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In JoVE (1)
Other Publications (12)
Articles by Karen J. Liu in JoVE
JoVE General
Electroporation of Craniofacial Mesenchyme
Department of Craniofacial Development, King's College London
Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.
Other articles by Karen J. Liu on PubMed
Cloning and Characterization of Xenopus Id4 Reveals Differing Roles for Id Genes
We have identified Xenopus Id4, a member of the Id (inhibitor of differentiation/DNA binding) class of helix-loop-helix proteins. Id factors dimerize with general bHLH factors, preventing their interaction with tissue-specific bHLH factors, to inhibit premature differentiation. The presence of several Id proteins could reflect simple redundancy in function, or more interestingly, might suggest different activities for these proteins. During embryonic development, Xenopus Id4 is expressed in a number of neural tissues, including Rohon-Beard neurons, olfactory placode, eye primordia, and the trigeminal ganglia. It is also expressed in other organs, such as the pronephros and liver primordium. As embryogenesis progresses, it is expressed in the migrating melanocytes and lateral line structures. We compare the expression of Id4 mRNA with that of Id2 and Id3 and find that the Id genes are expressed in complementary patterns during neurogenesis, myogenesis, kidney development, in the tailbud, and in the migrating neural crest. To examine the regulation of Id gene expression during Xenopus neural development, we show that expression of Id3 and Id4 can be induced by overexpression of BMP4 in the whole embryo and in ectodermal explants. Expression of Id2, Id3, and Id4 in these explants is unaffected by the expression of FGF-8 or a dominant-negative Ras (N17ras), suggesting that Id genes are not regulated by the FGF signaling pathway in naive ectoderm. We also show that Notch signaling can activate Id2 and Id3 expression in the whole embryo. In contrast, Id4 expression in the Rohon-Beard cells is inhibited by activated Notch and increased by a dominant-negative Delta. This may reflect an increase in Rohon-Beard cells in response to inhibition of Notch signaling rather than transcriptional regulation of Id4. Finally, to compare the activities of Id2, Id3, and Id4, we use animal cap explants and in vivo overexpression to show that Id proteins can differentially inhibit the activities of neurogenin and neuroD, both neurogenic bHLH molecules and MyoD, a myogenic bHLH protein. Id4 is able to inhibit the activity all these bHLH molecules, Id2 inhibits MyoD and neuroD, while Id3 blocks only neuroD activity in our assays.
Inhibition of Neurogenesis by SRp38, a NeuroD-regulated RNA-binding Protein
Although serine-arginine rich (SR) proteins have often been implicated in the positive regulation of splicing, recent studies have shown that one unusual SR protein, SRp38, serves, contrastingly, as a splicing repressor during mitosis and stress response. We have identified a novel developmental role for SRp38 in the regulation of neural differentiation. SRp38 is expressed in the neural plate during embryogenesis and is transcriptionally induced by the neurogenic bHLH protein neuroD. Overexpression of SRp38 inhibits primary neuronal differentiation at a step between neurogenin and neuroD activity. This repression of neuronal differentiation requires activation of the Notch pathway. Conversely, depletion of SRp38 activity results in a dysregulation of neurogenesis. Finally, SRp38 can interact with the peptidyltransferase center of 28S rRNA, suggesting that SRp38 activity may act, in part, via regulation of ribosome biogenesis or function. Strikingly, recent studies of several cell cycle regulators during primary neurogenesis have also revealed a crucial control step between neurogenin and neuroD. SRp38 may mediate one component of this control by maintaining splicing and translational silencing in undifferentiated neural cells.
