There is mechanistic support for this model based on the functional actions of RA and Fgf8. As we described earlier for forelimb field initiation, RA and Fgf8 exhibit an antagonistic relationship toward one another that is mediated on at least two levels: direct repression and the differential regulation of gene targets (see Figure 19.13A). RA functions as a direct transcriptional repressor of Fgf8 expression (Kumar and Duester 2014); therefore, as outgrowth of the limb bud progresses, the AER (and source of Fgf8) will move outside the reach of RA, enabling greater Fgf8 expression over time. In a more direct counter to RA, Fgf8 upregulates cytochrome P450 26 (Cyp26) proteins that degrade RA (Probst et al. 2011). In addition, RA and Fgf8 differentially regulate proximal-distal determining genes, such as Meis1/2, Hoxa11, and Hoxa13 in the stylopod, zeugopod, and autopod, respectively (Cooper et al. 2011). For instance, RA promotes the expression of the more proximal Meis1, whereas Fgf8 inhibits expression of this gene. The opposite relationship holds true for the more distal Hoxa13 (see Figure 19.13A, C). The upregulation of Meis1 by RA is protective because, aside from promoting extreme proximal cell fates, Meis1 protein also represses Cyp26b1 transcription. In fact, recent refinements of this model suggest that there are two distinct thresholds of RA-to-Fgf8 signaling. A relatively high RA-to-Fgf8 threshold determines the stylopod-to-zeugopod transition, while a low RA-to-Fgf8 threshold determines the zeugopod-to-autopod transition (Roselló-Díez et al. 2014). Moreover, there appears to be an epigenetic hold on autopod gene expression (Hoxa13) to allow the necessary time for zeugopod development. Pharmacological inhibition of histone deacetylases (HDACs) with a TSA-coated bead implanted into the early limb bud results in precocious Hoxa13 expression and specifically reduced skeletal elements of the zeugopod (Figure 1; Roselló-Díez et al. 2014).
Taken together, these data support a dual gradient model for proximal-to-distal patterning in the chick limb (reviewed by Tanaka 2013; Roselló-Díez et al. 2014; Cunningham and Duester 2015; Zúñiga 2015). The patterning stage is set prior to signs of limb formation in the lateral plate mesoderm, where RA is highly expressed and induces Meis1/2 expression throughout the limb field and early limb bud (thus supporting stylopod specification). Soon after, an opposing gradient of Fgf8 and Wnt from the AER antagonizes RA signaling along the proximal-distal axis. A distancing of the AER from the flank by proliferative outgrowth results in diminished RA signaling and enhancement of Fgf8, until a threshold of RA:Fgf8 signaling triggers Hoxa11 gene expression and Meis1/2 downregulation (yielding zeugopod differentiation). As a result of the reduction in Meis1/2 function, CYP26b1-mediated degradation of RA intensifies to reach the next threshold for autopod development. Although at this point the distal mesenchyme may be permissive for autopod fate specification, it is not until chromatin regulation permits access for the transcription of Hoxa13 that autopod differentiation commences (see Figure 19.13). This epigenetic regulation of delay in autopod development enables greater cell contributions to the zeugopod lineage, influencing its size, which may represent an important mechanism employed throughout development to shape the embryo.
Autonomous or nonautonomous yet again? It seems that the current challenge for the
limb field (and you) will be to further investigate the nature of, and interactions between, autonomous and nonautonomous mechanisms. The model should include mechanisms—epigenetic or otherwise—for controlling developmental time. Do you think the evidence is there to support a dual gradient model, or are distal gradients of FGF proteins alone sufficient?
Cooper, K. L., J. K. Hu, D. ten Berge, M. Fernandez-Teran, M. A. Ros and C. J. Tabin. 2011. Initiation of proximal-distal patterning in the vertebrate limb by signals and growth. Science 332: 1083–1086.
Cunningham, T. J. and G. Duester. 2015. Mechanisms of retinoic acid signalling and its roles in organ and limb development. Nat. Rev. Mol. Cell Biol. 16: 110–123.
Kumar, S. and G. Duester. 2014. Retinoic acid controls body axis extension by directly repressing Fgf8 transcription. Development 141: 2972–2977.
Probst, S., C. Kraemer, P. Demougin, R. Sheth, G. R. Martin, H. Shiratori, H. Hamada, D. Iber, R. Zeller and A. Zuniga. 2011. SHH propagates distal limb bud development by enhancing CYP26B1-mediated retinoic acid clearance via AER-FGF signalling. Development 138: 1913–1923.
Roselló-Díez, A., C. G. Arques, I. Delgado, G. Giovinazzo, and M. Torres. 2014. Diffusible signals and epigenetic timing cooperate in late proximo-distal limb patterning. Development 141: 1534–1543.
Tanaka, M. 2013. Molecular and evolutionary basis of limb field specification and limb initiation. Dev. Growth Differ. 55: 149–163.
Zúñiga, A. 2015. Next generation limb development and evolution: Old questions, new perspectives. Development 142: 3810–3820.
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