No description available.

No description available.

This project aimed to provide a greater understanding of limb development through the characterisation of Mendelian disorders. The more specific aim was to identify the developmental basis of the Post Axial Longitudinal Limb Reduction Deformity (PALLRD) seen in the autosomal recessive Miller syndrome caused by mutations in Dihydroorotate Dehydrogenase (DHODH)[1]. In addition whole exome sequence analysis was used to identify further causative variants in a group of individuals with Non Classical Miller syndrome. These individuals were negative for mutations in DHODH although they had a clinically overlapping PALLRD. A single novel variant was discovered in Fibroblast Growth Factor Receptor 1 gene (FGF1) in one individual in this cohort. Due to the known vital role of FGF signaling in limb bud development the functional significance of this variant was investigated further[2]. In vitro data suggested that this variant has a dominant negative effect. Finally I compared the differential gene expression profile of embryonic mouse forelimb and hindlimb at a later stage of development. Digital Gene Expression Serial Analysis of Gene Expression (DGE-SAGE) produced gene-expression profiles of the forelimbs and hind limbs from 14.5 days post conception (d.p.c) murine embryos. This data included known differentially expressed genes as well as novel candidate genes that are putative regulators of limb growth. Whole mount In Situ Hybrisation (WISH) and Quantitative Real Time Polymerase Chain Reaction (qRTPCR) provided corroborating evidence for the differential expression of a subset of these genes between the forelimbs and hind limbs. This project suggests a role for DHODH in limb bud cell proliferation. It also demonstrates a novel potentially dominant negative mutation within FGFR1 in an individual with a limb deformity. Finally a subset of genes involved in regulating the differential growth between the forelimb and hindlimb were presented.

Brachypod (bpH/bpH), an autosomal mutation in mice, is characterized by a shortening of the long bones and paws, and a delay or absence of ossification in some of the distal limb elements. The present study represents a detailed description of the brachypod phenotype in day 12 hindlimb buds maintained for 6 days in a submerged, serum-free organ culture system. Using this in vitro system, the proximal-to-distal effect on the severity of cartilage reduction was intensified in the brachypod explants with an intermediate expression in the heterozygotes. Immunofluorescent staining of the brachypod cartilage revealed a deficiency in and an abnormal distribution of the proteoglycans. Although there was no recognizable difference in the immunofluorescent staining for type II collagen between the mutant and wild-type, electron micrographs showed the presence of thick fibrils in the matrix. Other atypical structures in the brachypod cartilage included pleomorphic nuclei, reduced intracellular glycogen granules and profuse intercellular contacts. It is proposed that with the use of this in vitro system which supports the autonomous development of the individual limb elements, experiments concerning the pathogenesis of skeletal mutations such as brachypod should be more feasible.

No description available.

Tgf-β, Wnt, and Fgf signaling pathways are required for many developmental processes. Here, I investigated the requirement of these signaling pathways during tail regeneration and limb development in the Mexican axolotl (Ambystoma mexicanum). Using small chemical inhibitors during tail regeneration, I found that the Tgf-β signaling pathway was required from 0-24 and 48-72 hours post tail amputation (hpa), the Wnt signaling pathway was required from 0-120 hpa, and the Fgf signaling pathway was required from 0-12hpa. Tgf-β1 was upregulated after amputation and thus may mediate Tgf-β signaling pathway during tail regeneration. Both Smad-mediated and non-Smad mediated Tgf-β signaling were activated as early as 1hpa. Smad-mediated Tgf-β signaling via activated pSmad2 and pSmad3, and via phosphorylated Erk and Akt. Two different Tgf-β signaling pathway inhibitors, SB505124 and Naringenin, differentially regulated pSmad2, pSmad3, p-Erk, and p-Akt, while SB505124 and Naringenin both inhibited tail regeneration; only SB505124 reduced cell proliferation. Wnt/β-Catenin signaling was increased and was enhanced by Wnt-C59. Disruption of the Wnt signaling pathway directly or indirectly activated Erk and Akt signaling. Disruption of the Fgf signaling pathway decreased p-Erk and increased p-Akt. All three signaling pathways affected cell proliferation and mitosis during tail regeneration. The Wnt pathway inhibitor Wnt-C59 prevented forelimb bud outgrowth. The critical window for Wnt signaling regulating forelimb bud outgrowth was approximately developmental stage 40-42. Wnt signaling ligand Wnt3a and tight junction protein Zo-1 were expressed in the epidermis of the forelimb bud and both were down-regulated by Wnt-C59. Moreover, both Wnt and Fgf signaling pathways affected cell proliferation and mitosis of mesodermal cells during forelimb bud outgrowth. Overall, my results show that Tgf-β, Wnt, and Fgf signaling pathways are required for axolotl tail regeneration. All three pathways affect Erk and Akt signaling and guide cell proliferation and mitosis. The Wnt signaling pathway is required for forelimb bud outgrowth, and it appears to regulate expression of Wnt3a and Zo1, and control cell proliferation and mitosis of mesodermal cells underlying the forelimb epidermis. These data enrich understanding of signaling network dynamics that underlie tissue regeneration and vertebrate limb development.

