Wnt Signaling Interacts with Bmp and Edn1 to Regulate Dorsal-Ventral Patterning and Growth of the Craniofacial Skeleton

Craniofacial development requires signals from epithelia to pattern skeletogenic neural crest (NC) cells, such as the subdivision of each pharyngeal arch into distinct dorsal (D) and ventral (V) elements. Wnt signaling has been implicated in many aspects of NC and craniofacial development, but its roles in D-V arch patterning remain unclear. To address this we blocked Wnt signaling in zebrafish embryos in a temporally-controlled manner, using transgenics to overexpress a dominant negative Tcf3, (dntcf3), (Tg(hsp70I:tcf3-GFP), or the canonical Wnt inhibitor dickkopf1 (dkk1), (Tg(hsp70i:dkk1-GFP) after NC migration. In dntcf3 transgenics, NC cells in the ventral arches of heat-shocked embryos show reduced proliferation, expression of ventral patterning genes (hand2, dlx3b, dlx5a, msxe), and ventral cartilage differentiation (e.g. lower jaws). These D-V patterning defects resemble the phenotypes of zebrafish embryos lacking Bmp or Edn1 signaling, and overexpression of dntcf3 dramatically reduces expression of a subset of Bmp receptors in the arches. Addition of ectopic BMP (or EDN1) protein partially rescues ventral development and expression of dlx3b, dlx5a, and msxe in Wnt signaling-deficient embryos, but surprisingly does not rescue hand2 expression. Thus Wnt signaling provides ventralizing patterning cues to arch NC cells, in part through regulation of Bmp and Edn1 signaling, but independently regulates hand2. Similarly, heat-shocked dkk1+ embryos exhibit ventral arch reductions, but also have mandibular clefts at the ventral midline not seen in dntcf3+ embryos. Dkk1 is expressed in pharyngeal endoderm, and cell transplantation experiments reveal that dntcf3 must be overexpressed in pharyngeal endoderm to disrupt D-V arch patterning, suggesting that distinct endodermal roles for Wnts and Wnt antagonists pattern the developing skeleton.     

Positioning the expanded akirin gene family of Atlantic salmon within the transcriptional networks of myogenesis

Vertebrate akirin genes usually form a family with one-to-three members that regulate gene expression during the innate immune response, carcinogenesis and myogenesis. We recently established that an expanded family of eight akirin genes is conserved across salmonid fish. Here, we measured mRNA levels of the akirin family of Atlantic salmon (Salmo salar L.) during the differentiation of primary myoblasts cultured from fast-skeletal muscle. Using hierarchical clustering and correlation, the data was positioned into a network of expression profiles including twenty further genes that regulate myogenesis. akirin1(2b) was not significantly regulated during the maturation of the cell culture. akirin2(1a) and 2(1b), along with IGF-II and several igfbps, were most highly expressed in mononuclear cells, then significantly and constitutively downregulated as differentiation proceeded and myotubes formed/matured. Conversely, akirin1(1a), 1(1b), 1(2a), 2(2a) and 2(2b) were expressed at lowest levels when mononuclear cells dominated the culture and highest levels when confluent layers of myotubes were evident. However, akirin1(2a) and 2(2a) were first upregulated earlier than akirin1(1a), 1(1b) and 2(2b), when rates of myoblast proliferation were highest. Interestingly, akirin1(1b), 1(2a), 2(2a) and 2(2b) formed part of a module of co-expressed genes involved in muscle differentiation, including myod1a, myog, mef2a, 14-3-3β and 14-3-3γ. All akirin paralogues were expressed ubiquitously across ten tissues, although mRNA levels were regulated between cell-types and family members. Gene expression patterns were often highly correlated between akirin paralogues, suggesting that natural selection has maintained an intricate network of co-regulation among family members. We concluded that the Atlantic salmon akirin family performs a multifaceted role during myogenesis and has physiological functions spanning many cell-types.     

