Semaphorin 3f and post-embryonic regulation of retinal progenitors
Data files
Jul 07, 2025 version files 1.64 MB
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19_dpf_IPL_length_sema3faca304.xlsx
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ccnd1_7_dpf_ISH_data_analysis_area_sema3fa_D_V.pzfx
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Figure_2E_larval_eye_size_averages.prism
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Figure_2E_larval_eye_size_raw_data_Trial_1.xlsx
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Figure_2E_larval_eye_size_raw_data_Trial_2.xlsx
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Figure_2F_1_month_eye_area-head_area_averages.prism
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Figure_2F_Eye_measurments_1_month_raw.xlsx
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Figure_2I_J_pcna_analysis_72_hpf_averages.prism
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Figure_2I_J_pcna_area_areas_72_hpf_raw_data.xlsx
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Figure_2K_Cumulative_EdU_72_hpf_averages.prism
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Figure_2K_Cumulative_EdU_72_hpf_raw_data.xlsx
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Figure_2P_7_dpf_PCNA_averagemeasurements_PRISM_statistics.prism
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Figure_2P_PCNA_raw_measurements_7dpf.xlsx
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Figure_2Q_and_2R_EdU_CMZ_7_dpf_cell_counts_and_area_averages.prism
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Figure_2S_2T_pHH3_immunostaining_7_dpf_average_data.prism
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Figure_2S_pHH3_nearest_neighbour_raw_data.xlsx
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Figure_2T_nearest_neighbour_raw_data.xlsx
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Figure_2W_cmz_sema3fa-myc_Overexpression_averages.prism
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Figure_2W_sema3famyc_overexpression_raw_data.xlsx
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Figure_3E_F_vsx2_area_72_hpf_averages.prism
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Figure_3E_F_vsx2_area_72_hpf_raw_data.xlsx
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Figure_3G_H_high_vsx2_cmz_area_averages.prism
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Figure_3G_high_vsx2__naso_temporal_raw_data.xlsx
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Figure_3H_high_vsx2__dorso_ventral_raw_data.xlsx
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Figure_3K_L_CMZ_area_cell_counts_averages.prism
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Figure_3K_L_CMZ_area_cell_counts_raw_data.xlsx
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Figure_3O_DeLauney_neighbour_distance.prism
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Figure_4f_col15a1b_area_72_hpf_averages.prism
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Figure_4F_col15a1b_area_72_hpf_raw_data.xlsx
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Figure_4G_bmp4_area_72_hpf_averages.xlsx
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Figure_4G_bmp4_area_72_hpf_raw_data.prism
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Figure_4N_4P_5D_5H_qPCR_delta_ct_and_fold_change_averages.prism
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Figure_4N_4P_5D_5H_qPCR_fold_change_values_averages.xlsx
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Figure_4O_Q_cnd1_area_72_hpf_raw_data.xlsx
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Figure_4P_ccnd1_qPCR_averages.prism
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Figure_5C_cdkn1c_area_72_hpf_averages.prism
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Figure_5C_cdkn1c_area_72_hpf_raw_data.xlsx
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Figure_5G_atoh7_area_72_hpf_averages.prism
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Figure_5G_atoh7_area_72_hpf_raw_data.xlsx
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Figure_5Q_R_Hoescht_nuclei_in_ccnd1_and_atoh7_domains_averages.prism
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Figure_5Q_R_Hoescht_nuclei_in_ccnd1_and_atoh7_domains_raw.xlsx
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Figure_6_Edu_cohort_analysis_20_dpf_raw_data.xlsx
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Figure_6_EdU_pulse-chase_20_dpf.prism
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Figure_7C_cohort_movement_from_CMZ_14_day_chase.pzfx
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Figure_7E_F_cohort_movement_from_CMZ_3_day_chase_raw_data.xlsx
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Figure_7E_F_cohort_movement_from_CMZ_3_day_chase.prism
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Figure_7K_plxna3_area_raw_data.prism
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Figure_7K_plxna3_area_raw_data.xlsx
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Figure_7P_Cdh2_area_in_sema3fa_mutants_averages.prism
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Figure_7P_Cdh2_area_in_sema3fa_mutants_raw_data.xlsx
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Figure_7S_Cdh2_sema3fa_overexpression_averages.prism
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Figure_7S_Cdh2_sema3fa_overexpression_raw_data.xlsx
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hes6__of_eye_area_lateral_view.prism
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IPL_length_19_dpf_sema3fa_mutant.prism
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README.md
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TUNEL_sema3fa_mutant_7_dpf.prism
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Abstract
Neural progenitors produce specific cell types that form the circuits of the nervous system. Extrinsic signals regulate both progenitor proliferation and the production of specific neuron types. Where progenitors reside within a progenitor niche determines to which of these signals they are exposed, and thus likely has important consequences on the progeny they produce. Little is known, however, of the signals that determine progenitor location within the niche. Here we show that a member of the Class III family of secreted Semaphorins, Semaphorin3fa (Sema3fa), is required for the orderly arrangement of progenitors with a niche present in the periphery of the larval and adult retina of zebrafish, the ciliary marginal zone (CMZ). We used whole mount and fluorescent slide in situ hybridization to show that CMZ progenitors express mRNAs for various Sema3 receptors, including for nrp2a, nrp2band plxna1. We investigated the function and gene expression of progenitors in wild type fish and in a previously characterized CRISPR/Cas9-generated sema3fa mutant allele (sema3faca304). We found that mutant juvenile fish had a reduced eye size, implicating Sema3fa in the ongoing production of retinal cells by the CMZ. We used EdU labeling, and PCNA and pHH3 immunolabelling to show that larval mutant CMZ progenitors show altered cell cycle parameters. We also performed whole mount in situ hybridization and immunohistochemistry in retinal sections with various mRNA and protein markers of different domains of the CMZ. We found that the spatial organization of functionally distinct progenitors is disrupted. An EdU pulse-chase experiment revealed that the generation of retinal cell types in the appropriate proportions and numbers, as determined by laminar location of cells, was disrupted in the mutant fish. Our data support a model whereby Sema3fa secreted by CMZ progenitors reduces adhesive interactions, which allows for smooth progression of progenitors through the niche, ensuring progenitors receive the correct recipe of extrinsic signals to secure the proper generation of new retinal circuits.
