Abnormal Limb Development Research Articles

Tissue-specific requirement of sodium channel and clathrin linker 1 (Sclt1) for ciliogenesis during limb development.

Primary cilia have essential roles as signaling centers during development and adult homeostasis. Disruption of ciliary structure or function causes congenital human disorders called ciliopathies. Centriolar distal appendage (DAP) proteins are important for anchoring cilia to the membrane. However, the exact functions of DAP during in vivo ciliogenesis and animal development remain poorly understood. Here, we showed that the DAP component sodium channel and clathrin linker 1 (Sclt1) mutant mice had abnormal craniofacial and limb development with postnatal lethality. In mutant embryos, most of the affected tissues had defects in DAP recruitment to the basal body and docking to the membrane that resulted in reduced ciliogenesis and disrupted hedgehog (Hh) signaling in limb bud mesenchymal cells. However, limb digit formation and ciliogenesis in Sclt1 mutant mice were differentially affected between the fore- and hindlimb buds. The forelimbs developed normally in Sclt1 mutants, but the hindlimbs had preaxial polydactyly. Heterozygous loss of Cep83, another core DAP component, in Sclt1 mutant mice, caused forelimb and hindlimb polydactyly. These findings revealed the tissue-specific differential requirement of DAPs. Taken together, these results indicated that during limb development the ciliary base components, DAPs, play an essential role in ciliogenesis and Hh signaling in vivo in a position-dependent manner.

Exploring the impact of paediatric localized scleroderma on health-related quality of life: focus groups with youth and caregivers.

Paediatric localized scleroderma (LS) can negatively impact health-related quality of life (HRQoL) by causing skin fibrosis, abnormal limb development, disfigurement, and side-effects from immunosuppressive treatment. Studies to date have rarely included qualitative data gathered directly from paediatric patients with LS. To assess the impact of LS on HRQoL among affected youth and their caregivers using qualitative description. Youth with all subtypes of LS and their caregivers were purposively sampled to participate in age-appropriate focus groups (younger children, early adolescents, adolescents). Each group started with a drawing exercise followed by in-depth discussion of topics including skin symptoms (e.g. itch, pain, tightness), functional impairment, physical appearance, family and peer relationships, and treatment burden. Focus groups were transcribed verbatim and co-coded, with adjudication of differentially applied codes. The study findings were triangulated via comparison with adult reports and published literature. Eleven youth aged 9-16 years and 16 caregivers participated in three focus groups each. Major identified areas of impact included uncomfortable skin symptoms, physical functioning limitations, extracutaneous manifestations, body image, bullying and teasing, unwanted questioning from others, and treatment side-effects and burden. This is the first qualitative study of HRQoL in LS to include all major LS subtypes. We identified domains of HRQoL impacted by LS, some of which replicate earlier findings and some of which were novel. As impact also changed with developmental stage, our findings support the need for ongoing, formal evaluation of HRQoL in children and adolescents with LS. What is already known about this topic? Paediatric localized scleroderma (LS) negatively impacts health-related quality of life (HRQoL) via skin fibrosis, musculoskeletal and other extracutaneous manifestations from the disease process, and side-effects of systemic immunosuppression. The full impact of LS and its treatment on HRQoL is incompletely understood, with only one published qualitative study of youth with LS, which was limited to facial involvement. There are no qualitative studies of HRQoL in other LS subtypes to date. What does this study add? This is the first qualitative evaluation of HRQoL in youth with LS inclusive of all disease subtypes. Our study confirms that LS affects HRQoL across multiple distinct domains, including uncomfortable skin sensations, impacts on body image, bullying and teasing from peers, unwanted intrusive questioning, physical limitations, extracutaneous manifestations and high treatment burden. These results indicate the need for ongoing clinical assessment of paediatric patients in these domains. What are the clinical implications of the work? These results support the need to care for patients with LS holistically by synthesizing cutaneous, musculoskeletal and extracutaneous disease assessments with multidimensional evaluation of psychosocial impact and adverse effects of treatments. The development of an LS-specific HRQoL measure would advance such efforts.

Expression analysis of limb element markers during mouse embryonic development.

