In the vertebrate limb, appropriate growth and patterning is dependent on the activities of well-defined signaling centers. The apical ectodermal ridge (AER), a thickened epithelium at the apex of the distal limb, controls outgrowth along the proximal–distal axis through the activity of the fibroblast growth factors (FGFs; Saunders, 1948; Mariani and Martin, 2003). Fgfs from the AER permit continued outgrowth of the limb by preventing apoptosis and ensuring proliferation of responsive cells in the underlying subridge mesoderm (Rowe et al., 1982; Dudley et al., 2002). A group of mesodermal cells located along the posterior border of the developing limb called the zone of polarizing activity (ZPA) controls growth and patterning of the anterior–posterior (AP) axis. ZPA cells synthesize and secrete Sonic hedgehog (SHH), a protein critical for appropriate patterning of the AP axis (Echelard et al., 1993; Riddle et al., 1993; Lopez-Martinez et al., 1995; Marti et al., 1995; Pearse and Tabin, 1998). Additionally, descendants of ZPA cells directly contribute to mouse posterior digits (Harfe et al., 2004).
Limb skeletal determination occurs in a proximal–distal (PD) manner over time (Saunders, 1948; Summerbell, 1974; Rowe and Fallon, 1982). The observation that more proximal skeletal elements differentiate before more distal elements indicates the presence of an inherent timing mechanism in limb development (Searls, 1965; Thorogood and Hinchliffe, 1975). Interestingly, recent work has raised the possibility that a timing mechanism may be essential to understand patterning along the AP axis (Yang et al., 1997; Harfe et al., 2004). Originally, it was hypothesized that SHH secreted by ZPA cells acts as a morphogen to specify cell fate in a concentration dependent manner (Wolpert, 1969; Tickle et al., 1975; Riddle et al., 1993). Recently, a new model emphasizes the importance of developmental time as well as SHH concentration and proposes that the longer a cell expresses SHH, the more posterior its fate (Harfe et al., 2004).
Surprisingly little progress has been made in identifying genes that regulate the timing of developmental events in the vertebrate limb. Although progress has been made in other developing vertebrate systems (e.g., somitogenesis; reviewed by Pourquie, 2003), considerable insight has come from studying the heterochronic pathway in the nematode Caenorhabditis elegans (reviewed in Ambros, 2000; Rougvie, 2001; Pasquinelli and Ruvkun, 2002). Specifically, postembryonic development of C. elegans proceeds through four larval stages (L1–L4) before emergence of the adult. Orderly progression through these larval stages is critical for the realization of the adult phenotype. Each larval stage is characterized by the formation of specific cell types and execution of stage-specific programs. The heterochronic gene pathway controls the timing of these developmental events.
Progression from L4 to the adult developmental program is partially controlled by the heterochronic gene lin-29, which codes for a zinc-finger transcription factor (Rougvie and Ambros, 1995). Worms lacking LIN-29 reiterate the L4 program and do not initiate an adult program (Ambros and Horvitz, 1984). Overexpression of LIN-29 induces precocious initiation of adult programs before completion of the L4 program (Bettinger et al., 1996). Thus, temporal control of LIN-29 activity ensures timely completion of the L4 program and subsequent initiation of the adult program.
One factor known to temporally regulate LIN-29 function is Lin-41 (Slack et al., 2000). C. elegans LIN-41 contains an amino-terminus RING finger domain, two B-boxes, a Coiled-coil and NHL domain. This cluster of conserved motifs, excluding the NHL domain, places LIN-41 in the RING Finger, B-Box Coiled-Coil (RBCC) family of proteins that is now also called TRIpartite Motif (TRIM) proteins (Saurin et al., 1996; Borden, 1998; Reymond et al., 2001). RBCC/TRIM protein family members contain various combinations of the aforementioned domains, whereas the NHL protein domain is only associated with a subset of RBCC/TRIM proteins (Slack and Ruvkun, 1998).
Lin-41 was first isolated and functionally characterized in C. elegans, and loss of Lin-41 function results in precocious LIN-29 activity (Slack et al., 2000). Although the precise mechanism by which Lin-41 regulates Lin-29 is not known, regulation of Lin-41 is better defined. In the 3′-untranslated region (UTR) of lin-41 are complementary binding sites for the miRNAs let-7 and lin-4 (Slack et al., 2000). Recent work has shown that two let-7 complementary sites (LCSs; terminology of Vella et al., 2004a, b) are necessary and sufficient for down-regulation of a reporter construct fused to the 3′-UTR of lin-41 in C. elegans and zebrafish (Kloosterman et al., 2004; Vella et al., 2004a). These results indicate that regulation of lin-41 by let-7 may be conserved across species in vivo.
Two reports demonstrate that orthologues of C. elegans LIN-28 and let-7 are temporally expressed in vertebrate embryos (Moss and Tang, 2003; Pasquinelli et al., 2000). These data combined with the identification of probable vertebrate orthologues of lin-41 strongly suggest conservation of the heterochronic pathway during vertebrate embryogenesis (Slack et al., 2000; Kloosterman et al., 2004). Here, we describe the cloning and expression pattern of the chicken orthologue of C. elegans lin-41 in the developing embryo. We focus our analysis on the developing limb and report that clin-41 is expressed in three distinct phases during development. Furthermore, we perform functional analysis to determine the epistatic relationship between lin-41 and the main limb bud signaling centers in the chick and mouse. Finally, we use a bioinformatics approach to determine potential miRNA binding sites in the 3′-UTR of clin-41 and Northern analysis to determine whether these miRNAs are expressed in the chick limb.