Zone of Polarizing Activity Regulatory Sequence Mutations/Duplications with Preaxial Polydactyly and Longitudinal Preaxial Ray Deficiency in the Phenotype: A Review of Human Cases, Animal Models, and Insights Regarding the Pathogenesis

Clinicians and scientists interested in developmental biology have viewed preaxial polydactyly (PPD) and longitudinal preaxial ray deficiency (LPAD) as two different entities. Point mutations and duplications in the zone of polarizing activity regulatory sequence (ZRS) are associated with anterior ectopic expression of Sonic Hedgehog (SHH) in the limb bud and usually result in a PPD phenotype. However, some of these mutations/duplications also have LPAD in the phenotype. This unusual PPD-LPAD association in ZRS mutations/duplications has not been specifically reviewed in the literature. The author reviews this unusual entity and gives insights regarding its pathogenesis.

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The bHLH transcription factor dHAND controls Sonic hedgehog expression and establishment of the zone of polarizing activity during limb development

Development ◽

10.1242/dev.127.11.2461 ◽

2000 ◽

Vol 127 (11) ◽

pp. 2461-2470 ◽

Cited By ~ 9

Author(s):

J. Charite ◽

D.G. McFadden ◽

E.N. Olson

Keyword(s):

Transcription Factor ◽

Sonic Hedgehog ◽

Limb Development ◽

Target Genes ◽

Ectopic Expression ◽

Tissue Growth ◽

Limb Bud ◽

Bhlh Transcription Factor ◽

Expression Domain ◽

Zone Of Polarizing Activity

Limb outgrowth and patterning of skeletal elements are dependent on complex tissue interactions involving the zone of polarizing activity (ZPA) in the posterior region of the limb bud and the apical ectodermal ridge. The peptide morphogen Sonic hedgehog (SHH) is expressed specifically in the ZPA and, when expressed ectopically, is sufficient to mimic its functions, inducing tissue growth and formation of posterior skeletal elements. We show that the basic helix-loop-helix transcription factor dHAND is expressed posteriorly in the developing limb prior to Shh and subsequently occupies a broad domain that encompasses the Shh expression domain. In mouse embryos homozygous for a dHAND null allele, limb buds are severely underdeveloped and Shh is not expressed. Conversely, misexpression of dHAND in the anterior region of the limb bud of transgenic mice results in formation of an additional ZPA, revealed by ectopic expression of Shh and its target genes, and resulting limb abnormalities that include preaxial polydactyly with duplication of posterior skeletal elements. Analysis of mouse mutants in which Hedgehog expression is altered also revealed a feedback mechanism in which Hedgehog signaling is required to maintain the full dHAND expression domain in the developing limb. Together, these findings identify dHAND as an upstream activator of Shh expression and important transcriptional regulator of limb development.

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Variable expression of subclinical phenotypes instead of reduced penetrance in families with mild triphalangeal thumb phenotypes

Journal of Medical Genetics ◽

10.1136/jmedgenet-2019-106685 ◽

2020 ◽

Vol 57 (10) ◽

pp. 660-663

Author(s):

Jacob W P Potuijt ◽

Jeannette Hoogeboom ◽

Esther de Graaff ◽

Christianne A van Nieuwenhoven ◽

Robert Jan H Galjaard

Keyword(s):

