Zebrafish miR-214 modulates Hedgehog signaling to specify muscle cell fate

Numerous microRNAs (miRNAs) have been discovered in the genomes of higher eukaryotes, and functional studies indicate that they are important during development. However, little is known concerning the function of individual miRNAs. We approached this problem in zebrafish by combining identification of miRNA expression, functional analyses and experimental validation of potential targets. We show that miR-214 is expressed during early segmentation stages in somites and that varying its expression alters the expression of genes regulated by Hedgehog signaling. Inhibition of miR-214 results in a reduction or loss of slow-muscle cell types. We show that su(fu) mRNA, encoding a negative regulator of Hedgehog signaling, is targeted by miR-214. Through regulation of su(fu), miR-214 enables precise specification of muscle cell types by sharpening cellular responses to Hedgehog.     

A synonymous variant in IRGM alters a binding site for miR-196 and causes deregulation of IRGM-dependent xenophagy in Crohn's disease

Susceptibility to Crohn's disease, a complex inflammatory disease, is influenced by common variants at many loci. The common exonic synonymous SNP (c.313C>T) in IRGM, found in strong linkage disequilibrium with a deletion polymorphism, has been classified as non-causative because of the absence of an alteration in the IRGM protein sequence or splice sites. Here we show that a family of microRNAs (miRNAs), miR-196, is overexpressed in the inflammatory intestinal epithelia of individuals with Crohn's disease and downregulates the IRGM protective variant (c.313C) but not the risk-associated allele (c.313T). Subsequent loss of tight regulation of IRGM expression compromises control of intracellular replication of Crohn's disease-associated adherent invasive Escherichia coli by autophagy. These results suggest that the association of IRGM with Crohn's disease arises from a miRNA-based alteration in IRGM regulation that affects the efficacy of autophagy, thereby implicating a synonymous polymorphism as a likely causal variant.     

miRNA regulation of Sdf1 chemokine signaling provides genetic robustness to germ cell migration

microRNAs (miRNAs) function as genetic rheostats to control gene output. Based on their role as modulators, it has been postulated that miRNAs canalize development and provide genetic robustness. Here, we uncover a previously unidentified regulatory layer of chemokine signaling by miRNAs that confers genetic robustness on primordial germ cell (PGC) migration. In zebrafish, PGCs are guided to the gonad by the ligand Sdf1a, which is regulated by the sequestration receptor Cxcr7b. We find that miR-430 regulates sdf1a and cxcr7 mRNAs. Using target protectors, we demonstrate that miR-430-mediated regulation of endogenous sdf1a (also known as cxcl12a) and cxcr7b (i) facilitates dynamic expression of sdf1a by clearing its mRNA from previous expression domains, (ii) modulates the levels of the decoy receptor Cxcr7b to avoid excessive depletion of Sdf1a and (iii) buffers against variation in gene dosage of chemokine signaling components to ensure accurate PGC migration. Our results indicate that losing miRNA-mediated regulation can expose otherwise buffered genetic lesions leading to developmental defects.     

Distal ureter morphogenesis depends on epithelial cell remodeling mediated by vitamin A and Ret

Almost 1% of human infants are born with urogenital abnormalities, many of which are linked to irregular connections between the distal ureters and the bladder. During development, ureters migrate by an unknown mechanism from their initial integration site in the Wolffian ducts up to the base of the bladder in a process that we call ureter maturation. Rara(-/-) Rarb2(-/-) mice display impaired vitamin A signaling and develop syndromic urogenital malformations similar to those that occur in humans, including renal hypoplasia, hydronephrosis and mega-ureter, abnormalities also seen in mice with mutations in the proto-oncogene Ret. Here we show that ureter maturation depends on formation of the 'trigonal wedge', a newly identified epithelial outgrowth from the base of the Wolffian ducts, and that the distal ureter abnormalities seen in Rara(-/-) Rarb2(-/-) and Ret(-/-) mutant mice are probably caused by a failure of this process. Our studies indicate that formation of the trigonal wedge may be essential for correct insertion of the distal ureters into the bladder, and that these events are mediated by the vitamin A and Ret signaling pathways.     

