There and back again: development and regeneration of the zebrafish lateral line system

Abstract

The zebrafish lateral line is a sensory system used to detect changes in water flow. It is comprised of clusters of mechanosensory hair cells called neuromasts. The lateral line is initially established by a migratory group of cells, called a primordium, that deposits neuromasts at stereotyped locations along the surface of the fish. Wnt, FGF, and Notch signaling are all important regulators of various aspects of lateral line development, from primordium migration to hair cell specification. As zebrafish age, the organization of the lateral line becomes more complex in order to accommodate the fish's increased size. This expansion is regulated by many of the same factors involved in the initial development. Furthermore, unlike mammalian hair cells, lateral line hair cells have the capacity to regenerate after damage. New hair cells arise from the proliferation and differentiation of surrounding support cells, and the molecular and cellular pathways regulating this are beginning to be elucidated. All in all, the zebrafish lateral line has proven to be an excellent model in which to study a diverse array of processes, including collective cell migration, cell polarity, cell fate, and regeneration.

Figure 1

Anatomy of lateral line neuromasts. Top Panel) Image of an individual neuromast at 5 dpf. The lateral line nerve and afferent terminals are labeled in red by neuroD:tRFP (left). Support cells are labeled in green by ET20:GFP (middle). Bottom Panel) 5 dpf neuromast, with afferent terminals labeled in green marked by neuroD:GFP (left), and the hair cells in red, marked by ribeyeA:tRFP (middle). Yellow arrows indicate afferent terminals (left) and their synaptic partners (middle).

Figure 2

Organization of the zebrafish lateral line. Top Panel) Image of a 5 dpf zebrafish larva in which the neuromasts and interneuromast cells are labeled by the transgenic marker ET20:GFP. Red asterisks indicate both aLL and pLL neuromasts. Blue arrow indicates a melanocyte located in the horizontal myoseptum. Middle Panel) The lateral line nerve, labeled by neuroD:tRFP. Blue arrow indicates the same melanocyte as in the top panel, which obscures signal from the submerged nerve. Yellow arrows indicate individual afferent fibers projecting from the central bundle and innervating neuromasts. Bottom Panel) Composite image of the top and middle panels.

Figure 3

Migration of the pLLP. Top Panel) Image of the migrating pLLP in the Zebrabow transgenic line. The primordium is outlined by the dashed white line, and is traveling posteriorly (to the right). Middle Panel) Deposition of a mature protoneuromast at the trailing edge of the primordium. Bottom Panel) After deposition, the primordium continues its migration, leaving a string of interneuromast cells in its wake. Due to the random colorization of cells in the Zebrabow line, individual cells can be followed over time. As the pLLP migrates, the tan cell marked by the white asterisk is displaced anteriorly as it moves out of the leading zone and into the trailing zone. Conversely, the red cell marked by the gold plus remains relatively immobile, having already been incorporated into a protoneuromast. Anterior and posterior are marked in the top panel.

Figure 4

Signaling pathways regulating pLLP patterning. A) Primordium migration is regulated by asymmetric expression of chemokine receptors. Cxcr4b is expressed in the leading zone, whereas cxcr7b is expressed in the trailing zone. Cxcr4b expression is activated by Wnt signaling and the homeobox gene hoxb8a, which itself is activated by Wnt signaling. Hoxb8a also represses expression of cxcr7b, thus limiting its expression to the trailing zone. Conversely, the estrogen receptor Esr1 is expressed in the trailing zone and restricts expression of cxcr4b to the leading zone. Dashed arrows indicate suggested, yet unproven, interactions. B) Localization of Wnt and FGF signaling within the pLLP. Wnt signaling mediates proliferation near the leading edge, in part through the effectors lef1 and tcf7, whereas FGF signaling regulates the formation of epithelial rosettes closer to the trailing edge. Wnt-induced expression of the inhibitors Sef and Dusp6 restricts FGF signaling to the trailing zone, where the ligands Fgf3 and Fgf10a, themselves induced by Wnt signaling, can activate the Fgfr1 receptor. FGF signaling also drives the expression of the Wnt-inhibitor Dkk1b, which prevents Wnt activity from occurring in the trailing zone. Note that, while there is significant overlap between domains, the Wnt and FGF domains are not equivalent to the leading and trailing zones, respectively.

Figure 5

FGF signaling regulates apical constriction of protoneuromasts. Localized Fgf10a expression drives FGF signaling in surrounding cells. Activation of FGF-Ras-MAPK signaling drives the expression of the actin binding protein Shroom3, which in turn mediates the apical localization of Rock2a, which induces apical constriction through its interaction with non-muscle myosin.

Figure 6

Specification of hair cell precursors. Expression of atoh1a becomes restricted to centralized cells within developing protoneuromasts. Atoh1a expression activates atoh1b, which itself maintains expression of atoh1a. Atoh1a continues to drive expression of fgf10a and also activates expression of deltaD. Fgf10a diffuses to surrounding cells and, through activation of Fgfr1, maintains expression of notch3. This is prevented in the central cells by atoh1a-induced repression of fgfr1 expression. Binding of Notch3 to DeltaD represses the expression of atoh1a in surrounding cells. Atoh1a-expressing cells will ultimately differentiate into hair cells, whereas the surrounding cells lacking atoh1a expression will become support cells.

Figure 7

Planar polarization of lateral line neuromasts. Left Panel) Lateral DIC image of a neuromast at 5 dpf. Red arrow indicates kinocilia, whereas the green arrow indicates the location of the stereocilia (which are not visible in this image). Dashed blue line indicates the oblique transection across the apical surface of the hair cells shown in the right panel. Right Panel) The stereocilia (S, labeled in green) are localized dorsally or ventrally to their respective kinocilia (K, labeled in red), indicating that this neuromast has dorsoventral planar polarity.

Figure 8

Lateral line organization in adult zebrafish. Top Panel) Image of the anterior lateral line in an ET20:GFP transgenic adult fish. The eye is marked by “E.” Neuromasts are indicated by white asterisks. Note the expansion of neuromast patterning compared to that of Figure 1. Bottom Panel) Image of stitch patterning in the adult posterior L line. New neuromasts bud dorsally and ventrally off of existing neuromasts. Scales included for scale.

Figure 9

Time course of hair cell regeneration. A) A 5 dpf neuromast, with support cells labeled in green (ET20) and hair cells labeled in red (ribeyeA), prior to neomycin treatment. B) At 1 hour post neomycin treatment, the vast majority of the hair cells are gone, while the support cells are unaffected. C) At 24 hours post treatment, some hair cells have been restored. D) By 48 hours post treatment, most hair cells have regenerated. E) After 72 hours, the number of hair cells has returned to pre-treatment levels.