Chemical Rescue of Cleft Palate and Midline Defects in Conditional GSK-3beta Mice
Glycogen synthase kinase-3beta (GSK-3beta) has integral roles in a variety of biological processes, including development, diabetes, and the progression of Alzheimer's disease. As such, a thorough understanding of GSK-3beta function will have a broad impact on human biology and therapeutics. Because GSK-3beta interacts with many different pathways, its specific developmental roles remain unclear. We have discovered a genetic requirement for GSK-3beta in midline development. Homozygous null mice display cleft palate, incomplete fusion of the ribs at the midline and bifid sternum as well as delayed sternal ossification. Using a chemically regulated allele of GSK-3beta (ref. 6), we have defined requirements for GSK-3beta activity during discrete temporal windows in palatogenesis and skeletogenesis. The rapamycin-dependent allele of GSK-3beta produces GSK-3beta fused to a tag, FRB* (FKBP/rapamycin binding), resulting in a rapidly destabilized chimaeric protein. In the absence of drug, GSK-3beta(FRB)*(/FRB)* mutants appear phenotypically identical to GSK-3beta-/- mutants. In the presence of drug, GSK-3betaFRB* is rapidly stabilized, restoring protein levels and activity. Using this system, mutant phenotypes were rescued by restoring endogenous GSK-3beta activity during two distinct periods in gestation. This technology provides a powerful tool for defining windows of protein function during development.
Tissue Engineering in Cleft Palate and Other Congenital Malformations
Contributions from multidisciplinary investigations have focused attention on the potential of tissue engineering to yield novel therapeutics. Congenital malformations, including cleft palate, craniosynostosis, and craniofacial skeletal hypoplasias represent excellent targets for the implementation of tissue engineering applications secondary to the technically challenging nature and inherent inadequacies of current reconstructive interventions. Apropos to the search for answers to these clinical conundrums, studies have focused on elucidating the molecular signals driving the biologic activity of the aforementioned maladies. These investigations have highlighted multiple signaling pathways, including Wnt, fibroblast growth factor, transforming growth factor-beta, and bone morphogenetic proteins, that have been found to play critical roles in guided tissue development. Furthermore, a comprehensive knowledge of these pathways will be of utmost importance to the optimization of future cell-based tissue engineering strategies. The scope of this review encompasses a discussion of the molecular biology involved in the development of cleft palate and craniosynostosis. In addition, we include a discussion of craniofacial distraction osteogenesis and how its applied forces influence cell signaling to guide endogenous bone regeneration. Finally, this review discusses the future role of cell-based tissue engineering in the treatment of congenital malformations.
Cranial Osteogenesis and Suture Morphology in Xenopus Laevis: a Unique Model System for Studying Craniofacial Development
The tremendous diversity in vertebrate skull formation illustrates the range of forms and functions generated by varying genetic programs. Understanding the molecular basis for this variety may provide us with insights into mechanisms underlying human craniofacial anomalies. In this study, we provide evidence that the anuran Xenopus laevis can be developed as a simplified model system for the study of cranial ossification and suture patterning. The head structures of Xenopus undergo dramatic remodelling during metamorphosis; as a result, tadpole morphology differs greatly from the adult bony skull. Because of the extended larval period in Xenopus, the molecular basis of these alterations has not been well studied.
Dazap2 is Required for FGF-mediated Posterior Neural Patterning, Independent of Wnt and Cdx Function
The organization of the embryonic neural plate requires coordination of multiple signal transduction pathways, including fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs), and WNTs. Many studies have suggested that a critical component of this process is the patterning of posterior neural tissues by an FGF-caudal signaling cascade. Here, we have identified a novel player, Dazap2, and show that it is required in vivo for posterior neural fate. Loss of Dazap2 in embryos resulted in diminished expression of hoxb9 with a concurrent increase in the anterior marker otx2. Furthermore, we found that Dazap2 is required for FGF dependent posterior patterning; surprisingly, this is independent of Cdx activity. Furthermore, in contrast to FGF activity, Dazap2 induction of hoxb9 is not blocked by loss of canonical Wnt signaling. Functionally, we found that increasing Dazap2 levels alters neural patterning and induces posterior neural markers. This activity overcomes the anteriorizing effects of noggin, and is downstream of FGF receptor activation. Our results strongly suggest that Dazap2 is a novel and essential branch of FGF-induced neural patterning.
