In this study, we report the cloning of the chicken orthologue of C. elegans lin-41. During limb development, clin-41 is expressed in three phases, and we present data consistent with the hypothesis that clin-41 is regulated by FGFs from the AER and SHH from the ZPA. Furthermore, potential miRNA binding sites were identified in the 3′-UTR of chicken lin-41, including five sites for let-7 and one each for miR-125b, miR-225b, and miR-15a. These miRNAs are expressed in the chick limb at the appropriate developmental time consistent with the hypothesis that miRNAs regulate cLin-41 function.
The prevalence of annotation errors became clear to us from three observations. First, the mouse LIN-41 we have aligned is actually composed of two predicted genes that are adjacent to each other on mouse chromosome 9. In the mouse, only one is correctly identified as Lin-41. To construct the putative mouse LIN-41 protein sequence for our alignment, we combined the two protein sequences (accession no. XM357972 and accession no. XM356199). Second, using the nucleic acid sequence coding for the RING finger domain from human lin-41, we were able to identify homologous sequence in the mouse genome very near to the Lin-41 locus on chromosome 9. Third, the predicted chicken LIN-41 proteins from NCBI (GenBank accession no. XM426005) and ENSEMBL (GeneScan ID 00000012620) diverge from each other and with our predicted chicken LIN-41 protein beginning in the Coiled-coil domain. It is notable that our chicken LIN-41 shares greater overall similarity to human, mouse, and rat LIN-41 than do either of the chicken proteins predicted by NCBI and ENSEMBL. Further refinement of vertebrate genomic DNA sequence will help clarify this problem.
Does clin-41 Have a Role in Limb Development?
The Progress Zone model describes patterning events along the PD axis of the vertebrate limb and proposes that specification and determination of the limb occurs in a proximal–distal manner over time (Summerbell et al., 1973). In this model, the longer a cell remains in the Progress Zone (the undifferentiated, subridge mesoderm) under the influence of Fgfs from the AER, the more distal fate it acquires. Therefore, more distal limb structures are specified at progressively later times in development. Recently, new data and reanalysis of original studies related to PD growth challenge the idea that patterning occurs in a proximal–distal manner over time (Dudley et al., 2002; Sun et al., 2002). This work has led to a new model that proposes each region of the limb (stylopod, zeugopod, autopod) is specified very early in development, and expansion of these regions over time results in determination and growth of recognizable skeletal elements.
Whereas the concept of developmental time is explicit in the Progress Zone model, a timing mechanism also underlies the Expansion model. Based on experimental evidence, differentiation of the limb occurs in proximal–distal manner over time, such that more proximal skeletal elements differentiate before more distal elements (Saunders, 1948; Summerbell, 1974; Rowe and Fallon, 1982). With this in mind, we can speculate on a role for cLin-41 in vertebrate limb development.
In the limb, clin-41 is expressed in three phases whose timing correlate with other well-defined developmental and molecular events. Perhaps most significant is the correlation with events related to development of the autopod. According to the Progress Zone model, autopod specification begins between stages 21 and 25 (wing: Saunders, 1948; Summerbell et al., 1973; leg: Rowe and Fallon, 1982), a time that correlates with phase two of clin-41 expression. Also at this time, the Hoxd genes initiate a SHH-dependent expression pattern that also correlates with morphogenesis of the autopod (Nelson et al., 1996; Zákány et al., 2004). Phase changes for both clin-41 and Hoxd genes at similar times is not surprising as both are dependent upon Shh signaling for correct spatial expression. This point is further supported by the observation that the expression patterns of clin-41 and the Hoxd genes are similarly altered in ozd and ta2 mutants (compare Fig. 5 in this manuscript with Fig. 5B, F, and J in Ros et al., 2003, and Fig. 1J in Caruccio et al., 1999). For clin-41, the timing of the phase change and distal expression in the future autopod suggests an involvement in autopod morphogenesis.
