Skip to main content
. 2021 Dec 6;10:e75050. doi: 10.7554/eLife.75050

Figure 2. Tissue- and organelle-specific expression of seven CRISPR/Cas9 knock-in (KI) lines in medaka.

(A) eGFP-cbx1b F1 stage 40 medaka embryo. eGFP-Cbx1b labels all nuclei. Nuclei in the spinal cord of medaka are highlighted (yellow arrowhead). n > 10 embryos. SP = spinal cord. Scale bar = 30 µm. (B) mScarlet-pcna F1 stage 40 medaka embryo. mScarlet-Pcna is localized in the nuclei of cycling cells. mScarlet-Pcna is visible in skin epithelial cell nuclei located in the mid-trunk region of a medaka embryo. The localization of Pcna as speckles within the nucleus indicates cells in S phase of the cell cycle (yellow arrowheads). n = 10 embryos. EC = epithelial cells. Scale bar = 20 µm. (C) eGFP-rab11a F1 stage 40 medaka embryo. Expression of the membrane trafficking marker eGFP-Rab11a is evident in the caudal fin region. eGFP-Rab11a is strongly expressed in the spinal cord (yellow arrowhead) and lateral line neuromasts (magenta arrowhead). n = 6 embryos. CF = caudal fin. Scale bar = 30 µm. (D) mNG-myosinhc F1 stage 40 medaka embryo. mNG-Myosinhc is expressed solely in muscle cells. Myofibrils containing chains of individual sarcomere can be seen (mNG-Myosinhc labels the Myosin A band inside each sarcomere, yellow arrowheads). n > 10 embryos. MT = myotome. Scale bar = 30 µm. (E) cdh2-eGFP F1 stage 40 medaka embryo. Cdh2-eGFP is localized at cell membranes in several tissues, including the spinal cord (magenta arrowhead) and the notochord (yellow arrowhead). n = 5 embryos. NT = notochord. Scale bar = 50 µm. (F–F’) g3bp1-eGFP F1 stage 34–35 medaka embryo. Time-lapse imaging of G3bp1-eGFP dynamics under normal and stress conditions. (F) G3bp1-eGFP localizes to the cytoplasm under physiological conditions. (F’) Under stress conditions (temperature shock), G3bp1-eGFP localizes to stress granules (yellow arrowheads). Time in hours. n = 8 embryos. SG = stress granules. Scale bar = 50 µm. (G) mapre1b-mScarlet F1 stage 35 medaka embryo. Mapre1-mScarlet is expressed in a number of tissues and cell types including epithelial cells, muscle cells, the notochord, neuromasts and is highly expressed in the spinal cord. n = 5 embryos. SP = spinal cord. Scale bar = 50 µm.

Figure 2.

Figure 2—figure supplement 1. Ubiquitous nuclear expression of eGFP-cbx1b.

Figure 2—figure supplement 1.

(A) Anterior head region of eGFP-cbx1b F1 stage 40 medaka embryo. eGFP-Cbx1b is expressed ubiquitously with a nuclear localization pattern. n > 10 embryos. HE = head. Scale bar = 100 µm. (B) Maximum projection of spinal cord and notochord in the mid-trunk region of eGFP-cbx1b stage 40 medaka embryo. All nuclei are labelled by eGFP-Cbx1b. n > 10 embryos. SP = spinal cord, NT = notochord. Scale bar = 30 µm. (C) A subset of epithelial cell nuclei positive labelled by eGFP-Cbx1b. n > 10 embryos. EC = epithelial cells. Scale bar = 30 µm. (D) Partial view of the gut of eGFP-cbx1b KI stage 40 medaka embryo. Gut microvilli are positive for eGFP-Cbx1b (yellow arrowheads). MV = microvilli. n > 10 embryos. Scale bar = 30 µm.
Figure 2—figure supplement 2. Immunofluorescence of eGFP-cbx1b line confirms ubiquitous nuclear expression.

Figure 2—figure supplement 2.