The development of the vertebrate limb is a progressive process characterized by initial induction and patterning, concomitant growth and morphogenesis, and subsequent cell differentiation and tissue formation. Many of these processes are regulated by specific signaling centers and the environment they create. Through both classical approaches and recent molecular studies, we are beginning to understand the roles of these signaling events during limb development. However, several questions still remain and need to be further addressed. In this dissertation, I first examine how signaling molecules regulate the proximal-distal (PD) patterning of the limb. We demonstrate that early limb mesenchyme is initially maintained in a state capable of generating all limb segments by a combination of proximal and distal signals. As the limb grows, the proximal limb is established by exposure to flank-derived signal(s), whereas the distal segments are determined by distal signals when cells grow beyond the proximal influence. Thus, these results support the “two signal model” and contradict the classical view of PD patterning by a clock-based system that was postulated in the “progress zone model”. In the second part of this work, I focus on a later developmental event and study the cell- and non-cell-autonomous function of Sonic hedgehog (Shh) during limb muscle formation. Muscle progenitor cells migrate from the lateral somites into the developing limb, where they undergo patterning and differentiation in response to local signals. We find that Shh patterns limb musculature non-cell-autonomously, acting through adjacent non-muscle mesenchyme. However, Shh functions cell-autonomously to maintain cell survival in the dermomyotome and promote slow muscle differentiation. Finally, Shh signaling is required cell-autonomously to maintain Net1 expression, which in turn regulates directional muscle cell migration in the distal limb. The dissertation ends with three appendices, describing separate studies: first, mechanisms of limb loss in snakes, second, the role of Hippo signaling in limb development, and lastly a collaborative work with Dr. Jérôme Gros on limb morphogenesis. Taken together, this dissertation provides a glimpse into the diverse roles of signaling pathways during various stages of vertebrate limb development.

Proper embryonic patterning requires precise spatio-temporal regulation of retinoic acid (RA) activity. Morphogenesis can be regulated at the level of RA distribution, mainly via its synthesis and catabolism by the RALDH and CYP26 enzymes respectively, and at the level of RA-mediated transcription through activation of its cognate nuclear receptor, the retinoic acid receptors (RARs) α, β, and γ. Loss of Cyp26b1 leads to increased local levels of RA in tissues such as the limb and craniofacial structures, and results in neonatal lethality. Visible gross phenotypic defects in neonates include phocomelia (shortening of the limbs), adactyly (missing digits), micrognathia (shortened lower jaw), and open eyes at birth. In addition, these embryos exhibit cleft palate and have a paucity of vibrissal (whisker) and pelage (hair) follicles. We have previously shown that ablating the gene encoding RARγ in a Cyp26a1-null background was able to rescue the caudal abnormalities associated with improper RA exposure in these embryos by limiting aberrant RA signalling, and thus rescuing expression domains of target genes involved in caudal development. I show here that ablating Rarg in a Cyp26b1-null background is able to partially rescue the defects associated with loss of CYP26B1. These include a reduction in the severity of limb defects, rescued vibrissae, fused eyelids, and recovered aspects of axial skeletal development. This compound-null murine model illustrates that RARγ plays a specific role in transducing the RA signal within tissues that are affected by the loss of CYP26B1. Further molecular analysis of the pathways responsible for directing limb bud outgrowth and eyelid fusion provided insight into pathways regulated by RARγ in these rescued tissues. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2010-04-01 15:38:52.05

Retinoic acid, suspended in cottonseed oil, was administered via gavage to pregnant mice (ICR strain) on day 11 (E 11) of gestation at doses of either 20, 40, or 80 mg/kg. Fetuses were examined for external malformations on day 17 (E 17). Retinoic acid treatment induced micromelia (with the elimination of several long bones at higher doses) and digital defects (ectrodactyly and syndactyly) in a dose‐dependent manner in fetuses examined on day 17. Hindlimbs were affected more than forelimbs. In another group of experiments, limbs exposed to retinoic acid treatment in utero on E 11 were cultured on E 12 and maintained for 3 days in submerged culture. Cultured limbs were examined qualitatively for digital and long bone defects, and image analysis of the area and form of bone anlagen of cultured limbs was used to quantitatively evaluate the teratogenic potential of retinoic acid. The qualitative evaluation indicated that the retinoic acid‐induced effects obtained in vivo and with pretreated, cultured limbs were essentially the same, except that the severity of regional effects changed as a result of culture. The incidence of ectrodactyly was higher with cultured limbs than with E 17 fetal limbs, but fewer cultured limbs were missing long bones. These results suggest that culturing limbs, after they have been pretreated in utero, modifies their response to a teratogen and demonstrates that the paw skeleton is extremely sensitive to teratogen treatment under these experimental conditions. Therefore, care must be exercised when attempting to compare in vivo and in vitro teratogenic data. This study also clearly demonstrates the power and usefulness of image analysis for quantitative evaluation of both the area and form of a cultured specimen such as the developing limb bud. Quantitative, image analysis of cultured limbs showed a dose‐dependent decrease in area of both fore‐ and hindlimbs. The effect was most severe in hindlimbs. In the forelimb, the paw was affected more than the long bones; as the dose increased, this disparity of effect also increased. With the hindlimb, a greater effect on the paw occurred only at 80 mg/kg. Computing the soft tissue/bone ratio illustrated that retinoic acid had a greater effect on chondrogenic tissue than on soft tissue.

Maternal administration of a single dose of retinoic acid (vitamin A acid, 100 mg/kg) on either the 11 th, 11 1/2, 12th, 12 1/2, 13th or 13 1/2 day of gestation produced phocomelia or partial phocomelia in ICR/DUB fetuses. The results depended upon the time of treatment and two gradients of effect were produced: 1) cranio-caudal gradient, since forelimb defects resulted from treatment between days 11 and 13, while similar hindlimb abnormalities were produced by administration of retinoic acid 12 to 24 hours later: 2) proximo-distal gradient, due to the heterogenous sensitivity among individual bones of the limb. In the forelimb, early treatment (11th day) produced humero-ulnar defects and later treatment (12th day) ulnoradial defects. A similar proximo-distal gradient was observed in the hindlimb. The use of teratological studies as a tool to assist morphogenetic investigation is discussed.