3-1
rasd1 mutants do not show significant axon projection defect inhabenula
rasd1 is expressed in LHA in 3dpf and 4dpf larvae (Fig.2), and LHA neurons
project their axons to dorsal IPN at 4dpf. To determine whether rasd1 is involved in LHA axon projection, rasd1 mutant was generated by CRISPR/Cas9 system. Mutant rasd1 carries 7-base deletion in exon1, and the deletion causes frame shift mutation
leading to an immature stop codon introduction (Fig.3). Anti-Leftover (Lov) staining that labels LHA neurons and their axons is used for detecting habenula LHA axon projection into the IPN in zerafish larval brain (Gamse et al., 2005). Anti-Lov staining shows that there are no significant difference in habenula axon projection between rasd1-/- larvae and their WT siblings (Fig.4, A-D).
To block rasd1 signal, we designed rasd1Δ181-265 that is assumed as a dominant negative form and injected it into zebrafish embryos (Fig.4-H). The larvae that are injected with rasd1Δ181-265 RNA still do not show significant difference between injected larvae and no-injection control (Fig.4, E-G).
In Western blot analysis, I found the anti-Rasd1 antibodies generated in our laboratory could recognize over-expressed mCherry-tagged Rasd1, but it was hard to detect endogenous Rasd1 (Fig.5, A-B). Western blot result also indicated rasd1 MO is able to reduce Rasd1 expression in 3dpf larvae, and less effect in 4dpf larvae (Fig.5, C).
3-2 Ectopic rasd1 is induced in choroid plexus and hematocyte- like cells
Previous works show ectopic rasd1 expression is induced by DEX treatment in
Kemppainen and Behrend, 1998). To understand whether rasd1 expression in habenula is also induced by DEX treatment in zebrafish larvae, 3dpf larvae were treated by DEX.
Ectopic rasd1 is not induced in HA, but in cells in hindbrain and cells surrounding the hatching gland region (Fig.6, A-D), and ectopic rasd1is induced in almost all of larvae by 100μM to 500μM DEX treatment. Comparing to control, ectopic rasd1 expression is significantly induced in choroid plexus (CP) and hematocyte-like cells by 100μM to 500μM DEX treatment (Fig.6-E). Two-color in situ hybridization of rasd1 and clusterin (clu) confirms the co-expression of rasd1 and clu in CP (Fig.7, A-C).P-value of ectopic rasd1 induction rate in the CP is 0.035 between 100μM and 250μM DEX treatment, and is 0.620 between 250μM, and 500μM DEX treatment (Fig.6-F). It suggests that 250μM and 500μM DEX treatment have similar efficiency to induce ectopic rasd1 expression in CP.
Ectopic rasd1 is only induced in the surface of larvae by DEX treatment. Because DEX may not pass through the epidermis efficiently, I injected DEX into ventricular zone. Ectopic rasd1 expression is induced by DEX injection in 3dpf larvae, and the regions of ectopic rasd1 expression are similar to it in 3dpf larvae that are soaked in DEX (Fig.7, D-E). The resembling expression pattern suggests that DEX is able to infiltrate the epidermis.
To prove whether rasd1 is induced in habenula in 1dpf and 2dpf larvae with DEX treatment, we treated 1dpf and 2dpf larvae with 250μM DEX. DEX treatment induces ectopic rasd1expression in hematocyte-like cells in 1dpf and 2dpf larvae, and does not induce rasd1 expression in habenula (Fig.8).
Ectopic rasd1 expression is induced in CP and hematocyte-like cells in 3dpf and 4dpf larvae, and is only induced in hematocyte-like cells in 1dpf embryos. Ectopic rasd1 expression in CP is only induced in 10% of 2dpf embryos. DEX treatment does
not induce rasd1 expression in habenula in 1dpf and 2dpf embryos, and does not influence rasd1 expression in habenula in 3dpf and 4dpf larvae. It suggests that rasd1 expression in habenula is regulated by genetic programing.
