Residual microRNA expression dictates the extent of inner ear development in conditional Dicer knockout mice

Inner ear development requires coordinated transformation of a uniform sheet of cells into a labyrinth with multiple cell types. While numerous regulatory proteins have been shown to play critical roles in this process, the regulatory functions of microRNAs (miRNAs) have not been explored. To demonstrate the importance of miRNAs in inner ear development, we generated conditional Dicer knockout mice by the expression of Cre recombinase in the otic placode at E8.5. Otocyst-derived ganglia exhibit rapid neuron-specific miR-124 depletion by E11.5, degeneration by E12.5, and profound defects in subsequent sensory epithelial innervations by E17.5. However, the small and malformed inner ear at E17.5 exhibits residual and graded hair cell-specific miR-183 expression in the three remaining sensory epithelia (posterior crista, utricle, and cochlea) that closely corresponds to the degree of hair cell and sensory epithelium differentiation, and Fgf10 expression required for morphohistogenesis. The highest miR-183 expression is observed in near-normal hair cells of the posterior crista, whereas the reduced utricular macula demonstrates weak miR-183 expression and develops presumptive hair cells with numerous disorganized microvilli instead of ordered stereocilia. The correlation of differential and delayed depletion of mature miRNAs with the derailment of inner ear development demonstrates that miRNAs are crucial for inner ear neurosensory development and neurosensory-dependent morphogenesis.     

Sonic hedgehog signaling regulates Gli3 processing, mesenchymal proliferation, and differentiation during mouse lung organogenesis

Lack of Sonic hedgehog (Shh) signaling, mediated by the Gli proteins, leads to severe pulmonary hypoplasia. However, the precise role of Gli genes in lung development is not well established. We show Shh signaling prevents Gli3 proteolysis to generate its repressor forms (Gli3R) in the developing murine lung. In Shh(-/-) or cyclopamine-treated wild-type (WT) lung, we found that Gli3R level is elevated, and this upregulation appears to contribute to defects in proliferation and differentiation observed in the Shh(-/-) mesenchyme, where Gli3 is normally expressed. In agreement, we found Shh(-/-);Gli3(-/-) lungs exhibit enhanced growth potential. Vasculogenesis is also enhanced; in contrast, bronchial myogenesis remains absent in Shh(-/-);Gli3(-/-) compared with Shh(-/-) lungs. Genes upregulated in Shh(-/-);Gli3(-/-) relative to Shh(-/-) lung include Wnt2 and, surprisingly, Foxf1 whose expression has been reported to be Shh-dependent. Cyclins D1, D2, and D3 antibody labelings also reveal distinct expression patterns in the normal and mutant lungs. We found significant repression of Tbx2 and Tbx3, both linked to inhibition of cellular senescence, in Shh(-/-) and partial derepression in Shh(-/-); Gli3(-/-) lungs, while Tbx4 and Tbx5 expressions are less affected in the mutants. Our findings shed light on the role of Shh signaling on Gli3 processing in lung growth and differentiation by regulating several critical genes.     

Calpains expression during Xenopus laevis development

Calpains are cytoplasmic proteases activated by calcium, implicated in cell differentiation and apoptosis. The best characterized enzymes are calpains 1-3. The aim of this work was to localize calpains 1-3 during the development of Xenopus laevis in order to clarify the function of these three proteases. For the first time, we detected the localization of the three proteases at the protein level between one-cell stage and adult age. Their expression was weak at early stages, then increased at tadpole stage and decreased through metamorphosis and adult life. The calpain's expression was maximal during the period characterized by the appearance of organs and modelling process. These observations suggest that calpains play a crucial role during development.     

Differential activities of the core planar polarity proteins during Drosophila wing patterning

During planar polarity patterning of the Drosophila wing, a "core" group of planar polarity genes has been identified which acts downstream of global polarity cues to locally coordinate cell polarity and specify trichome production at distal cell edges. These genes encode protein products that assemble into asymmetric apicolateral complexes that straddle the proximodistal junctional region between adjacent cells. We have carried out detailed genetic analysis experiments, analysing the requirements of each complex component for planar polarity patterning. We find that the three transmembrane proteins at the core of the complex, Frizzled, Strabismus and Flamingo, are required earliest in development and are the only components needed for intercellular polarity signalling. Notably, cells that lack both Frizzled and Strabismus are unable to signal, revealing an absolute requirement for both proteins in cell-cell communication. In contrast the cytoplasmic components Dishevelled, Prickle and Diego are not needed for intercellular communication. These factors contribute to the cell-cell propagation of polarity, most likely by promotion of intracellular asymmetry. Interestingly, both local polarity propagation and trichome placement occur normally in mutant backgrounds where asymmetry of polarity protein distribution is undetectable, suggesting such asymmetry is not an absolute requirement for any of the functions of the core complex.     

