EMBO/soda-nel huggingface dataset:
Regional heterogeneity of astrocyte morphogenesis dictated by the formin protein, Daam2, modifies circuit function (2021)
Figure 5

Figure 5. Astrocyte-specific loss of Daam2 impairs olfaction behaviors.

A. Astrocyte-specific Daam2 cKO mice show delays in buried food finding. N=13, 15 mice per genotype. Data is presented as mean ± SEM. Student's t-test was used, *p<0.05.

B. Illustration of odor discrimination assay. For habituation, each mouse was habituated in a chamber with identical stimulation (odor A) on both sides for 2 minutes, repeated three times. To test for odor discrimination, a newly introduced odor (odor B) was randomly assigned to one of the sides.

C. Total travel distance was measured by tracing the nose and body of each mouse in each habituation session of the odor discrimination assay. Both genotypes show no difference in total travel distance. Total N=11, 13 mice were used for each genotype. Data is presented as mean ± SEM. Student's t-test was used.

D. Representative traces of nose position tracking of cHet and cKO mice during the discrimination test period. Habituated (odor A) and the new odor (odor B) were placed in the center of the indicated area (odor A: red circle, odor B: green circle). Nose positions within the circled area were indicated with the corresponding color for each odor.

E, F. Preference index was tracked and calculated using MATLAB and Optimouse. One sample t and Wilcoxon tests were used for statistical analysis. Data is presented as mean ± SEM. *** p<0.001. Total N=11, 13 mice were used for each genotype.
Regional heterogeneity of astrocyte morphogenesis dictated by the formin protein, Daam2, modifies circuit function (2021)
Figure 7

Figure 7. Slc4a4 regulates astrocyte maturation by interacting with Daam2

A. Double fluorescence in situ hybridization images indicating co-labeling of Slc4a4 (green) and GLAST (red). Scale bar: 100 μm.

B. Schematics showing induction of temporally-controlled astrocyte-specific conditional null alleles.

C. In situ hybridization confirms deletion of Slc4a4 in the cortex. GFAP immunostaining intensity was decreased significantly in Slc4a4 icKO at P12 whereas Sox9+ cell number remained unchanged. Scale bar: 100 μm.

D. Co-immunoprecipitation revealing that Slc4a4 associates with Daam2 in vivo.

E. Schematics showing induction of temporally-controlled astrocyte-specific conditional null alleles.

F-J. Representative images of GFAP immunostaining (F) and quantification (G-J) in controls, Daam2 icKO, Slc4a4 icKO, and double icKO 6 weeks after tamoxifen injection. Scale bar: 20 μm. Data is presented as mean ± SEM. n=10-24 images, N=3-4 mice per genotype. Scale bar: 20 μm. Two-way ANOVA is used for statistics. [*] and [#] indicates comparison with control and double icKO, respectively. **p<0.01, p***<0.001, ##p<0.01.

Extended View Figure Legends
Deep scanning lysine metabolism in Escherichia coli (2018)
Figure 3

Figure 3. Transporter route of AEC resistance. A. Mapping of enrichment across the positions targeted in lysP. The gray regions in the gene cartoon at the bottom highlights the windows containing targeted residues, with the enrichment map shown above for increasing AEC concentrations. Enrichments are color coded according the legend at the bottom. A histogram plot of enrichment is shown at the right. B. Growth of the reconstructed LysP T33F mutant compared to wild type cells transformed with a Non-Target gRNA. n=3. C. Growth of the reconstructed LysP Q219I mutant compared to wild type cells transformed with a Non-Target gRNA. n=3. D. Map of enriched mutations (green) to the modeled structure of LysP. E. Enrichment of synonymous mutations observed for LysP. Each synonymous mutation site is shown as a sphere in the structure and is color coded according to the enrichment score bar shown on the right. The bottom chart represents the fraction of all synonymous mutations in the gene that displayed positive enrichment scores under each AEC concentration tested. For comparison, data on LysR and DapF are also shown.
Deep scanning lysine metabolism in Escherichia coli (2018)
Figure 4