3-3 Efficiency of ectopic rasd1 expression induced by DEX is decreased gradually after DEX withdrawal
Effects of most of drugs decrease gradually after the treatment is terminated (Al Katheeri et al., 2004; Grady et al., 2010). To confirm whether ectopic rasd1 expression is induced by DEX treatment, we treated the larvae with 250μM DEX 3 hours and washed with fresh embryo water. Ectopic rasd1 expression significantly decreases in 1 hour withdrawal and less ectopic rasd1 expression is observed in 2 hours withdrawal (Fig.9). Finally, ectopic rasd1 expression resumes to almost normal level in 3 hours after DEX withdrawal.
3-4
rab6bb mutants are generated by CRISPR/Cas9 systemTo study the role of rab6bb in LHA axonal targeting process, I stared to generate rab6bb mutant with CRISPR/Cas9 system. Diagram in Fig.10 indicates the sgRNA
target site in exon1 of rab6bb (Fig.10-A). The target site is 30 base-pair downstream to the 5' end of the open reading frame. rab6bb DNA fragments of the adult fishes that has been injected were cut by T7E1. T7E1 assay indicates that one of the F0 and its descendants carry mutated rab6bb alleles (Fig.10-B). Capillary electrophoresis and sequencing show that five F1 carry 10-base-pair mutation and another five F1 carry 7-base-pair dleltion in fifteen F1 (Fig.10-C). rab6bb mutant is going to be utilized to confirm the role of rab6bb in LHA axon guidance once the line is established.
Chapter 4 Discussions and Conclusions
In this Master thesis project, the function of zygotic rasd1 and the mRNA expression regulation of rasd1 have been investigated. Although results from previous studies in our laboratory demonstrated that rasd1 and nrp1a morphants show similar LHA axon projection defect, the axon projecting onto the IPN seemed to be no difference between the rasd1 knockout fish and their WT siblings. Because previous works show mutants and morphants of several genes exhibit different phenotypes (Kok et al., 2015; Rossi et al., 2015). The discrepancy of the mutants and morphants could be caused by off-target effect of rasd1 MOs, or genetic compensation. LC-MS/MS or DNA microarray assay should be applied to further understand the discrepancy between the rasd1 morphants and mutants.
To block Rasd1 function, I injected rasd1Δ181-265 RNA which shall express dominant negative (DN) Rasd1 in embryos (Guo and Fang, 2006; Suzuki et al., 2006).
The injected larvae and WT control do not show significant difference in LHA projection volume and morphology. The reasons why the LHA axon projection pattern was unaffected in the rasd1Δ181-265 injected larvae could be that the truncated Rasd1 is able to block the signal cascade via Rasd1 but Rasd1 does not play a role in LHA axon targeting process. Alternatively, the rasd1Δ181-265 mRNAs injection did not express the DN Rasd1 as I had expected.
Even though Western blot could not indicate whether Rasd1 remains in rasd1 mutants, if rasd1 is involved in LHA axon target finding, minimum variation in LHA projection pattern formation may be cause by two possibilities. First possibility: Rasd1 is still expressed in zebrafish because of gene duplication. Undefined duplicated rasd1 in zebrafish genome is expressed and complements mutant rasd1. Undefined paralog of
able to be disrupted by rasd1 MOs. Nevertheless, this is unlikely the cause because 99%
of the Danio rerio genome sequences has been verified (ZFIN.org). Second possibility:
MOs knock-down causes disruption of the Rasd1 protein function, but an unknown protein complements the function of mutated Rasd1. This unknown protein has similar function as Rasd1, or it plays downstream to Rasd1 pathway and is up-regulated, so that the requirement of Rasd1 is bypassed.
