Search

Wilms tumour is a paediatric malignancy with features of halted kidney development. Here, we demonstrate that the Iroquois homeobox genes IRX3 and IRX5 are essential for mammalian nephrogenesis and govern the differentiation of Wilms tumour. Knock-out Irx3−/Irx5− mice showed a strongly reduced embryonic nephron formation. In human foetal kidney and Wilms tumour, IRX5 expression was already activated in early proliferative blastema, whereas IRX3 protein levels peaked at tubular differentiation. Accordingly, an orthotopic xenograft mouse model of Wilms tumour showed that IRX3−/− cells formed bulky renal tumours dominated by immature mesenchyme and active canonical WNT/β-catenin-signalling. In contrast, IRX5−/− cells displayed activation of Hippo and non-canonical WNT-signalling and generated small tumours with abundant tubulogenesis. Our findings suggest that promotion of IRX3 signalling or inhibition of IRX5 signalling could be a route towards differentiation therapy for Wilms tumour, in which WNT5A is a candidate molecule for enforced tubular maturation. (Less)

AIMS To characterize the urinary incontinence observed in adult Gli2+/- ; Gli3Δ699/+ female mice and identify the defects underlying the condition. METHODS Gli2+/- and Gli3Δ699/+ mice were crossed to generate: wild-type, mutant Gli2 (Gli2+/- ), mutant Gli3 (Gli3Δ699/+ ), and double mutant (Gli2+/- ; Gli3Δ699/+ ) female mice, verified via Polymerase Chain Reactions. Bladder functional studies including cystometrogram (CMG), leak point pressure (LPP), and voiding testing were performed on adult female mice. Female bladders and urethras were also analyzed via ink injection and histological assays. RESULTS CMG tracing showed no signal corresponding to the filling of the Gli2+/- ; Gli3Δ699/+ bladders. LPP were significantly reduced in Gli2+/- ; Gli3Δ699/+ mice compared to wild-type mice. CMG studies revealed a decrease in peak micturition pressure values in Gli2+/- ; Gli3Δ699/+ mice compared with all other groups. No significant differences between mutant and wild-type mice were detected in urinary output. Histological analyses revealed Gli2+/- ; Gli3Δ699/+ mice exhibited a widened urethra and a decrease in smooth muscle layer thickness in the bladder outlet and urethra, with increased mucosal folding. CONCLUSIONS Gli2+/- ; Gli3Δ699/+ adult female mice display persistent urinary incontinence due to the malformation of the bladder outlet and urethra. This presents a consistent and reliable genetic mouse model for female urinary incontinence and alludes to the key role of genetic factors involved in the condition.

Background: The vertebrate digit pattern is defined by the morphogen Sonic hedgehog (Shh), which controls the activity of Gli transcription factors. Gli1, 2 and 3 are dynamically expressed during patterning. Downstream of Shh, their activity is regulated by Sufu and Kif7, core components of the Shh signaling cascade. The precise roles of these regulators during limb development have not been fully described. We analyze the role of Sufu and Kif7 in the limb and demonstrate that their loss has distinct and synergistic effects on Gli activity and digit pattern. Results: Using a series of mouse mutants, we show that Sufu and Kif7 are expressed throughout limb development and their deletion has distinct effects on Gli levels and limb formation. Concomitant deletion of Sufu and Kif7 results in constitutive pathway activity and severe limb truncation. This is consistent with the recently published two-population model, which suggests that precocious activation of Shh signaling inhibits organizing center formation and limb outgrowth. Conclusions: Together, our findings demonstrate that perturbations of Sufu and Kif7 affect Gli activity and recapitulate the full spectrum of vertebrate limb defects, ranging from severe truncation to polydactyly. Developmental Dynamics 244:468–478, 2015. © 2014 Wiley Periodicals, Inc.

Functional annotation of mutations that cause human limb anomalies is enabled by basic developmental studies. In this study, we focus on the prepatterning stage of limb development and discuss a recent model that proposes anterior and posterior domains of the early limb bud generate two halves of the future skeleton. By comparing phenotypes in humans with those in model organisms, we evaluate whether this prepatterning concept helps to annotate human disease alleles. WIREs Dev Biol 2017, 6:e270. doi: 10.1002/wdev.270 For further resources related to this article, please visit the WIREs website.

