Neural crest invasion is a spatially-ordered progression into the head with higher cell proliferation at the migratory front as revealed by the photoactivatable protein, KikGR

Abstract

Neural crest cell (NCC) invasion is a complex sculpting of individual cells into organized migratory streams that lead to organ development along the vertebrate axis. Key to our understanding of how molecular mechanisms modulate the NCC migratory pattern is information about cell behaviors, yet it has been challenging to selectively mark and analyze migratory NCCs in a living embryo. Here, we apply an innovative in vivo strategy to investigate chick NCC behaviors within the rhombomere 4 (r4) migratory stream by combining photoactivation of KikGR and confocal time-lapse analysis of H2B-mRFP1 transfected NCCs. We find that the spatial order of r4 NCC emergence translates into a distal-to-proximal invasion of the 2nd branchial arch. Lead and trailing NCCs display similar average cell speeds and directionalities. Surprisingly, we find that lead NCCs proliferate along the migratory route and grow to outnumber trailing NCCs by nearly 3 to 1. A simple, cell-based computational model reproduces the r4 NCC migratory pattern and predicts the invasion order can be disrupted by slower, less directional lead cells or by environmental noise. Our results suggest a model in which NCC behaviors maintain a spatially-ordered invasion of the branchial arches with differences in cell proliferation between the migratory front and trailing NCCs.

Figure 1. Targeted fluorescent marking of subpopulations of neural crest cells within the fronts of r4 migratory streams

(A-D) A typical chick embryo was electroporated with KikGR, the lead 30% of the r4 NCC migratory stream (within the box) was photoactivated 10–14hrs after electroporation, and analyzed at +24hrs after egg re-incubation. (E) A line scan of the intensities (red and green) of multiple embryos (n=7) were averaged together to give a global representation of the line profile throughout the r4 stream at t=0+ (post-photoactivation). (F) The r4 NCC migratory stream was divided into 4 equal parts from the lateral edge of the neural tube to the most distal NCC of the emerging migratory front and the distributed number of photoactivated versus non-photoactivated NCCs. (G) The same embryo in (D) was re-incubated for 24hrs and the NCCs have invaded the 2^nd branchial arch. The photoactivated NCCs fill the distal portion of the 2^nd branchial arch. (H) A line scan of the intensities (red and green) were averaged together (n=7 embryos) to give a global representation of the line profile throughout the r4 NCC migratory stream at t=24hrs post-photoactivation. (I) The r4 NCC migratory stream at 24hrs post-photoactivation was divided into 4 equal parts from the lateral edge of the neural tube to the most distal NCC within the 2^nd branchial arch and shows the distributed number of photoactivated versus non-photoactivated NCCs. BA2=branchial arch 2, r=rhombomere, AFU=arbitrary fluorescence units. The scalebars are as marked and 500um in (B) and (C).

Figure 2. Targeted fluorescent marking of subpopulations of trailing neural crest cells within the r4 migratory streams

(A-D) A typical chick embryo was electroporated with KikGR, the back 60% of the r4 NCC migratory stream was photoactivated (within the dotted box) 10–14hrs after injection, and analyzed at +24hrs after egg re-incubation. (E) A line scan of the intensities (red and green) of multiple embryos (n=8) were averaged together to give a global representation of the line profile along the r4 NCC migratory stream. This profile shows that the trailing 60% of the migratory stream on average was photoactivated at t=0+. (F) The emerging r4 NCC migratory stream was divided into 4 equal parts from the lateral edge of the neural tube to the most distal NCC. The NCCs were counted red or green for each embryo (n=8) and averaged as a percentage of the total cell number versus percentage of the r4 stream length. (G) The same embryo in (D) was re-incubated for 24hrs and the photoactivated NCCs (red) are proximal to the 2^nd branchial arch entrance. (H) A line scan of the intensities (red and green) of multiple embryos (n=8) were averaged together to give a global representation of the line profile throughout the r4 stream at 24hrs. (I) The r4 NCC migratory stream at 24hrs was divided into 4 equal parts from the lateral edge of the neural tube to the most distal cell in the 2^nd branchial arch. The NCCs were counted red or green for each embryo (n=8). BA2=branchial arch 2, r=rhombomere, AFU=arbitrary fluorescence units. The scalebars are as marked and 500um in (B) and (C).