The dataset includes the measured values that formed the averaged data points in the graphs provided in the figures of the manuscript. Each point in the graphs comes from a separate animal (n) and the data is pooled between N=2-4 independent data sets. When areas were measured this was done on retinal sections, either plastic sections of whole mount in situ hybridization samples, or cryostat sections. For cryostat sections, samples were counter-stained with Hoescht to label nuclei. These sections came from the central retina that contained a lens. Two-four images/eye/larva of the central retina, with a lens, were collected. To normalize area measurements we measured the area of the CMZ, as defined by the peripheral edge of the inner plexiform and outer plexiform layers evident either in plastic sections, or as defined by the lack of Hoescht label in cryostat sections.
For the RT-qPCR data the values are from independent data sets, performed on mRNA isolated from 30 surgically isolated eyes. Three technical replicates were performed/data set for each primer set.
Dataset DOI: 10.5061/dryad.pc866t21r
Description of the data and file structure
Figure 2 (Data): Comparison of WT and *sema3faca304 *zebrafish eyes and CMZ
Figure 2E - Size of eyes normalized to body length (nose to posterior swim bladder). Measurements of the area of the eye of live 10-11 dpf larvae from a dorsal view. Eye area was normalized to the size of the larva. Data is pooled from 2 independent data sets. Raw data (Excel) and averaged data (Prism).
Figure 2F - Size of eyes, measured from a lateral view, normalized to the head size (nose to front of gills) at 1 month. Data is pooled from 2 independent data sets. Raw data (Excel) and averaged data (Prism).
Figure 2I-K - Cumulative EdU labelling at 72 hpf for 1, 2,4,6 and 12 hours;12 um frozen transverse 72 hpf retinal sections immunolabeled for PCNA and EdU and Hoescht counter-stained. Area of PCNA expression (I) and area of intense PCNA (J) expression represented as a percentage of the CMZ area. Data is pooled from 2 independent data sets. Raw data (Excel) and averaged data (Prism). Counts of EdU+ cells in the dorsal CMZ of frozen transverse retinal sections for the different periods of EdU pulse. Data for 1-4 hours pooled from 2 independent data sets, and 6 and 12 hour data is from 1 data set. Raw data (Excel) and averaged data (Prism).
Figure 2P: PCNA immunolabeling performed on transverse 7 dpf retinal sections. The area of the intense PCNA immunoreactivity was measured and normalized to the area of the CMZ. Data is pooled from 2 independent data sets. Raw data (Excel) and averaged data (Prism).
Figure 2Q,R: EdU labeling in 7 dpf horizontal retinal sections counterstained with Hoescht. The number of EdU+ cells in the CMZ, and the area they cover of the CMZ were measured. Data is pooled from 2 independent data sets. Data for individual larvae is in a prism file.
Figure 2S,T: Horizontal 7 dpf retinal sections cut and immunolabelled for pHH3 and counterstained with Hoescht. The number of pHH3+ cells in the CMZ, and the distance between their nearest pHH3+ neighbours were measured. Data is pooled from 3 independent data sets. Raw data (Excel) and averaged data (Prism).
Figure 2W: Heat-shocked at 5 dpf and EdU pulsed 48 hours later, and transverse retinal sections processed for EdU, Hoescht, and Myc immunolabelling to identify CMZ that overexpress Sema3fa. EdU+ cells counted in dorsal CMZ that expressed Myc or were Myc negative. Data is pooled from 3 independent data sets. Raw data (Excel) and averaged data (Prism).