The Hedgehog (Hh)-signaling pathway is essential for numerous developmental processes in Drosophila and vertebrate embryos. Hh signal transduction encompasses a complex series of regulatory events, including the generation of the mature Hh ligand, propagation of the ligand from source of production as well as the reception and interpretation of the signal in Hh-receiving cells. Many congenital malformations in humans are known to involve mutations in various components of the Hh-signaling pathway. This mini review summarizes some recent findings about the regulation of Hh signal transduction and describes the spectrum of human congenital malformations that are associated with aberrant Hh signaling. Based on a comparison of mouse-mutant phenotypes and human syndromes, we discuss how Hh-dependent Gli activator and repressor functions contribute to some of the congenital malformations.

The genetic polydactyly/arhinencephaly mouse, Pdn/Pdn, exhibits severe polydactyly both in the fore-and hindlimbs, agenesis of the olfactory bulbs, corpus callosum, anterior commissure, and hydrocephalus. A candidate gene for the Pdn mouse has been speculated to be Gli3, because Pdn has been considered to be an allele of Xt whose responsible gene has been clarified to be Gli3. Recently, it has been cleared that retro-transposons are inserted into nitron 3, upstream of zinc finger domain, of the Gli3 gene in the Pdn mouse, resulting to the severe suppression of Gli3 gene expression in Pdn/Pdn embryos. Meanwhile, XtJ/XtJ mice exhibit more severe polydactyly than that of Pdn/Pdn. Arhinencephaly and microholoprosencephaly including agenesis of the olfactory bulbs, corpus callosum, anterior commissure, hippocampal commissure, habenular commissure, and posterior commissure, and moreover, the cerebral cortical plates and hippocampus are not formed in the XtJ/XtJ mice. The XtJ/XtJ mouse has a large deletion in Gli3 structural gene and shows null expression. From these corroborations, we speculated that the differences in the Gli3 gene expression levels resulted in the phenotypic differences between the Pdn/Pdn and XtJ/XtJ mice.

VACTERL represents a non-random association of congenital anomalies in humans of poorly known etiology and pathogenesis. From our mutant analysis of Gli genes, which encode transcription factors mediating Sonic hedgehog (Shh) signal transduction, we observed that defective Shh signaling leads to a spectrum of developmental anomalies in mice strikingly similar to those of VACTERL. In this review, we will discuss the function of the three Gli transcription factors in Shh signaling and mammalian development. We propose that VACTERL could be caused by defective Shh signaling during human embryogenesis and suggest that the Gli mutant mice can serve as useful models for studying the pathogenesis of VACTERL.

Notch signaling plays an important role in cell-fate specification in multicellular organisms by regulating cell-cell communication. The Drosophila deltex gene encodes a modulator of the Notch pathway that has been shown to interact physically with the Ankyrin repeats of Notch. We isolated four distinct cDNAs corresponding to mouse homologs of deltex - mouse Deltex1 (MDTX1), mouse Deltex2 (MDTX2), mouse Deltex2DeltaE (MDTX2DeltaE), and mouse Deltex3 (MDTX3). Deduced amino acid sequences of these four cDNAs showed a high degree of similarity to Drosophila Deltex and its human homolog, DTX1 throughout their lengths, even though they possess distinct structural features. MDTX proteins formed homotypic and heterotypic multimers. We found that these genes were expressed in the central, peripheral nervous system and in the thymus, overlapping with those of mouse Notch1. In mammalian tissue culture cells, overexpression of any of the four mouse deltex homologs suppressed the transcriptional activity of E47, a basic helix-loop-helix (bHLH) protein, in a manner similar to suppression by an activated form of human Notch1 or human DTX1. In addition, overexpression of MDTX2 and MDTX2DeltaE in C2C12 cells under differentiation-inducing conditions suppressed the expression of myogenin, one of the myogenic transcriptional factors; this was also similar to a previously reported activity of constitutively activated Notch. Furthermore, misexpression of any of the MDTX genes in Xenopus embryos resulted in an expansion of the region expressing the neural cell adhesion molecule (N-CAM) gene, a marker for the neuroepithelium. Collectively, our results suggest that these mouse deltex homologs are involved in vertebrate Notch signaling and regulation of neurogenesis.

