Genetic analysis of Hedgehog signaling in ventral body wall development and the onset of omphalocele formation.
ABSTRACT: An omphalocele is one of the major ventral body wall malformations and is characterized by abnormally herniated viscera from the body trunk. It has been frequently found to be associated with other structural malformations, such as genitourinary malformations and digit abnormalities. In spite of its clinical importance, the etiology of omphalocele formation is still controversial. Hedgehog (Hh) signaling is one of the essential growth factor signaling pathways involved in the formation of the limbs and urogenital system. However, the relationship between Hh signaling and ventral body wall formation remains unclear.To gain insight into the roles of Hh signaling in ventral body wall formation and its malformation, we analyzed phenotypes of mouse mutants of Sonic hedgehog (Shh), GLI-Kruppel family member 3 (Gli3) and Aristaless-like homeobox 4 (Alx4). Introduction of additional Alx4(Lst) mutations into the Gli3(Xt/Xt) background resulted in various degrees of severe omphalocele and pubic diastasis. In addition, loss of a single Shh allele restored the omphalocele and pubic symphysis of Gli3(Xt/+); Alx4(Lst/Lst) embryos. We also observed ectopic Hh activity in the ventral body wall region of Gli3(Xt/Xt) embryos. Moreover, tamoxifen-inducible gain-of-function experiments to induce ectopic Hh signaling revealed Hh signal dose-dependent formation of omphaloceles.We suggest that one of the possible causes of omphalocele and pubic diastasis is ectopically-induced Hh signaling. To our knowledge, this would be the first demonstration of the involvement of Hh signaling in ventral body wall malformation and the genetic rescue of omphalocele phenotypes.
Project description:Although several syndromes include abnormalities of both the ventral body wall and external genitalia, the developmental bases of this correlation are largely unknown. Naturally occurring mutations in Aristaless-like 4 (Alx4, Strong's luxoid: Alx4Lst) have ventral body wall and pelvic girdle abnormalities. We sought to determine whether the development of the genital tubercle (GT) and its derivatives, the external genitalia, is affected by this mutation. We thus performed genetic and tissue labeling analyses in mutant mice. Alx4Lst/Lst mutants displayed hypoplasia of the dorsal GT and reduced expression of Fibronectin. We analyzed cell migration during GT formation by tissue labeling experiments and discovered that the cells located in the proximal segment of the umbilical cord (infra-umbilical mesenchyme) migrate toward the dorsal part of the GT. The Alx4Lst/Lst mutants also displayed augmented expression of Hh signal-related genes. Hence, we analyzed a series of combinatorial mutants for Alx4, Sonic hedgehog (Shh) and GLI-Kruppel family member 3 (Gli3). These phenotype-genotype analyses suggested a genetic interaction between Alx4 and Hh signaling during GT formation. Moreover, Hh gain-of-function mutants phenocopied some of these phenotypes. These observations reveal novel information regarding the pathogenic mechanisms of syndromic lower ventral body malformations, which are largely unknown.
Project description:Gli3 is a zinc-finger transcription factor whose activity is dependent on the level of hedgehog (Hh) ligand. Hh signaling has key roles during endochondral ossification; however, its role in intramembranous ossification is still unclear. In this study, we show that Gli3 performs a dual role in regulating both osteoprogenitor proliferation and osteoblast differentiation during intramembranous ossification. We discovered that Gli3Xt-J/Xt-J mice, which represent a Gli3-null allele, exhibit craniosynostosis of the lambdoid sutures and that this is accompanied by increased osteoprogenitor proliferation and differentiation. These cellular changes are preceded by ectopic expression of the Hh receptor Patched1 and reduced expression of the transcription factor Twist1 in the sutural mesenchyme. Twist1 is known to delay osteogenesis by binding to and inhibiting the transcription factor Runx2. We found that Runx2 expression in the lambdoid suture was altered in a pattern complimentary to that of Twist1. We therefore propose that loss of Gli3 results in a Twist1-, Runx2-dependent expansion of the sutural osteoprogenitor population as well as enhanced osteoblastic differentiation which results in a bony bridge forming between the parietal and interparietal bones. We show that FGF2 will induce Twist1, normalize osteoprogenitor proliferation and differentiation and rescue the lambdoid suture synostosis in Gli3Xt-J/Xt-J mice. Taken together, we define a novel role for Gli3 in osteoblast development; we describe the first mouse model of lambdoid suture craniosynostosis and show how craniosynostosis can be rescued in this model.