Rasd1 was first uncovered as a downstream response gene to DEX treatment. DEX treatment induces rasd1 expression in several types of mouse cells (Greenwood et al., 2016; Kemppainen and Behrend, 1998). In my DEX treating experiments, rasd1 expression in the larval habenula does not elevated in three different DEX treating conditions (100, 250, 500uM). DEX is a type of steroid medication which activates glucocorticoid receptor (Pandit et al., 2002). Under normal physiological conditions, glucocorticoid is released by stress stimulation (Popoli et al., 2012). It suggests glucocorticoid may increase in blood because of environmental change. rasd1 expression in habenula is not influenced by DEX treatment. This suggests that rasd1 expression in habenula is programmed by genetic algorithm. Interestingly, I found ectopic rasd1 expression is induced by DEX treatment in the larval CP and hematocyte-like cells. Double in situ hybridization of rasd1 and clusterin verified the ectopic rasd1 expression in the CP. CP is a major secretory tissue that produces cerebrospinal fluid (CSF) which protects neuron and cleans waste in the vertebrate brain (Lun et al., 2015).
It forms blood-CSF barrier (Lun et al., 2015) and plays a role in neurogenesis (Johansson, 2014). Previous studies also exhibit that stress enhances specific genes expression in CP (Martinho et al., 2012; Sathyanesan et al., 2012), but how CP influences CSF under stress is still unclear. DEX treatment induces ectopic rasd1 expression in CP suggesting that Rasd1 may play a role in the stress response process of
CP. Investigation of rasd1 in CP should help us to know how CP responses to stress.
Previous study exhibits DEX treatment induces ectopic rasd1 expression via glucocorticoid receptor (GR) and signal transducer and activator of transcription 5b (STAT5b) in rat (Lellis-Santos et al., 2012), but nr3c1 and stat5b is widely expressed in zebrafish larvae. DEX treatment only induces ectopic rasd1 expresion in CP and hematocyte-like cells in zebrafish. I propose three possibilities. First and second possibilities: because there are three GR isoforms and three STAT5b isoforms in zebrafish, rasd1 expression is induced by DEX only in two GR or STAT5b isoforms, or one of the GR or STAT5b isoforms functions as a dominate negative form and inhibits rasd1 expression in other tissue. Third possibility: tissue-specific transcription factor
and GR cooperatively enhance rasd1 expression in specific tissue under DEX treatment.
Rab6b is Rab6bb homology and is proposed to mediate the retrograde transport from Golgi to ER in human neuronal cells (Wanschers et al., 2007). Rab subfamily small GTPases localize on the surface of intracellular organelles and control membrane traffic (Brumell and Scidmore, 2007). If Rab6bb is involved in targeting process, mutated rab6bb will impact exocytosis or endocytosis. rab6ba is the paralog of rab6bb may be able to compensate mutated rab6bb. However, our pervious lab members found rab6ba is not expressed in LHA in 4dpf zebrafish larvae. The different expression regions of rab6bb and rab6ba should help us to understand the role of rab6bb in Nrp1a-mediated LHA axon path finding without severe whole body phenotype.
References
Aizawa, H., 2013. Habenula and the asymmetric development of the vertebrate brain.
Anat Sci Int 88, 1-9.
Al Katheeri, N.A., Wasfi, I.A., Lambert, M., Saeed, A., 2004. Pharmacokinetics and pharmacodynamics of dexamethasone after intravenous administration in camels:
effect of dose. Vet Res Commun 28, 525-542.
Amo, R., Aizawa, H., Takahoko, M., Kobayashi, M., Takahashi, R., Aoki, T., Okamoto, H., 2010. Identification of the zebrafish ventral habenula as a homolog of the mammalian lateral habenula. J Neurosci 30, 1566-1574.
Beretta, C.A., Dross, N., Guiterrez-Triana, J.A., Ryu, S., Carl, M., 2012. Habenula circuit development: past, present, and future. Front Neurosci 6, 51.
Bianco, I.H., Wilson, S.W., 2009. The habenular nuclei: a conserved asymmetric relay station in the vertebrate brain. Philos Trans R Soc Lond B Biol Sci 364, 1005-1020.
Brumell, J.H., Scidmore, M.A., 2007. Manipulation of rab GTPase function by intracellular bacterial pathogens. Microbiol Mol Biol Rev 71, 636-652.