Summary: The Nkx-6.1 homeodomain transcription factor was previously shown to be expressed in ventral neural progenitor cells and subsequently subsets of unidentified motor neurons during early neural development. In this study, we identify a specific subpopulation of motor neurons, the median half of the lateral motor neuron column (LMCm), that retain a strong expression of Nkx-6.1. In addition, we report novel patterns of Nkx-6.1 expression in several mesenchymal tissues surrounding Sonic hedgehog (Shh)-expressing cells, including ventral spinal meninges, esophageal mesenchyme, and dorsal tracheal mesenchyme. Whereas Shh signaling is required for Nkx-6.1 expression in the ventral neural tube and spinal meninges, an Shh-independent pathway appears to operate in regulating Nkx-6.1 expression in the foregut. The persistent and robust expression of Nkx-6.1 in motor neurons and mesenchymal cells suggests an important role for Nkx-6.1 in controlling cell fate specification and differentiation. genesis 27:6–11, 2000. © 2000 Wiley-Liss, Inc.

Several 10 bp AT-rich repeats containing a core sequence TTAATT or its complement, AATTAA, in the distal part of the Bombyx mori fibroin gene promoter are known to bind a group of silk gland factors and to be required for a maximal level of transcription in a posterior silk gland extract. These repeats are similar to a consensus sequence (TCAATTAAAT) of the binding sites deduced for a group of Drosophila homeodomain proteins. By DNasel footprinting assay, two of these proteins, ZEN (zerknullt) and EVE (even-skipped), were shown to bind these sites with high affinity. The “TTAATT” core sequence was found to be important for ZEN and EVE binding by an electrophoretic mobility shift assay. Though these homeodomain proteins apparently recognize the same AT-rich consensus sequence, we have identified a mutant sequence that enhances ZEN but not EVE binding. Alteration of sequence flanking the repeats was also found to affect the binding of these proteins. The binding of EVE and ZEN was shown to be facilitated by multiple repeats in the binding sites. Similar AT-rich repeats can be found in the promoter region of other silk protein genes and, as shown here, two regions in the sericin-1 gene promoter also bind these homeodomain proteins. The interaction of some homeodomain proteins and these homeodomain binding sites might be important for the developmental regulation of a group of silk protein genes.

Devices of the extraction procedues and a recovery of the final supernatant as tiers accompanied with a transcription ability assessment of the individual tiers have enabled us to develop a variety of cell-free transcription systems. The latter step revealed that the factors necessary for faithful transcription as well as enhanced transcription form aggregates or complexes indicating an intrinsic nature of these factors to interact each other. Altogether 14 cell-free transcription systems have been developed from Bombyx mori embryos and tissues of various developmental stages and a cultured cell line, and screened for activities enhancing the basal promoter levels of the silk genes. Using these extracts a differential transcription of plural tissue-specific genes was tried. The fibroin gene was preferentially transcribed than the sericin-1 gene in the extracts from the posterior silk glands where the fibroin gene is specifically transcribed in vivo, while the sericin-1 gene was dominant to the fibroin gene in the extracts from the middle silk glands where the sericin-1 gene is specifically transcribed. Thus, these nuclear extracts offer us a biochemical clue assaying factors responsible for the differential transcription.

Complementation of a posterior silk gland (psg) extract to a HeLa cell extract specifically enhances the transcription of the Bombyx mori fibroin gene. To map the regions responsible for this enhancement, the fibroin promoter was dissected and the transcriptional function of each region was analyzed. Besides the upstream promoter element 5' to the TATA box, two downstream elements were found to be important for the preferential transcription of the fibroin gene in the complementation system as well as in the psg extract. The minimal fibroin promoter from -37 to +10 (core-promoter) was preferentially transcribed in the psg extract, while the transcription efficiencies of other promoters like one of the Bombyx chorion genes and the adenovirus 2 major late promoter (Ad2MLP) were considerably lower. The transcription from the core-promoter was further enhanced when combined with either the intronic element from +156 to +454 or the upstream element. Both the upstream and intronic elements also stimulated the transcription from the Ad2MLP in an orientation independent manner. These results demonstrate that the transcription of the fibroin gene is mediated through an integration of multiple regulatory elements.