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Pownall ME, Isaacs HV. FGF Signalling in Vertebrate Development. San Rafael (CA): Morgan & Claypool Life Sciences; 2010.

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FGF Signalling in Vertebrate Development.

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FGF and Left-Right Asymmetry

Vertebrates, including humans, typically appear symmetrical from the outside; however, internally, there is clear left–right (LR) asymmetry: the heart, stomach, and spleen are on the left, while the liver and gall bladder are on the right; the right side of the lungs has more lobes than the left, and the intestines coil counterclockwise. This patterning is set up very early during development and requires the asymmetric expression of nodal in the left lateral plate mesoderm. This is a conserved aspect of normal LR patterning in all vertebrates studied; however, distinct mechanisms have been found to regulate left-sided nodal expression in different species (reviewed in Raya and Belmonte, 2006). In addition, many vertebrates have a ciliated laterality organ. In mouse and chick, it is the node, in zebrafish it is Kupffer’s vesicle (KV), and in frog it is the gastrocoel roof plate (GRP). The cilia in these laterality organs are motile and create a directional fluid flow. This directional flow is essential for LR patterning, and this has been dramatically demonstrated by exogenously manipulating the direction of the flow to impact LR asymmetry in cultured mouse embryos (Nonaka et al., 2002). Somewhat confusingly, this leftward flow of extraembryonic fluid is referred to as “nodal flow”; and although the TGFβ molecule nodal plays a conserved and essential role in LR asymmetry, “nodal flow” does not refer the protein nodal but rather to the node.

FGF8 has been implicated in the early processes that regulate LR asymmetry (Figure 15). Mice that are null for FGF8 do not develop through gastrula stages; however, in mice heterozygous for a hypomorphic allele (FGF8neo/–), about half were found to be abnormal in their LR patterning and lack nodal expression in the left lateral plate mesoderm (Meyers and Martin, 1999). In amniotes, FGF8 is expressed in the primitive streak, and in chick FGF8 expression extends anteriorly from the streak on the right-hand side of Hensen’s node during very early somite stages where it inhibits the expression of nodal (Boettger et al., 1999). Implanting FGF beads into the left lateral plate mesoderm in chick represses nodal expression and randomises the direction of heart looping. This is not seen in the mouse; indeed, the finding that FGF8 mutant mice do not express nodal and that implanting an FGF8 bead will induce nodal expression in the right lateral plate mesoderm is in striking contrast to the results in chick (Meyers and Martin, 1999). These contradictory data were probed further by investigating effects of FGF8 on LR asymmetry in cultured rabbit embryos, which, like chick, develop as a flat disc, while mice develop as a cylinder. Similar to mice, FGF8 was expressed symmetrically in the primitive streak in rabbits; however, similar to chick, FGF8 beads repressed endogenous nodal expression in the left lateral plate mesoderm. Application of SU5402 to the right lateral plate mesoderm resulted in bilateral nodal expression, indicating that in rabbit, as in chick, FGF8 acts to repress nodal expression (Fischer et al., 2002). The surprisingly different roles for FGF8 in two mammals point to the importance of the relative anatomy of the embryo in determining FGF8 function. Interestingly, the ability of FGF8 to repress nodal on the right in rabbits was found to require intercellular communication through gap junctions; the leftward nodal flow is thought to relieve FGF repression on the left by locally restricting gap junction communication (Feistel and Blum, 2008).

FIGURE 15. FGF8 regulates Nodal expression differently in chick and mouse embryos during LR axis specification.


FGF8 regulates Nodal expression differently in chick and mouse embryos during LR axis specification. In the mouse, FGF8 (shown in red) is expressed symmetrically in the primitive streak and is necessary and sufficient for the Expression of Nodal in the (more...)