The Planar Cell Polarity Effector Fuz is Essential for Targeted Membrane Trafficking, Ciliogenesis and Mouse Embryonic Development
The planar cell polarity (PCP) signalling pathway is essential for embryonic development because it governs diverse cellular behaviours, and 'core PCP' proteins, such as Dishevelled and Frizzled, have been extensively characterized. By contrast, the 'PCP effector' proteins, such as Intu and Fuz, remain largely unstudied. These proteins are essential for PCP signalling, but they have never been investigated in mammals and their cell biological activities remain entirely unknown. We report here that Fuz mutant mice show neural tube defects, skeletal dysmorphologies and Hedgehog signalling defects stemming from disrupted ciliogenesis. Using bioinformatics and imaging of an in vivo mucociliary epithelium, we established a central role for Fuz in membrane trafficking, showing that Fuz is essential for trafficking of cargo to basal bodies and to the apical tips of cilia. Fuz is also essential for exocytosis in secretory cells. Finally, we identified a Rab-related small GTPase as a Fuz interaction partner that is also essential for ciliogenesis and secretion. These results are significant because they provide new insights into the mechanisms by which developmental regulatory systems such as PCP signalling interface with fundamental cellular systems such as the vesicle trafficking machinery.
New Directions in Craniofacial Morphogenesis
The vertebrate head is an extremely complicated structure: development of the head requires tissue-tissue interactions between derivates of all the germ layers and coordinated morphogenetic movements in three dimensions. In this review, we highlight a number of recent embryological studies, using chicken, frog, zebrafish and mouse, which have identified crucial signaling centers in the embryonic face. These studies demonstrate how small variations in growth factor signaling can lead to a diversity of phenotypic outcomes. We also discuss novel genetic studies, in human, mouse and zebrafish, which describe cell biological mechanisms fundamental to the growth and morphogenesis of the craniofacial skeleton. Together, these findings underscore the complex interactions leading to species-specific morphology. These and future studies will improve our understanding of the genetic and environmental influences underlying human craniofacial anomalies.
Mesodermal Wnt Signaling Organizes the Neural Plate Via Meis3
In vertebrates, canonical Wnt signaling controls posterior neural cell lineage specification. Although Wnt signaling to the neural plate is sufficient for posterior identity, the source and timing of this activity remain uncertain. Furthermore, crucial molecular targets of this activity have not been defined. Here, we identify the endogenous Wnt activity and its role in controlling an essential downstream transcription factor, Meis3. Wnt3a is expressed in a specialized mesodermal domain, the paraxial dorsolateral mesoderm, which signals to overlying neuroectoderm. Loss of zygotic Wnt3a in this region does not alter mesoderm cell fates, but blocks Meis3 expression in the neuroectoderm, triggering the loss of posterior neural fates. Ectopic Meis3 protein expression is sufficient to rescue this phenotype. Moreover, Wnt3a induction of the posterior nervous system requires functional Meis3 in the neural plate. Using ChIP and promoter analysis, we show that Meis3 is a direct target of Wnt/beta-catenin signaling. This suggests a new model for neural anteroposterior patterning, in which Wnt3a from the paraxial mesoderm induces posterior cell fates via direct activation of a crucial transcription factor in the overlying neural plate.
Neural Crest Origin of Olfactory Ensheathing Glia
Olfactory ensheathing cells (OECs) are a unique class of glial cells with exceptional translational potential because of their ability to support axon regeneration in the central nervous system. Although OECs are similar in many ways to immature and nonmyelinating Schwann cells, and can myelinate large-diameter axons indistinguishably from myelination by Schwann cells, current dogma holds that OECs arise from the olfactory epithelium. Here, using fate-mapping techniques in chicken embryos and genetic lineage tracing in mice, we show that OECs in fact originate from the neural crest and hence share a common developmental heritage with Schwann cells. This explains the similarities between OECs and Schwann cells and overturns the existing dogma on the developmental origin of OECs. Because neural crest stem cells persist in adult tissue, including skin and hair follicles, our results also raise the possibility that patient-derived neural crest stem cells could in the future provide an abundant and accessible source of autologous OECs for cell transplantation therapy for the injured central nervous system.
Role of GSK-3β in the Osteogenic Differentiation of Palatal Mesenchyme
The function of Glycogen Synthase Kinases 3β (GSK-3β) has previously been shown to be necessary for normal secondary palate development. Using GSK-3ß null mouse embryos, we examine the potential coordinate roles of Wnt and Hedgehog signaling on palatal ossification.