Figure 3 Data:
Figure 3E,F,G,H: vsx2 from plastic sections of whole mount ISH at 72 hpf. CMZ area (3E), vsx2 ISH label normalized to CMZ area (3F), and intense vsx2 label in nasal-temporal CMZ (horizontal sections; 3G) and dorsal-ventral CMZ (transverse sections; 3H). Data is pooled from 3 independent data sets. Raw data (Excel) and averaged data (Prism).
Figure 3K: CMZ area of Hoescht-labelled 7 dpf horizontal sections. Data is pooled from 3 independent data sets. Raw data (Excel) and averaged data (Prism).
Figure 3L: Number of nuclei counted in the CMZ of Hoescht-labelled 7 dpf horizontal sections. Data is pooled from 3 independent data sets. Raw data (Excel) and averaged data (Prism).
Figure 3O: Neighbour distance for pHH3+ immunolabelled cells in 7 dpf horizontal retinal sections. Data is pooled from 3 independent data sets. Averaged data (Prism).
Figure 4 Data:
Figure 4F: col15a1b mRNA ISH domain area normalized to CMZ area in horizontal plastic sections of 72 hpf retinas. Data is pooled from 2 independent data sets. Raw data (Excel) and averaged data (Prism).
Figure 4G: bmp4 mRNA ISH domain area normalized to CMZ area in horizontal plastic sections of 72 hpf retinas. Data is pooled from 3 independent data sets. Raw data (Excel) and averaged data (Prism).
Figure 4N,P: qPCR data of mRNA isolated from 30 surgically dissected eyes; col15a1b (N) and ccnd1 (P). 3 independent replicates; 3 technical replicates/independent replicate. Statistics were performed on the delta Ct values.
Figure 4O,Q: ccnd1 mRNA ISH domain area normalized to CMZ area in horizontal plastic sections (O) and transverse plastic and cryostat sections (P) of 72 hpf retinas. Data are pooled from 3 independent data sets. Raw data (Excel) and averaged data (Prism).
Figure 5 Data:
Figure 5C: cdkn1c mRNA ISH domain area normalized to CMZ area in horizontal plastic sections of 72 hpf retinas. Data is pooled from 2 independent data sets. Raw data (Excel) and averaged data (Prism).
Figure 5G: atoh7 mRNA ISH domain area normalized to CMZ area in horizontal plastic sections of 72 hpf retinas. Data is pooled from 4 independent data sets. Raw data (Excel) and averaged data (Prism).
Figure 5Q,R: Counts of Hoescht-labelled nuclei within the atoh7 and ccnd1 FISH domains of the CMZ of 72 hpf transverse retinal sections. Data is pooled from 2 independent data sets. Raw data (Excel) and averaged data (Prism).
Figure 6 Data:
Figure 6E-H: EdU pulse-chase data. EdU pulse at 7 dpf and assessment of the laminar position (RGCL, INL,ONL) of EdU positive cells at 20 dpf. Sections were collected through the entire eye. The numbers of cells in each retinal layer for individual radial cohorts were counted and represented as percentages of the entire cohort. These layer percentages were averaged for all the cohorts of a single eye. These values are shown for the ONL (Figure 6F), INL (Figure 6G) and RGCL (Figure 6H) for each eye. The numbers of cells in each radial cohort were also averaged for each eye (Figure 6E). Data is pooled from 2 independent data sets. Raw data (Excel) and averaged data (Prism).
Figure 6I: The laminar distribution for individual cohorts of a single wild type and mutant eye.
Figure 6J-M: The variability of the laminar distribution for individual cohorts of individual wild type and mutant eyes, as represented by the standard deviation of these values for each eye (each dot represents cohort variability for an individual eye). Standard deviations are shown for the ONL (K), INL (L) and RGCL (M), and compared statistically by with a Levene’s test of the equality of variance.
Figure 7 Data:
Figure 7C,E,F: The EdU-pulse retinal sections analysed in Figure 6 were analysed in Figure 7C for the distance individual radial cohorts moved from the central edge of the CMZ. For E (horizontal cryostat sections) and F (transverse cryostat sections), Edu-pulse at 6 dpf and distance moved of most central radial cohort from the peripheral edge of the CMZ at 9 dpf was measured. For C,E and F, data were pooled from 3 independent data sets. Raw data (Excel) and averaged data (Prism).
Figure 7K: plxna3 mRNA ISH domain area normalized to CMZ area in transverse plastic and frozen (FISH) sections of 72 hpf retinas. Data is pooled from 3 independent data sets. Raw data (Excel) and averaged data (Prism).
Figure 7P,S: Intense Cdh2 immunoreactive domain area normalized to CMZ area in transverse plastic sections. Done on wild type and sema3fa-/- fish (Figure 7P; data pooled from 4 independent replicates), and CMZ expressing Sema3fa Myc or not (Figure 7S; data pooled from 2-3 independent replicates). The latter were generated through heat shock of 6 dpf larve injected at the one cell stage with hsp70:sema3famyc. Data is pooled from 2-3 independent data sets. Raw data (Excel) and averaged data (Prism).