Project description:Haploinsufficiency for the transcription factor SOX10 is associated with the pigmentary deficiencies of Waardenburg syndrome (WS) and is modeled in Sox10 haploinsufficient mice (Sox10(LacZ/+)). As genetic background affects WS severity in both humans and mice, we established an N-ethyl-N-nitrosourea (ENU) mutagenesis screen to identify modifiers that increase the phenotypic severity of Sox10(LacZ/+) mice. Analysis of 230 pedigrees identified three modifiers, named modifier of Sox10 neurocristopathies (Mos1, Mos2 and Mos3). Linkage analysis confirmed their locations on mouse chromosomes 13, 4 and 3, respectively, within regions distinct from previously identified WS loci. Positional candidate analysis of Mos1 identified a truncation mutation in a hedgehog(HH)-signaling mediator, GLI-Kruppel family member 3 (Gli3). Complementation tests using a second allele of Gli3 (Gli3(Xt-J)) confirmed that a null mutation of Gli3 causes the increased hypopigmentation in Sox10(LacZ/+);Gli3(Mos1/)(+) double heterozygotes. Early melanoblast markers (Mitf, Sox10, Dct, and Si) are reduced in Gli3(Mos1/)(Mos1) embryos, indicating that loss of GLI3 signaling disrupts melanoblast specification. In contrast, mice expressing only the GLI3 repressor have normal melanoblast specification, indicating that the full-length GLI3 activator is not required for specification of neural crest to the melanocyte lineage. This study demonstrates the feasibility of sensitized screens to identify disease modifier loci and implicates GLI3 and other HH signaling components as modifiers of human neurocristopathies.
Project description:Loss-of-function mutations in GLI3 and IHH cause craniosynostosis and reduced osteogenesis, respectively. In this study, we show that Ihh ligand, the receptor Ptch1 and Gli transcription factors are differentially expressed in embryonic mouse calvaria osteogenic condensations. We show that in both Ihh-/- and Gli3Xt-J/Xt-J embryonic mice, the normal gene expression architecture is lost and this results in disorganized calvarial bone development. RUNX2 is a master regulatory transcription factor controlling osteogenesis. In the absence of Gli3, RUNX2 isoform II and IHH are upregulated, and RUNX2 isoform I downregulated. This is consistent with the expanded and aberrant osteogenesis observed in Gli3Xt-J/Xt-J mice, and consistent with Runx2-I expression by relatively immature osteoprogenitors. Ihh-/- mice exhibited small calvarial bones and HH target genes, Ptch1 and Gli1, were absent. This indicates that IHH is the functional HH ligand, and that it is not compensated by another HH ligand. To decipher the roles and potential interaction of Gli3 and Ihh, we generated Ihh-/-;Gli3Xt-J/Xt-J compound mutant mice. Even in the absence of Ihh, Gli3 deletion was sufficient to induce aberrant precocious ossification across the developing suture, indicating that the craniosynostosis phenotype of Gli3Xt-J/Xt-J mice is not dependent on IHH ligand. Also, we found that Ihh was not required for Runx2 expression as the expression of RUNX2 target genes was unaffected by deletion of Ihh. To test whether RUNX2 has a role upstream of IHH, we performed RUNX2 siRNA knock down experiments in WT calvarial osteoblasts and explants and found that Ihh expression is suppressed. Our results show that IHH is the functional HH ligand in the embryonic mouse calvaria osteogenic condensations, where it regulates the progression of osteoblastic differentiation. As GLI3 represses the expression of Runx2-II and Ihh, and also elevates the Runx2-I expression, and as IHH may be regulated by RUNX2 these results raise the possibility of a regulatory feedback circuit to control calvarial osteogenesis and suture patency. Taken together, RUNX2-controlled osteoblastic cell fate is regulated by IHH through concomitant inhibition of GLI3-repressor formation and activation of downstream targets.
