Retinoic acid signaling in perioptic mesenchyme represses Wnt signaling via induction of Pitx2 and Dkk2

Morphogenesis during eye development requires retinoic acid (RA) receptors plus RA-synthesizing enzymes, and loss of RA signaling leads to ocular disorders associated with loss of Pitx2 expression in perioptic mesenchyme. Several Wnt signaling components are expressed in ocular tissues during eye development including Dkk2, encoding an inhibitor of Wnt/β-catenin signaling, which was previously shown to be induced by Pitx2 in the perioptic mesenchyme. Here, we investigated potential cross-talk between RA and Wnt signaling during ocular development. Genetic studies using Raldh1/Raldh3 double null mice deficient for ocular RA synthesis demonstrated that Pitx2 and Dkk2 were both down-regulated in perioptic mesenchyme. Chromatin immunoprecipitation and gel mobility shift studies demonstrated the existence of a DR5 RA response element upstream of Pitx2 that binds all three RA receptors in embryonic eye. Axin2, an endogenous readout of Wnt/β-catenin signaling, was up-regulated in cornea and perioptic mesenchyme of RA deficient embryos. Also, expression of Wnt5a was expanded in perioptic mesenchyme of RA deficient eyes. Our findings demonstrate excessive activation of Wnt signaling in the perioptic mesenchyme of RA deficient mice which may be responsible for abnormal development leading to defective optic cup, cornea, and eyelid morphogenesis.     

Wnt/beta-catenin signaling has an essential role in the initiation of limb regeneration

Anuran (frog) tadpoles and urodeles (newts and salamanders) are the only vertebrates capable of fully regenerating amputated limbs. During the early stages of regeneration these amphibians form a "blastema", a group of mesenchymal progenitor cells that specifically directs the regrowth of the limb. We report that wnt-3a is expressed in the apical epithelium of regenerating Xenopus laevis limb buds, at the appropriate time and place to play a role during blastema formation. To test whether Wnt/beta-catenin signaling is required for limb regeneration, we created transgenic X. laevis tadpoles that express Dickkopf-1 (Dkk1), a specific inhibitor of Wnt/beta-catenin signaling, under the control of a heat-shock promoter. Heat-shock immediately before limb amputation or during early blastema formation blocked limb regeneration but did not affect the development of contralateral, un-amputated limb buds. When the transgenic tadpoles were heat-shocked following the formation of a blastema, however, they retained the ability to regenerate partial hindlimb structures. Furthermore, heat-shock induced Dkk1 blocked fgf-8 but not fgf-10 expression in the blastema. We conclude that Wnt/beta-catenin signaling has an essential role during the early stages of limb regeneration, but is not absolutely required after blastema formation.     

Bmpr1a is required for proper migration of the AVE through regulation of Dkk1 expression in the pre-streak mouse embryo

Here, we report a novel mechanism regulating migration of the anterior visceral endoderm (AVE) by BMP signaling through BMPRIA. In Bmpr1a-deficient (Bmpr-null) embryos, the AVE does not migrate at all. In embryos with an epiblast-specific deletion of Bmpr1a (Bmpr1a^null/flox; Sox2Cre embryos), the AVE cells migrate randomly from the distal end of embryos, resulting in an expansion of the AVE. Dkk1, which is normally expressed in the anterior proximal visceral endoderm (PxVE), is downregulated in Bmpr-null embryos, whereas it is circumferentially expressed in Bmpr1a^null/flox; Sox2Cre embryos at E5.75-6.5. These results demonstrate an association of the position of Dkk1 expressing cells with direction of the migration of AVE. In Bmpr1a^null/flox; Sox2Cre embryos, a drastic decrease of WNT signaling is observed at E6.0. Addition of WNT3A to the culture of Bmpr1a^null/flox; Sox2Cre embryos at E5.5 restores expression patterns of Dkk1 and Cer1. These data indicate that BMP signaling in the epiblast induces Wnt3 and Wnt3a expression to maintain WNT signaling in the VE, resulting in downregulation of Dkk1 to establish the anterior expression domain. Thus, our results suggest that BMP signaling regulates the expression patterns of Dkk1 for anterior migration of the AVE.     