Files and variables
File: Figure_2E_larval_eye_size_averages.prism
Description: Normalized eye area data of 10 day post-fertilization live wild type and sema3fa-/- fish measured in images from a dorsal whole mount view
File: Figure_2E_larval_eye_size_raw_data_Trial_1.xlsx
Description: trial 1 - Eye area data of 10 day post-fertilization live wild type and sema3fa-/- fish measured in images from a dorsal whole mount view. Eye area and body length (nose to anterior swim bladder) in micron(2). NA placed in blank cells
Variables eye area, body length (nose to anterior swim bladder)
File: Figure_2E_larval_eye_size_raw_data_Trial_2.xlsx
Description: trial 2 - Eye area data of 10 day post-fertilization live wild type and sema3fa-/- fish measured in images from a dorsal whole mount view. Eye area and body length (nose to anterior swim bladder) in micron(2). NA placed in blank cells
Variables eye area, body length (nose to swim bladder)
File: Figure_2F_1_month_eye_area-head_area_averages.prism
Description: Normalized eye area data of 1 month live wild type and sema3fa-/- fish measured in images from a lateral whole mount view. Eye areas normalized to body length (nose to anterior edge of gills).
File: Figure_2F_Eye_measurments_1_month_raw.xlsx
Description: Eye and head areas in micron(2)
Variables Eye area, head area (nose to anterior gills)
File: Figure_2I_J_pcna_analysis_72_hpf_averages.prism
Description: Areas of PCNA immunostaining (total expression and intense expression) normalized to area of ciliary marginal zone as measured in retinal sections of one eye of individual wild type and sema3fa-/- 72 hour post fertilization fish
File: Figure_2I_J_pcna_area_areas_72_hpf_raw_data.xlsx
Description: Areas of PCNA immunostaining (intense and all) and ciliary marginal zone (CMZ) as measured in retinal sections of one eye of individual wild type and sema3fa-/- 72 hour post fertilization fish. NA placed in blank cells. Areas in micron(2)
Variables Total PCNA area, Intense PCNA area, CMZ area
File: Figure_2K_Cumulative_EdU_72_hpf_raw_data.xlsx
Description: EdU cell counts and hoescht-labelled nuclei numbers in the dorsal ciliary marginal zone (CMZ) as counted in retinal sections of one eye of individual wild type and sema3fa-/- 72 hour post fertilization fish. Fish were bathed in EdU for 1,2,4,6, and 12 hours. NA placed in blank cells.
Variables Numbers of EdU+ cells, Hoescht+ cells
File: Figure_2K_Cumulative_EdU_72_hpf_averages.prism
Description: EdU cell counts normalized to hoescht-labelled nuclei numbers in the dorsal ciliary marginal zone (CMZ) as counted in retinal sections of one eye of individual wild type and sema3fa-/- 72 hour post fertilization fish. Fish were bathed in EdU for 1,2,4,6, and 12 hours.
File: Figure_2P_7_dpf_PCNA_averagemeasurements_PRISM_statistics.prism
Description: Intense PCNA immunostaining area normalized to ciliary marginal zone (CMZ) area as measured in retinal sections for wild type and sema3fa-/- 7 day post fertilization fish.
File: Figure_2P_PCNA_raw_measurements_7dpf.xlsx
Description: Intense PCNA immunostaining area and ciliary marginal zone (CMZ) area as measured in retinal sections for wild type and sema3fa-/- 7 day post fertilization fish. Area in micron(2). NA placed in blank cells.
Variables Intense PCNA-positive area, CMZ area
File: Figure_2Q_and_2R_EdU_CMZ_7_dpf_cell_counts_and_area_averages.prism
Description: Average numbers of EdU positive cells in ciliary marginal zone (CMZ) and the percentage area of CMZ they occupy as measured in retinal sections for individual wild type and sema3fa-/- 7 day post fertilization fish
File: Figure_2S_2T_pHH3_immunostaining_7_dpf_average_data.prism
Description: Numbers of pHH3 immunostained cells in the ciliary marginal zone and the distance between cells and their nearest pHH3 positive neighbours as measured in retinal sections for individual wild type and sema3fa-/- 7 day post fertilization fish
File: Figure_2S_pHH3_nearest_neighbour_raw_data.xlsx, Figure_2T_nearest_neighbour_raw_data.xlsx
Description: distance between cells and their nearest pHH3 positive neighbours in the ciliary marginal zone (CMZ) as measured in retinal sections for individual wild type and sema3fa-/- 7 day post fertilization fish. Data in microns. NA placed in blank cells.
Variables Neighbour distance
File: Figure_2W_cmz_sema3fa-myc_Overexpression_averages.prism
Description: Average EdU+ cells counted in dorsal CMZ that expressed Myc or were Myc negative. Larvae expressing hsp70:semaefamyc were heat-shocked at 5 dpf and EdU pulsed 48 hours later, and transverse retinal sections processed for EdU, Hoescht, and Myc immunolabelling to identify CMZ that overexpress Sema3fa.