(A) Maximum intensity projection of Hoechst stained stage 30 medaka embryo focusing on the anterior head region. (A’) Maximum intensity projection of anti-GFP staining in the anterior head region of the eGFP-cbx1b KI line. (A’’) Merged image from (A) and (A’). Note the overlap of Hoechst and the anti-eGFP staining in the eGFP-cbx1b KI line. n = 5 embryos. HE = head. Scale bar = 40 µm. (B) Maximum intensity projection of Hoechst stained stage 30 medaka embryo focusing on the posterior trunk region. (B’) Maximum intensity projection of anti-GFP staining in the posterior trunk region of the eGFP-cbx1b KI line. (B’’) Merged image from (B) and (B’). Note the overlap of Hoechst and the anti-eGFP staining in the eGFP-cbx1b KI line. n = 5 embryos. TR = trunk. Scale bar = 50 µm. (C–C’’) (C) Maximum intensity projection of Hoechst stained stage 30 medaka embryo, a zoomed-in view on a section of the trunk from (B–B’’) shows individual nuclei. (C’) Maximum intensity projection of anti-GFP staining on zoomed-in view of the trunk in the eGFP-cbx1b KI line. (C’’) Merged image from (C) and (C’’). Note the overlap of Hoechst and the anti-eGFP staining in nuclei. n = 5 embryos. TR = trunk. Scale bar = 50 µm.
Figure 2—figure supplement 3. Tissue-specific expression of eGFP-rab11a and cdh2-eGFP KI lines.

Figure 2—figure supplement 3.

(A) Neuromasts of eGFP-rab11a F1 stage 40 medaka embryo. eGFP-rab11a is strongly expressed in the posterior lateral line neuromasts and posterior lateral line nerve. Ventral primary neuromasts strongly express eGFP-Rab11a (magenta arrowheads), as well as secondary neuromasts located at the horizontal myoseptum (yellow arrowheads). n = 10 embryos. pLL = posterior lateral line. Scale bar = 100 µm. (B) Anterior region of eGFP-rab11a F1 stage 40 medaka embryo. eGFP-Rab11a is strongly expressed in the optic tectum (magenta arrowhead) and in the otic vesicle sensory organs (yellow arrowhead). n = 10 embryos. OT = optic tectum, OV = otic vesicle. Scale bar = 50 µm. (C) Close-up on a ventral primary neuromast of eGFP-rab11a F1 stage 40 medaka embryo. Strong expression of eGFP-Rab11a in neuromast cells (magenta arrowhead) and the lateral line nerve (yellow arrowhead). n = 10 embryos. LN = lateral line nerve, NU = neuromast. Scale bar = 10 µm. (D) Spinal cord of eGFP-rab11a F1 stage 40 medaka embryo. eGFP-Rab11a is strongly expressed in the spinal cord. n = 10 embryos. SP = spinal cord. Scale bar = 10 µm. (E) Eye of cdh2-eGFP F1 stage 40 medaka embryo. Cdh2-eGFP is strongly expressed in elongated cells covering the retina. n = 10 embryos. EY = eye. Scale bar = 30 µm. (F) Expression of cdh2-eGFP F1 in stage 40 medaka embryo. Cdh2-eGFP is expressed in the spinal cord (magenta arrowhead) and notochord sheath cells (yellow arrowhead). n = 10 embryos. SP = spinal cord, SC = sheath cells. Scale bar = 50 µm. (G) Notochord of cdh2-eGFP F1 stage 40 medaka embryo. Cdh2-eGFP is weakly expressed in notochord vacuolated cells (yellow arrowhead). The contrast has been enhanced in panel (G) compared to (F) to highlight better the expression in VCs. n = 10 embryos. VC = vacuolated cells. Scale bar = 30 µm. (H) Neuromast support and hair cells of cdh2-eGFP F1 stage 40 medaka embryo. Neuromast support and hair cells within a mature primary neuromast organ express high levels of Cdh2-eGFP. n = 10 embryos. NU = neuromast. Scale bar = 10 µm.
Figure 2—figure supplement 4. Muscle-specific expression of mNG-myosinhc.

Figure 2—figure supplement 4.

(A) Muscle cells of mNG-myosinhc F1 stage 40 medaka embryo. mNG-Myosinhc is expressed in muscle cells of the posterior tail region and forms myofibrils, view of myofibrils from Figure 2D. n = 10 embryos. CF = caudal fin. Scale bar = 50 µm. Pectoral fin of mNG-myosinhc F1 stage 40 medaka embryo. mNG-Myosinhc is expressed in muscles of the pectoral fin, and myofibrils radiate from the base of the pectoral fin. (C) is a close-up of (B). n = 10 embryos. PC = pectoral fin. Scale bar = 50 µm. (D) Body trunk region of mNG-myosinhc F1 stage 40 medaka embryo. mNG-Myosinhc is expressed in muscle cells in the body trunk. Close-up of muscle cells from Figure 1D. n = 10 embryos. MT = myotome of trunk. Scale bar = 50 µm.