Cheah, J.H., Kim, S.F., Hester, L.D., Clancy, K.W., Patterson, S.E., Papadopoulos, V., Snyder, S.H., 2006. NMDA receptor-nitric oxide transmission mediates neuronal iron homeostasis via the GTPase Dexras1. Neuron 51, 431-440.
Chen, Y., Khan, R.S., Cwanger, A., Song, Y., Steenstra, C., Bang, S., Cheah, J.H., Dunaief, J., Shindler, K.S., Snyder, S.H., Kim, S.F., 2013. Dexras1, a Small GTPase, Is Required for Glutamate-NMDA Neurotoxicity. Journal of Neuroscience 33, 3582-3587.
Chou, M.Y., Amo, R., Kinoshita, M., Cherng, B.W., Shimazaki, H., Agetsuma, M.,
2016. Social conflict resolution regulated by two dorsal habenular subregions in zebrafish. Science 352, 87-90.
Colombo, A., Palma, K., Armijo, L., Mione, M., Signore, I.A., Morales, C., Guerrero, N., Meynard, M.M., Perez, R., Suazo, J., Marcelain, K., Briones, L., Haartel, S., Wilson, S.W., Concha, M.L., 2013. Daam1a mediates asymmetric habenular morphogenesis by regulating dendritic and axonal outgrowth. Development 140, 3997-4007.
Fakhoury, M., López, S.D., 2014. The Role of Habenula in Motivation and Reward.
Advances in Neuroscience Volume 2014, 6.
Gamse, J.T., Kuan, Y.S., Macurak, M., Brosamle, C., Thisse, B., Thisse, C., Halpern, M.E., 2005. Directional asymmetry of the zebrafish epithalamus guides dorsoventral innervation of the midbrain target. Development 132, 4869-4881.
Grady, J.A., Davis, E.G., Kukanich, B., Sherck, A.B., 2010. Pharmacokinetics and pharmacodynamics of dexamethasone after oral administration in apparently healthy horses. Am J Vet Res 71, 831-839.
Graham, T.E., Qiao, Z., Dorin, R.I., 2004. Dexras1 inhibits adenylyl cyclase. Biochem Biophys Res Commun 316, 307-312.
Greenwood, M.P., Greenwood, M., Mecawi, A.S., Antunes-Rodrigues, J., Paton, J.F., Murphy, D., 2016. Rasd1, a small G protein with a big role in the hypothalamic response to neuronal activation. Mol Brain 9, 1.
Guo, W., Fang, B.L., 2006. Dominant negative Ras as an anticancer agent. Cancer Biol Ther 5, 1492-1493.
Hikosaka, O., 2010a. The habenula: from stress evasion to value-based decision-making.
Nat Rev Neurosci 11, 503-513.
Hikosaka, O., 2010b. The habenula: from stress evasion to value-based decision-making.
Nature Reviews Neuroscience 11, 503-513.
Jesuthasan, S., 2012. Fear, anxiety, and control in the zebrafish. Dev Neurobiol 72, 395-403.
Jiao, S., Dai, W., Lu, L., Liu, Y.Z., Zhou, J.F., Li, Y., Korzh, V., Duan, C.M., 2011. The Conserved Clusterin Gene Is Expressed in the Developing Choroid Plexus Under the Regulation of Notch But Not IGF Signaling in Zebrafish. Endocrinology 152, 1860-1871.
Johansson, P.A., 2014. The choroid plexuses and their impact on developmental neurogenesis. Front Neurosci-Switz 8.
Kandel, E.R., 2012. The molecular biology of memory: cAMP, PKA, CRE, CREB-1, CREB-2, and CPEB. Mol Brain 5, 14.
Kemppainen, R.J., Behrend, E.N., 1998. Dexamethasone rapidly induces a novel ras superfamily member-related gene in AtT-20 cells. J Biol Chem 273, 3129-3131.
