No disease-related biochemical alterations in <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants.

<p><b>A-C</b>: <i>ctsd</i> mRNA expression and Ctsd protein levels in <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants. <b>A</b>: <i>ctsd</i> mRNA expression at 5dpf and in 7mpf as well as 22mpf brain samples derived from <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants compared to wildtype controls. 5dpf: p = 1.0. 7mpf: p = 0.4. 22mpf: p = 0.7. <b>B</b>: Western blots showing Ctsd and α-tubulin in 5dpf whole lysis samples as well as 7mpf and 22mpf brain samples from <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants and wildtype controls. <b>C</b>: Quantification of Ctsd from the Western blots shown in B normalized to α-tubulin. 5dpf: n = 4. 7mpf and 22mpf: n = 3. 5dpf: p = 0.8857. 7mpf: p = 1.0. 22mpf: p = 0.7. <b>D</b>: <i>stat3</i> mRNA levels in 5dpf <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants and 7mpf as well as 22mpf <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutant brains compared to wildtype control samples. 5dpf: p = 0.1. 7mpf: p = 0.4. 22mpf: p = 0.4. <b>E</b>: <i>flnca</i> mRNA levels in <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} larvae at 5dpf and 7mpf as well as 22mpf <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} brain samples in comparison to wildtype samples. 5dpf: p = 0.4. 7mpf: p = 0.7. 22mpf: p = 0.7. <b>F</b>: <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} larvae of 5dpf and 7mpf and 22mpf <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} brain samples are analysed for <i>flncb</i> mRNA expression levels and compared to wildtype. 5dpf: p = 0.1. 7mpf: p = 0.4. 22mpf: p = 0.4. A,D-E: Normalized to <i>actb1</i> and <i>tbp</i>. qPCR. 5dpf: n = 4. 7mpf and 22mpf: n = 3. A,C-E: S.E.M. Mann-Whitney test (two-tailed).</p

The number of MPCs is equal.

<p><b>A</b>: Schematic illustration of a zebrafish embryo at 24hpf (lateral view) and a detail of the region above the end of the yolk extension imaged for the analysis of the MPCs (lateral view). The four somites, which were considered for the quantification of MPCs are marked with red lines. <b>B</b>: Immunofluorescence staining with Pax7 at 24hpf in <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants and wildtype embryos. In each image two xanthophores are exemplarily marked with a red x. Images taken by spinning disk confocal microscopy. Anterior to the left. Lateral view. Orthogonal projections. Scale bar: 50μm. <b>C</b>: Quantification of Pax7-positive cells in the 4 somites (1–4) above the end of the yolk extension in <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants and wildtype embryos. Only Pax7-positive cells at the surface of the somites were counted. n = 15. S.E.M. Mann-Whitney test (two-tailed). All n.s. S1: p = 0.0854. S2: p = 0.7977. S3: p = 0.3489. S4: p = 0.2337.</p

No SpMN axonopathy in Grna and Grnb single and double KOs.

<p><b>A</b>: Schematic illustration of a zebrafish embryo at 28hpf (lateral view) and a detail of the region above the end of the yolk extension imaged for the analysis of SpMN axons (lateral view). <b>B:</b> In Grna and Grnb single and double KOs the SpMN axons show no extended branching. Whole-mount immunofluorescence staining of 28hpf embryos with znp1 antibody. The 5 SpMN axons above the end of the yolk extension are shown. Images taken by spinning disk confocal microscopy. Anterior to the left. Lateral view. Orthogonal projections. Scale bar: 100μm. <b>C-E</b>: Quantification of the SpMN axon length in homozygous and heterozygous Grna and Grnb single and double KOs and wildtype siblings. The SpMN axon length of the 5 SpMN axons (1–5) above the end of the yolk extension is measured from the exit point of the spinal cord to the tip of the growth cone. <b>C</b>: Homozygous and heterozygous Grna KOs and wildtype siblings. n = 30. <b>D</b>: Homozygous and heterozygous Grnb KOs and wildtype siblings. n = 30. <b>E</b>: Homozygous and heterozygous Grna and Grnb KOs and wildtype siblings. n = 25. S.E.M. Two-way ANOVA. Bonferroni post-test. All non-significant (n.s.).</p

Generation of Grna and Grnb KOs using ZFNs.