Background: While data regarding expression of limb element and tissue markers during normal mouse limb development exist, few studies show expression patterns in upper and lower limbs throughout key limb development stages. A comparison to normal developmental events is essential when analyzing development of the limb in mutant mice models. Results: Expression patterns of the joint marker Gdf5, tendon and ligament marker Scleraxis, early muscle marker MyoD1, and blood vessel marker Cadherin5 (Cdh5) are presented during the most active phases of embryonic mouse limb patterning. Anti‐neurofilament staining of developing nerves in the fore‐ and hindlimbs and cartilage formation and progression also are described. Conclusions: This study demonstrates and describes a range of key morphological markers and methods that together can be used to assess normal and abnormal limb development. Developmental Dynamics 247:1217–1226, 2018. © 2018 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists

Pathogenesis of Pregabalin-Induced Limb Defects in Mouse Embryos.

It is known that antiepileptic drugs might adversely affect neuronal function and thus influence brain development. However, we have reported that limb deformities are one of the most prominent disturbances caused by pregabalin (PGB) in the developing embryo. The aim of this work is to gain a better understanding of possible molecular mechanisms behind the musculoskeletal injuries and limb deformities associated with PGB. Pregnant mice divided into four groups. Each mouse received an intraperitoneal injection (IP) of 0, 20 (group I), 40 (group II) or 80 (group III) mg/kg/day of PGB during the organogenesis period. On gestational day 18, embryos were separated and their limbs were dissected. Levels of apoptotic proteins were analyzed by Western blotting. To establish whether apoptosis is present in the limbs, the specimens were examined by TUNEL. Pathological findings were also reported as a score ranging from 1 to 3 based on the level of differentiation. Western blot analysis demonstrated that PGB in all PGB-treated groups significantly upregulated the levels of cleaved caspase-3, 8 and 9. Also, the results showed that PGB exposure increased the percentage of TUNEL positive cells in different limb tissues especially the mesenchymal tissue. The histopathological findings revealed that PGB administration to pregnant mice inhibited limb tissue differentiation, albeit to varying degrees. The result of our study revealed that apoptosis and inhibition of limb tissue differentiation play an important role in the pathogenesis of PGB-induced limb malformations. Both intrinsic and extrinsic caspase-dependent pathways of cell death are important in mediating the abnormal limb development triggered by insult with the PGB. Evaluating the effect of PGB on molecules involved in the cross-talk between intrinsic and extrinsic apoptotic pathways and cell adhesion, migration, proliferation, and differentiation during embryonic development can further help to identify and clarify the involved mechanisms.

Analysis of spatiotemporal expression and function of the single-minded homolog in the branchiopod crustacean Daphnia magna

In insect Drosophila melanogaster, ventral midline cells are crucial to formation of the central nervous system (CNS) and have roles in the specification of ectodermal neuroblasts. Notably, midline cells also have more recently recognized roles in the formation of the higher crustacean Parhyale dorso-ventral axis. The single-minded is a master regulator of ventral midline cells and is required for these functions. Recently sim expression patterns have been reported in various arthropods. These results suggest that the midline precursors evolved from ventral neuroectoderm of common ancestor Mandibulata. However, sim function has been only analyzed in few organisms. To investigate whether these functions of sim, the gene encoding Single-minded, are conserved among insects and crustaceans, we examined the embryonic expression pattern of a lower crustacean Daphnia sim homolog (dma sim) and analyzed the function of dma sim during embryonic development. The Dma Sim protein was expressed in the ventral neuroectoderm (like in onychophoran and chelicerate) and midline (like in mandibulatan). In addition to this conserved ventral neuroectoderm and midline expression, Dma Sim was expressed outside the ventral midline; it was expressed in maxilla 2, presumptive shell glands, and other tissues. To investigate dma sim function, we used RNA interference (RNAi) to inhibit dma sim in Daphnia embryos. Embryos subjected to dma sim RNAi exhibited improper axon tract formation and abnormal limb and ventral development. Furthermore, RNAi-mediated knockdown of dma slit, a putative Dma Sim target gene, resulted in similar embryonic phenotypes. These results indicated that dma sim might be required for proper dma slit-mediated ventral development in addition to being required for a conserved role in the ventral midline. Our findings indicated that sim homologs might have provided different developmental functions to ventral midline cells during metazoan evolution.

