus Pulposus Is Required for the Postnatal Growth and Differentiation of the Mouse Intervertebral Disc

Intervertebral discs (IVD) are essential components of the vertebral column. They maintain separation, and provide shock absorbing buffers, between adjacent vertebrae, while also allowing movements between them. Each IVD consists of a central semi-liquid nucleus pulposus (NP) surrounded by a multi-layered fibrocartilagenous annulus fibrosus (AF). Although the IVDs grow and differentiate after birth along with the vertebral column, little is known about the mechanism of this. Understanding the signals that control normal IVD growth and differentiation would also provide potential therapies for degenerative disc disease, which is the major cause of lower back pain and affects a large proportion of the population. In this work, we show that during postnatal growth of the mouse, Sonic hedgehog (Shh) signaling from the NP cells controls many aspects of growth and differentiation of both the NP cells themselves and of the surrounding AF, and that it acts, at least partly, by regulating other signaling pathways in the NP and AF. Recent studies have shown that the NP cells arise from the embryonic notochord, which acts as a major signaling center in the embryo. This work shows that this notochord-derived tissue continues to carry out a major signaling function in the postnatal body and that the IVDs are signaling centers, in addition to their already known functions in the mechanics of vertebral column function.

XPACE4 is a localized pro-protein convertase required for mesoderm induction and the cleavage of specific TGFβ proteins in Xenopus development

XPACE4 is a member of the subtilisin/kexin family of pro-protein convertases. It cleaves many pro-proteins to release their active proteins, including members of the TGFβ family of signaling molecules. Studies in mouse suggest it may have important roles in regulating embryonic tissue specification. Here, we examine the role of XPACE4 in Xenopus development and make three novel observations: first, XPACE4 is stored as maternal mRNA localized to the mitochondrial cloud and vegetal hemisphere of the oocyte; second, it is required for the endogenous mesoderm inducing activity of vegetal cells before gastrulation; and third, it has substrate-specific activity, cleaving Xnr1, Xnr2, Xnr3 and Vg1, but not Xnr5, Derrière or ActivinB pro-proteins. We conclude that maternal XPACE4 plays an important role in embryonic patterning by regulating the production of a subset of active mature TGFβ proteins in specific sites.

FoxI1e activates ectoderm formation and controls cell position in the Xenopus blastula.

The segregation of the vertebrate embryo into three primary germ layers is one of the earliest developmental decisions. In Xenopus, where the process is best understood, the endoderm is specified by a vegetally localized transcription factor, VegT, which releases nodal signals that specify the adjacent marginal zone of the blastula to become mesoderm. However, little is known about how the ectoderm becomes specified. In this paper, we show that the forkhead protein FoxI1e (also known as Xema) is required at the blastula stage for normal formation of both the central nervous system and epidermis, the two early derivatives of the ectoderm. In addition, FoxI1e is required to maintain the regional identity of the animal cells of the blastula, the cells that are precursors of ectodermal structures. In its absence, they lose contact with the animal cap, mix with cells of other germ layers and differentiate according to their new positions. Because FoxI1e is initially expressed in the animal region of the embryo and is rapidly downregulated in the neural plate, its role in neural and epidermal gene expression must precede the division of the ectoderm into neural and epidermal. The work also shows that FoxI1e plays a role in the embryo in the poorly understood process of differential adhesion, which limits cell mixing as primary germ layers become specified.

The Paratenon Contributes to Scleraxis-Expressing Cells during Patellar Tendon Healing

The origin of cells that contribute to tendon healing, specifically extrinsic epitenon/paratenon cells vs. internal tendon fibroblasts, is still debated. The purpose of this study is to determine the location and phenotype of cells that contribute to healing of a central patellar tendon defect injury in the mouse. Normal adult patellar tendon consists of scleraxis-expressing (Scx) tendon fibroblasts situated among aligned collagen fibrils. The tendon body is surrounded by paratenon, which consists of a thin layer of cells that do not express Scx and collagen fibers oriented circumferentially around the tendon. At 3 days following injury, the paratenon thickens as cells within the paratenon proliferate and begin producing tenascin-C and fibromodulin. These cells migrate toward the defect site and express scleraxis and smooth muscle actin alpha by day 7. The thickened paratenon tissue eventually bridges the tendon defect by day 14. Similarly, cells within the periphery of the adjacent tendon struts express these markers and become disorganized. Cells within the defect region show increased expression of fibrillar collagens (Col1a1 and Col3a1) but decreased expression of tenogenic transcription factors (scleraxis and mohawk homeobox) and collagen assembly genes (fibromodulin and decorin). By contrast, early growth response 1 and 2 are upregulated in these tissues along with tenascin-C. These results suggest that paratenon cells, which normally do not express Scx, respond to injury by turning on Scx and assembling matrix to bridge the defect. Future studies are needed to determine the signaling pathways that drive these cells and whether they are capable of producing a functional tendon matrix. Understanding this process may guide tissue engineering strategies in the future by stimulating these cells to improve tendon repair.