Expression of cohesin and condensin genes during zebrafish development supports a non-proliferative role for cohesin

Cohesin and condensin are similar, but distinct multi-subunit protein complexes that have well-described roles in sister chromatid cohesion and chromosome condensation, respectively. Recently it has emerged that cohesin, and proteins that regulate cohesin function have additional developmental roles. To further understand the role of cohesin in development, we analyzed the expression of genes encoding cohesin and condensin subunits in developing zebrafish embryos and juvenile brain. We found that cohesin subunits are expressed in a pattern that is similar (but not quite identical) to the expression of condensin subunits. Cohesin genes smc1a, rad21, pds5b and smc3 were expressed in the forebrain ventricular zone, the tectum, the mid-hindbrain boundary, the fourth ventricle, branchial arches, the otic vesicle, the eye and faintly in the developing pectoral fins. Condensin genes smc2 and smc4 were expressed in the forebrain ventricular zone, the tectum, the mid-hindbrain boundary, the fourth ventricle, branchial arches, eye and pectoral fins. Condensin genes were additionally expressed in the hindbrain proliferative zone, an area in which cohesin genes were not detected. A comparison with pcna expression and BrdU incorporation revealed that the expression of cohesins and condensins closely overlap with zones of proliferation. Interestingly, cohesin genes were expressed in non-proliferating cells flanking rhombomere boundaries in the developing brain. In mature brain and eye, cohesin was expressed in both proliferating cells and in broad zones of post-mitotic cells. The distribution of cohesin and condensin mRNAs supports existing evidence for a non-cell cycle role for cohesin in the developing brain.     

Zebrafish Dkk3a Protein Regulates the Activity of myf5 Promoter through Interaction with Membrane Receptor Integrin α6b

Myogenic regulatory factor Myf5 plays important roles in muscle development. In zebrafish myf5, a microRNA (miR), termed miR-3906 or miR-In300, was reported to silence dickkopf-3-related gene (dkk3r or dkk3a), resulting in repression of myf5 promoter activity. However, the membrane receptor that interacts with ligand Dkk3a to control myf5 expression through signal transduction remains unknown. To address this question, we applied immunoprecipitation and LC-MS/MS to screen putative membrane receptors of Dkk3a, and Integrin α6b (Itgα6b) was finally identified. To further confirm this, we used cell surface binding assays, which showed that Dkk3a and Itgα6b were co-expressed at the cell membrane of HEK-293T cells. Cross-linking immunoprecipitation data also showed high affinity of Itgα6b for Dkk3a. We further proved that the β-propeller repeat domains of Itgα6b are key segments bound by Dkk3a. Moreover, when dkk3a and itgα6b mRNAs were co-injected into embryos, luciferase activity was up-regulated 4-fold greater than that of control embryos. In contrast, the luciferase activities of dkk3a knockdown embryos co-injected with itgα6b mRNA and itgα6b knockdown embryos co-injected with dkk3a mRNA were decreased in a manner similar to that in control embryos, respectively. Knockdown of itgα6b resulted in abnormal somite shape, fewer somitic cells, weaker or absent myf5 expression, and reduced the protein level of phosphorylated p38a in somites. These defective phenotypes of trunk muscular development were similar to those of dkk3a knockdown embryos. We demonstrated that the secreted ligand Dkk3a binds to the membrane receptor Itgα6b, which increases the protein level of phosphorylated p38a and activates myf5 promoter activity of zebrafish embryos during myogenesis.     

sept8a and sept8b mRNA expression in the developing and adult zebrafish

Septins are highly conserved GTP-binding proteins involved in numerous cellular processes. Despite a growing awareness of their roles in the cell biology, development and signal transmission in nervous systems, comparably little is known about precise septin expression. Here, we use the well-established model organism zebrafish (Danio rerio) to unravel the expression of sept8a and sept8b, with special focus on the CNS. We performed whole mount RNA in situ hybridization on zebrafish 1–4 dpf in combination with serial sectioning of epon-embedded samples as well as on brain sections of adult zebrafish to obtain precise histological mapping of gene expression. Our results show a common expression of both genes at embryonic stages, whereas sept8a is mainly restricted to the gill arches and sept8b to specific brain structures at later stages. Brains of adult zebrafish reveal a large spatial overlap of sept8a and sept8b expression with few regions uniquely expressing sept8a or sept8b. Our results indicate a neuronal expression of both genes, and additionally suggest expression of sept8b in glial cells. Altogether, this study provides a first detailed insight into the expression of sept8a and sept8b in zebrafish and contributes to a more comprehensive understanding of septin biology in vertebrate model systems.     