In the absence of Shh signaling, clin-41 expression is initially normal. It is then down-regulated and becomes undetectable by whole-mount in situ analysis at stage 23/24. Furthermore, loss of Gli3, the primary transcriptional effector of the Shh pathway in the limb, results in significant up-regulation of mlin-41 in the anterior limb mesoderm. Therefore, during normal limb development, we propose that Shh signaling is necessary to maintain appropriate clin-41 expression. We also propose that clin-41 is maintained by Fgf signaling from the AER. The specific removal or reduction of FGF-8 signaling from the AER also results in the absence or reduction of Shh signaling. The interaction between SHH and FGF-8 is the main component of the feedback loop between the AER and ZPA (Laufer et al., 1994; Niswander et al., 1994; Lewandoski et al., 2000; Moon and Capecchi, 2000). In cases where all Fgf signaling is lost and Shh is not expressed, for example in the limbless limb bud, clin-41 expression is rapidly lost. However, when FGF-8 and FGF-4 are removed, Shh signaling is lost but other FGFs remain active in the AER (Sun et al., 2002; Boulet et al., 2004). In this case, low, residual lin-41 expression is maintained. Together, the data indicate that lin-41 is genetically downstream of both Shh and Fgf, and normal lin-41 expression is dependent upon both signaling pathways for normal expression.
Beginning at phase two, clin-41 is down-regulated in Shh-expressing ZPA cells at a time when the autopod is being determined in the leg (see Rowe and Fallon, 1982). Because clin-41 down-regulation occurs in Shh-expressing cells beginning at phase two, we hypothesize that clin-41 down-regulation occurs in cells that have expressed high levels of SHH for long periods of time. It is possible, therefore, that clin-41 is reflecting the temporal gradient of SHH exposure proposed by Harfe and coworkers (2004; see also Yang et al., 1997).
In the limb, Shh signaling influences several aspects of muscle development, including specification, proliferation, and delayed differentiation (reviewed in Christ and Brand-Saberi, 2002). Recent studies have indicated that, in precursor muscle cells, Shh signaling controls muscle growth in the limb by influencing the balance between cell proliferation and differentiation, thus, ensuring the formation of appropriately sized muscle masses (Amthor et al., 1998). Delayed differentiation of precursor muscle cells results in overgrowth of muscle cells, whereas precocious differentiation results in muscle loss, two situations induced by SHH overexpression or loss of Shh signaling, respectively (Amthor et al., 1998; Bren-Mattison and Olwin, 2002). In C. elegans, LIN-41 ensures the completion of L4 larval stage and initiation of the adult program by inhibiting the activity of LIN-29, thus delaying the activation of adult cell specific differentiation (Slack et al., 2000). Based on its role in C. elegans and because clin-41 is likely dependent upon Shh signaling in the limb, we propose that cLIN-41 coordinates the proper timing of muscle cell proliferation and/or differentiation in the developing limb. cLIN-41 would be a Shh signaling effector molecule in the developing muscle precursor cells.
In the developing limb, correct spatial expression of clin-41 is dependent on both Shh and Fgfs. We have identified the presence of miRNA binding sites in the clin-41 3′-UTR and report the presence of miRNAs in the developing chick limb. Using a reporter assay, work from Mansfield and coworkers (2004) showed that let-7c and e are asymmetrically active in the developing mouse limb. Interestingly, the activity domain of let-7c is complementary to the expression pattern we show for mlin-41in the mouse limb at an equivalent stage (compare Fig. 4C or 5D of this manuscript with Fig. 2B in Mansfield et al., 2004). We also used the miRNA prediction algorithm on mouse lin-41 3′-UTR and let-7c scored highly, indicating that it may regulate mlin-41. Similar to C. elegans lin-41, there are several LCSs in vertebrate lin-41 3′-UTRs. Given the potential presence of LCSs in all vertebrate lin-41 3′-UTRs and that let-7 is expressed in the developing limbs of mouse and chick when lin-41 is expressed, our data support the hypothesis that let-7 regulates lin-41 in the developing vertebrate limb. miRNA regulation of vertebrate Lin-28, another C. elegans heterochronic gene orthologue, has also been proposed indicating that specific miRNA-target gene interactions can be highly conserved (Moss and Tang, 2003). A detailed comparison of lin-41 transcript distribution, protein distribution and let-7 activity will be necessary to better characterize this proposed regulatory interaction.
The function of Lin-41 in limb development is unknown. Based on its function in C. elegans, it is reasonable to propose that Lin-41 regulates the timing of cellular proliferation and/or differentiation in the developing limb. Whether removal of LIN-41 function would impact the developmental program of a specific region of the limb, e.g., the autopod, or whether it would affect the entire limb is an intriguing question. In the limb, we hypothesize that clin-41 expression is dependent upon Shh and Fgf signaling and most likely miRNAs. The possible regulation of Lin-41 by miRNAs in the limb suggests the presence of a highly conserved developmental pathway (reviewed in Harfe, 2005; Pasquinelli et al., 2005); this adds another level of complexity to understanding how the output of the Shh and Fgf pathways are ultimately translated into the proper patterning and growth of a developing limb.