<p><b>A</b>: Schematic illustration of human GRN and zebrafish Granulins. Human GRN has 7 ½ granulin domains, while 12 granulin domains are found in Grna, 9 in Grnb, and 1 ½ in Grn1 and Grn2. Grey: signal peptide. Black numbers: amino acids. Darker colour and white letters/numbers: granulin domains. <b>B-C:</b> Localisation of ZFN target sequences in <i>grna</i> and <i>grnb</i> and predicted protein sequence of selected alleles. The genomic structure of <i>grna</i> and <i>grnb</i> is depicted. ZFNs targeting <i>grna</i> and <i>grnb</i> are located in the first and fourth coding exon, respectively. ZFN-induced genomic lesions in <i>grna</i> can be detected with the restriction enzyme (RE) Eco91I and in <i>grnb</i> with the RE XcmI. Grey boxes: untranslated region (UTR). Coloured boxes: coding region. Light blue: ZFN binding sites in <i>grna</i>. Light red: ZFN binding sites in <i>grnb</i>. Green lines: binding sites of the RE. Dashed green line: cut site of the RE. Protein sequences of wildtype (wt) <i>grna</i> and 4 <i>grna</i> mutation alleles as well as wt <i>grnb</i> and 3 <i>grnb</i> mutation alleles are shown. *: Stop. <b>D-E</b>: Grna and Grnb protein is lost in all mutants. <b>D</b>: Grna signal is lost in all adult kidney samples from grna^−/− mutants, whereas a signal is present in wt. A Calnexin blot serves as a loading control. <b>E</b>: The Grnb signal observed in wt is lost in all 1.5dpf samples from <i>grnb</i>^−/− mutants. Injection of <i>grnb</i> mRNA leads to an increase in signal. The loading control α-tubulin is present in all samples.</p

No microgliosis and neurodegeneration in <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants.

<p><b>A</b>: Schematic illustration of a zebrafish larvae at 3dpf (lateral view) and a detail of the region (red line), dorsal view, imaged for the analysis of neutral red positive particles (B). The dashed red line marks the area that was imaged in the time lapse recordings of microglia (C). <b>B</b>: The number of neutral red positive particle in the region illustrated in A (Z-stack) is unchanged in wildtype and <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants. n = 15. S.E.M. Mann-Whitney test (two-tailed). p = 0.2884. <b>C-E</b>: Microglia in Tg(<i>apoeb</i>:lynEGFP) <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants and wildtype larvae at 3dpf are indistinguishable. <b>C:</b> Still images of the time lapse recordings in the optic tectum recorded by spinning disk confocal microscopy. Two microglia cells marked in each genotype by a white and yellow arrow. Dorsal view. Anterior to the left. n = 3. Scale bar: 50μm. Recording time: 60min. 1frame/min. <b>D:</b> The distance microglia move within one hour in <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants and wildtype larvae. Quantification of n = 3x5 randomly selected microglia from the time lapse recordings shown in C. S.E.M. Mann-Whitney test (two-tailed). p = 0.0671. <b>E:</b> Processes in the <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants and wildtype larvae persist for same durations. Quantification of n = 3x5 randomly selected processes from the time lapse recordings shown in C. S.E.M. Mann-Whitney test (two-tailed). p = 0.8296. <b>F:</b> Schematic illustration of a zebrafish larvae at 5dpf (lateral view) and a detail of the region, dorsal view, imaged for the analysis of acridine orange (AO) positive cells. <b>G</b>: The number of acridine orange positive cells in the region illustrated in C (Z-stack) is unchanged in wildtype and <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants. n = 15. S.E.M. Mann-Whitney test (two-tailed). p = 0.69.</p

<i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants swim like wildtype.

<p><b>A</b>: The swim path of wildtype, <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants, DMSO-treated, and PTZ-treated larvae is shown. PTZ treatment was used as a positive control. 5dpf. Movements < 2mm/s: black lines. Movements 2–6mm/s: green lines. Movements > 6mm/s: red lines. Recording time: 5min. <b>B</b>: The total distance moved within 5min in wildtype, <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants, DMSO-treated, and PTZ-treated larvae is shown. Wt-<i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>}: p = 0.2386. Wt-DMSO: p = 0.0534. Wt-PTZ: ***p = 0.0002. DMSO-PTZ: **p = 0.004. <b>C</b>: A graph of the mean velocity of wildtype, <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants, DMSO-treated, and PTZ-treated larvae is shown. Time frame: 5min. Wt-<i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>}: p = 0.5657. Wt-DMSO: p = 0.8081. Wt-PTZ: *p = 0.0137. DMSO-PTZ: **p = 0.0014. <b>D</b>: Percentage of time spent for movements with a velocity above 2mm/s in wildtype, <i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>} mutants, DMSO-treated, and PTZ-treated larvae is plotted. Time frame: 5min. Wt-<i>grna</i>^{<i>−/−</i>};<i>grnb</i>^{<i>−/−</i>}: p = 0.2585. Wt-DMSO: p = 0.0668. Wt-PTZ: **p = 0.0016. DMSO-PTZ: **p = 0.0037. <b>B-D:</b> n = 18. S.E.M. Mann-Whitney test (two-tailed).</p