Chronological expression of Wnt target genes Ccnd1, Myc, Cdkn1a, Tfrc, Plf1 and Ramp3

The canonical Wnt pathway regulates several biological processes including development, cell growth and proliferation via consecutive gene regulation. A high number of target genes of the Wnt pathway has been identified, but the chronological order of target gene expression is still elusive. This order is supposed to be crucial for the controlled course of events downstream of the activated Wnt pathway. Here we present the expression chronologies of the target genes Ccnd1 (encoding for cyclin D1), Myc (c-Myc), Cdkn1a (p21^CIP1/WAF1), Tfrc (Transferrin receptor 1), Plf1 (Proliferin-1) and Ramp3 (Receptor activity-modifying protein 3) in C57MG cells after stimulation with Wnt-3a. We discriminated between immediate (below 1 h), early (between 1 and 6 h), intermediate (between 6 and 12 h) and late (after 12 h) targets. According to this classification Myc and Tfrc belong to the immediate target genes; Ccnd1, Plf1 and Ramp3 are early target genes and Cdkn1a is an intermediate target gene.     

The interplay between SUCLA2, SUCLG2, and mitochondrial DNA depletion

SUCLA2-related mitochondrial DNA (mtDNA) depletion syndrome is a result of mutations in the β subunit of the ADP-dependent isoform of the Krebs cycle succinyl-CoA synthase (SCS). The mechanism of tissue specificity and mtDNA depletion is elusive but complementation by the GDP-dependent isoform encoded by SUCLG2, and the association with mitochondrial nucleoside diphosphate kinase (NDPK), is a plausible link.We have investigated this relationship by studying SUCLA2 deficient fibroblasts derived from patients and detected normal mtDNA content and normal NDPK activity. However, knockdown of SUCLG2 by shRNA in both patient and control fibroblasts resulted in a significant decrease in mtDNA amount, decreased NDPK and cytochrome c oxidase activities, and a marked growth impairment. This suggests that, SUCLG2, to a higher degree than SUCLA2, is crucial for mtDNA maintenance and that mitochondrial NDPK is involved. Although results pertain to a cell culture system, the findings might explain the pathomechanism and tissue specificity in mtDNA depletion caused by defective SUCLA2.     

Cyclical expression of the Notch/Wnt regulator Nrarp requires modulation by Dll3 in somitogenesis

Delta-like 3 (Dll3) is a divergent ligand and modulator of the Notch signaling pathway only identified so far in mammals. Null mutations of Dll3 disrupt cycling expression of Notch targets Hes1, Hes5, and Lfng, but not of Hes7. Compared with Dll1 or Notch1, the effects of Dll3 mutations are less severe for gene expression in the presomitic mesoderm, yet severe segmentation phenotypes and vertebral defects result in both human and mouse. Reasoning that Dll3 specifically disrupts key regulators of somite cycling, we carried out functional analysis to identify targets accounting for the segmental phenotype. Using microdissected embryonic tissue from somitic and presomitic mesodermal tissue, we identified new genes enriched in these tissues, including Limch1, Rhpn2, and A130022J15Rik. Surprisingly, we only identified a small number of genes disrupted by the Dll3 mutation. These include Uncx, a somite gene required for rib and vertebral patterning, and Nrarp, a regulator of Notch/Wnt signaling in zebrafish and a cycling gene in mouse. To determine the effects of Dll3 mutation on Nrarp, we characterized the cycling expression of this gene from early (8.5 dpc) to late (10.5 dpc) somitogenesis. Nrarp displays a distinct pattern of cycling phases when compared to Lfng and Axin2 (a Wnt pathway gene) at 9.5 dpc but appears to be in phase with Lfng by 10.5 dpc. Nrarp cycling appears to require Dll3 but not Lfng modulation. In Dll3 null embryos, Nrarp displayed static patterns. However, in Lfng null embryos, Nrarp appeared static at 8.5 dpc but resumed cycling expression by 9.5 and dynamic expression at 10.5 dpc stages. By contrast, in Wnt3a null embryos, Nrarp expression was completely absent in the presomitic mesoderm. Towards identifying the role of Dll3 in regulating somitogenesis, Nrarp emerges as a potentially important regulator that requires Dll3 but not Lfng for normal function.     