Project description:Greig cephalopolysyndactyly syndrome (GCPS) is an autosomal dominant disorder with polydactyly and syndactyly of the limbs and a broad spectrum of craniofacial abnormalities. Craniosynostosis of the metopic suture (interfrontal suture in mice) is an important but rare feature associated with GCPS. GCPS is caused by mutations in the transcription factor GLI3, which regulates Hedgehog signaling. The Gli3 loss-of-function (Gli3(Xt-J/Xt-J)) mouse largely phenocopies the human syndrome with the mice exhibiting polydactyly and several craniofacial abnormalities. Here we show that Gli3(Xt-J/Xt-J) mice exhibit ectopic ossification in the interfrontal suture and in the most severe cases the suture fuses already prior to birth. We show that abnormalities in frontal bones occur early in calvarial development, before the establishment of the interfrontal suture. It provides a model for the metopic suture pathology that can occur in GCPS.
Project description:Suppressor of Fused (SUFU) is an essential negative regulator of the Hedgehog (HH) pathway and involved in GLI transcription factor regulation. Due to early embryonic lethality of Sufu-/- mice, investigations of SUFU's role later in development are limited to conditional, tissue-specific knockout models. In this study we developed a mouse model (SufuEx456(fl)/Ex456(fl)) with hypomorphic features where embryos were viable up to E18.5, although with a spectrum of developmental defects of varying severity, including polydactyly, exencephaly and omphalocele. Development of certain tissues, like the skeleton, was more affected than that of others such as skin, which remained largely normal. Interestingly, no apparent changes in the dorso-ventral patterning of the neural tube at E9.0 could be seen. Thus, this model provides an opportunity to globally study SUFU's molecular function in organogenesis beyond E9.5. Molecularly, SufuEx456(fl)/Ex456(fl) embryos displayed aberrant mRNA splicing and drastically reduced levels of Sufu wild-type mRNA and SUFU protein in all tissues. As a consequence, at E9.5 the levels of all three different GLI proteins were reduced. Interestingly, despite the reduction of GLI3 protein levels, the critical ratio of the GLI3 full-length transcriptional activator versus GLI3 truncated repressor remained unchanged compared to wild-type embryos. This suggests that the limited amount of SUFU protein present is sufficient for GLI processing but not for stabilization. Our data demonstrate that tissue development is differentially affected in response to the reduced SUFU levels, providing novel insight regarding the requirements of different levels of SUFU for proper organogenesis.
Project description:The transcription factor GLI3 is a member of the Hedgehog (Hh/HH) signaling pathway that can exist as a full length (Gli3-FL/GLI3-FL) or repressor (Gli3-R/GLI3-R) form. In response to HH activation, GLI3-FL regulates HH genes by targeting the GLI1 promoter. In the absence of HH signaling, GLI3 is phosphorylated leading to its partial degradation and the generation of GLI3-R which represses HH functions. GLI3 is also involved in tissue development, immune cell development and cancer. The absence of Gli3 in mice impaired brain and lung development and GLI3 mutations in humans are the cause of Greig cephalopolysyndactyly (GCPS) and Pallister Hall syndromes (PHS). In the immune system GLI3 regulates B, T and NK-cells and may be involved in LPS-TLR4 signaling. In addition, GLI3 was found to be upregulated in multiple cancers and was found to positively regulate cancerous behavior such as anchorage-independent growth, angiogenesis, proliferation and migration with the exception in acute myeloid leukemia (AML) and medulloblastoma where GLI plays an anti-cancerous role. Finally, GLI3 is a target of microRNA. Here, we will review the biological significance of GLI3 and discuss gaps in our understanding of this molecule. Video Abstract.
