Expression profile of Laminin in relation with the development of the olfactory system.

A-D. Immunostaining for Laminin (magenta) on Tg(neurog1:GFP) embryos (green) at 18 and 22 hpf (A-B’, dorsal views), and on Tg(omp:meYFP) embryos (green) at 28 and 36 hpf (C-D’, frontal views). In A, white arrowheads = BM-like Laminin staining around the brain, grey arrowheads = spotty Laminin accumulation around the OP. In C, D, asterisks = interruptions in the OP’s and brain’s BM where the YFP+ axons exit the OP and enter the brain. arrows = distalmost extremity of the YFP+ axon bundle, which is in close contact with the internal side of the brain’s BM. In C’, D’, white arrowheads = ventro-medial unipolar neurons labelled by the Tg(omp:meYFP) line (see Miyasaka et al., 2005). All the images are single z-sections. E-J. Images extracted from confocal live imaging on TgBAC(lamC1:lamC1-sfGFP); Tg(cldnb:Gal4; UAS:RFP) embryos (frontal view, except for Fig. 1J, dorsal view). LamC1-sfGFP expression in green, and RFP expression (OP cells and peridermal skin cells) in magenta. Arrowheads = OP cells with cytoplasmic LamC1-sfGFP accumulation. Asterisks in F and H = axon exit point and entry point, respectively. Arrows in H and I = distalmost extremity of the RFP+ axon bundle, located close to the brain’s BM. In J, double headed arrow = gap in the LamC1-sfGFP observed at the interface with the periderm, where the nostril orifice opens in the skin, as previously reported (Baraban et al., 2023). All the images are single z-sections. K. Schematic representation of Laminin-containing BM (magenta) assembly during OP coalescence (a, b), of the formation of the exit/entry points, which often appear as zones with several, small BM interruptions (b, d) and the associated axonal behaviours: retrograde axon extension and lateral movement in the OP (a, b, grey arrows), growth as a fasciculated bundle, initially between the BMs of the OP and the brain (c), and then migration of the axonal tips along the internal side of the brain’s BM (d). Scale bar: 50 µm.

The integrity of the BMs of OP and brain tissues is strongly affected in sly mutants.

A-I. Immunostaining for Laminin (A-C), Nidogen (D-F) and Collagen IV (G-I) (magenta) on sly mutants and control siblings at 22 (dorsal view), 28, and 36 hpf (frontal view). For Laminin and Nidogen, the linear, BM-like staining seen in controls around the OP and brain tissues is not detected in sly mutants. In G, white arrowheads = BM-like linear Collagen IV staining, grey arrowheads = fibrous staining around the OP. In sly mutants, the linear Collagen IV pattern is dramatically disrupted, with, however, remaining fibrous patches of Collagen IV in some discrete areas located at the margin of the OP and/or between the OP and brain (arrows in G’, H’, I’). Scale bar: 50 µm. J, K. Examples of EM images of the intercellular space between NCC and the OP in control siblings (J, K) and the OP and the brain in sly mutants (J’, K’), at 24 (J, J’) and 32 hpf (K, K’). Arrows = plasma membranes. The pictures were taken in the areas depicted with red boxes in L, L’. L. Schematic view of the brain/OP boundary and of the areas (red boxes) where the pictures were taken in controls (L) and sly mutants (L’). OP, brain and NCC were identified by their position and shape: migrating NCC showed an elongated morphology along the AP axis, which differed from the round OP cell bodies and from brain neuroepithelial cells elongated along the ML axis. M, N. Thickness of the intercellular space in sly mutants (between OP and brain cells) and control siblings (between NCC and brain or OP cells) at 24 hpf (n = 3 controls; n = 3 mutants) and 32 hpf (n = 2 controls; n = 3 mutants). For 24 hpf, unpaired, two-tailed t test. For 32 hpf, Mann-Whitney test.

Analysis of OP coalescence in sly mutants and control siblings.