Recessive mutations in CAKUT and VACTERL association

Understanding the complex genetic makeup underlying congenital anomalies of the kidney and urinary tract (CAKUT) is of primary importance to improve diagnosis, stratify risk for later-onset complications, and develop therapeutic strategies. Saisawat et al. used homozygosity mapping coupled with next-generation sequencing to identify recessive mutations in TRAP1 in families with isolated CAKUT and with VACTERL association. This study points to a novel player in kidney development, possibly affecting apoptosis and endoplasmic reticulum stress signaling.

Amniotic fluid deficiency and congenital abnormalities both influence fluctuating asymmetry in developing limbs of human deceased fetuses.

Fluctuating asymmetry (FA), as an indirect measure of developmental instability (DI), has been intensively studied for associations with stress and fitness. Patterns, however, appear heterogeneous and the underlying causes remain largely unknown. One aspect that has received relatively little attention in the literature is the consequence of direct mechanical effects on asymmetries. The crucial prerequisite for FA to reflect DI is that environmental conditions on both sides should be identical. This condition may be violated during early human development if amniotic fluid volume is deficient, as the resulting mechanical pressures may increase asymmetries. Indeed, we showed that limb bones of deceased human fetuses exhibited increased asymmetry, when there was not sufficient amniotic fluid (and, thus, space) in the uterine cavity. As amniotic fluid deficiency is known to cause substantial asymmetries and abnormal limb development, these subtle asymmetries are probably at least in part caused by the mechanical pressures. On the other hand, deficiencies in amniotic fluid volume are known to be associated with other congenital abnormalities that may disturb DI. More specifically, urogenital abnormalities can directly affect/reduce amniotic fluid volume. We disentangled the direct mechanical effects on FA from the indirect effects of urogenital abnormalities, the latter presumably representing DI. We discovered that both factors contributed significantly to the increase in FA. However, the direct mechanical effect of uterine pressure, albeit statistically significant, appeared less important than the effects of urogenital abnormalities, with an effect size only two-third as large. We, thus, conclude that correcting for the relevant direct factors allowed for a representative test of the association between DI and stress, and confirmed that fetuses form a suitable model system to increase our understanding in patterns of FA and symmetry development.

Molecular studies of the congenital malformation induced by Largehead Atractylodes Rhizome, the most commonly used Chinese medicine for threatened miscarriage

Largehead Atractylodes Rhizome (LAR) is the most commonly used Chinese herbal medicine for threatened miscarriage. Potential reproductive toxicity of LAR was identified in early pregnancy in animals. Skeletal anomalies including loss of ulna and distal digits, shortening of humerus and radius were observed in higher clinical dose groups. Here, we aimed to study the molecular mechanism of the congenital malformation induced by LAR. In vitro whole mouse embryo culture was used to confirm the embryotoxicity effects of LAR on developing limb buds during early organogenesis. A pregnant mouse model was employed to study the developmental gene expression by quantitative PCR and whole hybridization and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling staining, in the forelimbs and hindlimbs during development in vivo. Severe growth retardation, multiple embryonic malformations and delayed limb bud development were observed. Limb-specific Tbx gene expressions in both developing forelimbs and hindlimbs were significantly decreased. Increased developmental apoptosis in apical ectodermal ridge and mesenchymal mesoderm of the developing limb buds was identified. Overexpressions of Tbx2 and Tbx3 in embryos in vitro rescued LAR-induced abnormal limb development and reduced apoptosis in the developing forelimb buds. In conclusion, LAR affects limb development by suppressing the expression of limb developmental genes and disturbing programmed cell death during limb formation in mice.