Figure 4. Investigating the biosynthetic gene dapF. A. Mapping of enrichment across the positions targeted in dapF. The gray regions in the gene cartoon at the bottom highlights the windows containing targeted residues, with the enrichment map shown above for increasing AEC concentrations. Enrichments are color coded according the legend at the bottom. A histogram plot of enrichment is shown at the right. B. Structure of the DapF dimer (PDB ID: 4IJZ) with the G210 and M260 sites highlighted in green. Diaminopimelate binding is shown in red. C. Zoom in the catalytic site highlighting the G210 and M260 sites relative to the catalytic cysteines. The G210D and M260Y substitutions are shown in the right panel. D. Absolute quantification of intracellular lysine concentration in wild type and the reconstructed DapF mutants. Quantification was performed using LC-MS, as described in the methods section. n=3. A two-sample student t-test assuming unequal variances was performed to calculate statistical significance. Concentrations are reported as fold-change relative to the wild-type control samples. E. DapF assay showing kinetics of the wild type, G210D and M260Y mutants. Assay was performed as described in the methods section. n=5. F. Differential gene expression quantified via qPCR for the dapF and lysA genes on a WT, DapF G210D and DapF M260Y backgrounds. Error bars represent 95% confidence intervals. A two-tailed student's t-distribution was used to calculate p-values, which were adjusted using the Benjamin-Hochberg statistical method for false discovery rates.
Deep scanning lysine metabolism in Escherichia coli (2018)
Figure 5

Figure 5. Investigating noise and real enrichment in the plasmid-barcodes. A. Mapping of enrichment across the positions targeted in lysR. The gray regions in the gene cartoon at the bottom highlights the windows containing targeted residues, with the enrichment map shown above for increasing AEC concentrations. Enrichments are color coded according the legend at the bottom. A histogram plot of enrichment is shown at the right. B. Modeled structure of LysR, with the HTH DNA-binding domains colored in blue and the co-inducer binding domain in gray. The S36 site is highlighted in green. The right panel zooms to the S36 site, showing close proximity to the DNA phosphate backbone. C. Absolute quantification of intracellular lysine levels in wild type and the reconstructed LysR S36R mutant. Quantification was performed using LC-MS, as described in the methods section. n=2. Concentrations are reported as fold-change relative to the wild-type control samples. D. Mapping of enrichment across the positions targeted in argP. Representation is the same as described in A. E. Modeled structure of ArgP, highlighting the E246 residue in the co-inducer binding domain (gray). F. Absolute quantification of intracellular lysine levels in wild type and the reconstructed ArgP E246 mutant. Quantification was performed using LC-MS, as described in the methods section. n=2. Concentrations are reported as fold-change relative to the wild-type control samples.
Transcytosis via the late endocytic pathway as a cell morphogenetic mechanism (2020)
Figure 1

Figure 1. Membrane organization during tube morphogenesis.

(A-B) Diagram of the organization of the tracheal system at the onset of terminal cell branching (A) and one hour later (B).

(C-C''') Terminal cell expressing reporters for membrane (C), ER (C') and Golgi apparatus (C'').

(D) Mean and 95% confidence interval of tube and branch length during the first hour of growth. Numbers represent the rate of growth estimated from the slope of the curve +/-SD, p=0.39, Mann-Whitney test, n=6.

(E) Distance of the tip of the tube, and the leading ER and Golgi accumulations from the cell junction that defines the base of the cell. Line and shading represent mean and 95% confidence interval, n=4.

(F-I) Terminal cells expressing the general membrane marker CD4::mIFP in combination with different membrane or polarity markers: PH::mCherry, a PIP2 sensor commonly used as apical plasma membrane marker (F'), an endogenously GFP-tagged Crb (G'), polarity proteins Par3::YFP (H') and Par6::mCherry (H''). The boxed region in (H''') is shown at higher magnification in (I) over 4 time points.

Data information: Red arrowheads: CD4::mIFP vesicles and associated markers at the tip of the cell. Anterior is left and dorsal is up. Scale bars: 5μm
Transcytosis via the late endocytic pathway as a cell morphogenetic mechanism (2020)
Figure 4

Figure 4. The role of endocytosis in terminal cell growth.