Kok, F.O., Shin, M., Ni, C.W., Gupta, A., Grosse, A.S., van Impel, A., Kirchmaier, B.C., Peterson-Maduro, J., Kourkoulis, G., Male, I., DeSantis, D.F., Sheppard-Tindell, S., Ebarasi, L., Betsholtz, C., Schulte-Merker, S., Wolfe, S.A., Lawson, N.D., 2015. Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish. Dev Cell 32, 97-108.
Kuan, Y.S., Yu, H.H., Moens, C.B., Halpern, M.E., 2007. Neuropilin asymmetry mediates a left-right difference in habenular connectivity. Development 134, 857-865.
Lee, A., Mathuru, A.S., Teh, C., Kibat, C., Korzh, V., Penney, T.B., Jesuthasan, S., 2010.
The habenula prevents helpless behavior in larval zebrafish. Curr Biol 20, 2211-2216.
Lellis-Santos, C., Sakamoto, L.H., Bromati, C.R., Nogueira, T.C., Leite, A.R.,
Yamanaka, T.S., Kinote, A., Anhe, G.F., Bordin, S., 2012. The regulation of Rasd1 expression by glucocorticoids and prolactin controls peripartum maternal insulin secretion. Endocrinology 153, 3668-3678.
Liu, Y., Berndt, J., Su, F., Tawarayama, H., Shoji, W., Kuwada, J.Y., Halloran, M.C., 2004. Semaphorin3D guides retinal axons along the dorsoventral axis of the tectum. J Neurosci 24, 310-318.
Lun, M.P., Monuki, E.S., Lehtinen, M.K., 2015. Development and functions of the choroid plexus-cerebrospinal fluid system. Nat Rev Neurosci 16, 445-457.
Martinho, A., Goncalves, I., Costa, M., Santos, C.R., 2012. Stress and glucocorticoids increase transthyretin expression in rat choroid plexus via mineralocorticoid and glucocorticoid receptors. J Mol Neurosci 48, 1-13.
Pandit, S., Geissler, W., Harris, G., Sitlani, A., 2002. Allosteric effects of dexamethasone and RU486 on glucocorticoid receptor-DNA interactions. J Biol Chem 277, 1538-1543.
Pasterkamp, R.J., 2012. Getting neural circuits into shape with semaphorins. Nature Reviews Neuroscience 13, 605-618.
Popoli, M., Yan, Z., McEwen, B.S., Sanacora, G., 2012. The stressed synapse: the impact of stress and glucocorticoids on glutamate transmission. Nat Rev Neurosci 13, 22-37.
Rossi, A., Kontarakis, Z., Gerri, C., Nolte, H., Holper, S., Kruger, M., Stainier, D.Y., 2015. Genetic compensation induced by deleterious mutations but not gene knockdowns. Nature 524, 230-233.
Sathyanesan, M., Girgenti, M.J., Banasr, M., Stone, K., Bruce, C., Guilchicek, E., Wilczak-Havill, K., Nairn, A., Williams, K., Sass, S., Duman, J.G., Newton, S.S., 2012. A molecular characterization of the choroid plexus and stress-induced gene
regulation. Transl Psychiatry 2, e139.
Shelly, M., Cancedda, L., Lim, B.K., Popescu, A.T., Cheng, P.L., Gao, H., Poo, M.M., 2011. Semaphorin3A regulates neuronal polarization by suppressing axon formation and promoting dendrite growth. Neuron 71, 433-446.
Shelton, L., Becerra, L., Borsook, D., 2012. Unmasking the mysteries of the habenula in pain and analgesia. Prog Neurobiol 96, 208-219.
Simonini, P.D.R., Breiling, A., Gupta, N., Malekpour, M., Youns, M., Omranipour, R., Malekpour, F., Volinia, S., Croce, C.M., Najmabadi, H., Diederichs, S., Sahin, O., Mayer, D., Lyko, F., Hoheisel, J.D., Riazalhosseini, Y., 2010. Epigenetically Deregulated microRNA-375 Is Involved in a Positive Feedback Loop with Estrogen Receptor alpha in Breast Cancer Cells. Cancer Res 70, 9175-9184.