BBS proteins interact genetically with the IFT pathway to influence SHH-related phenotypes

There are numerous genes for which loss-of-function mutations do not produce apparent phenotypes even though statistically significant quantitative changes to biological pathways are observed. To evaluate the biological meaning of small effects is challenging. Bardet–Biedl syndrome (BBS) is a heterogeneous autosomal recessive disorder characterized by obesity, retinopathy, polydactyly, renal malformations, learning disabilities and hypogenitalism, as well as secondary phenotypes including diabetes and hypertension. BBS knockout mice recapitulate most human phenotypes including obesity, retinal degeneration and male infertility. However, BBS knockout mice do not develop polydacyly. Here we showed that the loss of BBS genes in mice result in accumulation of Smoothened and Patched 1 in cilia and have a decreased Shh response. Knockout of Bbs7 combined with a hypomorphic Ift88 allele (orpk as a model for Shh dysfuction) results in embryonic lethality with e12.5 embryos having exencephaly, pericardial edema, cleft palate and abnormal limb development, phenotypes not observed in Bbs7−/− mice. Our results indicate that BBS genes modulate Shh pathway activity and interact genetically with the intraflagellar transport (IFT) pathway to play a role in mammalian development. This study illustrates an effective approach to appreciate the biological significance of a small effect.

Characterization of the Pathogenesis Mechanism after Pseudomonas aeruginosa Infection through Food Consumption Using Chick Embryo Model

This study introduced a chick embryos` infection model to elucidate the pathogenesis mechanism of Pseudomonas aeruginosa, which causes serious diseases in human after ingestion of P. aeruginosa-contaminated animal originated foods. The embryonic chick model is able to give a rapid and relatively inexpensive method to assess bacterial pathogenicity compared to embryos of other vertebrates. Embryos were infected with P. aeruginosa and elastase-deficient P. aeruginosa. After infection with P. aeruginosa cells, total bacterial cell numbers and gelatinase activities in the embryos were compared. Thereafter, precartilage condensation and chondrogenesis were assessed by peanut agglutinin (PNA) binding on day 3 and by Alcian blue staining for sulfated proteoglycans on day 5, respectively. P. aeruginosa significantly increased in embryos, resulting in abnormal limb development, whereas P. aeruginosa defective in elastase activity partly impaired proliferation. In addition, P. aeruginosa-infected chick embryos significantly stimulated the production of matrix metalloproteinases. Several analyses showed that elevated proteases suppressed the proliferation and survival of chondrogenic cells. The results show that this infection model was a useful assay to determine the virulence mechanism of P. aeruginosa in human after intake of microbiologically contaminated foods.

Familial Isolated Clubfoot Is Associated with Recurrent Chromosome 17q23.1q23.2 Microduplications Containing TBX4

Clubfoot is a common musculoskeletal birth defect for which few causative genes have been identified. To identify the genes responsible for isolated clubfoot, we screened for genomic copy-number variants with the Affymetrix Genome-wide Human SNP Array 6.0. A recurrent chromosome 17q23.1q23.2 microduplication was identified in 3 of 66 probands with familial isolated clubfoot. The chromosome 17q23.1q23.2 microduplication segregated with autosomal-dominant clubfoot in all three families but with reduced penetrance. Mild short stature was common and one female had developmental hip dysplasia. Subtle skeletal abnormalities consisted of broad and shortened metatarsals and calcanei, small distal tibial epiphyses, and thickened ischia. Several skeletal features were opposite to those described in the reciprocal chromosome 17q23.1q23.2 microdeletion syndrome associated with developmental delay and cardiac and limb abnormalities. Of note, during our study, we also identified a microdeletion at the locus in a sibling pair with isolated clubfoot. The chromosome 17q23.1q23.2 region contains the T-box transcription factor TBX4, a likely target of the bicoid-related transcription factor PITX1 previously implicated in clubfoot etiology. Our result suggests that this chromosome 17q23.1q23.2 microduplication is a relatively common cause of familial isolated clubfoot and provides strong evidence linking clubfoot etiology to abnormal early limb development.

Gene expression of sHsps, Hsp40 and Hsp60 families in normal and abnormal embryonic development of mouse forelimbs

Heat shock proteins (Hsps) are thought of as chaperones of morphologic development of cells and organisms, and believed to be closely related to normal and abnormal embryonic development. Previous studies only focused on a small number of HSPs and their relationships with embryogenesis, and roles of most HSPs in normal and abnormal embryonic development remain unclear. The present study detected expression of sHsps, Hsp40 and Hsp60 in normal and abnormal development of embryonic forelimbs in all-trans retinoic acid (atRA)-induced phocomelic, oligodactylic and atRA-induced abnormal limb bud development models in mice ex vivo. It was found that 17 Hsps of the three families were expressed in embryonic limb bud tissue of normal mice, though the expression patterns were different, showing a phase correlation with the development of embryo. There was a difference in the expression pattern and mRNA abundance of most genes between the atRA-indcued limb abnormal development model groups and the control group. Most members of the three families may play a stress-protective role in atRA-induced abnormal limb development, and abnormal expression of some genes ( Hsp25, HspB2, HspB3, HspB7, Hsp20, HspB9, HspB10 and Hsp40) may be related to atRA-induced phocomelic and other abnormalities.