(A-E) Distribution of the plasma membrane reporter PH::GFP in control cells (A-A'') and in cells where dynamin activity had been blocked using a temperature-sensitive allele of dynamin (shibirets, B, E). In (B), was inactivated at the onset of tube formation, whereas in (E) it was inactivated ~15 minutes after the tube had begun to form. (C-D) Distribution of PH::GFP fluorescence intensity in control and shibirets cells. Data from 1-2 minute-interval time lapses was collected in windows of 20 minutes each (except for t=0). Box plots represent median, interquartile range (IQR), and IQR*1.5 below and above the IQR. (C) Proportion of signal in the apical and in the basal membrane compartment over time in control cells (n=4) and also in cells where dynamin was inactivated (n=5), *p=0.015, Mann-Whitney test. (D) Total fluorescence intensity of PH::GFP over time in controls (n=4) and in cells where dynamin was inactivated (n=5), n.s.: p=0.063, Mann-Whitney test.

(F-G) Distribution of the luminal reporter Gasp::GFP in control cells (F) and in cells where dynamin was inactivated (G). Arrowheads point to protrusions sprouting from the lumen of the terminal cell.

(H-I) Distribution of the general plasma membrane marker CD4::mIFP in combination with the Clathrin light chain fused to GFP (CLC::GFP, H) and with Dynamin::YFP (I).

Data information: Scale bars: 5μm
Transcytosis via the late endocytic pathway as a cell morphogenetic mechanism (2020)
Figure 5

Figure 5. Effects of dynamin inactivation on membrane morphology.

(A-F) TEM tomograms and 3D reconstructions of terminal cells in shibirets embryos. (A) Control. (B-F) Kept at 34ºC for 1 (B, E) or for 2 hours (C, D, F). Boxed regions in (A) and (B) are magnified in (A'') and (B''). (A', B') Reconstructed 3D models. The tube membrane (cyan) is characterized by the electron-dense apical extracellular matrix (aECM). Invaginations with connections that can be traced to the tube membrane are red, invaginations from the basal plasma membrane are green. The basal membrane is light pink. (C-D') shibirets terminal cell after 2 hours at 34ºC. (C') 3D reconstruction, color coding for basal and tube membrane is the same as above. Membrane that is continuous with both the apical and the basal membrane is shown in green, purple and dark blue. (D-D') higher magnification of a region in the same cell at a level where membrane sheets (purple, dark blue) bridge the apical and basal plasma membrane domains. (E-E'') Two focal planes and model of a tomogram from the cell shown in (B) where a basal (pink arrowhead) and an apical invagination (blue) are seen in close proximity (closest distance is marked by cyan and blue arrowheads). (F-F') apical membrane overgrowth regions of the cell shown in (C). The genotype of the embryos was shibirets; btl>KDEL::RFP, Par3::YFP. The cell shown in (C-D) was found and acquired without the CLEM approach.

Data information: Scale bars: 500nm.
Transcytosis via the late endocytic pathway as a cell morphogenetic mechanism (2020)
Figure 6

Figure 6. Effect of dynamin inactivation on the distribution of basal proteins

(A-B) Terminal cells expressing the membrane marker CD4::mIFP under btl-gal4 and FGFR::GFP under its own promoter (from the fTRG library). (A) Time lapse imaging of a control cell. Blue arrowheads point to filopodia and basal plasma membrane and red ones point to puncta containing CD4::mIFP and FGFR::GFP. (B) shibirets cell imaged before dynamin inactivation, after 23 and 45 minutes of inactivation, and after 30 minutes of recovery. White arrowheads point to FGFR::GFP accumulation at the apical compartment.

(C-E) Single confocal planes of terminal cells expressing PH::mCherry and stained for βPS-Integrin. The outline of the cells was traced using the PH::mCherry signal and is shown as a blue dashed line. (C) Control. Blue arrowheads: βPS-Integrin signal in filopodia. (D) 1 hour at 34ºC, (E) 2 hours at 34ºC. (C''-E'') Orthogonal views of the lines shown in white in (C-E).

