The current study is designed to characterize the ontogeny of dactylin expression in the developing mouse embryo. Dactylin is an important gene for proper digit formation. It is a member of the F‐box/WD40 gene family and is involved in the ubiquitin ligase process that targets specific proteins for degradation. Mouse dactylaplasia has similar phenotypic characteristics to humans with split hand/ spilt foot malformation, furthermore dactylin, which has been mapped to chromosome 19 in mice, is syntenic to the SHFM3 region in humans on chromsome 10q24. Understanding the ontogeny of dactylin is important because it allows us to look at specific time periods critical for the development of digits. Mouse embryos were collected prior to digit formation (E8 and E10), during digit formation (E12), and after digit formation is complete (E14) to determine at what point dactylin expression was turned on and off. Once the embryos were collected the limb buds or entire limbs were removed, total mRNA isolated, and RT‐PCR was used to determine gene expression. We found dactylin expression to be present at all time periods examined. In order to get a more complete ontogeny, we are currently expanding the original time frame to E2 through E21. This work was supported by a Lander University Foundation Grant.
The formation of the vertebrate limb is a complex and tightly regulated developmental process. Outgrowth and patterning of the limb depends on two signaling centers in the limb bud, the Apical Ectodermal Ridge (AER) and the Zone of Polarizing Activity (ZPA). Outgrowth of the limb is controlled by the AER, which expresses the signals for Fibroblast Growth Factors, particularly Fibroblast Growth Factor 8 (FGF‐8). Anterior/posterior patterning of the limb is controlled by the ZPA, which expresses Sonic Hedge Hog (Shh). Previous studies have shown that both FGF‐8 and Shh signaling are crucial for limb development and outgrowth, as well as the expression of each other. In zebrafish, Shh mutants demonstrated a reduction in FGF signaling as well as a truncated limb. The aim of this study was to determine the mRNA expression pattern of FGF‐8 during limb development of the chicken (E 3–15) and the effect of cyclopamine, an inhibitor of Shh, on FGF‐8 mRNA expression using RT‐PCR. Although FGF‐8 expression was maintained in both the treated and untreated embryos throughout limb development, chicks exposed to cyclopamine demonstrated truncated limbs, consistent with previous data. Our data suggests that FGF‐8 expression is not affected by cyclopamine treatment. Future studies will investigate expression patterns of other genes, like BTRC, DLX‐5 and DLX‐6 that are involved in chick limb development after cyclopamine treatment. Sources for this research were provided by Lander University.
The split‐hand/split‐foot malformation (SHFM) is a congenital limb malformation that is characterized by absence or reduction of the central digital rays of the hands and feet. At one of the six well established loci, SHFM3 at chromosome 10q24, tandem duplications have been associated with the SHFM phenotype in humans; the smallest reported includes the genes POLL, BTRC, DPCD, and a portion of FBXW4. SUFU, a gene near the duplicated region, has been shown to be overexpressed in SHFM3 patients. RNA collected from chicken embryo limb buds (E2‐6), limbs (E7‐13), and wings (E14‐15) was analyzed for expression of the homologs of these genes by RT‐PCR. Poll expression was present in limb RNA at stages E2‐E15, although barely detectable. Expression of fbxw4 was detected in stages E3 through E15. Both sufu and dpcd expression was detected at E2, with no expression at E3, and the intensity of the bands varied for E4‐15. These results suggest that these four genes are expressed in the limb during development.To better understand how the expression of these genes correlates to the stages of limb development, E3‐15 cDNA was analyzed using quantitative PCR (qPCR) by comparing their expression to that of a control gene. Early results indicate the timing of the expression of these genes correlates to the end of digit formation on the chicken limb. Future studies will investigate the localization of these genes in the developing chicken limb.
Split‐hand/split‐foot malformation (SHFM), also known as ectrodactyly, is a congenital limb malformation that is characterized by absence or reduction of the central digital rays of the hands and feet. To date there are five well established loci for SHFM. At one of these loci, SHFM3 at chromosome 10q24, tandem duplications have been associated with the SHFM phenotype in humans. The smallest reported duplication includes the genes POLL, BTRC, DPCD, and a portion of FBXW4. Another gene near the duplicated region, SUFU, has been shown to be overexpressed in SHFM3 patients. RNA collected from chicken embryo limb buds (E2‐15) was analyzed for expression of poll, fbxw4, and sufu by reverse‐transcription polymerase chain reaction (RT‐PCR). Preliminary studies demonstrate that fbxw4 and sufu are first detected at E5 and are no longer detected after E13, which is consistent with the normal timing of digit patterning in the chicken. No poll expression was observed in limb bud RNA, which is not surprising, given that there is no previous correlation with limb development. These results suggest that both fbxw4 and sufu are expressed in the limb during the period of digit patterning, and while poll is in the commonly duplicated region, it is not expressed in a similar fashion. Future studies will explore the effects that overexpression of fbxw4 and sufu have on limb development in chicken and investigate the expression of other genes from the duplicated region.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.