Sonic Hedgehog ◽

Clinical Examination ◽

Regulatory Element ◽

Family Members ◽

Limb Bud ◽

Regulatory Sequence ◽

Variable Expression ◽

Zone Of Polarizing Activity ◽

Reduced Penetrance ◽

Triphalangeal Thumb

BackgroundThe of zone of polarizing activity regulatory sequence (ZRS) is a regulatory element residing in intron 5 of LMBR1 and regulates Sonic Hedgehog expression in the limb bud. Variants in the ZRS are generally fully penetrant and can cause triphalangeal thumb (TPT) and polydactyly in affected families.ObjectiveIn this report, we describe two families with mild phenotypical presentation.MethodsWe performed a field study for clinical evaluation and sequenced the ZRS for variantsusing Sanger sequencing.ResultsIn family I, a novel 165A>G variant in the ZRS (g.156584405A>G, GRCh37/Hg19) was found. In family II, we identified a 295T>C variant in the ZRS (g.156584535T>C, GRCh37/Hg19). Family members of both families who were presumed to be unaffected shared the variant in the ZRS with affected family members, suggesting reduced penetrance of the genotype. However, clinical examination of these unaffected family members revealed minor anomalies like broad thumbs and lack of thumb opposition. As the phenotype in affected patients is remarkably mild, we suggest that these ZRS variants are minimally disruptive for Sonic Hedgehog expression and therefore can result in subclinical phenotypes.ConclusionOur study underlines the importance of accurate clinical examination and appropriate genetic counselling in families with mild cases of TPT.

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Dual action of sonic hedgehog on chondrocyte hypertrophy:retrovirus mediated ectopic sonic hedgehog expression in limb bud micromass culture induces novel cartilage nodules that are positive for alkaline phosphatase and type X collagen

Journal of Cell Science ◽

10.1242/jcs.110.21.2691 ◽

1997 ◽

Vol 110 (21) ◽

pp. 2691-2701 ◽

Cited By ~ 4

Author(s):

N.S. Stott ◽

C.M. Chuong

Keyword(s):

Alkaline Phosphatase ◽

Gene Family ◽

Alcian Blue ◽

Sonic Hedgehog ◽

Ectopic Expression ◽

Limb Bud ◽

Blue Staining ◽

Alcian Blue Staining ◽

Type X Collagen ◽

Chondrocyte Maturation

Members of the vertebrate hedgehog gene family (HH) are involved in patterning and modulation of differentiation. Recently it has been shown that ectopic expression of HH gene family members in vivo blocks chondrocyte maturation through activation of a parathyroid hormone related peptide (PTHrP) dependent negative regulatory loop in the perichondrium. However, the direct effect of HH on chondrocyte maturation has not been tested. Here, we studied the effect of retroviral overexpression of the chicken sonic hedgehog gene (Shh) on the growth and maturation of limb bud cells in micromass cultures. Shh is neither expressed nor required for the initiation of cellular condensation in normal micromass cultures. With Shh over-expression, micromass cultures developed novel tightly whorled nodules in addition to the normal Alcian Blue positive cartilage nodules. We characterized the new nodules and showed that they are strongly positive for alkaline phosphatase, enriched in type X collagen and weakly positive for Alcian Blue staining. Shh overexpression also increased cell proliferation, but this cannot account for the formation of the new nodules. This current study shows that misexpression of Shh in in vitro chondrogenic cultures promotes characteristics of hypertrophic chondrocytes. Thus HH has two complementary functions; a direct positive effect on chondrocyte hypertrophy in the absence of PTHrP pathway, and an indirect negative feedback loop through PTHrP to prevent other less differentiated chondrocytes from becoming hypertrophic. These two complementary actions of HH coordinate the progression of cartilage maturation.

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Point mutations in a distant sonic hedgehog cis-regulator generate a variable regulatory output responsible for preaxial polydactyly

Human Molecular Genetics ◽

10.1093/hmg/ddm370 ◽

2007 ◽

Vol 17 (7) ◽

pp. 978-985 ◽

Cited By ~ 118

Author(s):

Laura A. Lettice ◽

Alison E. Hill ◽

Paul S. Devenney ◽

Robert E. Hill

Keyword(s):

Sonic Hedgehog ◽

Point Mutations ◽

Preaxial Polydactyly

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The mesenchymal factor, FGF10, initiates and maintains the outgrowth of the chick limb bud through interaction with FGF8, an apical ectodermal factor

Development ◽

10.1242/dev.124.11.2235 ◽

1997 ◽

Vol 124 (11) ◽

pp. 2235-2244 ◽

Cited By ~ 67

Author(s):

H. Ohuchi ◽

T. Nakagawa ◽

A. Yamamoto ◽

A. Araga ◽

T. Ohata ◽

...