Expanding functions of lipoprotein receptors

Lipoprotein receptors are evolutionarily ancient proteins that are expressed on the surface of many cell types. Beginning with the appearance of the first primitive multicellular organisms, several structurally and functionally distinct families of lipoprotein receptors evolved. Originally, these cell surface proteins were thought to merely mediate the traffic of lipids and nutrients between cells and, in some cases, by functioning as scavenger receptors, remove other kinds of macromolecules, such as proteases and protease inhibitors from the extracellular space and the cell surface. Over the last decade, this picture has fundamentally changed. We now appreciate that many of these receptors are not mere cargo transporters; they are deeply embedded in the machinery by which cells communicate with each other. By physically interacting and coevolving with fundamental signaling pathways, lipoprotein receptors have occupied essential and surprisingly diverse functions that are indispensable for integrating the complex web of cellular signal input during development and in differentiated tissues.

Osteogenesis imperfecta type III with intracranial hemorrhage and brachydactyly associated with mutations in exon 49 of COL1A2

Osteogenesis imperfecta (OI) is a heritable bone disorder characterized by fractures with minimal trauma. Intracranial hemorrhage has been reported in a small number of OI patients. Here we describe three patients, a boy (aged 15 years) and two girls (aged 17 and 7 years) with OI type III who suffered intracranial hemorrhage and in addition had brachydactyly and nail hypoplasia. In all of these patients, OI was caused by glycine mutations affecting exon 49 of the COL1A2 gene, which codes for the most carboxy-terminal part of the triple-helical domain of the collagen type I alpha 2 chain. These observations suggest that mutations in this region of the collagen type I alpha 2 chain carry a high risk of abnormal limb development and intracranial bleeding.

Recent insights into the role of IRF6 in craniofacial and skin development

Genetic variation in Interferon Regulatory Factor 6 (IRF6) causes, and contributes risk for, orofacial clefting disorders. In one of these disorders, in addition to orofacial clefts, individuals have skin webbing of the lower limbs. Thus, IRF6 is a key component of palatal and skin development in humans. In mice, Irf6 is expressed in the medial edge epithelium (MEE) that is dependent on Tgfb signaling. And, Irf6 is expressed in the spinous layer of the epidermis. Moreover, mice deficient for Irf6 have abnormal skin, limb and craniofacial development, including cleft palate. In the epidermis, in vivo and in vitro analyses demonstrate that Irf6 is necessary for keratinocyte differentiation. Furthermore, keratinocytes from Irf6–/– epidermis fail to close a scratch wound and display abnormal clonal growth, suggesting abnormal cellular proliferation and migration. In the palate, growth of the shelves is not defective, but they fail to elevate above the tongue, may be due to oral adhesions. The palatal epithelium is highly adherent, but the MEE fails to undergo its normal pattern of dissolution. Mice that are compound heterozygotes for the null and hypomorphic alleles (Irf6–/neo) display less severe abnormalities than the Irf6 deficient mice. In the oral cavity, Irf6–/neo mice have less severe oral adhesions. We are assessing the Irf6–/neo mice for defects in palatal development and epithelial differentiation.

