© 2001 Wiley-Liss, Inc.
genesis 30:149 –153 (2001)
Inhibition of skiA and skiB Gene Expression Ventralizes Zebrafish Embryos Zongbin Cui1,2, Karl J. Clark1, Christopher D. Kaufman1,†, and Perry B. Hackett1,3 1
Department of Genetics, Cell Biology and Development and The Arnold and Mabel Beckman Center for Transposon Research, University of Minnesota, St. Paul, Minnesota 2 Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, P. R. China 3 Discovery Genomics, Inc., St. Paul, Minnesota Received 1 May 2001; Accepted 13 June 2001 Published online 23 July 2001; DOI 10.1002/gene.1052
Dorsal–ventral (DV) patterning has been extensively studied in vertebrates. Signaling proteins identified in DV development include the bone morphogenetic proteins 2 and 4 (BMP2,4), members of the transforming growth factor (TGF)- superfamily, which have ventralizing activities, and their antagonists Chordin, Noggin and Follistatin (Thomsen, 1997; Heasman, 1997). Interactions among these signaling proteins appear to establish gradients of BMP activity that are responsible for DV patterning. Twisted gastrulation is a conserved protein that functions with Chordin to antagonize BMP signaling (Ross et al., 2001), indicating the existence of multilayered regulating pathways that govern one of the most fundamental events in vertebrate embryogenesis. Smad proteins mediate the intracellular transmission of BMP signals. Smad proteins can be sorted into three distinct functional classes: receptor-regulated Smad proteins acting as positive mediators of TGF- superfamily signaling (Smads 1, 2, 3, 5, and 8), global partners of receptor-regulated Smad proteins (Smad 4) and inhibitory Smads (6, 7, and 9) (Whitman, 1997; Kretzschmar and Massague´, 1998). In their inactive states, receptorregulated Smad proteins are cytoplasmic and form homotrimers that are activated by specific TGF- receptors via phosphorylation of a conserved SSXS motif in their MH2 domains. After phosphorylation, receptor-regulated Smad homotrimers aggregate with trimers of Smad 4 and translocate to the nucleus, where they interact with their cofactors to regulate the transcription of target genes. Biochemical analysis indicates that Ski proteins act as transcriptional co-repressors through interaction with Smads -1, -2, -3, and -4 in cells (Luo et al., 1999; Akiyoshi et al., 1999; Xu et al., 2000; Wang et al., 2000). Altered expression of Ski can lead to oncogenesis (Li et al., 1986; Stavnezer et al., 1986). In a previous study, we showed that there are two paralogues of the ski gene family in zebrafish, skiA and skiB. Overexpression of either gene disrupted gastrulation and resulted in a dorsalized phenotype, suggesting that Ski proteins participate in the DV patterning (Kaufman et al., 2000). Here, we significantly extend the initial conclusions by using a “knockdown” strategy
(Nasevicius and Ekker, 2000) to examine the roles of SkiA and SkiB during embryogenesis. Embryos injected with MO-skiA or MO-skiB have enlarged blood islands, expanded caudal somites, reduced or absent notochords, and little or no head structures after 24 h of development (Fig. 1 B–J), similar to the ventralized embryos described by Kishimoto et al. (1997). This confirms that SkiA and SkiB are involved in DV patterning in zebrafish. The level of ventralization was dose dependent and specific for each paralogue, as shown by secondary MOs directed against either skiA or skiB (Table 1). As expected (Nasevicius and Ekker, 2000), control MOs with four-base mismatches to either ski gene gave lower rates of ventralization when used at 60-ng levels (P ⬍ 0.0001 for both skiA and skiB) but not at the 10-ng levels (Table 1). However, both four-base mismatched MOs induced ventralization at significantly higher rates (P ⫽ 0.0095) at 60 ng than a random (standard) MO that was used to control for general MO toxicity. At comparable doses, MO-skiB injection generated more severely ventralized embryos than MO-skiA (Table 2). Co-injection of two different MO oligos for the same ski gene synergistically blocked gene expression and caused higher rates and degrees of ventralization (Table 2). We note that at the higher doses of MOs and mRNAs there is considerable lethality. Both agents can be toxic, especially when they affect such a fundamental process as DV patterning. Note that when both mRNA and MOs are injected, some embryos (D/V) have regions Contract grant sponsor: Arnold and Mabel Beckman Foundation, Contract grant sponsor: U.S. Department of Commerce-NOAA, Contract grant number: USDOC/NA46RG0101-02 (Minnesota Sea Grant publication JR477), Contract grant sponsor: Minnesota Sea Grant College Program supported by the NOAA Office of Sea Grant, United States Department of Commerce, Contract grant number: 1998-2001 NOAA-NA86-RG0033. *Correspondence to: Perry B. Hackett, Department of Genetics, Cell Biology and Development, University of Minnesota, 250 Biological Sciences Center, 1445 Gortner Avenue, St. Paul, MN 55108-1095. E-mail:
[email protected] †Current address: Max Delbru ¨ ck Center for Molecular Medicine, Robert Ro ¨ ssle Strasse 10, D-13092, Berlin, Germany. The U.S. Government is authorized to reproduce and distribute reprints for government purposes, not withstanding any copyright notation that may appear hereon.