Developmental expression of the amphioxus <Emphasis Type="Italic">Tbx1</Emphasis>/<Emphasis Type="Italic">10</Emphasis> gene illuminates the evolution of vertebrate branchial arches and sclerotome

We have isolated an amphioxus T-box gene that is orthologous to the two vertebrate genes, Tbx1 and Tbx10, and examined its expression pattern during embryonic and early larval development. AmphiTbx1/10 is first expressed in branchial arch endoderm and mesoderm of developing neurulae, and in a bilateral, segmented pattern in the ventral half of newly formed somites. Branchial expression is restricted to the first three branchial arches, and disappears completely by 4 days post fertilization. Ventral somitic expression is restricted to the first 10–12 somites, and is not observed in early larvae except in the most ventral mesoderm of the first three branchial arches. No expression can be detected by 4 days post fertilization. Integrating functional, phylogenetic and expression data from amphioxus and a variety of vertebrate model organisms, we have reconstructed the early evolutionary history of the Tbx1/10 subfamily of genes within the chordate lineage. We conclude that Tbx1/10-mediated branchial arch endoderm and mesoderm patterning functions predated the origin of neural crest, and that ventral somite specification functions predated the origin of vertebrate sclerotome, but that Tbx1 was later co-opted during the evolution of developmental programs regulating branchial neural crest and sclerotome migration.Edited by M. Akam     

Mutual antagonism between dickkopf1 and dickkopf2 regulates Wnt/β-catenin signalling

Wnts are secreted glycoproteins implicated in diverse processes during embryonic patterning in metazoans. They signal through seven-transmembrane receptors of the Frizzled (Fz) family [1] to stabilise β-catenin [2]. Wnts are antagonised by several extracellular inhibitors including the product of the dickkopf1 (dkk1) gene, which was identified in Xenopus embryos and is a member of a multigene family. The dkk1 gene acts upstream of the Wnt pathway component dishevelled but its mechanism of action is unknown [3]. Although the function of Dkk1 as a Wnt inhibitor in vertebrates is well established [3], [4], [5] and [6], the effect of other Dkks on the Wnt/β-catenin pathway is unclear. Here, we report that a related family member, Dkk2, activates rather than inhibits the Wnt/β-catenin signalling pathway in Xenopus embryos. Dkk2 strongly synergised with Wnt receptors of the Fz family to induce Wnt signalling responses. The study identifies Dkk2 as a secreted molecule that is able to activate Wnt/β-catenin signalling. The results suggest that a coordinated interplay between inhibiting dkk1 and activating dkk2 can modulate Fz signalling.     

Identification of dkk4 as a target of Eda-A1/Edar pathway reveals an unexpected role of ectodysplasin as inhibitor of Wnt signalling in ectodermal placodes

The development of epithelial appendages, including hairs, glands and teeth starts from ectodermal placodes, and is regulated by interplay of stimulatory and inhibitory signals. Ectodysplasin-A1 (Eda-A1) and Wnts are high in hierarchy of placode activators. To identify direct targets of ectodysplasin pathway, we performed microarray profiling of genes differentially regulated by short exposure to recombinant Eda-A1 in embryonic eda^−/− skin explants. Surprisingly, there were only two genes with obvious involvement in Wnt pathway: dkk4 (most highly induced gene in the screen), and lrp4. Both genes colocalized with Eda-A1 receptor Edar in placodes of ectodermal organs. They were upregulated upon Edar activation while several other Wnt associated genes previously suggested as Edar targets were unaffected. However, low dkk4 and lrp4 expression was retained in the absence of NF-κB signalling in eda^−/− hair placodes. We provide evidence that this expression was dependent on Wnt activity present prior to Eda-A1/Edar signalling. Dkk4 was recently suggested as a key Wnt antagonist regulating lateral inhibition essential for correct patterning of hair follicles. Several pieces of evidence suggest Lrp4 as a Wnt inhibitor, as well. The finding that Eda-A1 induces placode inhibitors was unexpected, and underlines the importance of delicate fine-tuning of signalling during placode formation.     

Regulation of Dlx gene expression in the zebrafish pharyngeal arches: from conserved enhancer sequences to conserved activity

The Dlx genes play an important role in the development of the pharyngeal arches and the structures derived from these tissues, including the craniofacial skeleton. They are typically expressed in a nested pattern along the proximo‐distal axis of the branchial arches in mice. This pattern is known as the “Dlx code” and has been proposed to be responsible for an early regional patterning of branchial arches in mammals. A number of cis‐ regulatory elements (CREs) have been identified within the Dlx loci, which target reporter expression to the developing pharyngeal arches of the mouse. Most of these CREs are located in the intergenic regions between the two genes constituting a Dlx bigene cluster. Therefore, the regionalized dlx expression in the branchial arches could be the result of the shared activities of these regulatory regions. Here we analyze previously published and new results showing these CREs are highly conserved in both their sequence and their activity in the pharyngeal arches of two distantly related vertebrates, the zebrafish and the mouse. A better understanding of Dlx gene regulation of the Dlx genes and of the genetic cascades in which they are involved can lead to clues explaining variations in morphology of the craniofacial regions of vertebrates.     