A, B. Images (dorsal views, 1 z-section) of representative OPs from a Tg(neurog1:GFP); sly -/-mutant (right) and a control Tg(neurog1:GFP) sibling (left) at the end of OP coalescence (22 hpf). The Tg(neurog1:GFP)+ OP clusters are surrounded by dotted lines. C-E. Graphs showing the anteroposterior (AP, in C), the mediolateral (ML, in D), and dorsoventral (DV, in E) dimensions of the Tg(neurog1:GFP)+ OP clusters in sly mutants (pink) and control siblings (blue) at 22 hpf (n = 62 controls and n = 32 mutants from 2 independent experiments). Ectopic fluorescent cells (cells that are physically separated from the main cluster) were not taken into account for the measurement of OP dimensions. Unpaired, two-tailed t test. F-F’’ and H-H’’. Images extracted from confocal live imaging on Tg(neurog1:GFP) control (F-F’’) and sly mutant (H-H’’) embryos during OP coalescence, dorsal view, average projection. Only the left side of the embryo is shown. G, I. Examples of 2D tracks (ML along X and AP along Y) of Tg(neurog1:GFP)+ OP cells (red) and Tg(neurog1:GFP)+ brain cells (green) in a control (G) and a sly mutant embryo (I). Only the left side of the embryo is shown. The time is color-coded: light colors at the beginning of the trajectory (17 hpf) towards dark colors for the end of the track (600 min later). J, K. MSD analysis for OP cells (J) and brain cells (K) in sly mutants and control siblings (n = 5 controls and n = 5 mutants from 3 independent experiments, 10 to 14 cells analysed in each tissue). L, M. Graphs showing the total lateral (L) and ventral (M) displacement of OP cells, starting at 17 hpf and during 600 min of time lapse (n = 5 control placodes and n = 5 mutant placodes from 3 independent experiments, 10 to 14 cells per placode, unpaired, two-tailed t test). N-P. Graphs showing the total anterior displacement of anterior, central and posterior OP cells (as defined in Breau et al., 2017), starting at 17 hpf and during 600 min of time lapse (n = 5 control placodes and n = 5 mutant placodes from 3 independent experiments, mean calculated from 1 to 12 cells per placode, unpaired, two-tailed t test). Note that in some of the OPs we could not find any trackable (i.e. expressing H2B-RFP) posterior OP cell, which explains why there are only 4 control points and 3 mutant points in the graph showing the anterior displacement of posterior cells).

Analysis of OP and brain morphogenesis in sly mutants and control siblings during the forebrain flexure.

A, B. Images (frontal view, 1 z-section) of representative placodes from a Tg(omp:meYFP); sly -/- mutant (right) and a control Tg(omp:meYFP) sibling (left) at 36 hpf. Laminin immunostaining in magenta. C-E. Graphs showing the anteroposterior (AP, in C), the mediolateral (ML, in D), and dorsoventral (DV, in E) dimensions of the Tg(omp:meYFP)+ OP clusters in sly mutants (pink) and control siblings (blue) at 36 hpf (n = 15 controls and n = 9 mutants from 4 independent experiments). Ectopic fluorescent cells (cells that are physically separated from the main cluster) were not taken into account for the measurement of OP dimensions. Unpaired, two-tailed t test. Similar measurements performed at younger stages are shown in Fig. S3A-L. F, G. Examples of images used for the analysis of ectopic cells, defined as Tg(omp:meYFP)+ cells being physically separated from the main YFP+ cluster. Arrowheads show instances of ectopic cells in a sly mutant. H. Table showing the % of control and mutant embryos with at least one ectopic cell located dorsally, ventrally, laterally, and medially to the main YFP+ cluster. The numbers of analysed embryos are indicated in the table. I-I’’ and K-K’’. Images extracted from confocal live imaging on Tg(omp:meYFP) control (I-I’’) and sly mutant (K-K’’) embryos during the forebrain flexure, from 22 hpf and over 1080 min, frontal view, maximum projection. Only the left side of the embryo is shown. J, L. Examples of 2D tracks (ML along X and DV along Y) of Tg(omp:meYFP)+ OP cells (red) and adjacent brain cells (green) in a control (J) and a sly mutant (L). The time is color-coded: light colors at the beginning of the trajectory (22 hpf) towards dark colors for the end of the track (1080 min later). Only the left side of the embryo is shown. M, N. 3D MSD analysis of OP (M) and brain cells (N) in sly mutants and control siblings (n = 4 controls and n = 3 mutants from 5 independent experiments, 10 to 14 cells analysed in each tissue). O. Rose plots indicating the orientation of the movement for control and mutant left OP cells (data pooled from n = 4 controls and n = 3 mutants from 5 independent experiments). Numbers = number of cells. Dorsal to the top, lateral to the left. There is a statistical difference in cell track orientations between controls and mutants (circular analysis of variance based on the likelihood ratio test: p = 1.233e-09 for the left OPs, and p = 3.439e-08 for the right OPs, the graphs for the rigth OPs are not shown).