Song, H., Ming, G., He, Z., Lehmann, M., McKerracher, L., Tessier-Lavigne, M., Poo, M., 1998. Conversion of neuronal growth cone responses from repulsion to attraction by cyclic nucleotides. Science 281, 1515-1518.
Stenmark, H., 2009. Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Bio 10, 513-525.
Suzuki, H., Kuzumaki, S., Nakagawa, K., Takimoto, M., Shichinohe, T., 2006.
Suppressive effect of modified dominant negative RAS mutant on human cancer by gene transfer with non-viral vector. Cancer Biol Ther 5, 1487-1491.
Taylor, R.W., Hsieh, Y.W., Gamse, J.T., Chuang, C.F., 2010. Making a difference together: reciprocal interactions in C. elegans and zebrafish asymmetric neural development. Development 137, 681-691.
Tojima, T., Kamiguchi, H., 2015. Exocytic and endocytic membrane trafficking in axon development. Dev Growth Differ 57, 291-304.
Wanschers, B.F., van de Vorstenbosch, R., Schlager, M.A., Splinter, D., Akhmanova, A.,
Hoogenraad, C.C., Wieringa, B., Fransen, J.A., 2007. A role for the Rab6B Bicaudal-D1 interaction in retrograde transport in neuronal cells. Exp Cell Res 313, 3408-3420.
Wolman, M.A., Liu, Y., Tawarayama, H., Shoji, W., Halloran, M.C., 2004. Repulsion and attraction of axons by semaphorin3D are mediated by different neuropilins in vivo. J Neurosci 24, 8428-8435.
Wu, B.T., Wen, S.H., Hwang, S.P.L., Huang, C.J., Kuan, Y.S., 2015. Control of Wnt5b secretion by Wntless modulates chondrogenic cell proliferation through fine-tuning fgf3 expression. J Cell Sci 128, 2328-2339.
Zerial, M., McBride, H., 2001. Rab proteins as membrane organizers. Nat Rev Mol Cell Bio 2, 107-117.
Figures
A B C
Figure 1. Rasd1 function and mRNA expression pattern. (A) Rasd1 mediates ferrous homeostasis in mouse cortical neurons. (Figure is modified from Cheah, Kim et al. 2006) (B) Rasd1 inhibits ESR1 expression in human breast cancer cells (Figure is modified form Wu, Liu et al. 2011) (C) Rasd1 regulates CREB phosphorylation via control PKA activity in Att-20 cells.
(Figure is modified form Greenwood, Greenwood et al. 2016) (D-F) rasd1 (in D) and rab6bb (in E) is coexpressed with nrp1a (in F) in 4dpf larval habenula. Arrows indicate the location of habenula. All images are dorsal views.
D E F
rasd1 rab6bb nrp1a
Figure 2. Expression patterns of rasd1 in various developmental stages. (A-D) Images showing mRNA staining of rasd1 in (A and A’) 24hpf, (B and B’) 48hpf, (C and C’)72hpf, and (D and D’) 96hpf. rasd1 expression level is significant increase in habenula and telencephalon from 72hpf to 96hpf. Arrows indicate the location of habenula. (A-D): Lateral views. (A’-D’):
Dorsal views. Scale bar:100μm.
24hpf
48hpf
72hpf
Lateral Dorsal
96hpf
A
B
C
D
B’
A’
C’
D’
9/11
10/19
38/47
39/47
Figure 3. CRISPR-Cas9-mediated rasd1 knockout line generation. (A) Sequencing result indicates a 7 base-pair deletion in exon1. This 7 base-pair deletion causes Hpy188III cleavage site loss and immature stop mutation. (B) Restriction enzyme test confirms Hpy188III cleavage site loss. DNA fragments are separated in 6% PAGE. (C) The schematic diagram
demonstrating the truncated Rasd1 predicted to be expressed in the rasd1 mutants.