Cilia-driven nodal flow is considered the earliest symmetry-breaking event during LR patterning because mice lacking motile cilia (Nonaka et al., 1998) have disrupted expression of LR markers and impaired directionality of heart looping. By labelling lipid membranes, Tanaka et al. (2005) were able to visualise remarkable particles less than 0.5 μm in diameter that carry Sonic hedgehog protein and RA that were being transported toward the left in the ventral node of one- to three-somite-stage mouse embryos. The release of these nodal vesicular particles (NVPs) from microvilli was found to be dependent on FGF signalling; as in embryos treated with SU5402, there is a failure to launch NVPs and they remain associated with the microvilli membrane in an intermediate form (Tanaka et al., 2005).

As in mice, fish mutant for fgf8 show defects in organ and pharyngeal skeleton laterality. In zebrafish, the ciliated laterality organ KV is absent in approximately one third of fgf8 mutants (acerebellar) pointing to a role for fgf8 in supporting the development of KV (Albertson and Yelick, 2005). KV is derived from the dorsal forerunner cells that express notail (ntl), a gene known to dependent on FGF signalling (Griffin et al., 1995), and the expression of ntl in the dorsal forerunner cells is deficient in ace mutant fish (Albertson and Yelick, 2005) suggesting that FGF8 is required in KV progenitors. In another study, AMOs were used to knock down fgf8 (as well as some downstream effectors of FGF signalling), which revealed a dramatic loss of cilia in KV and random expression of laterality markers including southpaw (spw) and lefty (lfty) in embryos deficient in FGF8 signalling (Hong and Dawid, 2009).

LR patterning was also investigated at a later time point, during the development of brain asymmetry in the diencephalic epithalamus (Regan et al., 2009). In zebrafish, parapineal precursors move from their bilateral positions at the midline and migrate leftward; this event leads to the left-specific organisation of this side of the brain. This migration is dependent on the left-sided nodal expression in the lateral plate mesoderm and, in the absence of nodal, there is equal likelihood of the parapineal precursors moving to the left or to the right (Concha et al., 2000). This indicates that there is still brain asymmetry without nodal, although it can have an opposite orientation. Ace(fgf8) mutant fish were found to have symmetrical epithalamus, and their pineal precursors do not migrate from the midline (Regan et al., 2009) indicating that fgf8 is important for brain asymmetry. Implanting an FGF bead into ace mutants allows some of the parapineal cells to migrate, and most of these migrate to the left, probably because there is still left expression of nodal in the epithalmus of the majority of ace embryos. The model proposed by the authors suggests that the midline expression of fgf8 makes the parapineal cells unstable so that they move. If there is no nodal, they lack direction and migrate either to the left or to the right; however, if there is no fgf8, then the cells do not migrate at all. This suggests that during the establishment of asymmetric patterning, simply generating asymmetry is a first step and determining the directionality of asymmetry is a subsequent event.

In zebrafish, FGFRs 1, 2, and 3 have been found on the cilia in KV using specific antibodies (Tanaka et al., 2005) and transcripts for FGFR-1 are localised to KV, the presomitic and lateral plate mesoderm, and distinct regions of the brain at the six-somite stage (Neugebauer et al., 2009). AMOs mediate knockdown of FGFR1 results in the bilateral expression of southpaw, which is normally expressed only in the left lateral plate mesoderm. Specifically depleting FGFR-1 in KV results in the same effects on spaw expression, and while there was no effect on the number or polarity of the cilia in KV, the cilia were significantly shorter in FGFR-1 knockdown zebrafish. The findings that cilia length was shortened by the disruption of FGF signalling in different developing organs that require FGF for their morphogenesis (such as the otic vesicle and the pronephros) and in laterality organs in another species (the Xenopus GRP) suggest that maintaining cilia length is an important mechanism through which FGF regulates developmental processes. In addition, cilia are known to play a role in modulating cell signalling through many pathways that include Wnt and Shh, and these pathways are known to interact with FGF signalling. This could mean that effects of FGF on cilia length may be another mechanism by which FGF signalling contributes to the regulation of other signal transduction pathways.

Copyright © 2010 by Morgan & Claypool Life Sciences.
Bookshelf ID: NBK53168


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