Keyword(s):

Sonic Hedgehog ◽

Limb Bud ◽

Fibroblast Growth ◽

Lateral Plate Mesoderm ◽

Lateral Plate ◽

Bud Formation ◽

Limb Mesenchyme ◽

Vertebrate Limb ◽

Fgf10 Expression ◽

Zone Of Polarizing Activity

Vertebrate limb formation has been known to be initiated by a factor(s) secreted from the lateral plate mesoderm. In this report, we provide evidence that a member of the fibroblast growth factor (FGF) family, FGF10, emanates from the prospective limb mesoderm to serve as an endogenous initiator for limb bud formation. Fgf10 expression in the prospective limb mesenchyme precedes Fgf8 expression in the nascent apical ectoderm. Ectopic application of FGF10 to the chick embryonic flank can induce Fgf8 expression in the adjacent ectoderm, resulting in the formation of an additional complete limb. Expression of Fgf10 persists in the mesenchyme of the established limb bud and appears to interact with Fgf8 in the apical ectoderm and Sonic hedgehog in the zone of polarizing activity. These results suggest that FGF10 is a key mesenchymal factor involved in the initial budding as well as the continuous outgrowth of vertebrate limbs.

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Relationship between retinoic acid and sonic hedgehog, two polarizing signals in the chick wing bud

Development ◽

10.1242/dev.120.11.3267 ◽

1994 ◽

Vol 120 (11) ◽

pp. 3267-3274 ◽

Cited By ~ 7

Author(s):

J. Helms ◽

C. Thaller ◽

G. Eichele

Keyword(s):

Retinoic Acid ◽

Sonic Hedgehog ◽

Positional Information ◽

Limb Bud ◽

Dependent Manner ◽

All Trans Retinoic Acid ◽

Wing Bud ◽

Zone Of Polarizing Activity ◽

Dose Dependent

Local application of all-trans-retinoic acid (RA) to the anterior margin of chick limb buds results in pattern duplications reminescent of those that develop after grafting cells from the zone of polarizing activity (ZPA). RA may act directly by conferring positional information to limb bud cells, or it may act indirectly by creating a polarizing region in the tissue distal to the RA source. Here we demonstrate that tissue distal to an RA-releasing bead acquires polarizing activity in a dose-dependent manner. Treatments with pharmacological (beads soaked in 330 micrograms/ml) and physiological (beads soaked in 10 micrograms/ml) doses of RA are equally capable of inducing digit pattern duplication. Additionally, both treatments induce sonic hedgehog (shh; also known as vertebrate hedgehog-1, vhh-1), a putative ZPA morphogen and Hoxd-11, a gene induced by the polarizing signal. However, tissue transplantation assays reveal that pharmacological, but not physiological, doses create a polarizing region. This differential response could be explained if physiological doses induced less shh than pharmacological doses. However, our in situ hybridization analyses demonstrate that both treatments result in similar amounts of mRNA encoding this candidate ZPA morphogen. We outline a model describing the apparently disparate effects of pharmacologic and physiological doses RA on limb bud tissue.

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Evidence that preaxial polydactyly in the Doublefoot mutant is due to ectopic Indian Hedgehog signaling

Development ◽

10.1242/dev.125.16.3123 ◽

1998 ◽

Vol 125 (16) ◽

pp. 3123-3132 ◽

Cited By ~ 4

Author(s):

Y. Yang ◽

P. Guillot ◽

Y. Boyd ◽

M.F. Lyon ◽

A.P. McMahon

Keyword(s):

