The regulation of Dkk1 expression during embryonic development

During embryogenesis, the Dkk1 mediated Wnt inhibition controls the spatiotemporal dynamics of cell fate determination, cell differentiation and cell death. Furthermore, the Dkk1 dose is critical for the normal Wnt homeostasis, as alteration of the Dkk1 activity is associated with various diseases. We investigated the regulation of Dkk1 expression during embryonic development. We identified nine conserved non-coding elements (CNEs), located 3′ to the Dkk1 locus. Analyses of the regulatory potential revealed that four of these CNEs in combination drive reporter expression very similar to Dkk1 expression in several organs of transgenic embryos. We extended the knowledge of Dkk1 expression during hypophysis, external genitalia and kidney development, suggesting so far to unexplored functions of Dkk1 during the development of these organs. Characterization of the regulatory potential of four individual CNEs revealed that each of these promotes Dkk1 expression in brain and kidney. In combination, two enhancers are responsible for expression in the pituitary and the genital tubercle. Furthermore, individual CNEs mediates craniofacial, optic cup and limb specific Dkk1 regulation. Our study substantially improves the knowledge of Dkk1 regulation during embryonic development and thus might be of high relevance for therapeutic approaches.     

Genetic interaction of Gsc and Dkk1 in head morphogenesis of the mouse

Mouse embryos lacking Gsc and Dkk1 function display severe deficiencies in craniofacial structures which are not found in either Dkk1 homozygous null or Gsc homozygous null mutant embryos. Loss of Gsc has a dosage-related effect on the severity of head truncation phenotype in Dkk1 heterozygous embryos. The synergistic effect of these mutations in enhancing head truncation provides direct evidence of a genetic interaction between Gsc and Dkk1, which display overlapping expression in the prechordal mesoderm. In the absence of Gsc activity, the expression of Dkk1, WNT genes and a transgenic reporter for WNT signalling are altered. Our results show that Gsc and Dkk1 functions are non-redundant in the anterior mesendoderm for normal anterior development and Gsc may influence Wnt signalling as a negative regulator.     

The Dkk1 dose is critical for eye development

During mammalian ocular development, several signaling pathways control the spatiotemporal highly defined realization of the three-dimensional eye architecture. Given the complexity of these inductive signals, the developing eye is a sensitive organ for several diseases.In this study, we investigated a Dkk1+/− haploinsufficiency during eye development, resulting in coloboma and anterior eye defects, two common developmental eye disorders. Dkk1 impacts eye development from a defined developmental time point on, and is critical for lens separation from the surface ectoderm via β-catenin mediated Pdgfrα and E-cadherin expression. Dkk1 does not impact the dorso ventral retina patterning in general but is critical for Shh dependent Pax2 extension into the midline region.The described results also indicate that the retinal Dkk1 dose is critical for important steps during eye development, such as optic fissure closure and cornea formation. Further analysis of the relationship between Dkk1 and Shh signaling revealed that Dkk1 and Shh coordinatively control anterior head formation and eye induction. During eye development itself, retinal Dkk1 activation is depending on cilia mediated Gli3 regulation. Therefore, our data essentially improve the knowledge of coloboma and anterior eye defects, which are common human eye developmental defects.     

The roles of Wnt antagonists Dkk1 and sFRP4 during adipogenesis of human adipose tissue‐derived mesenchymal stem cells

Abstract. Objectives: The canonical Wnt signalling pathway performs an important function in the control of adipogenesis. However, the mechanisms and mediators underlying these interactions have yet to be defined in detail. Thus, this study was performed in order to elucidate the roles of the Wnt family during adipogenic differentiation of human adipose tissue‐derived mesenchymal stem cells (hAMSCs). Materials and methods: We assessed several members of the Frizzled (FZD) family, the receptors of Wnts, inhibitors including the secreted frizzled‐related protein (sFRP) family and Dickkopfs (Dkks), and the downstream factor, β‐catenin. Expressional levels of adipogenic markers regulated by the small interfering RNA of Dkk1 (siDkk1) and sFRP4 (sisFRP4) were assessed using real‐time quantitative PCR and Western blot analysis. Results: The mRNA level of Dkk1 was expressed abundantly in the early stages of adipogenesis and decreased rapidly during the late stages of adipogenesis. However, sFRP4 mRNA was up‐regulated gradually during adipogenic differentiation in hAMSCs. Expression of FZD1, FZD7 and β‐catenin were reduced during adipogenic differentiation. Transfection of hAMSCs with siDkk1 or sisFRP4 partially inhibited differentiation of hAMSCs into adipocytes and restored levels of β‐catenin. Conclusions: We determined that Dkk1 was up‐regulated transiently in the early stages of adipogenesis, and that sFRP4 levels increased gradually during adipogeneis via inhibition of Wnt signalling. Collectively, these results show that Dkk1 and sFRP4 perform an important function in adipogenesis in hAMSCs.     