File: Figure_2W_sema3famyc_overexpression_raw_data.xlsx
Description: EdU+ cell counts in dorsal CMZ that expressed Myc or were Myc negative. Larvae expressing hsp70:semaefamyc were heat-shocked at 5 dpf and EdU pulsed 48 hours later, and transverse retinal sections processed for EdU, Hoescht, and Myc immunolabelling to identify CMZ that overexpress Sema3fa. NA placed in blank cells.
Variables EdU+ cells
File: Figure_3E_F_vsx2_area_72_hpf_averages.prism
Description: Average area of the ciliary marginal zone (CMZ) and area of vsx2 in situ hybridization expression normalized to CMZ area as measured in 7 um plastic sections of whole mount in situ hybridization of 72 hpf wild type and sema3fa-/- fish.
File: Figure_3E_F_vsx2_area_72_hpf_raw_data.xlsx
Description: Area of the ciliary marginal zone (CMZ) and area of vsx2 in situ hybridization expression normalized to CMZ area as measured in 7 um plastic sections of whole mount in situ hybridization of 72 hpf wild type and sema3fa-/- fish. Data in microns(2). NA placed in blank cells.
Variables CMZ area, vsx2 expression area
File: Figure_3G_H_high_vsx2_cmz_area_averages.prism
Description: Area of intense vsx2 in situ hybridization expression normalized to the area of the ciliary marginal zone (CMZ) in transverse (dorso-ventral) and horizontal (nano-temporal) plastic retinal sections of vsx2 whole mount in situ hybridization.
File: Figure_3G_high_vsx2__naso_temporal_raw_data.xlsx, Figure_3H_high_vsx2__dorso_ventral_raw_data.xlsx
Description: Area of intense vsx2 in situ hybridization expression normalized to the area of the ciliary marginal zone (CMZ) in transverse (dorso-ventral) and horizontal (nano-temporal) plastic retinal sections of vsx2 whole mount in situ hybridization. Data in microns(2). NA placed in blank cells.
Variables vsx2 expression area, cmz area
File: Figure_3K_L_CMZ_area_cell_counts_averages.prism
Description: Averages of area of ciliary marginal zone (CMZ) and numbers of Hoescht-labelled cells in the CMZ as measured from 12 um frozen retinal sections of 7 day post-fertilization wild type and sema3fa-/- fish
File: Figure_3K_L_CMZ_area_cell_counts_raw_data.xlsx
Description: Averages of area of ciliary marginal zone (CMZ) and numbers of Hoescht-labelled cells in the CMZ as measured from 12 um frozen retinal sections of 7 day post-fertilization wild type and sema3fa-/- fish. Areas in microns(2). NA placed in blank cells.
Variables Hoescht+ cell numbers, CMZ area
File: Figure_3O_DeLauney_neighbour_distance.prism
Description: Average distance between ciliary marginal zone (CMZ) nuclei and their nearest neighbours as measured from 12 um frozen retinal sections of 7 day post-fertilization wild type and sema3fa-/- fish.
File: Figure_4f_col15a1b_area_72_hpf_averages.prism
Description: Area of the col15a1b in situ hybridization expression domain normalized to the area of the ciliary marginal zone as measured in 7 um plastic sections of whole mount in situ hybridization of 72 hpf wild type and sema3fa-/- fish.
File: Figure_4F_col15a1b_area_72_hpf_raw_data.xlsx
Description: Area of the col15a1b in situ hybridization expression domain and the area of the ciliary marginal zone (CMZ) as measured in 7 um plastic sections of whole mount in situ hybridization of 72 hpf wild type and sema3fa-/- fish. Areas in microns(2). NA placed in blank cells.
Variables col15a1b expression area, cmz area
File: Figure_4G_bmp4_area_72_hpf_raw_data.prism
Description: Average areas of the bmp4 in situ hybridization expression domain and the area of the ciliary marginal zone (CMZ) as measured in 7 um plastic sections of whole mount in situ hybridization of 72 hpf wild type and sema3fa-/- fish.
File: Figure_4G_bmp4_area_72_hpf_averages.xlsx
Description: Areas of the bmp4 in situ hybridization expression domain and the area of the ciliary marginal zone (CMZ) as measured in 7 um plastic sections of whole mount in situ hybridization of 72 hpf wild type and sema3fa-/- fish. Areas in microns(2). NA placed in blank cells.
Variable bmp4 expression area, cmz area
File: Figure_4N_col15a1b_qPCR_averages.prism
Description: Delta ct values and fold change comparison for col15a1b for RT-qPCR performed on mRNA isolated from 72 hour post fertilization surgically dissected eyes
File: Figure_4P_ccnd1_qPCR_averages.prism
Description: Delta ct values and fold change comparison for ccnd1 for RT-qPCR performed on mRNA isolated from 72 hour post fertilization surgically dissected eyes
File: Figure_4O_Q_ccnd1_area_72_hpf_averages.prism
Description: Average areas of the ccnd1 in situ hybridization expression domain normalized to the area of the ciliary marginal zone (CMZ) as measured in 7 um plastic sections of whole mount in situ hybridization of 72 hpf wild type and sema3fa-/- fish.