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FIG. 1. Morphological characteristics of ski-morphants and altered gene expression detected by in situ hybridization. Panels show 24-h embryos. (A) uninjected embryo; (B–E) V1, V2, V3, and V4 embryos injected with 10 ng of MO-skiAI and 10 ng of MO-skiAII; (F–J) V1, V2, V3, and V4 embryos injected with 5 ng of MO-ski BI and 5 ng of MO-skiBII. (K–P) Dorsal views of in situ hybridization patterns of embryos at 70% epiboly (K–M) and nine-somite stage (N–P). K, shh, wild-type embryo; L, shh, MO-skiA-injected embryo; M, shh, MO-skiB-injected embryo; N, myoD, wild-type embryo; O, myoD, MO-skiA-injected embryo; P, myoD, MO-skiB-injected embryo.
that are dorsalized and others that are ventralized. This is probably because of unequal distribution of the MOs and/or rescuing mRNAs, which should not be unexpected given the variations seen by everyone after injections of either mRNA or MO alone. In situ hybridization analyses indicate that the pattern of sonic hedgehog (shh) expression is of normal width in the rostral part of the embryo, but posteriorly becomes progressively narrower and denser in the MO-skiA and MO-skiB-injected embryos (Fig. 1). The rostral somites of MO-skiA and MO-skiB-injected embryos contain little, if any, paraxial myoD-expressing cells, although adaxial staining is normal. However, myoD expression was unchanged in the caudal regions of embryos (Fig.1). The altered expression patterns of shh during gastrulation and myoD in mesodermal tissues during somitogenesis are consistent with those found with dorsal gene mutations (Hammerschmidt et al., 1996).
We tested the specificity of the MO injections by coinjecting skiA and skiB mRNAs to examine further whether the ventralized embryos from MO-skiA or MOskiB injection could be rescued by their mRNAs and to investigate their differences in function. We deleted the 5⬘ untranslated sequences and changed a few bases in the coding regions of the skiA and skiB genes in pBluescript constructs to make mRNAs that would not be repressed by the MOs. Co-injection of skiA mRNA with MO-skiA shifted some of the ventralized embryos to normal and the same results were found with skiB mRNA plus MO-skiB (Table 2). However, co-injection of skiA mRNA with MO-skiB or skiB mRNA with MO-skiA did not shift ventralized embryos to normal (Table 2). Most importantly, injection of skiB mRNA dorsalized most embryos as expected. However, although injection of skiA mRNA also dorsalized most of the embryos, a few remained ventralized (Table 2). We conclude that skiA
Table 1 Microinjection of skiA and skiB MOs into One-Cell Zebrafish Embryos Oligo type NE Standard skiA I skiA II 4-mis skiA I skiB I skiB II 4-mis skiB I
Injection (ng)
N
% Lethality
(%) Ventralized
(%) Normal
10 60 10 30 60 10 30 60 10 60 10 30 60 10 30 60 10 60
1,115 155 108 278 263 375 112 195 382 180 192 184 204 297 132 165 233 186 163
9 13 18 9 16 27 5 10 22 8 26 12 12 21 18 21 29 15 18
0 0 0a 8 48 70 2 57 76 6b 14c 9 55 79 34 73 71 14b 17c
91 87 82 83 36 3 93 33 2 86 60 79 33 0 48 6 0 71 65
