DOI: 10.5061/dryad.pc866t21r
title: Semaphorin 3f and post-embryonic regulation of retinal progenitors

description: 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.
description: <strong>Methods</strong>
 <strong><em>Animals:</em></strong> Larvae
 were used from the respective in-crosses of wild type (WT) Tupfel Long Fin
 (TL) fish, <em>sema3fa<sup>ca304/ca304</sup></em>
 zebrafish, and for the latter a corresponding WT line generated from the
 same clutch that gave rise to the specific
 <em>sema3fa<sup>ca304/ca304</sup></em> 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
 sema3fa<sup>ca304 </sup>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. <strong><em>Sema3fa
 Overexpression:</em></strong><em> </em>We
 engineered a <em>sema3fa</em> heat shock construct,
 <em>hsp70:sema3fa:p3E-MTpA</em> in <em>pdestTol2CG2
 </em>with <em>myl7:egfp</em> 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. <strong><em>Cdh2
 analysis</em></strong> Embryos injected
 with <em>hsp70:sema3fa:p3E-MTpA</em> were heat shocked at 52
 hpf and 24 hours later embryos were fixed.Cryostat sections (12 µm) of 72
 hpf WT and <em>sema3fa<sup>ca304</sup>
 </em>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. <strong><em>Mutant
 CMZ analysis:</em></strong><strong>
 </strong>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 <em>sema3fa</em> mutant
 larve included: <em>1) Nearest Neighbour
 Analysis:<strong> </strong></em>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.
 <em>2) In Situ Hybridization: </em>We performed
 either whole mount in situ hybridization (ISH) or fluorescent ISH on
 retinal sections for 72 hpf and 7 dpf fish. Probes included:
 <em>sema3fa</em>, <em>nrp2a</em>,
 <em>nrp2b</em>, <em>plxna1a</em>,
 <em>plxna1b, plxna2, plxna4</em>, <em>atoh7</em>,
 <em>neurod4</em> and <em>vsx2</em>,
 <em>plxna3</em>; and probes generated by PCR
 (<em>col15a1b, cdkn1c</em> F, <em>ccnd1</em>,
 <em>hes6</em>, <em>bmp4</em>,
 <em>nrp1a</em>, <em>nrp1b</em>) 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. <em>3)
 Immunohistochemistry</em><em>:</em> Immunohistochemistry
 was performed on 12 µm cryostat cut frozen retinal sections.
 <em>Antibodies included those against </em>Phosphohistone H3,
 Proliferating Cell Nuclear Antigen<em>, atypical PKC,
 Crumbs2a,<strong> </strong></em>c-Myc, and
 ß-catenin.<strong><em> </em></strong>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.
description: # Dryad dataset Dataset DOI:
 [10.5061/dryad.pc866t21r](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