Project description:Mammary gland development starts in utero with one or several pairs of mammary rudiments (MRs) budding from the surface ectodermal component of the mammalian embryonic skin. Mice develop five pairs, numbered MR1 to MR5 from pectoral to inguinal position. We have previously shown that Gli3(Xt-J/Xt-J) mutant embryos, which lack the transcription factor Gli3, do not form MR3 and MR5. We show here that two days after the MRs emerge, Gli3(Xt-J/Xt-J) MR1 is 20% smaller, and Gli3(Xt-J/Xt-J) MR2 and MR4 are 50% smaller than their wild type (wt) counterparts. Moreover, while wt MRs sink into the underlying dermis, Gli3(Xt-J/Xt-J) MR4 and MR2 protrude outwardly, to different extents. To understand why each of these five pairs of functionally identical organs has its own, distinct response to the absence of Gli3, we determined which cellular mechanisms regulate growth of the individual MRs, and whether and how Gli3 regulates these mechanisms. We found a 5.5 to 10.7-fold lower cell proliferation rate in wt MRs compared to their adjacent surface ectoderm, indicating that MRs do not emerge or grow via locally enhanced cell proliferation. Cell-tracing experiments showed that surface ectodermal cells are recruited toward the positions where MRs emerge, and contribute to MR growth during at least two days. During the second day of MR development, peripheral cells within the MRs undergo hypertrophy, which also contributes to MR growth. Limited apoptotic cell death counterbalances MR growth. The relative contribution of each of these processes varies among the five MRs. Furthermore, each of these processes is impaired in the absence of Gli3, but to different extents in each MR. This differential involvement of Gli3 explains the variation in phenotype among Gli3(Xt-J/Xt-J) MRs, and may help to understand the variation in numbers and positions of mammary glands among mammals.
Project description:Hedgehog (Hh) signaling in vertebrates depends on intraflagellar transport (IFT) within primary cilia. The Hh receptor Patched is found in cilia in the absence of Hh and is replaced by the signal transducer Smoothened within an hour of Hh stimulation. By generating antibodies capable of detecting endogenous pathway transcription factors Gli2 and Gli3, we monitored their kinetics of accumulation in cilia upon Hh stimulation. Localization occurs within minutes of Hh addition, making it the fastest reported readout of pathway activity, which permits more precise temporal and spatial localization of Hh signaling events. We show that the species of Gli3 that accumulates at cilium tips is full-length and likely not protein kinase A phosphorylated. We also confirmed that phosphorylation and betaTrCP/Cul1 are required for endogenous Gli3 processing and that this is inhibited by Hh. Surprisingly, however, Hh-dependent inhibition of processing does not lead to accumulation of full-length Gli3, but instead renders it labile, leading to its proteasomal degradation via the SPOP/Cul3 complex. In fact, full-length Gli3 disappears with faster kinetics than the Gli3 repressor, the latter not requiring SPOP/Cul3 or betaTrCP/Cul1. This may contribute to the increased Gli3 activator/repressor ratios found in IFT mutants.
Project description:Truncating GLI3 mutations in Pallister-Hall Syndrome with renal malformation suggests a requirement for Hedgehog signaling during renal development. HH-dependent signaling increases levels of GLI transcriptional activators and decreases processing of GLI3 to a shorter transcriptional repressor. Previously, we showed that Shh-deficiency interrupts early inductive events during renal development in a manner dependent on GLI3 repressor. Here we identify a novel function for GLI3 repressor in controlling nephron number. During renal morphogenesis, HH signaling activity, assayed by expression of Ptc1-lacZ, is localized to ureteric cells of the medulla, but is undetectable in the cortex. Targeted inactivation of Smo, the HH effector, in the ureteric cell lineage causes no detectable abnormality in renal morphogenesis. The functional significance of absent HH signaling activity in cortical ureteric cells was determined by targeted deletion of Ptc1, the SMO inhibitor, in the ureteric cell lineage. Ptc1(-/-UB) mice demonstrate ectopic Ptc1-lacZ expression in ureteric branch tips and renal hypoplasia characterized by reduced kidney size and a paucity of mature and intermediate nephrogenic structures. Ureteric tip cells are remarkable for abnormal morphology and impaired expression of Ret and Wnt11, markers of tip cell differentiation. A finding of renal hypoplasia in Gli3(-/-) mice suggests a pathogenic role for reduced GLI3 repressor in the Ptc1(-/-UB) mice. Indeed, constitutive expression of GLI3 repressor via the Gli3(Delta699) allele in Ptc1(-/-UB) mice restores the normal pattern of HH signaling, and expression of Ret and Wnt11 and rescued the renal phenotype. Thus, GLI3 repressor controls nephron number by regulating ureteric tip cell expression of Wnt11 and Ret.