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Collection : Elife (E-Journal - Via Crossref)

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Figure 6. Expression and function of alternative pangolin isoforms in Anopheles. ; (A) Differential expression analysis of maternal transcripts between anterior and posterior halves of 1 hr-old Anopheles embryos. logFC: log fold-change. (B) Sketches of Aga-panMat and Aga-panZyg transcripts based on RNA-seq and RACE experiments (see also legend to Figure 1A). Blue lines the position of in situ hybridization probes and dsRNA. (C) RNA in situ hybridization of Aga-panMat and Aga-panZyg transcripts in 1 hr-old preblastoderm and germband extending embryos of A.gambiae. Anterior is left and dorsal up. Scale bar: 100μm. (D) 1 st instar A.coluzzii larval cuticle of wild type (top). Cuticle phenotypes of double abdomen (middle; 3/37) and intermediate, anterior truncation phenotype (bottom; 8/37) following Aco-panMat RNAi. Scale bar: 100μm. (E) Differential expression analysis of maternal transcripts between anterior and posterior halves of 1 hr-old Nephrotoma (Tipulidae) embryos. logFC: log fold-change. (F) RNA in situ hybridization of Nsu-panMat.

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Figure 4—figure supplement 2. Gene expression patterns at the Gli3 and Nkx3-2 candidate intervals. ; (A) Gli3 expression was determined using in situ hybridization. Gli3 was robustly expressed during limb development in both developing fore- and hindlimb buds, especially in the autopod (hand/foot plate). Lower panels show expression of Nkx3-2 and its neighboring genes Rab28 and Bod1l. The stronger expression of Nkx3-2 in the developing limb buds as well as the known role of Nkx3-2 in bone maturation (Sivakamasundari et al., 2012) strongly argues for Nkx3-2 being the gene underlying the selection response at the chromosome 5 locus. Scale bars: 1 mm for whole-mounts; 0.5 mm for limb buds. FL, forelimb; HL, hind limb; unless otherwise indicated by ‘L’, all images were taken from the right side. (B) We collected E12.5 embryos from each line and performed in situ hybridization to determine the sites and level of expression of Nkx3-2 and Rab28 in the Longshanks (right columns) and Control (left column) lines. Both genes are expressed in similar sites overall and specifically in the developing fore- and hind limb buds in the region of the presumptive zeugopods. These data indicate common sites of expression and rule out qualitative presence/absence differences in expression. Note that although the N1 enhancer pattern appear to differ from endogenous Nkx3-2 expression (Figure 4E, details in limb buds), it matches the pattern of early Nkx3-2 expression, detectable through the use of lineage-tracing via the combined effect of a Nkx3-2 Cre-driver line and the lacZ reporter line Rosa26R (see Figure 2B in Sivakamasundari et al., 2012).

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Figure 2. pappaa is expressed by neuromast support cells and motor neurons. ; (A) Whole mount in situ hybridization shows pappaa mRNA expression at four dpf by lateral line neuromasts (arrowheads). (B) Fluorescent in situ hybridization of pappaa (green) and brn3c:GFP labeled hair cells (white) shows pappaa mRNA expression by the support cells that surround hair cells. (C) RT-PCR of fluorescently sorted brn3c:GFP labeled hair cells and mnx1:GFP labeled motor neurons shows pappaa expression by motor neurons, but not hair cells. RT-PCR products represent pappaa cDNA fragment.

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Figure 1—figure supplement 1. Generation of Nxph4-βgeo knock-in mouse and characterization of Nxph4 expression by β-galactosidase staining and in situ hybridization. ; (A) Targeting scheme to generate Nxph4-βgeo allele. Doted lines indicate homologous recombination sites. ES cells with correct Nxph4-βgeo allele were obtained from KOMP. (B) Genotype analysis of tail DNA showed a 180 bp band from Nxph4 wild type allele and a 150 bp band from Nxph4-βgeo allele. PCR products were separated on 3% MetaPhor agarose gel. (C) A parasagittal section of an adult Nxph4βgeo/+ mouse brain processed for β-galactosidase staining, resulting in a blue reaction product where Nxph4 is expressed. Arrows indicate regions with strong Nxph4 expression. Scale bar: 1 mm. (D) Double in situ staining of adult wild type mice with probes against Nxph4, Vglut1/2 (excitatory neuron markers), and Gad1 (an inhibitory neuron marker). Nxph4 co-localized with Vglut2 in the olfactory bulb, vestibular nuclei, and cerebral cortex, but also showed overlapping signal with Gad1 in the vestibular nuclei. Scale bar, 100 μm. (E) β-galactosidase staining of adult Nxph4βgeo/+ mice shows Nxph4 expression in the glomerular layer (gl) and mitral cell layer (ml) of the main olfactory bulb, ventral cochlear nucleus (VCN), pontine nuclei (PN), and locus coeruleus (LC). Scale bars: Eii, 200 μm; others, 500 μm. (F) RNA in situ hybridization with a probe against Nxph4 detected Nxph4 in several regions of adult mouse brain. Scale bars: Cerebellum, 1 mm; OB (Olfactory Bulb), 500 μm; Amygdala, 100 μm; SFO, 200 μm; MPO, 200 μm; MB, 300 μm; PrS, 100 μm; AP, 100 μm.