Flrt2 and Flrt3 have overlapping and non-overlapping expression during craniofacial development

Craniofacial morphogenesis is a complex multi-step process that involves numerous biological processes to coordinate the growth, proliferation, migration, and subsequent differentiation of the cranial neural crest cells. Members of the Fibronectin Leucine-Rich Transmembrane (Flrt) gene family have been previously reported to be widely expressed in the developing embryo. We mapped the expression of Flrt2 and Flrt3 at critical stages of craniofacial development and found that, during early craniofacial development, Flrt2 was highly expressed initially in the cranial neural crest cells and Flrt3 in the midbrain. Later both genes were expressed in the developing pharyngeal region. Flrt2 expression predominated in the neural crest-derived mesenchyme in the medial aspect of the developing frontonasal region in close relationships with the expression of Fgfr2, Shh, and Msx1, three genes shown previously to play critical roles in craniofacial development. Flrt2 was also present in the vomero-nasal organ, mandibular primodia, and the posterior aspects of the unfused and fused secondary palatal shelves. Flrt3, however, had a more restrictive expression, being present in the mesenchyme underlying the ectoderm of the medial nasal process and in the mandibular primordium and in regions undergoing outgrowth, in a pattern that overlapped with Bmp4 expression. Both Flrt2 and Flrt3 were later found to be present at sites of epithelial–mesenchymal interactions such as the developing tooth buds, hair follicles, and eye. Together the data suggested important roles for Flrt2 and Flrt3 in mediating events such as NCC migration, chondrogenesis and epithelial–mesenchymal interactions during craniofacial development.     

Dickkopf (Dkk) 1 promotes the differentiation of mouse embryonic stem cells toward neuroectoderm

Wnt signaling has been demonstrated to have extensive roles during embryogenesis. The Wnt family is highly conserved. In mice, there are 19 Wnt genes. Dickkopf (Dkk), through its interactions with Wnt co-receptors, low-density lipoprotein receptor-related protein (LRP), Frizzled and Kremen, can act as a negative regulator to block the Wnt-signaling pathway. There are four Dkk genes in the human genome, and three in that of the mouse. Dkk1 is involved in a variety of craniofacial developmental processes and behaves as a strong head inducer and limb regulator. Dkk1 mutant mice are embryonic-lethal. Here, we investigated the effects of Dkk1 on the differentiation of murine ESCs in both the ESC and embryoid body (EB) states. The results demonstrate that Dkk1 overexpression can initiate the differentiation program of ESCs toward neuroectoderm. We believe this finding can augment our understanding of mouse ESC differentiation.     

BMP signalling in craniofacial development

The BMP signalling pathway is conserved throughout evolution and essential for mammalian embryonic and postnatal development and growth. In the vertebrate head, this signal is involved in the development of a variety of structures and shows divergent roles. During early head development, BMP signalling participates in the induction, formation, determination and migration of the cranial neural crest cells, which give rise to most of the craniofacial structures. Subsequently, it is also important for patterning and formation of facial primordia. During craniofacial skeletogenesis, BMP signalling is an early inductive signal required for committed cell migration, condensation, proliferation and differentiation. Thereafter, BMP signalling maintains regulatory roles in skeletons and skeletal growth centres. For myogenesis, BMP signalling is a negative regulator. Importantly, myostatin has been identified as a key mediator in this process. During palatogenesis, the crucial role of BMP signalling is demonstrated by mouse models with Alk2 or Alk3 (BMP type I receptors) deletion from the neural crest or craniofacial region, in which cleft palate is one of the major anomalies. BMP signalling is also an important participant for tooth development, regulating early tooth morphogenesis and subsequent odontoblast differentiation. In this review these aspects are discussed in detail with a focus on recent advances.     

