Journal: bioRxiv

Article Title: A parental transcriptional response to microsporidia infection induces inherited immunity in offspring

doi: 10.1101/2020.10.11.335117

Figure Lengend Snippet: P0 populations of N2 C. elegans were either not infected or infected with a moderate dose of N. parisii spores at the L1 stage (doses defined in methods). At 72 hpi, animals were treated with sodium hypochlorite solution to release F1 embryos. Naïve uninfected larvae served as a negative control for N. parisii detection. Naïve infected larvae exposed to a maximal dose of N. parisii served as a positive control for N. parisii detection. At 2 hpi F1 animals were fixed and stained with DY96 to detect N. parisii spores (green) as well as a FISH probe to detect N. parisii 18S RNA (red). N. parisii was never observed in primed, uninfected animals. Representative images of F1 populations are shown. Scale bars, 50 μm.

Article Snippet: Worms were then washed once in 1 ml hybridization buffer (900 mM NaCl, 20 mM Tris pH 8.0, 0.01% SDS), and incubated overnight at 46°C in 100 μl hybridization buffer containing 5-10 ng/μl of FISH probe conjugated to a Cal Fluor 610 dye (LGC Biosearch Technologies).

Techniques: Infection, Negative Control, Positive Control, Staining

Journal: bioRxiv

Article Title: A parental transcriptional response to microsporidia infection induces inherited immunity in offspring

doi: 10.1101/2020.10.11.335117

Figure Lengend Snippet: (A-D) P0 populations of N2 C. elegans were either not infected or infected with a low dose of N. parisii spores at the L1 stage (doses defined in methods). At 72 hpi, animals were treated with sodium hypochlorite solution to release F1 embryos. Naïve and primed F1 larvae were exposed to a maximal dose of N. parisii at the L1 stage. At 30 mpi, animals were fixed and stained with DY96 to detect N. parisii spores (green) as well as a FISH probe to detect N. parisii 18S RNA (red). (A) Representative images of worms stained with FISH probe to detect invaded sporoplasms, marked by asterisks. Scale bars, 25 μm. (B) The number of sporoplasms per animal was quantified by microscopy. Each circle represents a count from a single worm. Mean ± SEM (horizontal bars) is shown. Data pooled from 3 independent experiments using n = 16-20 worms per condition per experiment. (C) Representative images of worms stained with DY96 to detect spores, marked by asterisks, in the intestinal lumen. Scale bars, 25 μm. (D) The number of spores per animal was quantified by microscopy. Each circle represents a count from a single worm. Mean ± SEM (horizontal bars) is shown. Data pooled from 3 independent experiments using n = 20-21 worms per condition per experiment. (E) Naïve and primed F1 larvae were fed fluorescent beads at the L1 stage. After 30 min, animals were fixed and imaged. Fluorescence from beads was thresholded to determine the amount of beads eaten by each worm (% of body filled with beads). Each circle represents a measurement from a single worm. Mean ± SEM (horizontal bars) is shown. Data pooled from 3 independent experiments using n = 24-30 worms per condition per experiment. (F) Naïve and N. parisii -primed F1 L1 larvae were maintained on slow-killing plates with wild-type P. aeruginosa and survival monitored. Animal survival at an 84 hpi end point is shown. Mean ± SEM (horizontal bars) is shown. Data pooled from 4 independent experiments each comprising 2-4 technical replicates, using n = 13-37 worms per condition per experiment. (G-H) Naïve and N. parisii -primed F1 L1 larvae were maintained on slow-killing plates with dsRed- P. aeruginosa and fixed at 48 hpi. (G) Representative images of worm populations grown on dsRed- P. aeruginosa . Scale bars, 200 μm. (H) Images of worms were analysed and fluorescence thresholded to determine bacterial burdens of individual worms (% of body filled with P. aeruginosa ). Each circle represents a measurement from a single worm. Mean ± SEM (horizontal bars) is shown. Data pooled from 3 independent experiments using n = 21-47 worms per condition per experiment. (I) Naïve and primed F1, and pals-22 mutant L1 larvae were infected with Orsay virus. At 16 hpi, worms were fixed and stained with FISH probe to detect Orsay virus RNA. Worms with one or more cells stained by FISH were counted as infected. Mean ± SEM (horizontal bars) is shown. Data pooled from 4 independent experiments with n > 52 worms per condition per experiment. The p-values were determined by unpaired two-tailed Student’s t-test. (B-F, H) Significance was defined as p < 0.05; **, p < 0.01; ***, p < 0.001. (I) Significance with Bonferroni correction was defined as *, p < 0.025; ****, p < 0.0001.