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Figure 6—figure supplement 1. Expression of Lepr/lepr mRNA in zebrafish and mouse endochondral bone. ; (A) Sections through the Ch bone were processed for RNAscope in situ hybridization using probes against lepr (blue) and the hypertrophic chondrocyte and osteoblast marker runx2b (red). Images with DIC are shown above. Arrows indicate lepr +cells in the marrow. Magnifications corresponding to the boxed regions show runx2b+ hypertrophic zones flanking a central lepr+ proliferative zone within the growth plate (bidirectional arrows indicate zones). By 27 mm, a distinct lepr+ proliferative zone is no longer apparent. n = 3 for each stage. (B) Sections through the femur in 8 week old mice were processed for RNAscope in situ hybridization using a LepR probe (magenta), and counterstained with Hoechst to label nuclei (blue). Magnified regions of the growth plate show broader expression in columnar chondrocytes and only rare expression in the hypertrophic zone. Strong LepR expression is also seen within and near the perichondrium. Comparable LepR expression was observed in sections from three independent femurs. Scale bars = 50 μm.

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Figure 2. Reelin protein in the ventral midbrain at E13.5 and E14.5. ; (A–C) Double immunolabeling for TH and Reelin shows Reelin protein in the region of the red nucleus (RN, dashed outline) and in the lateral TH+ mDA domain (arrowhead) at E13.5. (D–G) Double immunolabeling for TH and Reelin (D–F) and RNA in situ hybridization for Reelin mRNA (G) at E14.5. Reelin mRNA and Reelin protein are strongly expressed in the RN. Reelin protein is also localized ventral and lateral to the RN, overlapping with the lateral mDA domain (arrowheads). Note that a brightfield image was acquired for the in situ hybridization signal of Reelin. The brightfield image was inverted to obtain the image shown in (G). Asterisks indicate ventral midline. Scale bar: 50 μm.

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Figure 2—figure supplement 1. cxcr4b promoter activity during somitogenesis and primordium migration. ; (A, B) Images of cxcr4b:h2a-EGFP embryos fixed at 13 hpf (A) and 33 hpf (B) and stained by in situ hybridization against EGFP mRNA. (C, D) Images of cxcr4b:h2a-mCherry embryos fixed at 13 hpf (C) and 33 hpf (D) and stained by in situ hybridization against mCherry mRNA. Scale Bars: 200 µm.

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Figure 4. Normal embryogenesis of lnc-setd1ba mutants. ; (A) The relative position of lnc-setd1ba and the protein-coding gene setd1ba. The gene deletion region is marked by dashed red line. Arrows flanking black dotted line mark the primer-binding sites for qRT-PCR product. (B) Maternal and zygotic lnc-setd1ba mutants were not different from wild-type embryos at 1-dpf. (C) Representative images of in situ hybridization for lnc-setd1ba at four- to eight-cell stage mutant (18/18) and wild-type (25/25) embryos. (D) In situ hybridization for the protein-coding mRNA, setd1ba (9/11) in lnc-setd1ba mutants compared to the wild-type embryos (15/15). (E) qRT-PCR at 1 cell stage and 1-dpf for the lncRNA and its neighboring genes rhoF and setd1ba. The statistical significance of the observed changes was determined using t-test analysis and represented by star marks (ns, , , , and **** respectively mark p-values≥0.05,

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Supplementary file 3. Clpex mutants display defects in expression of anterior/posterior patterning genes. ; E9.5 WT (A,C) and Clpex mutant (B, D) RNA in situ hybridization with α-sense Alx3 probe, an anterior pattering gene. E9.5 WT (E) and Clpex mutant (F) RNA in situ hybridization with α-sense Lhx8 probe, an anterior patterning gene. E9.5 WT (G) and Clpex mutant (H) RNA in situ hybridization with α-sense Tbxt (Brachyury) probe, a posterior patterning gene. E8.5 WT (I) and Clpex mutant (J) RNA in situ hybridization with α-sense Tbxt (Brachyury) probe, a posterior patterning gene.

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Figure 1—figure supplement 1. Two-color fluorescent in-situ hybridization validates transgenic line expression patterns. ; Confocal images (z-projection of ~5 µm) showing fluorescent in-situ hybridization for endogenous RNA (magenta, left), transgenic fluorophore (yellow, middle) and overlaid two-color image (right). White arrowheads indicate colocalization. (A) Tg(gata3:loxP-dsRed-loxP:GFP); (B) Tg(gata3:Gal4; UAS:GFP); (C) Tg(gad1b:GFP); (D) Tg(glyt2:loxP-mCherry-loxP:GFP). Scale bars = 10 μm.

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