Cultured Limb Bud Cells Research Articles

A new cell culture resource for investigations of reptilian gene function.

The establishment of CRISPR/Cas9 gene editing in Anolis sagrei has positioned this species as a powerful model for studies of reptilian gene function. To enhance this model, we developed an immortalized lizard fibroblast cell line (ASEC-1) for the exploration of reptilian gene function in cellular processes. We demonstrate the use of this cell line by scrutinizing the role of primary cilia in lizard Hedgehog (Hh) signaling. Using CRISPR/Cas9 mutagenesis, we disrupted the ift88 gene, which is required for ciliogenesis in diverse organisms. We determined that loss of itf88 from lizard cells leads to an absence of primary cilia, a partial derepression of gli1 transcription, and an inability of the cells to respond to the Smoothened agonist, SAG. Through a cross-species analysis of SAG-induced transcriptional responses in cultured limb bud cells, we further determined that ∼46% of genes induced as a response to Hh pathway activation in A. sagrei are also SAG responsive in Mus musculus limb bud cells. Our results highlight conserved and diverged aspects of Hh signaling in anoles and establish a new resource for investigations of reptilian gene function.

Shh, Bmp-2 and Hoxd-13 gene expression in chick limb bud cells in culture.

The anteroposterior axis of the vertebrate limb bud is determined by signals from the zone of polarizing activity (ZPA). Sonic hedgehog (Shh) is expressed in the posterior mesoderm, which corresponds closely to ZPA activity. Moreover, Bmp-2 and HoxD genes are expressed in the broader posterior mesoderm, and it is thought that the ZPA signaling pathway consists of these gene products. Limb outgrowth and patterning, including expression of these genes, depend on the apical ectodermal ridge (AER). Fibroblast growth factors (FGF) have been identified as candidates for signal molecules from the AER. To further understand the ZPA signaling pathway and the participation of FGF, expressions of these genes were examined by reverse transcription-polymerase chain reaction in chick limb bud cells cultured with FGF-4. The present results indicate that FGF-4 cannot maintain Shh expression but can maintain Hoxd-13 expression in cultured posterior cells; moreover, Bmp-2 is expressed independently of FGF-4. These results suggest that Bmp-2 and Hoxd-13 expressions do not require a continuous expression of Shh. Further, it was demonstrated that posterior cells cultured with FGF-4 recovered Shh expression when grafted to the limb bud, indicating that FGF-4 maintains not Shh expression itself but competence of Shh expression.

Hoxd-12 differentially affects preaxial and postaxial chondrogenic branches in the limb and regulates Sonic hedgehog in a positive feedback loop.

Several 5' members of the Hoxd cluster are expressed in nested posterior-distal domains of the limb bud suggesting a role in regulating anteroposterior pattern of skeletal elements. While loss-of-function mutants have demonstrated a regulatory role for these genes in the developing limb, extensive functional overlaps between various different Hox genes has hampered elucidation of the roles played by individual members. In particular, the function of Hoxd-12 in the limb remains obscure. Using a gain-of-function approach, we find that Hoxd-12 misexpression in transgenic mice produces apparent transformations of anterior digits to posterior morphology and digit duplications, while associated tibial hemimelia and other changes indicate that formation/growth of certain skeletal elements is selectively inhibited. If the digital arch represents an anterior bending of the main limb axis, then the results are all reconcilable with a model in which Hoxd-12 promotes formation of postaxial chondrogenic condensations branching from this main axis (including the anteriormost digit) and selectively antagonizes formation of 'true' preaxial condensations that branch from this main axis (such as the tibia). Hoxd-12 misexpression can also induce ectopic Sonic hedgehog (Shh) expression, resulting in mirror-image polydactyly in the limb. Misexpression of Hoxd-12 in other lateral plate derivatives (sternum, pelvis) likewise phenocopies several luxoid/luxate class mouse mutants that all share ectopic Shh signalling. This suggests that feedback activation of Shh expression may be a major function of Hoxd-12. Hoxd-12 can bind to and transactivate the Shh promoter in vitro. Furthermore, expression of either exogenous Hoxd-11 or Hoxd-12 in cultured limb bud cells, together with FGF, induces expression of the endogenous Shh gene. Together these results suggest that certain 5' Hoxd genes directly amplify the posterior Shh polarizing signal in a reinforcing positive feedback loop during limb bud outgrowth.

環境中有機塩素化合物の相互作用の解析 ラット胎仔肢芽培養法を用いて

Interactions of binary mixutues among organochlorine compounds detected in the water and sediment of urban rivers were analysed in cultured limb bud cells using Toxic Unit analysis and isobole diagram.Type and mode of action of mixtures vary depedns on the mixture ratio and end points (chondrogenesis, cell proliferation) which resulted in varied potential of developmental toxicity among mixtures.As limb bud cell culture (LBC) assesses cell differentiation and cell proliferation simultaneously, LBC combined with Toxic Unit analysis and isobole diagram provides useful tool for analysis of interation of mixture.

FGF-4 maintains polarizing activity of posterior limb bud cells in vivo and in vitro.

The polarizing region is a major signalling tissue involved in patterning the tissues of the vertebrate limb. The polarizing region is located at the posterior margin of the limb bud and can be recognized by its ability to induce additional digits when grafted to the anterior margin of a chick limb bud. The signal from the polarizing region operates at the tip of the bud in the progress zone, a zone of undifferentiated mesenchymal cells, maintained by interactions with the apical ectodermal ridge. A number of observations have pointed to a link between the apical ectodermal ridge and signalling by the polarizing region. To test this possibility, we removed the posterior apical ectodermal ridge of chick wing buds and assayed posterior mesenchyme for polarizing activity. When the apical ectodermal ridge is removed, there is a marked decrease in polarizing activity of posterior cells. The posterior apical ectodermal ridge is known to express FGF-4 and we show that the decrease in polarizing activity of posterior cells of wing buds that normally follows ridge removal can be prevented by implanting a FGF-4-soaked bead. Furthermore, we show that both ectoderm and FGF-4 maintain polarizing activity of limb bud cells in culture.