br regulates the expression of the ecdysone biosynthesis gene npc1

The growth and metamorphosis of insects are regulated by ecdysteroid hormones produced in the ring gland. Ecdysone biosynthesis-related genes are both highly and specifically expressed in the ring gland. However, the intrinsic regulation of ecdysone biosynthesis has received little attention. Here we used the Drosophila npc1 gene to study the mechanism of ring gland-specific gene expression. npc1 is important for sterol trafficking in the ring gland during ecdysone biosynthesis. We have identified a conserved ring gland-specific cis-regulatory element (RSE) in the npc1 promoter using promoter fusion reporter analysis. Furthermore, genetic loss-of-function analysis and in vitro electrophoretic mobility shift assays revealed that the ecdysone early response gene broad complex (br) is a vital factor in the positive regulation of npc1 ring gland expression. Moreover, br also affects the ring gland expression of many other ecdysone biosynthetic genes as well as torso and InR, two key factors in the regulation of ecdysone biosynthesis. These results imply that ecdysone could potentially act through its early response gene br to achieve positive feedback regulation of ecdysone biosynthesis during development.     

The homeobox gene Hhex is essential for proper hepatoblast differentiation and bile duct morphogenesis

Hhex is required for early development of the liver. A null mutation of Hhex results in a failure to form the liver bud and embryonic lethality. Therefore, Hhex null mice are not informative as to whether this gene is required during later stages of hepatobiliary morphogenesis. To address this question, we derived Hhex conditional null mice using the Cre-loxP system and two different Cre transgenics (Foxa3-Cre and Alfp-Cre). Deletion of Hhex in the hepatic diverticulum (Foxa3-Cre;Hhex(d2,3/-)) led to embryonic lethality and resulted in a small and cystic liver with loss of Hnf4alpha and Hnf6 expression in early hepatoblasts. In addition, the gall bladder was absent and the extrahepatic bile duct could not be identified. Loss of Hhex in the embryonic liver (Alfp-Cre;Hhex(d2,3/-)) caused irregular development of intrahepatic bile ducts and an absence of Hnf1beta in many (cystic) biliary epithelial cells, which resulted in a slow, progressive form of polycystic liver disease in adult mice. Thus, we have shown that Hhex is required during multiple stages of hepatobiliary development. The altered expression of Hnf4alpha, Hnf6 and Hnf1beta in Hhex conditional null mice suggests that Hhex is an essential component of the genetic networks regulating hepatoblast differentiation and intrahepatic bile duct morphogenesis.     

hecate, a zebrafish maternal effect gene, affects dorsal organizer induction and intracellular calcium transient frequency

A zebrafish maternal effect mutation, in the gene hecate, results in embryos that have defects in the formation of dorsoanterior structures and altered calcium release. hecate mutant embryos lack nuclear accumulation of beta-catenin and have reduced expression of genes specific to the dorsal organizer. We found that hecate mutant embryos exhibit a nearly 10-fold increase in the frequency of intracellular Ca2+ transients normally present in the enveloping layer during the blastula stages. Inhibition of Ca2+ release leads to ectopic expression of dorsal genes in mutant embryos suggesting that Ca2+ transients are important in mediating dorsal gene expression. Inhibition of Ca2+ release also results in the expression of dorsal-specific genes in the enveloping layer in a beta-catenin-independent manner, which suggests an additional function for the Ca2+ transients in this cellular layer. The mutant phenotype can be reversed by the expression of factors that activate Wnt/beta-catenin signaling, suggesting that the Wnt/beta-catenin pathway, at least as activated by an exogenous Wnt ligand, is intact in hec mutant embryos. Our results are consistent with a role for the hecate gene in the regulation of Ca2+ release during the cleavage stages, which in turn influences dorsal gene expression in both marginal cells along the dorsoventral axis and in the enveloping layer.     

