A framework for understanding morphogenesis and migration of the zebrafish posterior Lateral Line primordium

Abstract

A description of zebrafish posterior Lateral Line (pLL) primordium development at single cell resolution together with the dynamics of Wnt, FGF, Notch and chemokine signaling in this system has allowed us to develop a framework to understand the self-organization of cell fate, morphogenesis and migration during its early development. The pLL primordium migrates under the skin, from near the ear to the tip of the tail, periodically depositing neuromasts. Nascent neuromasts, or protoneuromasts, form sequentially within the migrating primordium, mature, and are deposited from its trailing end. Initially broad Wnt signaling inhibits protoneuromast formation. However, protoneuromasts form sequentially in response to FGF signaling, starting from the trailing end, in the wake of a progressively shrinking Wnt system. While proliferation adds to the number of cells, the migrating primordium progressively shrinks as its trailing cells stop moving and are deposited. As it shrinks, the length of the migrating primordium correlates with the length of the leading Wnt system. Based on these observations we show how measuring the rate at which the Wnt system shrinks, the proliferation rate, the initial size of the primordium, its speed, and a few additional parameters allows us to predict the pattern of neuromast formation and deposition by the migrating primordium in both wild-type and mutant contexts. While the mechanism that links the length of the leading Wnt system to that of the primordium remains unclear, we discuss how it might be determined by access to factors produced in the leading Wnt active zone that are required for collective migration of trailing cells. We conclude by reviewing how FGFs, produced in response to Wnt signaling in leading cells, help determine collective migration of trailing cells, while a polarized response to a self-generated chemokine gradient serves as an efficient mechanism to steer primordium migration along its relatively long journey.

Figure 1

Overview of pLL primordium migration and morphogenesis. A. 32hpf zebrafish embryo, showing position of the deposited L1 neuromast and the migrating pLL primordium. Inset shows a magnified view of an example pLL primordium. B. 48hpf zebrafish embryo showing neuromast spacing (L1–L6) after completion of migration, as well as the position of the future terminal neuromast cluster (TNM). C. Frames from a timelapse movie showing where cells that will make up the L2–L7 neuromasts reside in the pLL primordium. D. Frame from a timelapse movie showing the position of both deposited and prospective neuromast (blue) and interneuromast (orange) cells. Scale bar in A and C, 200μm.

Figure 2

Overview of Wnt and Fgf signaling in the morphogenesis of the pLL primordium. A. Initiation of pLL primordium migration. The pLL placode is initially dominated by Wnt activity (yellow). Subsequently, an FGF responsive center (blue) is established at the trailing end of the prospective pLL primordium. It initiates formation of the first protoneuromast. Additional FGF responsive centers and associated protoneuromasts form sequentially as migratory behavior is initiated. B. As the Wnt system shrinks so does the primordium. Cells incorporated into protoneuromasts are deposited as neuromasts while those that were not are deposited as interneuromast cells. C. Eventually, the Wnt system shrinks below some threshold and the primordium resolves to form terminal neuromasts after depositing 5–6 neuromasts. D. Schematic of signaling within the forming and mature protoneuromasts within the pLL primordium. E. Cross section through a mature protoneuromast showing the relationship between central Atoh1a-positive hair cell progenitor and surrounding cells. F. Detail of Fgf (shown in blue) and Notch/Delta signaling in early patterning of the neuromast.

Figure 3

Modeling neuromast deposition patterns by the pLL primordium. A. Total length of the pLL primordium over time (averaged from n = 9 embryos). B. Length of the Wnt-active domain (as reported by the Tcf/Lef-miniP:dGFP reporter line). C. Ratio of Wnt-active domain length to total pLL primordium length. Red line indicates the best linear fit. D. Three frames from a model run showing the progressive shrinking of the Wnt-active zone together with the pLL primordium. E. Neuromast deposition pattern at 48hpf in wild-type ClaudinB:lynGFP embryos (top) and model results (scaled to the length of the embryo outline) for wild-type parameters. F. Neuromast deposition pattern at 48hpf in embryos injected with 2ng lef1 morpholino (top) and model results for lef1 morphant parameters (for details, see text and table S1). G. Quantification of the neuromast deposition pattern (mean and standard deviation) for n = 20 model runs using both wild-type (left) and lef1 (right) parameters.

Figure 4

Schematic of guidance cues in pLL migration. A. Fgfs secreted by leading Wnt-active cells (yellow) can act as a directional cue for the migration of trailing cells. B. Polarized expression of Cxcr4b and Cxcr7b chemokine receptors in the pLL primordium. Cxcl12a gradient generated by ligand internalization is shown below in blue. Trailing, Cxcr7b-expressing cells are unable to respond to Cxcl12a, while leading cells expressing Cxcr4b are able to. C. Comparison of externally imposed gradient with a self-generated gradient on the migration of the pLL primordium. Blue represents the levels of Cxcl12 along the migratory path in each condition.