File: Figure_4O_Q_cnd1_area_72_hpf_raw_data.xlsx
Description: Areas of the ccnd1 in situ hybridization expression domain and the areas of the ciliary marginal zone (CMZ) as measured in 7 um plastic sections of whole mount in situ hybridization of 72 hpf wild type and sema3fa-/- fish. Areas in microns(2). NA placed in blank cells.
Variables ccnd1 expression area, cmz area
File: Figure_4N_4P_5D_5H_qPCR_delta_ct_and_fold_change_averages.prism
Description: Delta ct values and fold change comparison for col15a1b, atoh7, ccnd1 and cdkn1c in 72 hour post fertilization surgically dissected eyes
File: Figure_4N_4P_5D_5H_qPCR_fold_change_values_averages.xlsx
Description: Delta ct values and fold change comparison for col15a1b, atoh7, ccnd1 and cdkn1c for RT-qPCR performed on mRNA isolated from 72 hour post fertilization surgically dissected eyes (N=30)
Variables Fold change, Delta Ct
File: Figure_5C_cdkn1c_area_72_hpf_averages.prism
Description: Areas of the cdkn1c in situ hybridization expression domain normalized to the area of the ciliary marginal zone (CMZ) as measured in 7 um plastic sections of whole mount in situ hybridization of 72 hpf wild type and sema3fa-/- fish.
File: Figure_5C_cdkn1c_area_72_hpf_raw_data.xlsx
Description: Areas of the cdkn1c in situ hybridization expression domain and the area of the ciliary marginal zone (CMZ) as measured in 7 um plastic sections of whole mount in situ hybridization of 72 hpf wild type and sema3fa-/- fish. Areas in microns(2). NA placed in blank cells.
Variables cdkn1c expression domain area, cmz area
File: Figure_5G_atoh7_area_72_hpf_averages.prism
Description: Areas of the atoh7 in situ hybridization expression domain normalized to the area of the ciliary marginal zone (CMZ) as measured in 7 um plastic sections of whole mount in situ hybridization of 72 hpf wild type and sema3fa-/- fish.
File: Figure_5G_atoh7_area_72_hpf_raw_data.xlsx
Description: Areas of the atoh7 in situ hybridization expression domain and the areas of the ciliary marginal zone (CMZ) as measured in 7 um plastic sections of whole mount in situ hybridization of 72 hpf wild type and sema3fa-/- fish. Areas in microns(2). NA placed in blank cells.
Variables atoh7 expression domain area, cmz area
File: Figure_5Q_R_Hoescht_nuclei_in_ccnd1_and_atoh7_domains_averages.prism
Description: Average numbers of Hoescht nuclei in ccnd1 and atoh7 in situ hybridization expression domains within the ciliary marginal zone (CMZ) as measured in 12 um frozen retinal sections of fluorescent in situ hybridization of 72 hpf wild type and sema3fa-/- fish.
File: Figure_5Q_R_Hoescht_nuclei_in_ccnd1_and_atoh7_domains_raw.xlsx
Description: Average numbers of Hoescht nuclei in ccnd1 and atoh7 in situ hybridization expression domains within the ciliary marginal zone (CMZ) as measured in 12 um frozen retinal sections of fluorescent in situ hybridization of 72 hpf wild type and sema3fa-/- fish. NA placed in blank cells.
Variables Hoescht+ nuclei numbers
File: Figure_6_Edu_cohort_analysis_20_dpf_raw_data.xlsx
Description: Numbers of cells in the outer nuclear layer (ONL), inner nuclear layer (INL), and retinal ganglion cell layer (RGCL) for individual radial cohorts of EdU labelled cells generated by a pulse of EdU at 7 days post fertilization followed by a two week chase. Comparison between wild type and sema3fa-/- larvae. Cell counts performed in every second retinal section through the central retina containing the lens. Numbers represented as % of cells within the cohort. Standard deviations of the percentage data for ONL, INL and RGCL for cohorts of individual eyes. NA placed in blank cells.
Variables Numbers cell numbers, percentage of cells, standard deviation
File: Figure_6_EdU_pulse-chase_20_dpf.prism
Description: Average percentage for individual larvae of cells in the outer nuclear layer (ONL), inner nuclear layer (INL), and retinal ganglion cell layer (RGCL) for individual radial cohorts of EdU labelled cells generated by a pulse of EdU at 7 days post fertilization followed by a two week chase. Comparison between wild type and sema3fa-/- larvae. Cell counts performed in every second retinal section through the central retina containing the lens. Average standard deviations of the percentage data for ONL, INL and RGCL for cohorts of individual eyes.
File: Figure_7C_cohort_movement_from_CMZ_14_day_chase.pzfx
Description: Average distance of individual EdU-labelled cohorts, generated by a pulse of EdU at 7 days post fertilization followed by a two week chase, from the central edge of the ciliary marginal zone (CMZ) for individual eyes. Distances (microns) measured for radial cohorts as observed in 12 µm frozen central retinal sections. Comparison between wild type and sema3fa-/- larvae.