Non-invasive recordings using a patch pipette on the surface of the optic tectum.

<p>(A) Photo of the non-invasive EEG ZF recording set-up. To the left is a surface recording pipette, filled with 1 M NaCl. In the upper right is the reference electrode. Both are connected to the amplifier, as shown in the schematic in (B). The mounted fish is positioned on a microscope with which ZF viability can be monitored continuously. (C), long-term EEG recording of a kcnj10a morphant fish initially paralyzed in 20 mM D-tubocurarine. Fish were generally viable for over an hour, but paralysis appeared to wear off after 50 to 60 min. Movement artifacts ensued, associated with electrical activity and visible twitching (arrowhead). A 5 s period indicated by a box in C is represented above the trace in higher resolution.</p

<i>cln3</i> ATG MO morphants have abnormal brain and heart morphology.

(A, A’) 32 hpf normal development of the WT forebrain, midbrain, hindbrain, retina and fourth ventricle. (B, B’) 32 hpf 1.6 ng cln3 ATG MO morphants display abnormal development of all parts of the brain, a smaller retina, and enlargement of the fourth ventricle. (C, C’) normal development of the WT tail, yolk, pericardial sac and heart at 4 dpf. The WT brain completely fills the cranium. (D, D’) 1.6 ng cln3 ATG MO morphants have a curved tail, and a larger yolk and pericardial sac at 4 dpf. The heart has an elongated appearance and is lacking pigmented erythrocytes. The fourth ventricle is enlarged and the mid- and hindbrain appear smaller. Lateral views. Anterior is to left. Dorsal is up. Abbreviations: v, fourth ventricle; hb, hindbrain; mhb, mid-hindbrain boundary; mb, midbrain, fb, forebrain, r, retina; t, tail; y, yolk; ps, pericardial sac; h, heart; (n = 4 per group). Lateral views. Scale bars: A-D 250 μm; A’-D’ 100 μm.</p

Invasive recordings using a patch pipette inserted into the optic tectum of 120 hpf old ZF larvae.

<p>(A), 22 min recording of a buffer-injected control larva. Frequent spiking and also low level activity is present throughout the trace. (B), higher temporal and voltage resolution of the area marked in A. (C), kcnj10a morphant larva, showing genuine epileptic activity (marked with stars). Low-level activity is followed by a spontaneous large transient (arrowhead). Similar transients were seen while the fish showed brief total body contractions and could also be produced by a light tap on the recording setup (vertical arrow). The square marked “D” in C is shown in (D) at higher resolution.</p

Cellular proliferation is abnormal in <i>cln3</i> ATG MO morphant zebrafish.

<p>(A, A’) Proliferation, assayed at 4 dpf using anti-PH3 (a marker of proliferative cells in mitotic M phase), is observed throughout the 4 dpf WT retina, jaw and the brain. (B, B’) A marked reduction in the amount of cellular proliferation throughout the retina can be seen in the 1.6 ng <i>cln3</i> ATG MO morphant. Although not quantified, it appears that proliferation in the morphant brain (B) is increased compared to WT. Confocal images are Z-projections. Scale bar: 100 μm (A, A’, B) and 50 μm (B’). Lateral views. Anterior is to the left. Dorsal is up. (C) Quantification of these data show that the number of proliferating cells in the morphant retina is significantly reduced from 100.3 cells in WT to 50.3 cells in morphants; ***<i>p</i><0.0006 (<i>n</i> = 3 zebrafish per group). (D) Quantification demonstrating a significantly reduced mean retinal area in the morphants (0.0566 mm² for WT retinae compared to 0.0135 mm² for morphant retinae; ****<i>p</i><0.0001 (n = 10 zebrafish per group)). (C, D) Data represent mean ±SD; results were evaluated using a 2-tailed unpaired Student’s <i>t</i>-test.</p