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. 2016 Apr 18:7:11288.
doi: 10.1038/ncomms11288.

Repulsive cues combined with physical barriers and cell-cell adhesion determine progenitor cell positioning during organogenesis

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Repulsive cues combined with physical barriers and cell-cell adhesion determine progenitor cell positioning during organogenesis

Azadeh Paksa et al. Nat Commun. .

Abstract

The precise positioning of organ progenitor cells constitutes an essential, yet poorly understood step during organogenesis. Using primordial germ cells that participate in gonad formation, we present the developmental mechanisms maintaining a motile progenitor cell population at the site where the organ develops. Employing high-resolution live-cell microscopy, we find that repulsive cues coupled with physical barriers confine the cells to the correct bilateral positions. This analysis revealed that cell polarity changes on interaction with the physical barrier and that the establishment of compact clusters involves increased cell-cell interaction time. Using particle-based simulations, we demonstrate the role of reflecting barriers, from which cells turn away on contact, and the importance of proper cell-cell adhesion level for maintaining the tight cell clusters and their correct positioning at the target region. The combination of these developmental and cellular mechanisms prevents organ fusion, controls organ positioning and is thus critical for its proper function.

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Figures

Figure 1
Figure 1. PGCs are motile at the gonad region.
(a) PGCs migrate from four different positions in the embryo (green clusters in 6 hpf image) towards the developing gonads to form two separate cell clusters by end of the first day of embryonic development (lateral and dorsal views). Insets display higher magnification of the gonad region (white boxes). Scale bars represent 50 μm. (b) A schematic cross-section of a 1-day-old zebrafish embryo showing the somites (magenta), the two separate PGC clusters (green cells) located on either side of the developing gut (red structure), as well as the expression of cxcl12a at this stage (yellow). (c) Snapshots from a time-lapse movie (Supplementary Movie 2) showing a lateral view of a PGC cluster starting at 24 hpf In the first three time points posterior migration of a PGC is highlighted (green track) and lateral–medial migration of the same PGC is presented in the following panels (yellow track). Scale bar, 25 μm.
Figure 2
Figure 2. Expression patterns of cxcl12a and lpp variants.
(a) cxcl12a is expressed at the site where the gonad develops within a 24 hpf embryo (green box and red arrowhead in the inset in the left panel), but not in 28 hpf embryos (right panel). Higher expression level of cxcl12a is detected in the lateral line (blue arrows). Scale bar, 50 μm. (b) lpp1 (variants X1 and X2) and lpp3 are expressed in the somites and developing vessels. See also Supplementary Fig. 3.
Figure 3
Figure 3. Abnormal positioning of PGCs in embryos treated with Cas9 and sgRNAs set against lpps.
(a) Optical cross-sections (plane marked by dashed line in the embryo scheme) of whole-mount 28 hpf embryos (Tg(kop:egfp-f-3′nos3) expressing EGFP in their PGCs following RNAscope procedure labelling myoD expression in somites (magenta, border marked in white). In contrast with control embryos (left panels), in embryos treated with Cas9 and a set of sgRNAs targeting 6 lpps (right panels) PGCs detach from the yolk and can contact somites (arrows). Scale bars, 20 μm. Dorsal is up. (b) The percentages of PGCs per embryo detached from the yolk (left graph) and percentage of those in contact with the somites (right graph) is significantly elevated in LPPs-depleted embryos. The statistical significance was evaluated using the Mann–Whitney U-test (*P≤0.05, **P≤0.01). Green lines signify the mean, error bars the standard error of the mean (s.e.m), N the number of embryos and n the number of PGCs examined.
Figure 4
Figure 4. PGCs avoid regions expressing LPP proteins.
(a) Generation of embryos lacking Cxcl12a, whose PGCs are labelled by EGFP and overexpress either LPP proteins or a Control protein in mCherry-labelled half of the embryo. (b) PGCs avoid cellular domains of the embryos, which overexpress LPPs (middle row), as compared to control domains (top row) or those overexpressing phosphatase-inactive versions of LPPs (lower row). (c) A significant reduction in the percentage of PGCs located within the LPPs-overexpressing domain in 10 hpf embryos (one-way analysis of variance; *P≤0.05). Error bars designate minimum to maximum range of the data points. N and n show the number of embryos and PGCs, respectively. See also Supplementary Fig. 8. (d) mCherry-labelled cells overexpressing Cxcl12a with either a Control Protein or LPPs were transplanted into embryos lacking Cxcl12a (medny054) whose PGCs express EGFP. (e) Images and a graph demonstrating the association of PGCs with Cxcl12a-expressing cells in control embryos (upper image, magenta point in graph) and the lack of interaction with cells expressing Cxcl12a and LPPs (starred red cells in lower image, blue points in graph, 77% of embryos showed absolutely no cell association). The statistical significance was evaluated using the Mann–Whitney U-test (****P≤0.0001). Scale bar, 15 μm. See also Supplementary Movie 3 and Supplementary Fig. 9.