Abnormal skin, limb and craniofacial morphogenesis in mice deficient for interferon regulatory factor 6 (Irf6)

Transcription factor paralogs may share a common role in staged or overlapping expression in specific tissues, as in the Hox family. In other cases, family members have distinct roles in a range of embryologic, differentiation or response pathways (as in the Tbx and Pax families). For the interferon regulatory factor (IRF) family of transcription factors, mice deficient in Irf1, Irf2, Irf3, Irf4, Irf5, Irf7, Irf8 or Irf9 have defects in the immune response but show no embryologic abnormalities. Mice deficient for Irf6 have not been reported, but in humans, mutations in IRF6 cause two mendelian orofacial clefting syndromes, and genetic variation in IRF6 confers risk for isolated cleft lip and palate. Here we report that mice deficient for Irf6 have abnormal skin, limb and craniofacial development. Histological and gene expression analyses indicate that the primary defect is in keratinocyte differentiation and proliferation. This study describes a new role for an IRF family member in epidermal development.

Mutations in the gene encoding the low-density lipoprotein receptor LRP4 cause abnormal limb development in the mouse

Positional cloning of two recessive mutations of the mouse that cause polysyndactyly ( dan and mdig—Chr 2) confirmed that the gene encoding MEGF7/LRP4, a member of the low-density lipoprotein receptor family, plays an essential role in the process of digit differentiation. Pathologies observed in the mutant mice provide insight into understanding the function(s) of LRP4 as a negative regulator of the Wnt–β-catenin signaling pathway and may help identify the genetic basis for common human disorders with similar phenotypes.

P63 Is an Essential Proapoptotic Protein during Neural Development

The p53 family member p63 is required for nonneural development, but has no known role in the nervous system. Here, we define an essential proapoptotic role for p63 during naturally occurring neuronal death. Sympathetic neurons express full-length TAp63 during the developmental death period, and TAp63 levels increase following NGF withdrawal. Overexpression of TAp63 causes neuronal apoptosis in the presence of NGF, while cultured p63-/- neurons are resistant to apoptosis following NGF withdrawal. TAp63 is also essential in vivo, since embryonic p63-/- mice display a deficit in naturally occurring sympathetic neuron death. While both TAp63 and p53 induce similar apoptotic signaling proteins and require BAX expression and function for their effects, TAp63 induces neuronal death in the absence of p53, but p53 requires coincident p63 expression for its proapoptotic actions. Thus, p63 is essential for developmental neuronal death, likely functioning both on its own, and as an obligate proapoptotic partner for p53.

Regulation of limb development by the sphingosine 1-phosphate receptor S1p1/EDG-1 occurs via the hypoxia/VEGF axis

Angiogenesis, also known as new blood vessel formation, is regulated coordinately with other tissue differentiation events during limb development. Although vascular endothelial cell growth factor (VEGF) is important in the regulation of angiogenesis, chondrogenesis and osteogenesis during limb development, the role of other angiogenic factors is not well understood. Sphingosine 1-phosphate, a platelet-derived lipid mediator, regulates angiogenesis and vascular maturation via its action on the G-protein-coupled receptor S1P 1 (also known as EDG-1). In addition to vascular defects, abnormal limb development was also observed in S1p 1 −/− mice. Here we show that strong induction of S1P 1 expression is observed in the blood vessels and the interdigital mesenchymal cells during limb development. Deletion of S1P 1 results in aberrant chondrocyte condensation and defective digit morphogenesis. Interestingly, the vasculature in the S1p 1 −/− limbs was hyperplastic and morphologically altered. In addition, the hypoxia inducible factor (HIF)-1α and its response gene VEGF were induced in S1p 1 −/− limbs. However, aberrant regulation of HIF-1α and VEGF were not observed in embryonic fibroblasts derived from S1p 1 −/− mice, suggesting a non-cell autonomous effect of S1P 1 on VEGF expression. Indeed, similar limb defects were observed in endothelium-specific S1P 1 null mice in vivo. These data suggest that the function of S1P 1 in the developing vasculature is essential for proper limb development.

An introduction to congenital hand anomalies.

The intention of this introduction is to provide a platform from which ensuing articles may deal with the details of management of specific entities. A basic knowledge of the process of limb development and an appreciation of our current knowledge of the causations of abnormal limb development are necessary to assist the surgeon in explanation as to how and why the child has the limb anomaly. Undoubtedly it is from this knowledge that prevention and improved management will evolve in the future. Some words about classification are appropriate. This is always a contentious issue, as classification cannot currently be precisely related to causation but is necessary such that those working in the field can speak in one language and understand each other. Finally, I will outline a philosophy on which to base both indications for, and timing of surgical intervention.