Expression of otd orthologs in the amphipod crustacean, Parhyale hawaiensis

The arthropod head is a complex metameric structure. In insects, orthodenticle (otd) functions as a ‘head gap gene’ and plays a significant role in patterning and development of the anterior head ectoderm, the protocerebrum, and the ventral midline. In this study, we characterize the structure and developmental deployment of two otd paralogs in the amphipod crustacean, Parhyale hawaiensis. Photd1 is initially expressed at gastrulation through germband stages in a bilaterally symmetric, restricted region of the anterior head ectoderm and also in a single column of cells along the ventral midline. Late in embryogenesis, Photd1 is expressed within the developing anterior brain and the expression along the embryonic midline has become restricted to a stereotypic group of segmentally reiterated cells. The second ortholog Photd2, however, has a unique temporal–spatial expression pattern and is not detected until after the head lobes have been organized in the developing ectoderm of the germband during late germband stages. Anteriorly, Photd2 is coincident with the Photd1 head expression domain; however, Photd2 is not detected along the ventral midline during formation of the germband and only appears in the ventral midline late in embryonic development in a restricted group of cells distinct from those expressing Photd1. The early expression of Photd1 in the anterior head ectoderm is consistent with a role as a head gap gene. The more posterior expression of Photd1 is suggestive of a role in patterning the embryonic ventral midline. Photd2 expression appears too late to play a role in early head patterning but may contribute to latter patterning in restricted regions of both the head and the ventral midline. The comparative analysis of otd reveals the divergence of gene expression and gene function associated with duplication of this important developmental gene.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Essential role of Dkk3 for head formation by inhibiting Wnt/β‐catenin and Nodal/Vg1 signaling pathways in the basal chordate amphioxus

To dissect the molecular mechanism of head specification in the basal chordate amphioxus, we investigated the function of Dkk3, a secreted protein in the Dickkopf family, which is expressed anteriorly in early embryos. Amphioxus Dkk3 has three domains characteristic of Dkk3 proteins—an N‐terminal serine rich domain and two C‐terminal cysteine‐rich domains (CRDs). In addition, amphioxus Dkk3 has a TGFβ‐receptor 2 domain, which is not present in Dkk3 proteins of other species. As vertebrate Dkk3 proteins have been reported to regulate either Nodal signaling or Wnt/β‐catenin signaling but not both in the same species, we tested the effects of Dkk3 on signaling by these two pathways in amphioxus embryos. Loss of function experiments with an anti‐sense morpholino oligonucleotide (MO) against amphioxus Dkk3 resulted in larvae with truncated heads and concomitant loss of expression of anterior gene markers. The resemblance of the headless phenotype to that from upregulation of Wnt/β‐catenin signaling with BIO, a GSK3β inhibitor, suggested that Dkk3 might inhibit Wnt/β‐catenin signaling. In addition, the Dkk3 MO rescued dorsal structures in amphioxus embryos treated with SB505124, an inhibitor of Nodal signaling, indicating that amphioxus Dkk3 can also inhibit Nodal signaling. In vitro assays in Xenopus animal caps showed that Nodal inhibition is largely due to domains other than the TGFβ domain. We conclude that amphioxus Dkk3 regulates head formation by modulating both Wnt/β‐catenin and Nodal signaling, and that these functions may have been partitioned among various vertebrate lineages during evolution of Dkk3 proteins.     

EZH2‐mediated repression of Dkk1 promotes hepatic stellate cell activation and hepatic fibrosis

EZH2, a histone H3 lysine‐27‐specific methyltransferase, is involved in diverse physiological and pathological processes including cell proliferation and differentiation. However, the role of EZH2 in liver fibrosis is largely unknown. In this study, it was identified that EZH2 promoted Wnt pathway‐stimulated fibroblasts in vitro and in vivo by repressing Dkk‐1, which is a Wnt pathway antagonist. The expression of EZH2 was increased in CCl₄‐induced rat liver and primary HSCs as well as TGF‐β1‐treated HSC‐T6, whereas the expression of Dkk1 was reduced. Silencing of EZH2 prevented TGF‐β1‐induced proliferation of HSC‐T6 cells and the expression of α‐SMA. In addition, knockdown of Dkk1 promoted TGF‐β1‐induced activation of HSCs. Moreover, silencing of EZH2 could restore the repression of Dkk‐1 through trimethylation of H3K27me3 in TGF‐β1‐treated HSC‐T6 cells. Interestingly, inhibition of EZH2 had almost no effect on the activation of HSC when Dkk1 was silenced. Collectively, EZH2‐mediated repression of Dkk1 promotes the activation of Wnt/β‐catenin pathway, which is an essential event for HSC activation.     