The Caenorhabditis elegans pumilio homolog, puf-9, is required for the 3'UTR-mediated repression of the let-7 microRNA target gene, hbl-1

The Puf family of RNA-binding proteins directs cell fates by regulating gene expression at the level of translation and RNA stability. Here, we report that the Caenorhabditis elegans pumilio homolog, puf-9, controls the differentiation of epidermal stem cells at the larval-to-adult transition. Genetic analysis reveals that loss-of-function mutations in puf-9 enhance the lethality and heterochronic phenotypes caused by mutations in the let-7 microRNA (miRNA), while suppressing the heterochronic phenotypes of lin-41, a let-7 target and homolog of Drosophila Brat. puf-9 interacts with another known temporal regulator hbl-1, the Caenorhabditis elegans ortholog of hunchback. We present evidence demonstrating that puf-9 is required for the 3'UTR-mediated regulation of hbl-1, in both the hypodermis and the ventral nerve cord. Finally, we show that this regulation is dependent on a region of the hbl-1 3'UTR that contains putative Puf family binding sites as well as binding sites for the let-7 miRNA family, suggesting that puf-9 and let-7 may mediate hypodermal seam cell differentiation by regulating common targets.     

Differential Delta expression underlies the diversity of sensory organ patterns among the legs of the Drosophila adult

Many studies have shown that morphological diversity among homologous animal structures is generated by the homeotic (Hox) genes. However, the mechanisms through which Hox genes specify particular morphological features are not fully understood. We have addressed this issue by investigating how diverse sensory organ patterns are formed among the legs of the Drosophila melanogaster adult. The Drosophila adult has one pair of legs on each of its three thoracic segments (the T1-T3 segments). Although homologous, legs from different segments have distinct morphological features. Our focus is on the formation of diverse patterns of small mechanosensory bristles or microchaetae (mCs) among the legs. On T2 legs, the mCs are organized into a series of longitudinal rows (L-rows) precisely positioned along the leg circumference. The L-rows are observed on all three pairs of legs, but additional and novel pattern elements are found on T1 and T3 legs. For example, at specific positions on T1 and T3 legs, some mCs are organized into transverse rows (T-rows). Our studies indicate that the T-rows on T1 and T3 legs are established as a result of Hox gene modulation of the pathway for patterning the L-row mC bristles. Our findings suggest that the Hox genes, Sex combs reduced (Scr) and Ultrabithorax (Ubx), establish differential expression of the proneural gene achaete (ac) by modifying expression of the ac prepattern regulator, Delta (Dl), in T1 and T3 legs, respectively. This study identifies Dl as a potential link between Hox genes and the sensory organ patterning hierarchy, providing insight into the connection between Hox gene function and the formation of specific morphological features.     

Differential expression of two small Hsps during diapause in the corn stalk borer Sesamia nonagrioides (Lef.)

We isolated and characterized two members of the α-crystallin/sHsp family, SnoHsp19.5 and SnoHsp20.8 from Sesamia nonagrioides (Lepidoptera: Noctuidae). The cDNAs encoded proteins of 174 and 185 amino acids, with calculated molecular weights of 19.5 and 20.8 kDa, respectively. The deduced amino acid sequences of SnoHsp19.5 and SnoHsp20.8 showed highest homology to Hsp19.7 of Mamestra brassicae and to Bombyx mori Hsp20.4, respectively. Expression patterns of SnoHsp19.5 and SnoHsp20.8 in non-diapausing individuals under different environmental conditions (heat or cold) showed different accumulation profiles for the two genes after heat and cold treatment. SnoHsp19.5 was consistently expressed, while SnoHsp20.8 gene was down-regulated in deep diapause and was up-regulated at the termination of diapause. Our results suggest that these two genes play distinctive roles in the regulation of diapause.     