(F-G) Quantification of fluorescence intensity from stained embryos of FGFR::GFP (F) and of βPS-Integrin (G). Data are plotted as mean +/- SD; significance was assessed using one-way ANOVA with Dunnett's correction for multiple comparisons. **p=0.0022, ***p=0.0003, ****p<0.0001. Number of cells analyzed, for (F): Control n=8, 1hr at 34ºC n=6, Recovery n=8. (G). For (G): Control= 7, 1hr at 34ºC= 8, 2hrs at 34ºC= 8.

Data information: Scale bars: 5μm
Transcytosis via the late endocytic pathway as a cell morphogenetic mechanism (2020)
Figure 7

Figure 7. Composition and distribution of endosomal compartments during terminal cell growth

(A-D) High time resolution imaging of the membrane marker CD4::mIFP expressed under btl-gal4. (A) Example of a large CD4 vesicle (arrowhead) rapidly moving towards the tip of the terminal cell. (B-D) Analysis of 28 CD4 vesicles in five terminal cells. (B) Trajectories of CD4 vesicles with their original position mapped to the origin of the plot. (C) Rose diagram of the trajectories shown in (B). (D) Speed of CD4 vesicles compared to speed of tube growth measured over 25 min. Data are plotted as mean +/-SD. *p<0.001, Mann-Whitney test.

(E-H) Terminal cells expressing the membrane reporter CD4::mIFP together with markers for vesicles of the endocytic pathway. (E) Rab5::YFP, red arrowheads point to a Rab5-positive vesicle; the white arrowhead shows the same vesicle once it lost the Rab5 signal. (F) FYVE::GFP, a PI3P reporter and Spin::mRFP , a lysosomal marker. Black arrowheads point to small FYVE::GFP vesicles and the squared area shows large ones at the tip. (G) Par6::mCherry and endogenously labelled Rab7::YFP. (H) Percentage of CD4::mIFP vesicles that carry the indicated markers. Data are plotted as mean +/-SD. For each marker we analyzed at least 20 minutes of cell growth with 10-20 timepoints. Number of cells analyzed: Par3, n=2; Rab5, n=3; FYVE::GFP, n=4; FYVE::GFP-Spin::RFP (which includes single- and double-positive vesicles), n=3; dLamp1, n=2; Rab7, n=5.

Data information: Scale bars: 5μm
Transcytosis via the late endocytic pathway as a cell morphogenetic mechanism (2020)
Figure 8

Figure 8. Distribution of late endosomal markers upon disruption of the endocytic pathway

(A-F) Terminal cells expressing CD4::mIFP and FYVE::GFP under btl-gal4. The outline of the cells was traced using the CD4::mIFP signal and is shown as a blue dashed line. (A-C) Control cell. (D-F) shibirets cell before 34ºC (D), after 30 minutes at 34ºC (E) and after 30 minutes of recovery (F). Red arrowheads: FYVE::GFP puncta.

(G-H) Number of FYVE::GFP vesicles in 4 control cells (G) and in 6 shibirets cells (H). Box plots represent median, interquartile range (IQR), and IQR*1.5 below and above the IQR. Significance in (H) was assessed using one-way ANOVA with Geisser-Greenhouse correction for paired data and Dunnett's multiple comparisons test. *p=0.0288, ***p=0.0016.

(I) shrbO3/G5 mutant cell expressing CD4::mIFP under btl-gal4. Arrowhead: CD4::mIFP aggregation.

(J-K) Terminal cells over-expressing Shrb::GFP under btl-gal4 together with PH::mCherry (J), or together with Spin::RFP and CD4::mIFP (K). Arrowheads: Shrb::GFP accumulations.

(L-M) Dorsal trunk cells stained for Serp. In control embryos (L), Serp is seen exclusively in the tracheal lumen. The cells themselves are not visible in these images. In embryos expressing Shrb::GFP under btl-gal4 (M), Serp is also seen inside the cells, usually in association with or surrounded by Shrb::GFP. Boxed regions are magnified in (L'-M') and shown as single confocal planes.