Crispld2 is required for neural crest cell migration and cell viability during zebrafish craniofacial development

The CAP superfamily member, CRISPLD2, has previously been shown to be associated with nonsyndromic cleft lip and palate (NSCLP) in human populations and to be essential for normal craniofacial development in the zebrafish. Additionally, in rodent models, CRISPLD2 has been shown to play a role in normal lung and kidney development. However, the specific role of CRISPLD2 during these developmental processes has yet to be determined. In this study, it was demonstrated that Crispld2 protein localizes to the orofacial region of the zebrafish embryo and knockdown of crispld2 resulted in abnormal migration of neural crest cells (NCCs) during both early and late time points. An increase in cell death after crispld2 knockdown as well as an increase in apoptotic marker genes was also shown. This data suggests that Crispld2 modulates the migration, differentiation, and/or survival of NCCs during early craniofacial development. These results indicate an important role for Crispld2 in NCC migration during craniofacial development and suggests involvement of Crispld2 in cell viability during formation of the orofacies. genesis 53:660–667, 2015. © 2015 Wiley Periodicals, Inc.     

Dkk1 regulates ventral midbrain dopaminergic differentiation and morphogenesis

Dickkopf1 (Dkk1) is a Wnt/β-catenin inhibitor that participates in many processes during embryonic development. One of its roles during embryogenesis is to induce head formation, since Dkk1-null mice lack head structures anterior to midbrain. The Wnt/β-catenin pathway is also known to regulate different aspects of ventral midbrain (VM) dopaminergic (DA) neuron development and, in vitro, Dkk1-mediated inhibition of the Wnt/β-catenin pathway improves the DA differentiation in mouse embryonic stem cells (mESC). However, the in vivo function of Dkk1 on the development of midbrain DA neurons remains to be elucidated. Here we examined Dkk1(+/-) embryos and found that Dkk1 is required for the differentiation of DA precursors/neuroblasts into DA neurons at E13.5. This deficit persisted until E17.5, when a defect in the number and distribution of VM DA neurons was detected. Furthermore, analysis of the few Dkk1(-/-) embryos that survived until E17.5 revealed a more severe loss of midbrain DA neurons and morphogenesis defects. Our results thus show that Dkk1 is required for midbrain DA differentiation and morphogenesis.     

Dkk3 is required for TGF-beta signaling during Xenopus mesoderm induction

Abstract The Dickkopf (Dkk) family is composed of four main members (Dkk1–4), which typically regulate Wnt/β-catenin signaling. An exception is Dkk3, which does not affect Wnt/β-catenin signaling and whose function is poorly characterized. Here, we describe the Xenopus dkk3 homolog and characterize its expression and function during embryogenesis. Dkk3 is maternally expressed and zygotically in the cement gland, head mesenchyme, and heart. We show that depletion of Dkk3 in Xenopus embryos by Morpholino antisense oligonucleotides induces axial defects as a result of Spemann organizer and mesoderm inhibition. Dkk3 depletion leads to down-regulation of Activin/Nodal signaling by reducing levels of Smad4 protein. Dkk3 overexpression can rescue phenotypic effects resulting from overexpression of the Smad4 ubiquitin ligase Ectodermin. Furthermore, depletion of Dkk3 up-regulates FGF signaling, while Dkk3 overexpression reduces it. These results indicate that Dkk3 modulates FGF and Activin/Nodal signaling to regulate mesoderm induction during early Xenopus development.     

Growth of cranial synchondroses and sutures requires polycystin-1

In vertebrates, coordinated embryonic and postnatal growth of the craniofacial bones and the skull base is essential during the expansion of the rostrum and the brain. Identification of molecules that regulate skull growth is important for understanding the nature of craniofacial defects and for development of non-invasive biologically based diagnostics and therapies.Here we report on spatially restricted growth defects at the skull base and in craniofacial sutures of mice deficient for polycystin-1 (Pkd1). Mutant animals reveal a premature closure of both presphenoid and sphenooccipital synchondroses at the cranial base. Furthermore, knockout mice lacking Pkd1 in neural crest cells are characterized by impaired postnatal growth at the osteogenic fronts in craniofacial sutures that are subjected to tensile forces. Our data suggest that polycystin-1 is required for proliferation of subpopulations of cranial osteochondroprogenitor cells of both mesodermal and neural crest origin during skull growth. However, the Erk1/2 signalling pathway is up-regulated in the Pkd1-deficient skeletal tissue, similarly to that previously reported for polycystic kidney.