Analysis of brain width and brain/placode boundary in sly mutants and control siblings.

A, A’. Immunostaining for HuC (cyan) at 36 hpf on Tg(cldnb:Gal4; UAS:RFP) (magenta) control and sly mutant embryos (frontal view). Similar immunostainings performed at 28 hpf are shown in Fig. S4A, A’. B, B’. Images of Tg(elementC:gfp); Tg(cldnb:Gal4; UAS:RFP) control and mutant embryos at 36 hpf (frontal view), similar images acquired at 28 hpf are shown in Fig. S4B, B’. GFP (green) is expressed by the forebrain and a few OP cells. Arrows indicate where the brain width was measured (in 3 distinct positions along the DV axis). Measurements were also carried out at 3 distinct AP levels (through the z-stack). C, D. Width of the forebrain in 36 hpf controls and sly mutants, at 3 different DV and 3 different AP levels (n = 9 controls and n = 8 mutants from 4 independent experiments, unpaired, two-tailed t test). Quantifications for the 28 hpf stage are shown in Fig. S4C, D. E-F’. Immunostaining for the OP marker Dlx3b (cyan) was performed on 36 hpf sly mutants and control siblings (frontal view). The signal was segmented using deep learning approaches (white signal), and the distortion index (see Material and Methods) of the OP/brain boundary was calculated in the regions outlined with red boxes. G, H. Graphs showing the distortion indexes in controls and mutants at 36 hpf, for the left and right OPs (n = 3 controls and n = 3 mutants). ANOVA test (mixed models, with animals as random effect and genotype and side as fixed effects).

Quantitative live imaging of axonal behaviours in sly mutants and control siblings.

A, B. Images extracted from confocal live imaging on control (A-A’’’’) and sly mutant (B-B’’’’) embryos injected with the omp:meYFP plasmid to obtain a mosaic labelling of OP neurons and their axons (frontal view, maximum projections). The OP neurons and their axons were imaged every 10 min over 1000 min from 22 hpf. Here, only the 200-400 min time window is shown as an example. Arrowheads = positions of individual growth cones over time. C-N. Individual YFP+ growth cones, as well as YFP+ cell bodies in the OP, were tracked during 4 consecutive periods of 200 min each (from 200 min of imaging, since before no growth cone could be detected, n = 5 mutants and n = 5 controls from 6 independent experiments). The mean movement of OP cell bodies was substracted from the growth cone tracks to get rid of the global flexure movement. 200-400 min: 6 growth cones in controls, 4 in mutants; 400-600 min: 15 growth cones in controls, 4 in mutants; 600-800 min: 18 growth cones in controls, 4 in mutants; 800-1000 min: 16 growth cones in controls, 7 in mutants. C, F, I, L. Tracks of the growth cones merged at their origin for the 4 consecutive periods of 200 min. For each time window, the difference in the orientation of the tracks was analysed using the circular analysis of variance based on the likelihood ratio test. D, G, J, M. Mean speed of the growth cones. Unpaired, two-tailed t tests. E, H, K, N. Persistence of the growth cones, defined as the distance between the initial and final positions of the growth cones divided by the total length of their trajectory. Unpaired, two-tailed t tests, except for the analysis of the persistence at 400-600 min, and for the speed and persistence at 600-800 min, where Mann Whitney tests were performed.