Data information: Scale bars: 5μm

Expanded View figure legends
Fine Tuning of Histone Demethylase KDM6A/B Improves the Development of Nuclear Transfer Embryo (2018)
Figure 2

Figure. 2 The effect of ZGA on the SCNT embryo quality.

A. Schematics of the live-cell imaging experiments. The embryo imaged from pronuclei until blastocyst stage and transferred to pseudo-pregnant females.

B. Representative live-cell images of dynamics MERVL::tdTomato expression during SCNT embryos preimplantation development. The selected images from a series acquired every 15 min. The time after starting observation is shown on the upper right corner of each image. Scale bar, 50 μm.

C. Representative DAPI staining of blastocysts of tdTomato+ and tdTomato- SCNT embryos after 115 hr. of culture in vitro. Scale bar, 50 μm.

D. The tdTomato+ and tdTomato- SCNT blastocyst cell numbers were determined by counting the DAPI-stained cells. Three independent experiment replicates were performed. N, total number of embryos analyzed for each condition. Red bars indicated the mean value.

E. Immunofluorescence images of tdTomato+ and tdTomato- blastocysts derived from SCNT. Nanog (ICM) and Cdx2 (TE) were used as lineage markers. Representative images from ≥ 55 embryos analyzed in four independent micromanipulations are shown. Scale bar, 50 μm.

F. Representative image of tdTomato+ and tdTomato- SCNT embryos retrieved at E6.5. The tdTomato+ SCNT embryo displays normal egg-cylinder morphology. By contrast, the tdTomato- SCNT embryo shows abnormal morphology. Epi, embryonic epiblast; ExEm, extraembryonic ectoderm; EPC, ectoplacental cone. Scale bar, 50 μm.

G. The bar chart showing the efficiency of attachment to the feeder cells of SCNT blastocysts. The efficiency was calculated based on the total number of blastocysts used for ntES derivation. N, total number of embryos analyzed for each condition. Error bars, s.d., n ≥ 3. **P < 0.01 by two-tailed Student's  t-test.

H. The bar chart shows the efficiency of ntES derivation. The efficiency was calculated based on the total number of attached blastocysts for ntES derivation. N, total number of embryos analyzed for each condition. Error bars, s.d., n ≥ 3. **P < 0.01 by two-tailed Student's  t-test.

I. RT-qPCR analysis for somatic cell genes in SCNT 1-cell embryos, 2-cell tdTomato+, and 2-cell tdTomato- embryos. Results were normalized based on the geometric mean of the expression levels of two reference genes (Ywhaz and Gapdh). Error bars, s.e.m., n = 3. ***P < 0.001 by two-tailed Student's  t-test.
Fine Tuning of Histone Demethylase KDM6A/B Improves the Development of Nuclear Transfer Embryo (2018)
Figure 4

Figure. 4 Overexpression of KDM6A only improves the blastocyst formation rate of SCNT embryos, but not full-term development.

A, B RT-qPCR analysis of KDM6A (A) and KDM6B (B) mRNA levels in SCNT 2-cell embryo. Data shown are mean expression values relative to Gapdh. The value in ICSI control was set as 1. Error bars, s.e.m., n ≥ 3. **P < 0.01 by Student's t-test.

C. The sketch of KDM6A and KDM6B in vitro transcription vector (right), and the integrity of in vitro transcripted mRNA was confirmed by electrophoresis with formaldehyde gels (left). M, marker; T7, in vitro transcription promoter; HA, hemagglutinin epitope tag.

D. Immunostaining of SCNT embryo for H3K27me3 and HA epitope tag after injection of different mRNA as indicated. Representative images from ≥ 187 embryos analyzed in four independent micromanipulations for each condition are shown. Scale bar, 20 μm.

E. Schematic illustration of mRNA injection into oocytes and SCNT.

F. Representative images of SCNT embryos at 115 h after injection of different mRNA as indicated. Scale bar, 50 μm.

G. The bar chart showing the efficiency of blastocyst formation. Injection of KDM6A mRNA improved the preimplantation development rate of SCNT embryos. Error bars, s.e.m., n ≥ 3. **P < 0.01, ***P < 0.001 by two-tailed Student's  t-test. n.s., not significant.

