110
M. Maden
acts via fgf-4 through the chick AER to induce shh, but there is no AER in amphibians), or, they may be due to the concentration and stage-dependent effects of RA
(that produce deletions or duplications when administered at different times or at
different concentrations).
Current State of the Field
Complexities of Limb Formation
While many studies have demonstrated that all three axes of the limb are specified/respecified by RA, mechanistic studies have been invariably conducted on a
single axis—either the PD axis involving tbx5—meis—cyp26b1 or the AP pathway
involving dHand—shh—hoxd-12. Surprisingly little attention has been paid to the
DV axis or to the concerted coordination of all three axes. However, the existence
of a coordinated elaboration has been seen in studies where increasing doses of RA
were used to rescue raldh2
−/− mutant limb buds. Gradually increasing RA leads to
different degrees of development in both the AP and PD, and by implication, the DV.
An excellent example of an interaction between axes with regard to RA has been
revealed by a transcriptome analysis of shh-deficient mouse forelimb buds (Probst
et al. 2011). In these shh-deficient limb buds, many genes, including RARβ and meis2,
that are normally expressed proximally, were up-regulated and expressed ectopically
in a distal domain. Conversely, cyp26b1 was down-regulated. The analysis nicely
shows how shh and RARβ serve as a crucial link between the PD and AP axis during
limb outgrowth, with RA inducing shh, and then, shh down-regulating RA using an
AER-FGF-CYP26B1 loop to allow distal outgrowth. Similar analyses in the future
will surely be very productive for understanding the interactions between limb axes.
In addition, contemporary models of gene interactions in limb development
include a recognition of the very different conditions and roles that the same molecule
can exhibit during the course of limb formation (Zeller et al. 2009; Bénazet et al.
2009). For example, BMP is first required for AER formation at initially high expression levels. Then, at reduced levels, it regulates AER length during proliferative
expansion. Finally, at elevated expression levels, BMP participates in the determination of digit identities. Similarly, relatively high levels of RA are required to induce
limb and shh induction, but normal outgrowth only proceeds if RA levels are kept
low by AER-FGF- mediated up-regulation of cyp26b1. Relatively high levels of RA
are required for interdigital cell death.
The Contribution of Modeling to Understanding RA Pathways
Currently, mathematical models are being developed and tested to investigate RA
signaling actions and gene interactions. Recently, reaction-diffusion modelling has
M. Maden
acts via fgf-4 through the chick AER to induce shh, but there is no AER in amphibians), or, they may be due to the concentration and stage-dependent effects of RA
(that produce deletions or duplications when administered at different times or at
different concentrations).
Current State of the Field
Complexities of Limb Formation
While many studies have demonstrated that all three axes of the limb are specified/respecified by RA, mechanistic studies have been invariably conducted on a
single axis—either the PD axis involving tbx5—meis—cyp26b1 or the AP pathway
involving dHand—shh—hoxd-12. Surprisingly little attention has been paid to the
DV axis or to the concerted coordination of all three axes. However, the existence
of a coordinated elaboration has been seen in studies where increasing doses of RA
were used to rescue raldh2
−/− mutant limb buds. Gradually increasing RA leads to
different degrees of development in both the AP and PD, and by implication, the DV.
An excellent example of an interaction between axes with regard to RA has been
revealed by a transcriptome analysis of shh-deficient mouse forelimb buds (Probst
et al. 2011). In these shh-deficient limb buds, many genes, including RARβ and meis2,
that are normally expressed proximally, were up-regulated and expressed ectopically
in a distal domain. Conversely, cyp26b1 was down-regulated. The analysis nicely
shows how shh and RARβ serve as a crucial link between the PD and AP axis during
limb outgrowth, with RA inducing shh, and then, shh down-regulating RA using an
AER-FGF-CYP26B1 loop to allow distal outgrowth. Similar analyses in the future
will surely be very productive for understanding the interactions between limb axes.
In addition, contemporary models of gene interactions in limb development
include a recognition of the very different conditions and roles that the same molecule
can exhibit during the course of limb formation (Zeller et al. 2009; Bénazet et al.
2009). For example, BMP is first required for AER formation at initially high expression levels. Then, at reduced levels, it regulates AER length during proliferative
expansion. Finally, at elevated expression levels, BMP participates in the determination of digit identities. Similarly, relatively high levels of RA are required to induce
limb and shh induction, but normal outgrowth only proceeds if RA levels are kept
low by AER-FGF- mediated up-regulation of cyp26b1. Relatively high levels of RA
are required for interdigital cell death.
The Contribution of Modeling to Understanding RA Pathways
Currently, mathematical models are being developed and tested to investigate RA
signaling actions and gene interactions. Recently, reaction-diffusion modelling has