Article Snippet: Worms were then washed once in 1 ml hybridization buffer (900 mM NaCl, 20 mM Tris pH 8.0, 0.01% SDS), and incubated overnight at 46°C in 100 μl hybridization buffer containing 5-10 ng/μl of FISH probe conjugated to a Cal Fluor 610 dye (LGC Biosearch Technologies).

Techniques: Infection, Staining, Microscopy, Fluorescence, Mutagenesis, Two Tailed Test

Journal: bioRxiv

Article Title: A parental transcriptional response to microsporidia infection induces inherited immunity in offspring

doi: 10.1101/2020.10.11.335117

Figure Lengend Snippet: P0 populations of N2 C. elegans were either not infected or infected with a low dose of N. parisii spores at the L1 stage (doses defined in methods). At 72 hpi, animals were treated with sodium hypochlorite solution to release F1 embryos. (A-B) Naïve and primed F1 larvae were exposed to a very high dose of N. parisii at the L1 stage. At 3 hpi, animals were fixed and stained with DY96 to detect N. parisii spores (green) as well as a FISH probe to detect N. parisii 18S RNA and reveal intracellular sporoplasms (red). (A) The number of sporoplasms per animal was quantified by microscopy. Each circle represents a count from a single worm. Mean ± SEM (horizontal bars) is shown. Data pooled from 2 independent experiments using n = 20-26 worms per condition per experiment. (B) The number of spores per animal was quantified by microscopy. Each circle represents a count from a single worm. Mean ± SEM (horizontal bars) is shown. Data pooled from 2 independent experiments using n = 20 worms per condition per experiment. (C) Naïve and primed F1 larvae were fed on fluorescent beads at the L1 stage. After 3 hours, animals were fixed and imaged. Fluorescence from beads was thresholded to determine the amount of beads eaten by each worm (% of body filled with beads). Each circle represents a measurement from a single worm. Mean ± SEM (horizontal bars) is shown. Data pooled from 2 independent experiments using n = 20-30 worms per condition per experiment. (D) Naïve and primed F1 larvae were exposed to a very high dose of N. parisii at the L1 stage. At 3 hpi, populations were split and half of the animals fixed. The remaining animals were maintained in the absence of spores until a 24 h end point before fixing. Animals were stained with a FISH probe to detect N. parisii 18S RNA (red) and the number of sporoplasms per animal was quantified by microscopy. Each circle represents a count from a single worm. Mean ± SEM (horizontal bars) is shown. Data pooled from 2 independent experiments using n = 20 worms per condition per experiment. (E) Naïve and N. parisii -primed F1 L1 larvae were maintained on slow-killing plates with dsRed- P. aeruginosa and fixed at 48 hpi. Representative images of worms grown on dsRed- P. aeruginosa are shown. Scale bars, 100 μm. (F) N2 and pals-22 F1 larvae were exposed to a very high dose of N. parisii at the L1 stage. At 3 hpi, populations were split and half of the animals fixed. The remaining animals were maintained in the absence of spores until a 24 h end point before fixing. Animals were stained with a FISH probe to detect N. parisii 18S RNA (red) and the number of sporoplasms per animal was quantified by microscopy. Three independent experiments with n = 100 worms per experiment. The p-values were determined by unpaired two-tailed Student’s t-test. Significance with Bonferroni correction was defined as p < 0.05. ***, p < 0.001.

Article Snippet: Worms were then washed once in 1 ml hybridization buffer (900 mM NaCl, 20 mM Tris pH 8.0, 0.01% SDS), and incubated overnight at 46°C in 100 μl hybridization buffer containing 5-10 ng/μl of FISH probe conjugated to a Cal Fluor 610 dye (LGC Biosearch Technologies).