H. Phenotypic analysis of E8.5 SCNT mouse embryos injection with 20 ng/μl or 50 ng/μl KDM6A mRNA (left). Representative images of the KDM6A injected embryos at E19.5 (right). The injected SCNT embryos were only obtained degenerated embryos. AI, allantois; SM, somite. Scale bar, 50 μm.

I. The retrieved rate of embryo assessed at E8.5. Five independent experiment replicates were performed. The numbers at  the bottom of the bars indicated the total number of transferred embryos.

J. Schematic diagram depicts the position of highly conserved TPR and JmjC domain. The KDM6A are broadly expressed proteins characterized by N-terminal TPRs and C-terminal JmjC domain. In KDM6B the only clearly identifiable domain is the C-terminal JmjC domain. White asterisk indicates the mutation point.

K. Preimplantation development rates in the KDM6A-HA, KDM6B-HA, or KDM6A-cM/-nM/-ncM-HA mRNA-injected and non-injected control SCNT groups. The efficiency was calculated based on the number of cleavage embryo. Error bars, s.d., the total numbers of cleavage embryos in each condition (KDM6A-HA, KDM6B-HA, KDM6A-cM/-nM/-ncM-HA, and Control) from three independent experiments were 275, 199, 290, 221, 244, and 286, respectively.
Fine Tuning of Histone Demethylase KDM6A/B Improves the Development of Nuclear Transfer Embryo (2018)
Figure 5

Figure. 5 KDM6B knockdown  greatly improved the preimplantation development rate of SCNT embryos.

A. RT-qPCR analysis of KDM6A/B in SCNT embryos injected with siRNA-6A or siRNA-6B. The SCNT embryos were subject to injection of 10 μM or 20 μM  siRNA as indicated. Data are mean expression relative to Gapdh with siRNA-control normalized to 1. Error bars, s.e.m., n = 3. **P < 0.01, ***P < 0.001 according to two-tailed Student's t-test. n.s., not significant.

B. The bar chart shows the KDM6A/B transcript levels in SCNT embryo with 20 μM siRNA-6A/B. All data are mean expression relative to Gapdh with control siRNA injected SCNT embryos normalized to 1.  Error bars, s.e.m., n = 3, **P < 0.01 by Student's t-test.

C. Immunofluorescence staining results showing H3K27me3 of 2-stage SCNT embryos. Embryo was injected with siRNA as indicated. The H3K27me3 levels between control and double injected with siRNA-6A-6B cannot observe any difference, but a marked decrease was observed when injected with either siRNA-6A or siRNA-6B. Scale bar, 20 μm.

D. Western blot showing the expression of KDM6A and KDM6B in SCNT embryos after injection of different siRNA as indicated. Protein lysates from 1,500 embryos were loaded in each lane. The results of one representative of two independent experiments are presented. The Ponceau S staining demonstrates equivalent loading of each lane are shown in Appendix Fig S5C.

E. Schematic illustration of siRNA injection into oocytes and SCNT.

F. Representative images of SCNT embryos at 115 h after injection of different siRNA as indicated. These images are representative examples of the quantification shown in Fig 5G. n ≥ 3. Scale bar, 50 μm.

G. Injection of siRNA-6B improved the preimplantation development rate of SCNT embryos. Both cumulus cells, Sertoli cells, and C57 MEF cells were used as donor cells. The bovine intraspecies SCNT embryos derived from bovine ear fibroblast cells. Shown is the percentage of embryos that reached the indicated stages. ♂ and ♀ indicated the male and female, respectively. Error bars, s.d., n ≥ 3.

H. RT-qPCR analysis for select ZGA genes in ICSI and SCNT embryos. The SCNT embryo was injected with siRNA as indicated. Results were normalized based on the geometric mean of the expression levels of two reference genes (Ywhaz and Gapdh). Error bars, s.e.m., n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 according to two-tailed Student's t-test. n.s., not significant.