Protein tyrosine phosphatase 1B deficiency or inhibition delays ErbB2-induced mammary tumorigenesis and protects from lung metastasis

We investigated the role of protein tyrosine phosphatase 1B (PTP1B) in mammary tumorigenesis using both genetic and pharmacological approaches. It has been previously shown that transgenic mice with a deletion mutation in the region of Erbb2 encoding its extracellular domain (referred to as NDL2 mice, for 'Neu deletion in extracellular domain 2') develop mammary tumors that progress to lung metastasis. However, deletion of PTP1B activity in the NDL2 transgenic mice either by breeding with Ptpn1-deficient mice or by treatment with a specific PTP1B inhibitor results in significant mammary tumor latency and resistance to lung metastasis. In contrast, specific overexpression of PTP1B in the mammary gland leads to spontaneous breast cancer development. The regulation of ErbB2-induced mammary tumorigenesis by PTB1B occurs through the attenuation of both the MAP kinase (MAPK) and Akt pathways. This report provides a rationale for the development of PTP1B as a new therapeutic target in breast cancer.     

Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation

The composite structure of the mammalian skull, which forms predominantly via intramembranous ossification, requires precise pre- and post-natal growth regulation of individual calvarial elements. Disturbances of this process frequently cause severe clinical manifestations in humans. Enhanced DNA binding by a mutant MSX2 homeodomain results in a gain of function and produces craniosynostosis in humans. Here we show that Msx2-deficient mice have defects of skull ossification and persistent calvarial foramen. This phenotype results from defective proliferation of osteoprogenitors at the osteogenic front during calvarial morphogenesis, and closely resembles that associated with human MSX2 haploinsufficiency in parietal foramina (PFM). Msx2-/- mice also have defects in endochondral bone formation. In the axial and appendicular skeleton, post-natal deficits in Pth/Pthrp receptor (Pthr) signalling and in expression of marker genes for bone differentiation indicate that Msx2 is required for both chondrogenesis and osteogenesis. Consistent with phenotypes associated with PFM, Msx2-mutant mice also display defective tooth, hair follicle and mammary gland development, and seizures, the latter accompanied by abnormal development of the cerebellum. Most Msx2-mutant phenotypes, including calvarial defects, are enhanced by genetic combination with Msx1 loss of function, indicating that Msx gene dosage can modify expression of the PFM phenotype. Our results provide a developmental basis for PFM and demonstrate that Msx2 is essential at multiple sites during organogenesis.     

Target mimicry provides a new mechanism for regulation of microRNA activity

MicroRNAs (miRNA) regulate key aspects of development and physiology in animals and plants. These regulatory RNAs act as guides of effector complexes to recognize specific mRNA sequences based on sequence complementarity, resulting in translational repression or site-specific cleavage. In plants, most miRNA targets are cleaved and show almost perfect complementarity with the miRNAs around the cleavage site. Here, we examined the non-protein coding gene IPS1 (INDUCED BY PHOSPHATE STARVATION 1) from Arabidopsis thaliana. IPS1 contains a motif with sequence complementarity to the phosphate (Pi) starvation-induced miRNA miR-399, but the pairing is interrupted by a mismatched loop at the expected miRNA cleavage site. We show that IPS1 RNA is not cleaved but instead sequesters miR-399. Thus, IPS1 overexpression results in increased accumulation of the miR-399 target PHO2 mRNA and, concomitantly, in reduced shoot Pi content. Engineering of IPS1 to be cleavable abolishes its inhibitory activity on miR-399. We coin the term 'target mimicry' to define this mechanism of inhibition of miRNA activity. Target mimicry can be generalized beyond the control of Pi homeostasis, as demonstrated using artificial target mimics.     

Eya1-deficient mice lack ears and kidneys and show abnormal apoptosis of organ primordia.

Haploinsufficiency for human EYA1, a homologue of the Drosophila melanogaster gene eyes absent (eya), results in the dominantly inherited disorders branchio-oto-renal (BOR) syndrome and branchio-oto (BO) syndrome, which are characterized by craniofacial abnormalities and hearing loss with (BOR) or without (BO) kidney defects. To understand the developmental pathogenesis of organs affected in these syndromes, we inactivated the gene Eya1 in mice. Eya1 heterozygotes show renal abnormalities and a conductive hearing loss similar to BOR syndrome, whereas Eya1 homozygotes lack ears and kidneys due to defective inductive tissue interactions and apoptotic regression of the organ primordia. Inner ear development in Eya1 homozygotes arrests at the otic vesicle stage and all components of the inner ear and specific cranial sensory ganglia fail to form. In the kidney, Eya1 homozygosity results in an absence of ureteric bud outgrowth and a subsequent failure of metanephric induction. Gdnf expression, which is required to direct ureteric bud outgrowth via activation of the c-ret Rtk (refs 5, 6, 7, 8), is not detected in Eya1-/- metanephric mesenchyme. In Eya1-/- ear and kidney development, Six but not Pax expression is Eya1 dependent, similar to a genetic pathway elucidated in the Drosophila eye imaginal disc. Our results indicate that Eya1 controls critical early inductive signalling events involved in ear and kidney formation and integrate Eya1 into the genetic regulatory cascade controlling kidney formation upstream of Gdnf. In addition, our results suggest that an evolutionarily conserved Pax-Eya-Six regulatory hierarchy is used in mammalian ear and kidney development.     