File: Figure_7E_F_cohort_movement_from_CMZ_3_day_chase.prism
Description: Average distance of individual EdU-labelled cohorts, generated by a pulse of EdU at 7 days post fertilization followed by a 3 day chase, from the peripheral edge of the ciliary marginal zone (CMZ) for individual eyes. Distances (microns) measured for radial cohorts as observed in 12 µm frozen central retinal sections. Comparison between wild type and sema3fa-/- larvae.
File: Figure_7E_F_cohort_movement_from_CMZ_3_day_chase_raw_data.xlsx
Description: Distances of individual EdU-labelled cohorts, generated by a pulse of EdU at 7 days post fertilization followed by a 3 day chase, from the peripheral edge of the ciliary marginal zone (CMZ) for individual eyes. Distances (microns) measured for radial cohorts as observed in 12 µm frozen central retinal sections. Comparison between wild type and sema3fa-/- larvae. NA placed in blank cells.
Variables Distance
File: Figure_7K_plxna3_area_raw_data.xlsx
Description: Areas of the plxna3 in situ hybridization expression domain normalized to the areas of the ciliary marginal zone (CMZ) as measured in either 7 um plastic sections of whole mount in situ hybridization of 72 hpf wild type and sema3fa-/- fish, or fluorescent in situ hybridization on frozen retinal sections. Areas in microns(2). NA placed in blank cells.
Variables plxna3 in situ hybridization expression domain area, cmz area
File: Figure_7K_plxna3_area_raw_data.prism
Description: Average areas of the plxna3 in situ hybridization expression domain and the areas of the ciliary marginal zone (CMZ) as measured in either 7 um plastic sections of whole mount in situ hybridization of 72 hpf wild type and sema3fa-/- fish, or fluorescent in situ hybridization on frozen retinal sections.
File: Figure_7P_Cdh2_area_in_sema3fa_mutants_averages.prism
Description: Average areas of the intense Cdh2 immunoreative domain normalized to the area of the ciliary marginal zone (CMZ) as measured in 12 um frozen retinal sections of 72 hpf wild type and sema3fa-/- fish.
File: Figure_7P_Cdh2_area_in_sema3fa_mutants_raw_data.xlsx
Description: Areas of the intense Cdh2 immunoreative domain and the areas of the ciliary marginal zone (CMZ) as measured in 12 um frozen retinal sections of 72 hpf wild type and sema3fa-/- fish. Areas in microns(2). NA placed in blank cells.
Variables Area of the intense Cdh2 immunoreative domain, CMZ area
File: Figure_7S_Cdh2_sema3fa_overexpression_averages.prism
Description: Average areas of the intense Cdh2 immunoreative domain normalized to the area of the ciliary marginal zone (CMZ) in the CMZ expressing or not expressing Sema3fa-myc of embryos injected at the 1-cell stage with a hsp70:sema3famyc construct. Embryos were heat-shocked at 52 hpf and analysis performed 24 hours later; transverse retinal sections were processed for Cdh2 and Myc immunolabelling.
File: Figure_7S_Cdh2_sema3fa_overexpression_raw_data.xlsx
Description: Areas of the intense Cdh2 immunoreative domain normalized to the area of the ciliary marginal zone (CMZ) in the CMZ expressing or not expressing Sema3fa-myc of embryos injected at the 1-cell stage with a hsp70:sema3famyc construct. Embryos were heat-shocked at 52 hpf and analysis performed 24 hours later; transverse retinal sections were processed for Cdh2 and Myc immunolabelling. Areas in microns(2). NA placed in blank cells.
Variables intense Cdh2 immunoreative domain area, cmz area
File: TUNEL_sema3fa_mutant_7_dpf.prism
Description: Average numbers of TUNEL positive cells counted in whole mount eyes of 7 day post fertilization sema3fa-/- and wild type eyes PTU-treated at 24 hours
File: ccnd1_7_dpf_ISH_data_analysis_area_sema3fa_D_V.pzfx
Description: Average area of the atoh7 in situ hybridization expression domain normalized to the area of the ciliary marginal zone (CMZ) as measured in 7 um plastic retinal sections of whole mount in situ hybridization of wild type and sema3fa-/- 7 day post-fertilization fish
File: hes6__of_eye_area_lateral_view.prism
Description: Average area of hes6 in situ hybridization expression in the area ringing the lens normalized to the area of the eye as measured from lateral views of whole mount wild type and sema3fa-/- fish
File: IPL_length_19_dpf_sema3fa_mutant.prism
Description: Average circumferential length of the inner plexiform layer (IPL) in Hoescht-labelled sections of wildtype and sema3fa-/- 19 day post fertilization retinas
File: 19_dpf_IPL_length_sema3faca304.xlsx
Description: Circumferential lengths of the inner plexiform layer (IPL) in Hoescht-labelled sections of wildtype and sema3fa-/- 19 day post fertilization retinas. Distances in microns. NA placed in blank cells.