Chondrogenesis in Chick Limb Bud Mesodermal Cells: Reciprocal Modulation by Activin and Inhibin

Activin and inhibin are members of the transforming growth factor-β superfamily sharing a common β-sub-unit, but with opposite biological activities on the release of follicle-stimulating hormone from pituitary. Recent studies have demonstrated that activin is the long sought after mesoderm induction factor in amphibians. To determine if activin and inhibin affect the proliferation and differentiation of chick limb bud mesodermal cells during cartilage formation of the limb, recombinant human activin A and inhibin A were tested on stage 24 chick limb bud mesodermal cells. The results showed that activin A has an inhibitory effect on chondrogenic differentiation, as indicated by Alcian blue staining and decreased [35S]sulfate incorporation into proteoglycans. In addition, the expression of type II collagen mRNA, a specific phenotypic marker of cartilage, was greatly inhibited when cultures were exposed to activin A for 5 days at a dose of 10 ng/ml. In contrast, inhibin A stimulated cartilage formation, as indicated by increased expression of type II collagen mRNA and synthesis of proteoglycans. These results imply that activin A and inhibin A can modulate chondrogenesis during differentiation of limb bud cells in culture.

Teratogenicity of phenylhydantoins in an in vitro system: molecular orbital-generated quantitative structure-toxicity relationships.

1. The ability of 20 mono- and di-phenylhydantoin derivatives to inhibit differentiation of rat embryo mid-brain and limb bud cells in culture has been used as an index of the teratogenic hazard represented by these compounds. 2. Molecular orbital calculations on these compounds, using the MINDO-3 (modified intermediate neglect of differential overlap) and CNDO-2 (complete neglect of differential overlap) methods, were combined with indices of teratogenicity in the two cell types, to generate a coherent structure-toxicity relationship. 3. Teratogenicity correlated with frontier orbital electron density of the N1 hydantoin ring atom (HOMO-N1) in a sub-series of 12 monophenylhydantoins, whereas the corresponding toxicity for both mono- and di-phenylhydantoins related more to the molecular polarizability (alpha mol) of the molecule. 4. Furthermore the same structural parameter (alpha mol) exhibited a parallelism with log P values of these 20 compounds, indicating the importance of lipophilicity in the toxicity of these compounds. 5. Overall, the data emphasize the ability of electronic structural calculations to identify chemical descriptors of toxicity.

Effects of benzimidazoles on mouse and rat limb bud cells in culture

Benzimidazoles exhibited an inhibitory action on the chondrogenesis in a mouse limb bud cell culture system; further, nitro and chloro groups at the 5 position in 2-(2-pyridyl)benzimidazole were found to be potent substituents. However, 2-(2-pyridyl)benzoxazole and 5-methoxy-2-(2-pyridyl)benzothiazole exhibited no inhibitory action under our experimental conditions. Thus, imidazole NH proton seemed to be important in the inhibitory action in the mouse cell system. The concentrations of thiabendazole (TBZ), 5-hydroxy-TBZ (5-HY-TBZ) and N-methyl-TBZ necessary to reduce the amounts of cartilage proteoglycan by 50% (TP 50) were estimated to be about 0.35, 0.25 and 0.70 mM, respectively, in the rat limb bud cell culture system. The TP 50 of TBZ and 5-HY-TBZ in the rat cell system were 3- to 4-fold higher than those in the mouse cell system.

Cell shape and cartilage differentiation of early chick limb bud cells in culture

The onset of chondrogenesis in the embryonic chick is preceded by a prechondrogenic condensation of the prospective cartilage cells. Similarly, in culture, the chondrogenic phenotype is only expressed by limb mesoderm cells plated at densities above confluence. This has led to the proposal that chondrogenic differentiation requires immediate histogenic interactions prior to overt chondrogenesis (Solursh et al., 1978, In Vitro 14, 51–61; and, 1980, Dev. Biol. 78, 141–150). It is noticeable that in both the in vivo and in vitro situations cells secreting a cartilaginous matrix are rounded in shape. Recently, Glowacki, Trepman and Folkman (personal communication) have shown a dependence of phenotypic expression by mature chondrocytes on cell shape. Cells maintained in a rounded configuration by culturing on a semi-adhesive substratum (poly(HEMA)) synthesised more sulphur-containing extracellular matrix than cells allowed to flatten on normal tissue culture plastic. We have investigated whether there is a similar shape-dependent relationship in the differentiation of chick embryonic mesoblasts into actively secreting chondroblasts. Our results show that a rounded cell shape is conducive to the synthesis of a sulphated matrix.

Chondrogenesis: a model developmental system for measuring teratogenic potential of compounds.

A simple test for determining the teratogenic potential of compounds is described using embryonic limb bud cells in culture. These mesenchyme cells multiply and differentiate into chondrocytes during a 6-day culture period. The extent of chondrogenesis is assessed by staining for the cartilage specific proteoglycan with alcian blue. The amount of stain is then measured spectrophotometrically. Compounds which interfere with growth or differentiation reduce the amount of proteoglycan and as a consequence, reduce alcian blue staining. Compounds can be added directly to the media or be activated using several different metabolizing systems. The dose of a compound needed to reduce alcian blue staining by 50% is designated the teratogenic potential (TP50) of that compound. TP50's of proven teratogens compare favorably with in vivo teratogenic doses of the teratogens.