Fgf8 signalling from the AER is essential for normal limb development

Vertebrate limb development depends on signals from the apical ectodermal ridge (AER), which rims the distal tip of the limb bud. Removal of the AER in chick results in limbs lacking distal skeletal elements. Fibroblast growth factor (FGF) proteins can substitute for the AER (refs 4-7), suggesting that FGF signalling mediates AER activity. Of the four mouse Fgf genes (Fgf4 , Fgf8, Fgf9, Fgf17) known to display AER-specific expression domains within the limb bud (AER-Fgfs), only Fgf8 is expressed throughout the AER. Moreover, Fgf8 expression precedes that of other AER-Fgfs (refs 8-13), suggesting that Fgf8 may perform unique functions early in limb development. In mice, loss of function of Fgf4 (refs 13,14), Fgf9 (D. Ornitz, pers. comm.) or Fgf17 (ref. 15) has no effect on limb formation. We report here that inactivating Fgf8 in early limb ectoderm causes a substantial reduction in limb-bud size, a delay in Shh expression, misregulation of Fgf4 expression, and hypoplasia or aplasia of specific skeletal elements. Our data identify Fgf8 as the only known AER-Fgf individually necessary for normal limb development, and provide insight into the function of Fgf signalling from the AER in the normal outgrowth and patterning of the limb.     

Indian hedgehog is a major mediator of progesterone signaling in the mouse uterus

The hedgehog family of morphogens are regulators of cell proliferation, differentiation and cell-cell communication. These morphogens have been shown to have important roles in organogenesis, spermatogenesis, stem cell maintenance and oncogenesis. Indian hedgehog (encoded by Ihh) has been shown to be expressed in the uterine epithelium under the control of the steroid hormone, progesterone. Although in vivo and in vitro studies have shown that progesterone achieves its effects on uterine function through epithelial-stromal cross-talk, molecular mediator(s) for this cellular communication pathway have not been elucidated. Using new experimental approaches that ablate Ihh specifically in Pgr-positive uterine cells of the mouse, we demonstrate that Ihh is an essential mediator of Pgr action in the uterus, and expression of this factor is critical in mediating the communication between the uterine epithelium and stroma required for embryo implantation.     

Requirement of Bmpr1a for Müllerian duct regression during male sexual development

Elimination of the developing female reproductive tract in male fetuses is an essential step in mammalian sexual differentiation. In males, the fetal testis produces the transforming growth factor beta (TGF-beta) family member anti-Müllerian hormone (Amh, also known as Müllerian-inhibiting substance (Mis)), which causes regression of the Müllerian ducts, the primordia of the oviducts, uterus and upper vagina. Amh induces regression by binding to a specific type II receptor (Amhr2) expressed in the mesenchyme surrounding the ductal epithelium. Mutations in AMH or AMHR2 in humans and mice disrupt signaling, producing male pseudohermaphrodites that possess oviducts and uteri. The type I receptor and Smad proteins that are required in vivo for Müllerian duct regression have not yet been identified. Here we show that targeted disruption of the widely expressed type I bone morphogenetic protein (BMP) receptor Bmpr1a (also known as Alk3) in the mesenchymal cells of the Müllerian ducts leads to retention of oviducts and uteri in males. These results identify Bmpr1a as a type I receptor for Amh-induced regression of Müllerian ducts. Because Bmpr1a is evolutionarily conserved, these findings indicate that a component of the BMP signaling pathway has been co-opted during evolution for male sexual development in amniotes.     