Figure 5
Figure 5. Lack of the developing gut causes PGC cluster fusion.
(a) Whole-mount in situ hybridization on 30 hpf wild-type (N=420) and sox32 mutant embryos (N=89). PGCs are labelled with nanos3 (nos3, arrows) and the gut with foxa3 probe (arrowhead, missing in sox32 mutant embryos), both in blue. Unlike the separated PGC clusters in wild-type embryos, clusters are fused at the midline in sox32 mutant embryos (upper and lower panel images, respectively). Lateral (left panels) and dorsal (right panels) views are shown. (b) Cross-sections of 28 hpf sox17:dsred transgenic embryos whose gut is labelled in red and the PGCs membrane with EGFP. In control embryos (upper panel; N=5) bilateral PGC clusters form on either side of the gut tube. In embryos lacking the gut (Sox32-deficient; N=4) the PGC clusters fuse (lower panel). Nuclei counterstained with Hoechst. Scale bars, 25 μm. (c) Generation of mosaic embryos lacking Sox32 function in all cells whose endoderm is restored by providing Sox17 function to a group of cells (Scheme). The Sox32-deficient PGC clusters (green) are separated by the gut tissue at 28 hpf (red in lower panel, arrow; N=13), while in control embryos lacking endodermal tissues fused PGC clusters are observed (upper panel; N=24). Anterior is up. (d) PGC clusters in wild-type (N=20) and sox32 mutant (N=7) embryos lacking the gut tissue at 18, 24 and 28 hpf showing the dynamics of the fusion. N is the number of embryos analysed. Scale bar, 50 μm. Anterior is up.
Figure 6
Figure 6. The developing gut functions as a physical barrier.
(a) PGCs exhibiting dynamic movements within separated clusters (control, upper), while in embryos lacking the gut (lower panels) PGCs migrate over the midline to form one cluster (Supplementary Movie 4). Time point 0 corresponds to 24.5 hpf Scale bars, 100 μm. (b) Four representative migration tracks of PGCs relative to the gut (Supplementary Movie 5). PGC tracking using ImageJ. Scale bar, 25 μm. (c) Interaction of a PGC with the gut tube (Supplementary Movie 6). Polarity change in actin distribution is observed on contact. Time point 0 corresponds to 25 hpf The white arrow displays the direction of actin polarity. Scale bar, 5 μm. (d) PGC behaviours. On touching the gut, the main behaviour observed (29/42 encounters for 28 PGCs) is a rapid (14 min) polarity inversion away from the barrier and a change in the direction of migration. In the remaining encounters (13/42) PGCs exhibited a prolonged contact with the gut (69 min) without stable polariziation. (e) Cell crowding prolongs the time required for moving away from the barrier (45 versus 14 min; Mann–Whitney U-test, **P≤0.01). (f) Increased interaction time among PGCs that do not touch (right), as compared with the interaction time large and small clusters of the PGCs with the barrier (left). (df) Error bars display interquartile range, green lines median values, N and n number of embryos and PGCs respectively. The PGCs are shown in green and the gut in red (gut not presented in the right schematic drawing in f where PGC–PGC interaction time is displayed).
Figure 7
Figure 7. Boundary conditions and cell–cell adhesion level control cell cluster size and positioning.
The steady-state distribution of 14 particles across the gonad region (black box in the schematic zebrafish embryo; five cell diameter wide) that is confined by non-reflective (a) or reflective boundaries (b) for different cell–cell adhesion levels (ɛ=0–0.3 (a.u.)). The y axes in the graphs represent the probability density to find a given particle at a certain position at the site of gonad. Snapshots from Supplementary Movies 9–10 (t=4850, min) for different ɛ values are provided on the right in a and b, respectively. (c) The distribution of cells at the gonad site in 24–25 hpf zebrafish embryos. N is the number of embryos and n that of PGCs.
Figure 8
Figure 8. Progenitor cell positioning at target site.
An illustration demonstrating the interplay of repulsive cues and physical barriers within the embryo that govern the positioning of PGCs at the site of the developing gonad. At the time of PGC clusters initial formation, the guidance cue cxcl12a RNA is expressed at the migration target (yellow). In the following stages the chemokine is not expressed, calling for other mechanisms maintaining the position of the germline progenitors. In wild-type embryos dorsal repulsive tissues (somites expressing LPPs in magenta) inhibit the dorsal migration of PGC clusters, while the developing gut (red) acts as a physical barrier separating the clusters, thereby contributing to the formation of distinct cell clusters at the site of developing organ. In embryos deficient for the function of LPPs or lacking the physical barrier, the PGCs exhibit abnormal positioning.

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