Techniques: Infection, Staining, Microscopy, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: A parental transcriptional response to microsporidia infection induces inherited immunity in offspring

doi: 10.1101/2020.10.11.335117

Figure Lengend Snippet: (A-B) P0 populations of N2 C. elegans were either not infected or infected with a low dose of N. parisii spores at the L1 stage (doses defined in methods). At 72 hpi, animals were treated with sodium hypochlorite solution to release F1 embryos. F1 embryo populations were then split and either tested for immunity, or maintained under non-infection conditions for the collection and subsequent testing of F2 embryos. Both naïve and primed F1 and F2 larvae were exposed to a high dose of N. parisii at the L1 stage. At 72 hpi, F1 and F2 animals were fixed and stained with DY96 to visualize N. parisii spores and embryos. (A) Individual DY96 stained worms were imaged to determine infection status. Mean ± SEM (horizontal bars) is shown. Data pooled from 3 independent experiments using n = 16-20 worms per condition per experiment. (B) Images of DY96 stained worms were analysed and worms possessing 1 or more embryos were considered gravid. Mean ± SEM (horizontal bars) is shown. Data pooled from 3 independent experiments using n = 16-20 worms per condition per experiment. (C) Naïve and primed F1 larvae were obtained as above and challenged with a maximal dose of N. parisii spores at either the L1, L2/L3 or L4 stage. Animals were fixed at 30 mpi and stained with a FISH probe to detect N. parisii 18S RNA. The number of sporoplasms per animal was quantified by microscopy. Shown is the average number of sporoplasms in primed worms relative to the naïve control. Mean ± SEM (horizontal bars) is shown. Data pooled from 3 independent experiments using n = 12-20 worms per condition per experiment. (D-E) Briefly, N2 C. elegans (P0, F1, F2) were infected at the L1 stage with N. parisii for one, two or three successive generations. Each infection period lasted 72 h before treating with sodium hypochlorite solution to obtain the next generation of embryos (see schematic ). F3 larvae were exposed to a high dose of N. parisii at the L1 stage. At 72 hpi, F3 animals were fixed and stained with DY96 to visualize N. parisii spores and embryos. (D) Images of DY96 stained F3 worms were analysed and fluorescence from N. parisii spores thresholded to determine parasite burdens of individual worms (% of body filled with spores). Each circle represents a measurement from a single worm. Mean ± SEM (horizontal bars) is shown. Data pooled from 2 independent experiments using n = 25 worms per condition per experiment. (E) Images of DY96 stained F3 worms were analysed and embryos per worm quantified. Each circle represents a count from a single worm. Mean ± SEM (horizontal bars) is shown. Data pooled from 2 independent experiments using n = 30 worms per condition per experiment. (F-G) P0 populations of N2 C. elegans were either not infected or infected with a low dose of N. parisii spores at the L1 stage (for 72 h), L2/L3 stage (for 48 h) or L4 stage (for 24 h). At 72 h post L1, animals were treated with sodium hypochlorite solution to release F1 embryos. Both naïve and primed F1 larvae were exposed to a high dose of N. parisii at the L1 stage. At 72 hpi, animals were fixed and stained with DY96 to visualize N. parisii spores and embryos. (F) Individual DY96 stained worms were imaged to determine infection status. Mean ± SEM (horizontal bars) is shown. Data pooled from 3 independent experiments using n = 100-197 worms per condition per experiment. (G) Images of DY96 stained worms were analysed and worms possessing 1 or more embryos were considered gravid. Mean ± SEM (horizontal bars) is shown. Data pooled from 4 independent experiments using n = 100-197 worms per condition per experiment. The p-values were determined by unpaired two-tailed Student’s t-test. (A-B, G) Significance was defined as: *, p < 0.05; **, p < 0.01; ***, p < 0.001. (C, F) Significance with Bonferroni correction was defined as: *, p < 0.025; **, p < 0.005; ***, p < 0.0005. (D-E). Significance with Bonferroni correction was defined as: *, p < 0.016.