Syndecan-1 is required for Wnt-1-induced mammary tumorigenesis in mice

Syndecan-1 is a cell-surface, heparan-sulphate proteoglycan (HSPG) predominantly expressed by epithelial cells. It binds specifically to many proteins, including oncoproteins. For example, it induces the assembly of a signalling complex between FGF ligands and their cognate receptors. But so far there has been no direct evidence that this proteoglycan contributes to tumorigenesis. Here we have examined the role of syndecan-1 (encoded by Sdc1) during mammary tumour formation in response to the ectopic expression of the proto-oncogene Wnt1. We crossed syndecan-1-deficient mice with transgenic mice that express Wnt1 in mammary gland (TgN(Wnt-1)1Hev; ref. 2). Ectopic Wnt-1 expression induces generalized mammary hyperplasia, followed by the development of solitary tumours (median time 22 weeks). We show that in Sdc1-/- mice, Wnt-1-induced hyperplasia in virgin mammary gland was reduced by 70%, indicating that the Wnt-1 signalling pathway was inhibited. Of the 39 tumours that developed in a test cohort of mice, only 1 evolved in the Sdc1-/- background. In addition, we show that soluble syndecan-1 ectodomain purified from mouse mammary epithelial cells stimulates the activity of a Wnt-1 homologue in a tissue culture assay. Our results provide both genetic and biochemical evidence that syndecan-1 can modulate Wnt signalling, and is critical for Wnt-1-induced tumorigenesis of the mouse mammary gland.     

A novel member of the F-box/WD40 gene family, encoding dactylin, is disrupted in the mouse dactylaplasia mutant.

Early outgrowth of the vertebrate embryonic limb requires signalling by the apical ectodermal ridge (AER) to the progress zone (PZ), which in response proliferates and lays down the pattern of the presumptive limb in a proximal to distal progression. Signals from the PZ maintain the AER until the anlagen for the distal phalanges have been formed. The semidominant mouse mutant dactylaplasia (Dac) disrupts the maintenance of the AER, leading to truncation of distal structures of the developing footplate, or autopod. Adult Dac homozygotes thus lack hands and feet except for malformed single digits, whereas heterozygotes lack phalanges of the three middle digits. Dac resembles the human autosomal dominant split hand/foot malformation (SHFM) diseases. One of these, SHFM3, maps to chromosome 10q24 (Refs 6,7), which is syntenic to the Dac region on chromosome 19, and may disrupt the orthologue of Dac. We report here the positional cloning of Dac and show that it belongs to the F-box/WD40 gene family, which encodes adapters that target specific proteins for destruction by presenting them to the ubiquitination machinery. In conjuction with recent biochemical studies, this report demonstrates the importance of this gene family in vertebrate embryonic development.     

A cooperative microRNA-tumor suppressor gene network in acute T-cell lymphoblastic leukemia (T-ALL)

The importance of individual microRNAs (miRNAs) has been established in specific cancers. However, a comprehensive analysis of the contribution of miRNAs to the pathogenesis of any specific cancer is lacking. Here we show that in T-cell acute lymphoblastic leukemia (T-ALL), a small set of miRNAs is responsible for the cooperative suppression of several tumor suppressor genes. Cross-comparison of miRNA expression profiles in human T-ALL with the results of an unbiased miRNA library screen allowed us to identify five miRNAs (miR-19b, miR-20a, miR-26a, miR-92 and miR-223) that are capable of promoting T-ALL development in a mouse model and which account for the majority of miRNA expression in human T-ALL. Moreover, these miRNAs produce overlapping and cooperative effects on tumor suppressor genes implicated in the pathogenesis of T-ALL, including IKAROS (also known as IKZF1), PTEN, BIM, PHF6, NF1 and FBXW7. Thus, a comprehensive and unbiased analysis of miRNA action in T-ALL reveals a striking pattern of miRNA-tumor suppressor gene interactions in this cancer.     

Submucosal glands are the predominant site of CFTR expression in the human bronchus.

We have used in situ hybridization and immunocytochemistry to characterize the cellular distribution of cystic fibrosis (CF) gene expression in human bronchus. The cystic fibrosis transmembrane conductance regular (CFTR) was primarily localized to cells of submucosal glands in bronchial tissues from non-CF individuals notably in the serous component of the secretory tubules as well as a subpopulation of cells in ducts. Normal distribution of CFTR mRNA was found in CF tissues while expression of CFTR protein was genotype specific, with delta F508 homozygotes demonstrating no detectable protein and compound heterozygotes expressing decreased levels of normally distributed protein. Our data suggest mechanisms whereby defects in CFTR expression could lead to abnormal production of mucus in human lung.