Altered expression of Autism-associated genes in the brain of Fragile X mouse model

Autism is a severe neurodevelopmental disorder, which typically emerges in early childhood. Most cases of autism have not been linked to mutations in a specific gene, and the etioloty of the disorder remains to be established [S.S. Moy, J.J. Nadler, T.R. Magnuson, J.N. Crawley, Mouse models of autism spectrum disorders: the challenge for behavioral genetics, Am. J. Med. Genet. 142 (2006) 40-51]. Fragile X syndrome is caused by mutation in the FMR1 gene and is characterized by mental retardation, physical abnormalities, and, in most case, autistic-like behavior [R.J. Hagerman, A.W. Jackson, A. Levitas, B. Rimland, M. Braden, An analysis of autism in fifty males with the Fragile X syndrome, Am. J. Med. Genet. 23 (1986) 359-374, C.E. Bakker, C. Verheij, R. Willemsen, R. van der Helm, F. Oerlemans, M. Vermeij, A. Bygrave, A.T. Hoogeveen, B.A. Oostra, E. Reyniers, K. De Boulle, R. D’Hooge, P. Cras, D. van Velzen, G. Nagels, J.J. Marti, P. De Deyn, J.K. Darby, P.J. Willems, Fmr1 knockout mice: a model to study Fragile X mental retardation, Cell 78 (1994) 23-33]. The FMR1 knockout (KO) mouse is one of the best characterized animal models for human disorders associated with autism [S.S. Moy, J.J. Nadler, T.R. Magnuson, J.N. Crawley, Mouse models of autism spectrum disorders: the challenge for behavioral genetics, Am. J. Med. Genet. 142 (2006) 40-51]. We have used real-time PCR to investigate changes in expression levels of three genes: WNT2, MECP2, and FMR1 in different brain regions of Fagile X mice and litter mate controls. We found major changes in the expression pattern for the three genes examined. FMR1, MECP2, and WNT2 expression were drastically down regulated in the Fragile X mouse brain.     

XsFRP5 modulates endodermal organogenesis in Xenopus laevis

Canonical Wnt signalling is known to be involved in the regulation of differentiation and proliferation in the context of endodermal organogenesis. Wnt mediated β-catenin activation is understood to be modulated by secreted Frizzled-related proteins, such as XsFRP5, which is dynamically expressed in the prospective liver/ventral pancreatic precursor cells during late neurula stages, becoming liver specific at tailbud stages and shifting to the posterior stomach/anterior duodenum territory during tadpole stages of Xenopus embryogenesis. These expression characteristics prompted us to analyse the function of XsFRP5 in the context of endodermal organogenesis. We demonstrate that XsFRP5 can form a complex with and inhibit a multitude of different Wnt ligands, including both canonical and non-canonical ones. Knockdown of XsFRP5 results in transient pancreatic hypoplasia as well as in an enlargement of the stomach. In VegT-injected animal cap explants, XsFRP5 can induce expression of exocrine but not endocrine pancreatic marker genes. Both, its expression characteristics as well as its interactions with XsFRP5, define Wnt2b as a putative target for XsFRP5 in vivo. Knockdown of Wnt2b results in a hypoplastic stomach as well as in hypoplasia of the pancreas. On the basis of these findings we propose that XsFRP5 exerts an early regulatory function in the specification of the ventral pancreas, as well as a late function in controlling stomach size via inhibition of Wnt signalling.     

En1 and Wnt7a interact with Dkk1 during limb development in the mouse

Wnt signaling plays an essential role in induction and development of the limb. Missing digits are one consequence of the reduced Wnt signaling in Wnt7a null mice, while extra digits result from excess Wnt signaling in mice null for the Wnt antagonist Dkk1. The extra digits and expanded apical ectodermal ridge (AER) of Dkk1-deficient mice closely resemble En1 null mice. To evaluate the in vivo interaction between En1 and the canonical Wnt signaling pathway, we generated double and triple mutants combining the hypomorphic doubleridge allele of Dkk1 with null alleles of En1 and Wnt7a. Reducing Dkk1 expression in Dkk1d/+Wnt7a-/- double mutants prevented digit loss, indicating that Wnt7a acts through the canonical pathway during limb development. Reducing Dkk1 levels in Dkk1d/dEn1-/- double mutants resulted in severe phenotypes not seen in either single mutant, including fused bones in the autopod, extensive defects of the zeugopod, and loss of the ischial bone. The subsequent elimination of Wnt7a in Dkk1d/dEn1-/-Wnt7a-/- triple mutants resulted in correction of most, but not all, of these defects. The failure of Wnt7a inactivation to completely correct the limb defects of Dkk1d/dEn1-/- double mutants indicates that Wnt7a is not the only gene regulated by En1 during development of the mouse limb.