Embryos from line AB were microinjected with morpholinos (MOs) from Gene Tools, LLC (Corvallis, OR). MOs were resuspended in 1 ⫻ Danieau buffer (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5.0 mM HEPES). N is the number of embryos injected. An average of two experiments were done for each condition, except NE. Lethality was measured at 24 h after fertilization. The samples were injected into the middle area between yolk and cell. A “standard” (random) MO was used to demonstrate that the MO dosage was not toxic. Chi-square statistical analysis of the data showed that: a The four-base mismatched MOs at 60 ng caused significantly more ventralization than the standard MO (P ⫽ 0.00095). b No significant differences between MOs and four-base mismatched MOs at 10 ng (P ⬎ 0.1). c Significant differences in ventralization rates between MOs and four-base mismatched MOs at 60 ng (P ⬍ 0.0001). In the four-based mismatched MOs, the mismatched bases are underlined in the MO sequences below. Standard, 5⬘-CCTCTTACCTCAGTTACAATTTATA-3⬘; skiA I, 5⬘-CGCTACTGTTTCCATGTTTCGCGCA-3⬘; 4-mis skiA I, 5⬘CGCAគ ACTC គ TTTCCATGTTAគ CGCC គ CA-3⬘; skiA II, 5⬘-GGCAAGAAATGCGACTCAAAGGTCC-3⬘; skiB I, 5⬘-AACCTGGGCAGTTTAATCACAACAG-3⬘; 4-mis skiB I, 5⬘-AACG គ TGGTគ CAGTTTAATCTគ CAAG គ AG-3⬘; skiB II, 5⬘-AACAGATTCATAAATGTGAGTCGC-3⬘. Table 2 Morphological Rescue of skiA and skiB MO-Induced Effects by Their mRNAs
Material injected AI ⫹ AII skiA mRNA AI ⫹ AII⫹ A mRNA BI ⫹ BII skiB mRNA BI ⫹ BII ⫹ B mRNA AI ⫹ AII ⫹ B mRNA BI ⫹ BII ⫹ A mRNA
Amount injected 5 ng each 10 ng each 50 pg 100 pg 400 pg 2 ⫻ 15 ng ⫹ 100 pg 2 ⫻ 15 ng ⫹ 400 pg 1 ng each 2.5 ng each 5 ng each 50 pg 100 pg 200 pg 2 ⫻ 5 ng ⫹ 50 pg 2 ⫻ 5 ng ⫹ 100 pg 2 ⫻ 5 ng ⫹ 200 pg 2 ⫻ 10 ng ⫹ 100 pg 2 ⫻ 15 ng ⫹ 100 pg 2 ⫻ 15 ng ⫹ 200 pg 2 ⫻ 5 ng ⫹ 100 pg 2 ⫻ 5 ng ⫹ 200 pg
N 89 172 190 429 506 277 280 608 1,043 548 122 134 221 421 213 298 337 300 181 175 226
% Dead
V1
11 17 43 51 44 58 49 14 18 17 12 15 38 15 18 19 34 55 76 46 36
32 59 16 6 7 5 0 24 37 61 0 0 0 40 22 11 12 10 3 6 12
% Ventralized V2 V3 2 7 3 2 1 4 0 4 6 16 0 0 0 18 9 0 7 5 0 7 8
6 2 0 0 0 0 0 2 4 2 0 0 0 0 0 0 0 0 0 0 0
V4
% Normal
% D/V
% D
2 2 0 0 0 0 0 3 5 4 0 0 0 0 0 0 0 0 0 0 0
47 13 32 6 3 12 13 53 30 0 48 17 6 6 6 49 8 0 0 0 0
0 0 0 0 0 0 6 0 0 0 0 0 0 16 23 3 7 5 0 3 23
0 0 6 35 45 20 32 0 0 0 40 68 56 5 22 18 32 25 21 38 21
5⬘ - untranslated regions (UTR)-modified pSKA-S041/ and pSKB-M042/ (Kaufman et al., 2000) were used for transcription in vitro. Alterations in the initiation codon-proximal bases were made by polymerase chain reaction (PCR) without altering the encoded amino acids in the coding region. Lethality was measured at 24 h after fertilization. AI, AII, BI, and BII are morpholinos (MOs). For SkiA mRNA, the first round PCR was performed with two primers, skiA-3 (5⬘-ATGGAG គ ACC គ GTG គ GCC គ CGACAGAGTTTCCAGCCTC-3⬘) and skiA-5 (5⬘-GTTTACGCGTATCGAATTCCTGCAGCCCG-3⬘), to mutate 4 bases underlined in the primer sequence. The second round PCR was performed with two primers, skiA-4 (5⬘-CGATACGCGTAAACATGGAGACCGTGG-3⬘) and skiA-5. The PCR products were digested with MluI, followed by ligation, transformation and sequencing. The same strategy was used to modify the skiB 5⬘UTR by primers, skiB-2 (5⬘-CGATACGCGTAAACATGGAGGCTCCTTC-3⬘) and skiA-5, and a MluI site was also generated upstream its initiation codon. Both skiA and skiB without 5⬘ UTRs were linearized with XhoI, and synthetic RNA was transcribed from the T3 promoter using mMachine kit (Ambion Austin, TX) following the manufacturer’s directions. Zebrafish embryos were microinjected at the one-cell stage. D/V embryos have mixtures of dorsalized and ventralized domains, suggesting unequal distribution of MOs and/or mRNA. %D is the percentage of dorsalized embryos.