A B
C
Figure 4. Loss- and gain-of-Rasd1 functional analyses. (A-C) Images showing anti-Lov antibody staining in the IPN. WT (in A), rasd1 heterozygous and homozygous mutant (in B and C) do not show significant difference in IPN projection morphology. (D) IPN projection
volumes from images in (A-C). (E-F) Images showing IPN of rasd1Δ181-265RNA injected larvae.
(G) Quantitative result of the IPN from (E and F) showing no significant difference in their morphology. (H) The diagram of Rasd1Δ181-265, truncated Rasd1 remains 180 amino-acid and C-terminal has been deleted. Scale bar:10μm.
A B C
D
E F
G
H
Lov
Lov
Figure 5. Rasd1 antibodies can detect overexpressed Rasd1. (A) Western blot result from Rasd1-mCherry RNA-injected embryo lysates indicates that anti-mCherry labeled band can be detected by anti-Rasd1 produced in our laboratory. The anti-b-actin was used as loading control.
(B) Western blot result from 4dpf total larval lysates indicated the endogenous Rasd1 level is hardly detected by our anti-Rasd1 antibodies. (C) Western blot result indicated that knocking down rasd1 expression by antisense morpholino (MO) could reduce Rasd1 expression in 3dpf injected larvae, and it showed less effect in 4dpf.
A B
C
75hpf
Figure 6. Tissue-specific ectopic expression of rasd1 can be induced in dexamethasone (DEX) treated larvae. (A-D) Images showing rasd1 expressions in larvae treated with (A-A’’) 0μM, (B-B’’) 100μM, (C-C’’) 250μM, and (D-D’’) 500μM DEX for 3 hours. Ectopic rasd1 expression is indicated by arrows (hematocyte-like cells) and arrow heads (choroid plexus). (E-F) Statistical results showing the percentages of rasd1 ectopic expression induction rate (E), and distribution of expression regions (F). (A-D): Lateral views. (A’-D’): Dorsal views. (A’’-D’’): Ventral views. Scale bar:100μm.
E F
Figure 7. Dexamethasone (DEX) treatment induced ectopic rasd1 expression in the
choroid plexus. (A-C) Images showing double mRNA in situ hybridization results of rasd1 and clusterin (clu) expressions in the DEX treated 75hpf larvae. Arrows indicate the CP. (D-D’’) PBS-injected larvae or (E-E’’) DEX-injected larvae. (A, D-E): Lateral views. (B, D’-E’):
Dorsal views. (D’’-E’’): Ventral views. (C): Enlarged dorsal view. Scale bar:100μm.
clu
Figure 8. Effects of dexamethasone (DEX) treatment in earlier embryonic stages. (A-D) rasd1 expression in 27hpf (A-A’’) control or DEX treated (B-B’’) embryos, or in 51hpf (C-C’’) control or DEX treated (D-D’’) larvae incubated in 250μM DEX for 3hrs. Ectopic rasd1
expression is indicated by arrows. (A-D): Lateral views. (A’-D’): Dorsal views. (A’’-D’’):
Ventral views. Scale bar:100μm.
Figure 9. Ectopic rasd1 expression diminished after DEX withdraw. (A-E) Images showing rasd1 expression in 75hpf control (A-A’’), or 75-78hpf with different treating conditions
indicated in the left column. Ectopic rasd1 expression is indicated by arrows (hematocyte-like cells) and arrow heads (choroid plexus). (F) Statistical chart showing the percentage of ectopic rasd1 expression induction rates. Ectopic rasd1 expression induction rate decreases gradually after the treatment is terminated. (A-E): Lateral views. (A’-E’): Dorsal views. (A’’-E’’): Ventral views. Scale bar:100μm.
Figure 10. CRISPR-Cas9-mediated rab6bb knockout line generation. (A) The diagram of rab6bb locus in zebrafish genome. CRISPR/Cas9 target site is indicated by arrow. (B) rab6bb PCR products contain target site are cut by T7E1. DNA fragments are separated in 6% PAGE.
(C) Snapshop showing the sequencing results from the F1 embryos produced by the F0 fish.
B
A
C