Expression of zebrafish hip: Response to Hedgehog signalling,comparison with ptc1 expression,and possible role in otic patterning

In zebrafish, Hedgehog (Hh) signalling is required to specify posterior otic identity. This presents a conundrum, as the nearest source of Hh to the developing inner ear is the ventral midline, in the notochord and floorplate. How can a source of Hh that is ostensibly constant with respect to the anteroposterior axis of the otic vesicle specify posterior otic identity? One possibility is that localised inhibition of Hh signalling is involved. Here we show that genes coding for three inhibitors of Hh signalling, su(fu), dzip1 and hip, are expressed in and around the developing otic vesicle. su(fu) and dzip1 are ubiquitously expressed and unaffected by Hh levels. The expression of hip, however, is positively regulated by Hh signalling and has a complex, dynamic pattern. It is detectable in the neural tube, otic vesicle, statoacoustic ganglion, brain, fin buds, mouth, somites, pronephros and branchial arches. These expression domains bear some similarity, but are not identical, to those of ptc1, a Hh receptor gene that is also positively regulated by Hh signalling. In the neural tube, for instance, hip is expressed in a subset of the ptc1 expression domain, while in other regions, including the otic vesicle, hip and ptc1 expression domains differ. Significantly, we find that initial expression of hip is higher in and adjacent to anterior otic regions, while ptc1 expression becomes progressively restricted to the posterior of the ear. Hip-mediated inhibition of Hh signalling may therefore be important in restricting the effects of Hh to posterior regions of the developing inner ear.     

Stability of the analytical solution of Penna model of biological aging

There are some analytical solutions of the Penna model of biological aging; here, we discuss the approach by Coe et al. (Phys. Rev. Lett. 89, 288103, 2002), based on the concept of self-consistent solution of a master equation representing the Penna model. The equation describes transition of the population distribution at time t to next time step (t + 1). For the steady state, the population n(a, l, t) at age a and for given genome length l becomes time-independent. In this paper we discuss the stability of the analytical solution at various ranges of the model parameters—the birth rate b or mutation rate m. The map for the transition from n(a, l, t) to the next time step population distribution n(a + 1, l, t + 1) is constructed. Then the fix point (the steady state solution) brings recovery of Coe et al. results. From the analysis of the stability matrix, the Lyapunov coefficients, indicative of the stability of the solutions, are extracted. The results lead to phase diagram of the stable solutions in the space of model parameters (b, m, h), where h is the hunt rate. With increasing birth rate b, we observe critical b ₀ below which population is extinct, followed by non-zero stable single solution. Further increase in b leads to typical series of bifurcations with the cycle doubling until the chaos is reached at some b _c. Limiting cases such as those leading to the logistic model are also discussed.     

Sprouty genes are essential for the normal development of epibranchial ganglia in the mouse embryo

Fibroblast growth factor (FGF) signalling has important roles in the development of the embryonic pharyngeal (branchial) arches, but its effects on innervation of the arches and associated structures have not been studied extensively. We investigated the consequences of deleting two receptor tyrosine kinase (RTK) antagonists of the Sprouty (Spry) gene family on the early development of the branchial nerves. The morphology of the facial, glossopharyngeal and vagus nerves are abnormal in Spry1−/−;Spry2−/− embryos. We identify specific defects in the epibranchial placodes and neural crest, which contribute sensory neurons and glia to these nerves. A dissection of the tissue-specific roles of these genes in branchial nerve development shows that Sprouty gene deletion in the pharyngeal epithelia can affect both placode formation and neural crest fate. However, epithelial-specific gene deletion only results in defects in the facial nerve and not the glossopharyngeal and vagus nerves, suggesting that the facial nerve is most sensitive to perturbations in RTK signalling. Reducing the Fgf8 gene dosage only partially rescued defects in the glossopharyngeal nerve and was not sufficient to rescue facial nerve defects, suggesting that FGF8 is functionally redundant with other RTK ligands during facial nerve development.