Article Snippet: Worms were then washed once in 1 ml hybridization buffer (900 mM NaCl, 20 mM Tris pH 8.0, 0.01% SDS), and incubated overnight at 46°C in 100 μl hybridization buffer containing 5-10 ng/μl of FISH probe conjugated to a Cal Fluor 610 dye (LGC Biosearch Technologies).

Techniques: Infection, Staining, Microscopy, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: A parental transcriptional response to microsporidia infection induces inherited immunity in offspring

doi: 10.1101/2020.10.11.335117

Figure Lengend Snippet: (A) Transgenic C. elegans expressing a fluorescent germline protein (GFP::3xFLAG::CSR-1) were infected with a low dose of N. parisii spores at the L1 stage (doses defined in methods). At 72 hpi, animals were fixed and stained with a FISH probe to detect N. parisii 18S RNA (red). Representative images of the germline of an infected worm are shown. Inset images: Np , N. parisii ; (1) DG, distal gonad; (2) PG, posterior gonad; (3) E, embryo. Scale bars, 20 μm. (B-C) Transgenic C. elegans expressing GFP under IPR gene promoters were either uninfected or exposed to a low dose of heat-killed or live N. parisii spores at the L1 stage. At 5 hpi, animals were imaged. Scale bars, 100 μm. (B) Representative images of transgenic pals-5p::gfp worms. (C) Representative images of transgenic f26f2.1p::gfp worms. (D) Table comparing genes previously reported to be upregulated in N. parisii infected animals with gene expression changes in other published data sets. p-values calculated using Fisher’s Exact test. (E) N2 and rde-1 mutant L1 larvae were infected with Orsay virus and fixed at 72 hpi. Representative images of worms stained with FISH probe to detect Orsay virus RNA. Scale bars, 400 μm. (F) P0 populations of N2 C. elegans were either untreated, exposed to 50 mM cadmium from the L4 stage, or infected with a low dose of N. parisii spores at the L4 stage. After 24 h, animals were treated with sodium hypochlorite solution to release F1 embryos. F1 larvae were exposed to 50 mM cadmium at the L4 stage. After 24 h, animals were fixed and stained with DY96 to visualize worm embryos. Images of DY96 stained worms were analysed and embryos per worm quantified. Each circle represents a count from a single worm. Mean ± SEM (horizontal bars) is shown. Data pooled from 3 independent experiments using n = 25-26 worms per condition per experiment. The p-values were determined by unpaired two-tailed Student’s t-test. Significance with Bonferroni correction was defined as p < 0.025. ***, p < 0.0005.

Article Snippet: Worms were then washed once in 1 ml hybridization buffer (900 mM NaCl, 20 mM Tris pH 8.0, 0.01% SDS), and incubated overnight at 46°C in 100 μl hybridization buffer containing 5-10 ng/μl of FISH probe conjugated to a Cal Fluor 610 dye (LGC Biosearch Technologies).

Techniques: Transgenic Assay, Expressing, Infection, Staining, Mutagenesis, Two Tailed Test

Journal: iScience

Article Title: Differential fates of Kazald gene quartet: Ancestral roles in skeletogenesis and regeneration to putative innovations in fish and birds