Hedgehog Signaling ◽

Interspecific Backcross ◽

Limb Bud ◽

Chromosome 1 ◽

Indian Hedgehog ◽

Posterior Limb ◽

Preaxial Polydactyly ◽

Ectopic Activation ◽

Zone Of Polarizing Activity ◽

Anterior Posterior

Patterning of the vertebrate limb along the anterior-posterior axis is controlled by the zone of polarizing activity (ZPA) located at the posterior limb margin. One of the vertebrate Hh family members, Shh, has been shown to be able to mediate the function of the ZPA. Several naturally occurring mouse mutations with the phenotype of preaxial polydactyly exhibit ectopic Shh expression at the anterior limb margin. In this study, we report the molecular characterization of a spontaneous mouse mutation, Doublefoot (Dbf). Dbf is a dominant mutation which maps to chromosome 1. Heterozygous and homozygous embryos display a severe polydactyly with 6 to 8 digits on each limb. We show here that Shh is expressed normally in Dbf mutants. In contrast, a second Hh family member, Indian hedgehog (Ihh) which maps close to Dbf, is ectopically expressed in the distal limb bud. Ectopic Ihh expression in the distal and anterior limb bud results in the ectopic activation of several genes associated with anterior-posterior and proximal-distal patterning (Fgf4, Hoxd13, Bmp2). In addition, specific components in the Hedgehog pathway are either ectopically activated (Ptc, Ptc-2, Gli1) or repressed (Gli2). We propose that misexpression of Ihh, and not a novel Smoothened ligand as recently suggested (Hayes et al., 1998), is responsible for the Dbf phenotype. We consider that Ihh has a similar activity to Shh when expressed in the early Shh-responsive limb bud. To determine whether Dbf maps to the Ihh locus, which is also on chromosome 1, we performed an interspecific backcross. These results demonstrate that Dbf and Ihh are genetically separated by approximately 1.3 centimorgans, suggesting that Dbf mutation may cause an exceptionally long-range disruption of Ihh regulation. Although this leads to ectopic activation of Ihh, normal expression of Ihh in the cartilaginous elements is retained.

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Embryonic retinoic acid synthesis is required for forelimb growth and anteroposterior patterning in the mouse

Development ◽

10.1242/dev.129.15.3563 ◽

2002 ◽

Vol 129 (15) ◽

pp. 3563-3574 ◽

Cited By ~ 11

Author(s):

Karen Niederreither ◽

Julien Vermot ◽

Brigitte Schuhbaur ◽

Pierre Chambon ◽

Pascal Dollé

Keyword(s):

Retinoic Acid ◽

Sonic Hedgehog ◽

Acid Synthesis ◽

Limb Bud ◽

Anteroposterior Patterning ◽

Limb Morphogenesis ◽

Functional Zone ◽

Mutant Mouse Embryos ◽

Bud Growth ◽

Zone Of Polarizing Activity

Numerous studies, often performed on avian embryos, have implicated retinoic acid (RA) in the control of limb bud growth and patterning. Here we have investigated whether the lack of endogenous RA synthesis affects limb morphogenesis in mutant mouse embryos deficient for the retinaldehyde dehydrogenase 2 (Raldh2/Aldh1a2). These mutants, which have no detectable embryonic RA except in the developing retina, die at E9.5-E10 without any evidence of limb bud formation, but maternal RA supplementation through oral gavage from E7.5 can extend their survival. Such survivors exhibit highly reduced forelimb rudiments, but apparently normal hindlimbs. By providing RA within maternal food, we found both a stage- and dose-dependency for rescue of forelimb growth and patterning. Following RA supplementation from E7.5 to 8.5, mutant forelimbs are markedly hypoplastic and lack anteroposterior (AP) patterning, with a single medial cartilage and 1-2 digit rudiments. RA provided until E9.5 significantly rescues forelimb growth, but cannot restore normal AP patterning. Increasing the RA dose rescues the hypodactyly, but leads to lack of asymmetry of the digit pattern, with abnormally long first digit or symmetrical polydactyly. Mutant forelimb buds are characterized by lack of expression or abnormal distal distribution of Sonic hedgehog (Shh) transcripts, sometimes with highest expression anteriorly. Downregulation or ectopic anterior expression of Fgf4 is also seen. As a result, genes such as Bmp2 or Hoxd genes are expressed symmetrically along the AP axis of the forelimb buds, and/or later, of the autopod. We suggest that RA signaling cooperates with a posteriorly restricted factor such as dHand, to generate a functional zone of polarizing activity (ZPA).