Genetic interactions between Pax9 and Msx1 regulate lip development and several stages of tooth morphogenesis

Developmental abnormalities of craniofacial structures and teeth often occur sporadically and the underlying genetic defects are not well understood, in part due to unknown gene-gene interactions. Pax9 and Msx1 are co-expressed during craniofacial development, and mice that are single homozygous mutant for either gene exhibit cleft palate and an early arrest of tooth formation. Whereas in vitro assays have demonstrated that protein-protein interactions between Pax9 and Msx1 can occur, it is unclear if Pax9 and Msx1 interact genetically in vivo during development. To address this question, we compounded the Pax9 and Msx1 mutations and observed that double homozygous mutants exhibit an incompletely penetrant cleft lip phenotype. Moreover, in double heterozygous mutants, the lower incisors were consistently missing and we find that transgenic BMP4 expression partly rescues this phenotype. Reduced expression of Shh and Bmp2 indicates that a smaller “incisor field” forms in Pax9^+/−;Msx1^+/− mutants, and dental epithelial growth is substantially reduced after the bud to cap stage transition. This defect is preceded by drastically reduced mesenchymal expression of Fgf3 and Fgf10, two genes that encode known stimulators of epithelial growth during odontogenesis. Consistent with this result, cell proliferation is reduced in both the dental epithelium and mesenchyme of double heterozygous mutants. Furthermore, the developing incisors lack mesenchymal Notch1 expression at the bud stage and exhibit abnormal ameloblast differentiation on both labial and lingual surfaces. Thus, Msx1 and Pax9 interact synergistically throughout lower incisor development and affect multiple signaling pathways that influence incisor size and symmetry. The data also suggest that a combined reduction of PAX9 and MSX1 gene dosage in humans may increase the risk for orofacial clefting and oligodontia.     

The nuclear receptor gene nhr-25 plays multiple roles in the Caenorhabditis elegans heterochronic gene network to control the larva-to-adult transition

Developmental timing in the nematode Caenorhabditis elegans is controlled by heterochronic genes, mutations in which cause changes in the relative timing of developmental events. One of the heterochronic genes, let-7, encodes a microRNA that is highly evolutionarily conserved, suggesting that similar genetic pathways control developmental timing across phyla. Here we report that the nuclear receptor nhr-25, which belongs to the evolutionarily conserved fushi tarazu-factor 1/nuclear receptor NR5A subfamily, interacts with heterochronic genes that regulate the larva-to-adult transition in C. elegans. We identified nhr-25 as a regulator of apl-1, a homolog of the Alzheimer's amyloid precursor protein-like gene that is downstream of let-7 family microRNAs. NHR-25 controls not only apl-1 expression but also regulates developmental progression in the larva-to-adult transition. NHR-25 negatively regulates the expression of the adult-specific collagen gene col-19 in lateral epidermal seam cells. In contrast, NHR-25 positively regulates the larva-to-adult transition for other timed events in seam cells, such as cell fusion, cell division and alae formation. The genetic relationships between nhr-25 and other heterochronic genes are strikingly varied among several adult developmental events. We propose that nhr-25 has multiple roles in both promoting and inhibiting the C. elegans heterochronic gene pathway controlling adult differentiation programs.     

High efficient gene targeting on the AGAMOUS gene in an ArabidopsisAtLIG4 mutant

Gene targeting induced by homologous integration of a foreign DNA segment into a chromosomal target sequence enables precise disruption or replacement of genes of interest and provides an effective means to analyze gene function, and also becomes an useful technique for breeding. But, integration of introduced DNA fragments is predominantly non-homologous in most species. However, we presented high-efficient homologous integration in disruptants of non-homologous end joining (NHEJ), that is, the Ku70-, Ku80- or Lig4-homologs deficient strain, in a model fungus Neurospora crassa. When the effect of NHEJ-defective plants for gene targeting was therefore examined in a model plant Arabidopsis (Arabidopsis thaliana), the efficiencies of gene targeting in the Atlig4/Atlig4 plant were 2/7 (28.6%) against calli obtained a selection-marker gene, 2/16 (12.5%) against selected calli, and about 2/540 (0.004%) against total cell particles at the starting point for transformation. The results of this paper show that the NHEJ-deficient system might cause a decrease in the efficiency of transformation but gives true targeted transformants with high efficiency in plant cell.