Elsevier

Gene

Volume 222, Issue 1, 5 November 1998, Pages 119-124
Gene

Identification of newt connective tissue growth factor as a target of retinoid regulation in limb blastemal cells

https://doi.org/10.1016/S0378-1119(98)00478-8Get rights and content

Abstract

In order to analyse target genes regulated by retinoic acid in urodele limb regeneration, we have used pseudotyped retroviruses to obtain stably transfected newt limb blastemal (progenitor) cells in culture which express chimeric retinoic acid/thyroid hormone receptors δ1 or δ2. After treatment with thyroid hormone to activate the chimeric receptors, we used a polymerase chain reaction (PCR)-based subtraction method to identify target genes which are retinoid regulated. Newt connective tissue growth factor, a secreted protein recognised in several vertebrates, has been identified in this way and found to be expressed in the limb blastema and regulated by retinoic acid. This approach should permit a systematic analysis of retinoid target genes in limb regeneration.

Introduction

Limb regeneration in urodele amphibians, such as the newt and axolotl, proceeds via the formation of a blastema – a structure containing dividing mesenchymal progenitor cells (for review see Brockes, 1997). The blastema regenerates just those structures which are distal to its level of origin on the proximodistal axis – thus a wrist blastema gives rise to a hand, a shoulder blastema to an arm. The positional identity of the blastema is respecified in a proximal direction by RA, its precursor retinoids such as retinol, and synthetic retinoids such as TTNPB, all of which are able to activate the RAR family of nuclear receptors (Maden, 1982; Kim and Stocum, 1986). The action of retinoids on the limb blastema is of significance for our understanding of the molecular basis of positional identity, and also for the identity of other target genes which may be implicated in the diverse effects of retinoids on limb cells.

The newt limb and limb blastema express at least five members of the RAR family in both the epidermis and mesenchyme, and all five may be activated on exposure to RA (Ragsdale et al., 1989, Ragsdale et al., 1992, Ragsdale et al., 1993). In order to overcome this problem of receptor multiplicity, we have constructed five chimeric RARs (χRARs) by replacing the RA binding domain (EF regions) of each RAR with the corresponding region of the Xenopus T3 receptor-α (Schilthuis et al., 1993; Gann et al., 1996). If a particular χRAR is transfected into a newt limb cell and exposed to T3, then a single RAR isoform is activated. This approach has revealed that inhibition of blastemal cell division by RA is mediated by RARα1 (Schilthuis et al., 1993), that induction of the WE3 antigen in the epidermis is mediated by δ1 (Pecorino et al., 1994), while activation of δ2 changes proximodistal identity, as assayed by the distribution of transfected cells in intercalary regeneration (Pecorino et al., 1996).

In order to systematically investigate the target genes which are up- or down-regulated by specific RAR isoforms in blastemal cells, it would be desirable to stably transfect a single χRAR into cultured newt cells, although the stable transfection of urodele cells has not previously been demonstrated. In the present account we describe the use of pseudotyped retroviral vectors (Burns et al., 1993; Yee et al., 1994) to accomplish this for χRARs δ2 and δ1. The stably transfected blastemal cells have been used to identify a new target gene for RA in this context – the newt homologue of human connective tissue growth factor (CTGF).

Section snippets

Cells and cell culture

Newt (Notophthalmus viridescens) B1H1 hindlimb blastemal cells were maintained in culture essentially as described (Ferretti and Brockes, 1988; Brockes, 1992) in supplemented 63% Minimal Eagle's Medium with 10% selected fetal bovine serum. Cells were propagated in gelatin-coated flasks and were initially infected with pseudotyped retrovirus (see below) between passages 19 and 24. The newt cells have an indefinite life span under these conditions of culture (Ferretti and Brockes, 1988; Powell et

Construction and verification of blastemal cell lines expressing chimeric receptors

The cultured newt cells used in these experiments were derived originally from mesenchymal explants of an adult hind limb blastema, and were maintained in culture as dividing populations of indefinite life span (Ferretti and Brockes, 1988; Powell et al., 1998). They express markers typical of blastemal cells, including the 22/18 antigen (Kintner and Brockes, 1985; Ferretti and Brockes, 1988) and the K8/18 cytokeratin pair (Ferretti et al., 1989). The transfection efficiency for B1H1 cells is

Discussion

The pseudotype retroviruses have proved effective vectors for the isolation of stably transfected blastemal cell lines. Although it is not possible at present to obtain clonal growth with these cells, the neo selection is apparently effective in that essentially all the cells can be shown to express the chimeric receptors after selection. The variation in the level of expression in independent isolates (Fig. 1) is consistent with a relatively low number of clones being present in the final

Acknowledgements

We thank Dr Jane Burns for much helpful advice and for the gift of strains for construction of pseudotyped retroviruses, Dr Alex Gann for his participation in early experiments with pseudotypes, and Dr Anoop Kumar for his help with Fig. 2. David E. Cash is a recipient of a Burroughs Wellcome Fund Hitchings-Elion Fellowship. This work was supported by a Programme Grant from the Medical Research Council.

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    Present address: Department of Zoology, 3029 Cordley Hall, Oregon State University, Corvallis, OR 97331, USA.

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