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A Novel ZRS Mutation in a Chinese Patient with Preaxial Polydactyly and Triphalangeal Thumb

Cytogenetic and Genome Research ◽

10.1159/000448820 ◽

2016 ◽

Vol 149 (3) ◽

pp. 171-175 ◽

Cited By ~ 9

Author(s):

Pan-Feng Wu ◽

Shuai Guo ◽

Xue-Feng Fan ◽

Liang-Liang Fan ◽

Jie-Yuan Jin ◽

...

Keyword(s):

De Novo ◽

Novel Mutation ◽

Genetic Diagnosis ◽

Chinese Patient ◽

Regulatory Sequence ◽

Preaxial Polydactyly ◽

Thumb Duplication ◽

Zone Of Polarizing Activity ◽

The Right ◽

Triphalangeal Thumb

Preaxial polydactyly (PPD; OMIM 603596), which is characterized as having supernumerary fingers, is an unusual congenital hand abnormality. Triphalangeal thumb (TPT; OMIM 190600) is identified by an extra phalangeal bone and is often found in association with PPD. When in combination, the disease is referred to as PPD type II (PPD II; OMIM 174500). Previous studies have demonstrated that variations in the zone of polarizing activity regulatory sequence (ZRS; chr7:156,583,796-156,584,569; hg19) region are associated with PPD II. In this study, our patient was diagnosed with PPD II, having bilateral thumb duplication and unilateral TPT (on the right hand). Further investigation of possible causative genes identified a de novo heterozygous ZRS mutation (ZRS 428T>A). This novel mutation was neither found in 200 normal controls nor reported in online databases. Moreover, the bioinformatics program Genomic Evolutionary Rate Profiling (GERP) revealed this site (ZRS428) to be evolutionarily highly conserved, and the 428T>A point mutation was predicted to be deleterious by MutationTaster. In conclusion, the affected individual shows bilateral thumb duplication, but unilateral TPT making this case special. Thus, our findings not only further support the important role of ZRS in limb morphogenesis and expand the spectrum of ZRS mutations, but also emphasize the significance of genetic diagnosis and counseling of families with digit number and identity alterations as well.

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Slug, a zinc finger gene previously implicated in the early patterning of the mesoderm and the neural crest, is also involved in chick limb development

Development ◽

10.1242/dev.124.9.1821 ◽

1997 ◽

Vol 124 (9) ◽

pp. 1821-1829

Author(s):

M.A. Ros ◽

M. Sefton ◽

M.A. Nieto

Keyword(s):

Zinc Finger ◽

Sonic Hedgehog ◽

Limb Development ◽

Limb Bud ◽

Mesenchymal Phenotype ◽

Signalling Molecules ◽

Slug Expression ◽

Snail Family ◽

Zone Of Polarizing Activity ◽

Signalling Systems

The great advances made over the last few years in the identification of signalling molecules that pattern the limb bud along the three axes make the limb an excellent model system with which to study developmental mechanisms in vertebrates. The understanding of the signalling networks and their mutual interactions during limb development requires the characterisation of the corresponding downstream genes. In this study we report the expression pattern of Slug, a zinc-finger-containing gene of the snail family, during the development of the limb, and its regulation by distinct axial signalling systems. Slug expression is highly dynamic, and at different stages of limb development can be correlated with the zone of polarizing activity, the progress zone and the interdigital areas. We show that the maintenance of its expression is dependent on signals from the apical ectodermal ridge and independent of Sonic Hedgehog. We also report that, in the interdigit, apoptotic cells lie outside of the domains of Slug expression. The correlation of Slug expression with areas of undifferentiated mesenchyme at stages of tissue differentiation is consistent with its role in early development, in maintaining the mesenchymal phenotype and repressing differentiation processes. We suggest that Slug is involved in the epithelial-mesenchymal interactions that lead to the maintenance of the progress zone.

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