Variables: Length of inner plexiform layer
Code/software
1) .prism files to be viewed with GraphPad Prism 10
2) .xlxs files to be viewed with Microsoft Excel 16
Access information
Other publicly accessible locations of the data:
Not applicable
Data was derived from the following sources:
- not applicable
Methods
Animals: Larvae were used from the respective in-crosses of wild type (WT) Tupfel Long Fin (TL) fish, sema3faca304/ca304 zebrafish, and for the latter a corresponding WT line generated from the same clutch that gave rise to the specific sema3faca304/ca304 fish line (Halabi et al., 2021). For whole mount in situ hybridization, pigment synthesis was inhibited by adding 1-phenyl-2-thiourea (PTU) to the E3 solution at 12-24 hpf.
Eye Size Measurements: Live sema3faca304 and WT siblings were imaged from a dorsal orientation at 10 days post fertilization (dpf). Separate sets of one-month old fish were imaged from a lateral orientation. The areas of the pigmented eyes of larvae (dorsal view) and juveniles (lateral view) were measured in a masked fashion and normalized to the nose to swim bladder length and the size of the head (measured to the back of the gills), respectively.
Sema3fa Overexpression: We engineered a sema3fa heat shock construct, hsp70:sema3fa:p3E-MTpA in pdestTol2CG2 with myl7:egfp as the transgenesis marker (Kwan et al., 2007). The heat shock construct was injected into one cell-stage zebrafish embryos alongside transposase mRNA. Larvae (5 dpf) with eGFP positive heart expression were heat-shocked, and two days later processed for EdU labeling. Sema3fa overexpressing cells were identified by immunolabelling retinal cryostat sections for Myc and EdU cells by Click-iT. Numbers of EdU-positive cells (3-4 sections/larva) were counted in dorsal CMZ that showed no (Myc-) or considerable Myc (Myc+) immunolabelling.
Cdh2 analysis
Embryos injected with hsp70:sema3fa:p3E-MTpA were heat shocked at 52 hpf and 24 hours later embryos were fixed.Cryostat sections (12 µm) of 72 hpf WT and sema3faca304 retinas, or heat-shocked Sema3fa overexpression larvae, were immunostained with a polyclonal antibody against Cdh2. The area of the bright Cdh2 immunopositive label was represented as a percentage of the area of the CMZ, and the percentages for several sections averaged for each individual embryo.
Mutant CMZ analysis: Larvae were collected at 72 hours post-fertilization (hpf) and 7 days post-fertilization (dpf). Cell counts and gene expression domain areas were measured in plastic or frozen cryostat retinal sections and then normalized to the area of the CMZ. Frozen sections were counterstained with Hoescht to label cell nuclei. The CMZ domain was defined by the anatomical landmarks of the edges of the inner and outer plexiform layers. CMZ measurements were made in 2-3 sections through the central retina of a single eye for each larva, and data was pooled for 2-4 independent experiments. Each point in the graphs is from a single larva. Analysis of the CMZ in wild type and sema3fa mutant larve included:
1) Nearest Neighbour Analysis: Hoechst-labelled nuclei within the CMZ of retinal sections of 7 dpf fish, We used the Delaunay Triangulation plugin available in Fiji (Schindelin et al., 2012) to determine distances between the centres of nuclei of neighbouring Hoescht-labelled nuclei of retinal sections of 7 dpf fish.
2) In Situ Hybridization: We performed either whole mount in situ hybridization (ISH) or fluorescent ISH on retinal sections for 72 hpf and 7 dpf fish. Probes included: sema3fa, nrp2a, nrp2b, plxna1a, plxna1b, plxna2, plxna4, atoh7, neurod4 and vsx2, plxna3; and probes generated by PCR (col15a1b, cdkn1c F, ccnd1, hes6, bmp4, nrp1a, nrp1b) of cDNA generated from zebrafish embryos. Whole mount preparations were plastic sectioned (7 µm). In sections the areas of ISH signal were measured and normalized to the CMZ area. CMZ measurements were made in 2-3 sections through the central retina of a single eye for each larva, and data was pooled for 2-4 independent experiments.
3) Immunohistochemistry: Immunohistochemistry was performed on 12 µm cryostat cut frozen retinal sections. Antibodies included those against Phosphohistone H3, Proliferating Cell Nuclear Antigen, atypical PKC, Crumbs2a, c-Myc, and ß-catenin. PCNA area was measured in sections and normalized to the CMZ area, pHH3 cells were counted within the CMZ, and the distances between neighbouring PCNA cells were measured.
4) Progenitor Proliferation: We used Click-iT chemistry to label proliferating cells in the CMZ of retinal cryosections. Larvae at the indicated ages were bathed in Edu for 1,2,4,6 or 12 hours at 28°C and then processed either immediately (progenitor analysis) or after 3-14 days (radial cohort analysis). For the radial cohort analysis, we restricted our analysis to those cell cohorts where the participating sections were fully intact and cells could be counted across all three layers.