Figure 3. Cell division does not significantly alter the red-to-green fluorescence ratio in KikGR photoactivated NCCs and NCCs at the migratory stream front show higher cell proliferation than trailing NCCs

KikGR-photoactivated embryos were monitored for short term time-lapse confocal imaging sessions of approximately 4hrs (n=8; multiple cell divisions per time-lapse session). During this time, several (>3) photoactivated NCCs divided and were analyzed for fluorescent intensity measurements before and after the cell division. (A) In a typical time-lapse imaging session, a subpopulation of r4 KikGR photoactivated NCCs were followed over time (at 2min intervals). (B,F) Prior to dividing, NCCs appeared to collapse projections and round-up. An intensity line profile through the NCC shows the red and green fluorescence intensities as a function of the distance along the line (dotted line through the cell). (C,G) NCC division was apparent when a neighboring daughter cell appeared. An intensity line profile through the NCC progeny shows only a minor decrease in the red and green fluorescence intensities as a function of the distance along the line (dotted line through the cell). (D,H) As the daughter cells move apart from each other, the NCCs begin to segregate and (E,I) extend protrusions to move along the migratory route. The scale bar is 10um and the time is in mins. (J) The average number of NCCs are plotted at t=0+ (immediately after photoactivation) for KikGR-expressing NCCs photoactivated at the front of the r4 migratory stream (red) and photoactivated trailing NCCs (green) and at t=24hr later in re-incubated embryos (n=7). (K) The data from the photoactivated front, and photoactivated back are plotted (green and red dots correspond to green and red bars in (J), respectively) and fit to the general equation for cell growth; N(t) = N(0)*2^(kt), N(0)=the number of NCCs that were originally photoactivated and (1/k)=the cell cycle rate. r4=rhombomere 4, AFU=arbitrary fluorescence units, um=microns. The number of NCCs versus time are plotted for the photoactivated front (solid red line, red data points) and photoactivated back (dotted red line, green data points).

Figure 4. Time-lapse imaging of H2B-mRFP1 labeled NCCs confirms a spatial order is maintained within the r4 migratory stream and reveals differences in cell proliferation

(A-D) Selected images from a typical time-lapse imaging session (n=7) show H2B-mRFP1 transfected NCCs (represented by colored spheres) migrating in the r4 NCC stream after cell tracking analysis of raw data. The first 30% of emerging NCCs are pseudo-colored red, while later emerging NCCs are marked in green. (D) Cell trajectories are marked by red or green colored lines over the course of the entire time-lapse. (E) The distribution of the lead (red) and trailing (green) NCCs at the end of the time-lapse sessions, graphed as the percentage of NCCs in 4 different quadrants of the r4 NCC migratory stream. (F) The average total distance an individual NCC traveled along the migratory route is plotted for the lead and trailing subgroups of NCCs as the average track displacement. (G) The calculated average NCC speed and directionality (spotted bars) for each subgroup (lead-red and trailing-green colored bars). r4=rhombomere 4. The scalebar is 50um.

Figure 5. Time-lapse analysis reveals differences in cell proliferation between the migratory front and trailing NCCs

(A) Quantitative measurements of the percentage of dividing NCCs in the lead 30% (red) and the trailing NCCs (green) (from cell tracking data in n=7 time-lapse sessions). (B) Analysis of the number of cell divisions occurring in the subgroup of cells that do undergo division in both lead (red) and trailing cells (green). (C) A plot of the start and final positions of the lead NCCs emerging lateral to r4 and their distribution within the migratory front at the end of the time-lapse sessions, as determined from tracing the backward trajectories of lead NCCs. NCCs are pseudo-colored based on their start position at t=0, as purple (rostral r4), blue (mid-r4), and yellow (caudal r4). The migratory front is segregated into 3 subdomains of rostral, mid, and caudal, and the NCCs distribution as a percentage of the number of NCCs in that particular region is shown as a pie chart. *, significantly different, p<0.1, **, significantly different, p<0.05, (n=7 time-lapse sessions analyzed). r4=rhombomere 4.

Figure 6. Computational model simulations of r4 NCC migratory stream patterning

(A) The 2D domain of the computational model was derived from the layout of the NCC migratory stream that emerges lateral to r4 with contribution from r3 and r5. The Y-axis was labeled to correspond to the antero-posterior axis along the neural tube midline, in 50um increments, with Y=0 designated as the axial level of midr4. The X-axis was labeled to correspond to the length along the migratory route from the neural tube to the entrance to the 2^nd branchial arch. (B) The environmental signal modeled as an attraction function had the shape of an increasing function with a peak at (X=L,Y=0), (C) that was a normal distribution, f(X,Y). The computational model simulation results for the (D) normal wildtype (WT) pattern, (E) when lead NCCs are more efficient (sample the environment twice as many times as normal), and (F) when trailing cells are more efficient. (G-H) Simulation results of perturbation to the environment (in the form of adding noise to the attraction function f(X,Y) values, (G) 10% noise, and (H) 25% noise. r=rhombomere.