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Table 3 Gene Expression Rescue of skiA and skiB MO-Induced Effects by Their mRNAs Material injected
Amount injected
Antisense RNA probes
N
% Normal
%V
% D/V
% D
15 ng each 15 ng each 2 ⫻ 15 ng ⫹ 200 pg 2 ⫻ 15 ng ⫹ 200 pg 5 ng each 5 ng each 2 ⫻ 5 ng ⫹ 200 pg 2 ⫻ 5 ng ⫹ 200 pg
shh myoD shh myoD shh myoD shh myoD shh myoD
24 41 45 39 80 107 91 43 112 108
100 100 16 18 20 42 4 0 18 26
0 0 84 82 49 16 96 100 48 22
0 0 0 0 14 19 0 0 18 31
0 0 0 0 19 23 0 0 16 21
NE AI ⫹ AII AI ⫹ AII ⫹ A mRNA BI ⫹ BII BI ⫹ BII ⫹ B mRNA
Antisense RNA probes were made with a MAXIscript kit from Ambion and labeled with Digoxigenin-11-UTP from Roche Diagnostics Corporation (Indianapolis, IN). In situ hybridization was performed as described by Thisse et al. (1993). V, ventralized embryos; D/V, embryos with both dorsalized and ventralized domains; D, dorsalized embryos.
and skiB have distinctive roles in DV patterning such that neither gene can complement the other. To determine further the specificity of the ski MOs, we examined the expression patterns of shh and myoD in embryos co-injected with skiA or SkiB MOs plus their corresponding mRNAs. The expression profiles of shh and myoD in some embryos were largely rescued by the corresponding ski mRNAs (Table 3), supporting the model that skiA and skiB are involved in DV patterning of zebrafish. There are two different BMP2 proteins in zebrafish, BMP2a and BMP2b. BMP2b is expressed earlier and appears to be critical for ventral patterning (Schier and Talbot, 1998). Hild et al. (1999) suggested that there are three phases of DV patterning in zebrafish. They are: 1) an early Smad5- and Bmp2b-independent phase when a coarse initial DV pattern is set up, 2) an intermediate Smad5- and Bmp2b-dependent phase during which the putative morphogenetic Bmp2/4 gradient is established, and 3) a later Smad5-independent phase during gastrulation when the Bmp2/4 gradient is interrupted and cell fates are specified. Apart from BMP2/BMP4, the wntsignaling pathway is required for dorsal mesoderm formation (Nasevicius et al., 1998). Five smad genes (1–5) have been identified in zebrafish, of which smads 1, 2, and 5 show distinct roles during DV patterning (Dick et al., 1999; Mu ¨ ller et al., 1999; Dick et al., 2000). Nevertheless, little is known about their relations and their interactions with the Ski proteins. The most interesting conclusion we draw is that skiA and skiB play crucial and nearly identical roles in DV patterning but that they functionally cannot replace each other even though they are very similar. This suggests that for body axis determination, either SkiA and SkiB interact as heterodimers for activity, that they act sequentially in the same pathways, or that they are involved in parallel signaling pathways. Whichever it is, the data clearly indicate the power of MO-based, functional genetics in zebrafish to elucidate developmental processes in vertebrates.
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INHIBITION OF SKIA AND SKIB IN ZEBRAFISH
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