doi: 10.1016/j.isci.2026.114934

Figure Lengend Snippet: Kazald3 may have a connection to skeletogenesis and regeneration in teleost fish, but has no clear role in other lineages (A) Quantification of Kazald3 expression in regenerating limbs, lower jaw, brain, and retina of adult axolotl, the uninjured skin of non-tetrapod/teleost fish, and in regenerating fins of adult bichir. Kazald3 was duplicated in the lineage leading to paddlefish with no subsequent loss, creating Kazald3a and Kazald3b . L.fish = Lungfish. (B) Quantification of Kazald3 expression in whole embryos of developing axolotl and turtle. (C) Quantification of Kazald gene expression in the bone of adult zebrafish, bone of adult medaka, adult bone and the larvae of eel, and the developing tooth of the mbuna cichlid. Kazald2 was duplicated in the lineage leading to eel with no subsequent loss, creating Kazald2a and Kazald2b . (D–F) Whole-mount in situ hybridization of kazald3 in regenerating caudal fins of adult zebrafish from 2 to 4 dpa. Dashed line indicates the amputation planes. Scale bars, 100 μm. (G–G″) HCR of kazald3 (G), gfp (G′), and their overlap (G″) in 4 dpa regenerating caudal fin of adult runx2 :GFP transgenic zebrafish. Dashed line indicates the amputation plane; solid white lines outline individual fin rays. Scale bar, 100 μm. (H and I) Whole-mount in situ hybridization of kazald3 in 3 (H) and 5 (I) dpf zebrafish embryos. Scale bars, 500 μm. (I′) Inset displaying the boxed region of the embryo in I. Arrowheads indicate areas of kazald3 expression that associate with the locations of ossifying skeletal elements (i.e., the opercle and cleithrum). Scale bar, 100 μm. PRJ IDs indicate the publicly available RNA-Seq datasets that generated the raw data for the listed tissues. Dots represent biological replicates in examined datasets, and error bars represent standard deviation when calculable. CPM = Counts Per Million, St. = stage, dpa = days post amputation, hpi = hours post injury, Reg. = regenerating, Rep. = replaced. Data are represented as Mean ± SD.

Article Snippet: Samples were washed again in PBT, incubated in pre-warmed Hybridization Buffer (Molecular Instruments, #BPH01726) for 5 min and then pre-hybridized in new Hybridization Buffer for 30 min at 37°C.

Techniques: Expressing, Gene Expression, In Situ Hybridization, Transgenic Assay, RNA Sequencing, Generated, Standard Deviation

Journal: iScience

Article Title: Differential fates of Kazald gene quartet: Ancestral roles in skeletogenesis and regeneration to putative innovations in fish and birds

doi: 10.1016/j.isci.2026.114934

Figure Lengend Snippet: Kazald2 is expressed during regeneration in species throughout the osteichthyan lineage (A) Quantification of Kazald1 and Kazald2 expression over the course of axolotl limb regeneration. (A′ and A″) Whole-mount in situ hybridization of Kazald2 in 5 dpa and intact axolotl limb. Scale bar, 500 μm. (B) Quantification of Kazald2 expression in the regenerating fins and tail of lungfish and bichir. (C) Quantification of Kazald2 expression in the regenerating mandible, spine, brain, and retina of axolotl, and the regenerating lens of newt. (D) RT-qPCR examining Kazald2 expression in the regenerating limbs of the lungless salamander. (E) Quantification of Kazald expression over the course of lamprey spine regeneration. (F) Quantification of Kazald expression over the course of amphioxus tail regeneration. (G) Quantification of kazald2 and kazald3 expression in the regenerating heart and caudal fin of zebrafish, and Kazald1 and Kazald4 expression in the regenerating tails of green anole and tokay gecko. (H) Whole-mount in situ hybridization of kazald2 in regenerating caudal fin of adult zebrafish at 2 dpa. Dashed line indicates the amputation planes. Scale bar, 250 μm. (I) Quantification of Kazald1 and Kazald2 expression over the course of axolotl embryo development, and in the developing limb bud of axolotl larvae. PRJ IDs indicate the publicly available RNA-Seq datasets that generated the raw data for the listed tissues. Dots represent biological replicates in examined datasets, and error bars represent standard deviation when calculable. CPM = counts per million, hpa = hours post-amputation, dpa = days post-amputation, wpa = weeks post-amputation, hpi = hours post-injury, dpi = days post-injury, Reg. = regenerating, Uninj. = uninjured, Dor. = dorsal, Ventr. = ventral. Data are represented as mean ± SD.

Article Snippet: Samples were washed again in PBT, incubated in pre-warmed Hybridization Buffer (Molecular Instruments, #BPH01726) for 5 min and then pre-hybridized in new Hybridization Buffer for 30 min at 37°C.

Techniques: Expressing, In Situ Hybridization, Quantitative RT-PCR, RNA Sequencing, Generated, Standard Deviation