Formation of the Posterior Lateral Line system in zebrafish is pioneered by the Posterior Lateral Line primordium (PLLp), a group of about 150 cells that forms near the ear. While leading cells in the PLLp have a relatively mesenchymal morphology, trailing cells are more epithelial; they have distinct apical basal polarity and they reorganize to sequentially form nascent neuromasts or protoneuromasts. The PLLp begins migration toward the tip of the tail at about 22 hours post fertilization (hpf). Proliferation adds to the growth of the PLLp, nevertheless, as the PLLp migrates, the length of the column of cells undergoing collective migration progressively shrinks as cells stop migrating are deposited from the trailing end: cells that were incorporated into protoneuromasts are deposited as neuromasts, while cells that were not, are deposited between neuromasts as interneuromast cells. Eventually, the PLLp ends its migration a day later after depositing 5-6 neuromasts and by resolving into 2-3 terminal neuromasts. Establishment of polarized Wnt and FGF signaling systems coordinates morphogenesis and migration of the PLLp. Wnt signaling dominates at the leading end of the migrating primordium and is thought to determine the relatively mesenchymal morphology of leading cells. Wnt signaling promotes its own activity and at the same time drives expression of fgf3 and fgf10. However, leading cells do not respond to these FGF ligands because Wnt signaling simultaneously promotes expression of intracellular inhibitors of the FGF receptor. Instead, the FGFs activate FGF receptors and initiate FGF signaling at the trailing end of the PLLp, where Wnt signaling is weakest. There, FGF signaling determines expression of the diffusible Wnt antagonist Dkk1b, which counteracts Wnt signaling to help establish stable FGF responsive centers. Once established, the trailing FGF signaling system coordinates morphogenesis of nascent neuromasts by simultaneously promoting the reorganization of cells into epithelial rosettes and by initiating expression of factors that help specify a sensory hair cell progenitor at the center of each forming neuromast. Over time, the leading domain with active Wnt signaling shrinks closer to the leading edge and additional FGF signaling centers form sequentially in its wake, each associated with formation of additional protoneuromasts. In past year our group has examined mechanisms that determine the dynamics of Wnt-FGF signaling in the pLL primordium, including those that determine its initial polarization, progressive shrinking of the leading Wnt system and sequential formation of trailing FGF signaling centers in its wake. A. Sox2 inhibits lef1 expression in the trailing zone to facilitate FGF signaling-dependent neuromast formation The establishment of a new FGF signaling center at the trailing end of the leading Wnt system is facilitated by FGF signaling-dependent expression of Dkk1b. However, dkk1b is only transiently expressed in nascent protoneuromasts. Because its expression is likely to require some minimal amount of Wnt activity, dkk1b expression is absent in more mature protoneuromasts located closer to the trailing end, where there is much less or no Wnt signaling. If trailing protoneuromasts do not express dkk1b, what makes sure Wnt signaling is not allowed to dominate again and suppress FGF signaling in these protoneuromasts? We have now shown that the transcription factor Sox2 is expressed in maturing protoneuromasts, where it inhibits lef1 expression to facilitate FGF signaling. Interestingly, while it remains unclear what promotes sox2 expression, Lef1 dependent Wnt signaling inhibits sox2 expression in the leading zone. This mutually inhibitory relationship between Sox2 and Lef1, we suggest, helps cells switch, from a state dominated by Wnt activity in the leading zone to one dominated by FGF in the trailing zone. B. A role for HSPGS in effective reception of FGF signals? As Heparan Sulphate proteoglycans (HSPGs) can influence both range and efficacy of signaling, we examined the role of an HSPG, Syndecan 4 that is expressed in a tissue specific manner in the migrating pLL primordium. sdc4 is not expressed in the leading zone but at progressively higher levels in maturing protoneuromasts and its expression persists in deposited neuromasts. Consistent with its absence in the leading zone, its expression is inhibited by Wnt signaling. In addition, despite being expressed in trailing protoneuromasts, its expression is also inhibited by FGF signaling and exposure to SU5402, a small molecule inhibitor of the FGF receptor, results in expansion in its domain and level of expression. Interference with sdc4 function, with morpholinos or by generation of CRSPR mutants, reveals that it is required for effective FGF signaling and loss of its function is accompanied by a decrease in FGF signaling, delayed formation of protoneuromasts and slower primordium migration. While changes in the spacing of deposited neuromasts and slower primordium migration was obvious in embryos injected with sdc4 morpholinos, these changes were not initially observed in sdc4 mutants. Neverthless, both sdc4 mutants and morphants showed a similar reduction in the expression of FGF-signaling dependent pea3 expression and a trailward expansion in Wnt-dependent lef1 expression, both changes that demonstrate ineffective FGF signaling in the trailing zone. The absence of overt problems with neuromast formation and primordium migration in sdc4 mutants suggested that, despite FGF signaling being compromised, something was compensating for loss of sdc4 in the mutant and preventing overt problems with pLL primordium morphogenesis. Investigation of this possibility revealed that sdc3, also expressed in the primordium, was likely to be part of the compensating mechanism as knockdown of sdc3 in sdc4 mutants resulted in slower migration, revealing a change previously seen only in sdc4 morphants. Why sdc3 is able to partially compensate for loss of sdc4 in sdc4 mutants but not in sdc4 morphants remain unclear but similar differences in morphant and mutant phenotypes have now also been reported for a number of other genes. If sdc4 is required to make FGF signaling more effective, why do both Wnt and FGF signaling systems inhibit its expression and ensure its expression is only permitted in the most trailing protoneuromasts? We suggest that sdc4 expression in trailing cells may be part of feedback mechanism that has evolved to optimize the level of FGF signaling in the primordium. Ensuring expression in the most trailing cells may facilitate activation of FGF receptors in a region where access to FGFs, primarily produced by leading cells becomes progressively more difficult. Why not allow sdc4 expression throughout the primordium? One possibility is that, in the context of their cross inhibitory relationship, excessive FGF signaling in more leading parts of the primordium may allow too much inhibition of Wnt signaling, premature shrinking of the Wnt system, and eventully premature termination of the pLL primordium system. This prediction will need to be tested in future studies by determining if ectopic expression of sdc4 in the primordium results in premature termination.
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