Drs. Shen and Ozacar contributed equally to this work.
Heat-shock–mediated conditional regulation of hedgehog/gli signaling in zebrafish
Article first published online: 27 MAR 2013
Copyright © 2013 Wiley Periodicals, Inc.
Volume 242, Issue 5, pages 539–549, May 2013
How to Cite
Shen, M.-C., Ozacar, A. T., Osgood, M., Boeras, C., Pink, J., Thomas, J., Kohtz, J. D. and Karlstrom, R. (2013), Heat-shock–mediated conditional regulation of hedgehog/gli signaling in zebrafish. Dev. Dyn., 242: 539–549. doi: 10.1002/dvdy.23955
- Issue published online: 17 APR 2013
- Article first published online: 27 MAR 2013
- Accepted manuscript online: 26 FEB 2013 06:58AM EST
- Manuscript Accepted: 14 JAN 2013
- Manuscript Revised: 16 DEC 2012
- Manuscript Received: 5 AUG 2012
- NIH . Grant Number: NS039994
- Sonic Hedgehog;
- heat shock;
- Top of page
- RESULTS AND DISCUSSION
- EXPERIMENTAL PROCEDURES
Background: Hedgehog (Hh) signaling is required for embryogenesis and continues to play key roles postembryonically in many tissues, influencing growth, stem cell proliferation, and tumorigenesis. Systems for conditional regulation of Hh signaling facilitate the study of these postembryonic Hh functions. Results: We used the hsp70l promoter to generated three heat-shock–inducible transgenic lines that activate Hh signaling and one line that represses Hh signaling. Heat-shock activation of these transgenes appropriately recapitulates early embryonic loss or gain of Hh function phenotypes. Hh signaling remains activated 24 hr after heat shock in the Tg(hsp70l:shha-EGFP) and Tg(hsp70l:dnPKA-BGFP) lines, while a single heat shock of the Tg(hsp70l:gli1-EGFP) or Tg(hsp70l:gli2aDR-EGFP) lines results in a 6- to 12-hr pulse of Hh signal activation or inactivation, respectively. Using both in situ hybridization and quantitative polymerase chain reaction, we show that these lines can be used to manipulate Hh signaling through larval and juvenile stages. A ptch2 promoter element was used to generate new reporter lines that allow clear visualization of Hh responding cells throughout the life cycle, including graded Hh responses in the embryonic central nervous system. Conclusions: These zebrafish transgenic lines provide important new experimental tools to study the embryonic and postembryonic roles of Hh signaling. Developmental Dynamics 242:529–539, 2013. © 2013 Wiley Periodicals, Inc.
- Top of page
- RESULTS AND DISCUSSION
- EXPERIMENTAL PROCEDURES
Hedgehog (Hh)/Gli signaling is required for tissue patterning, cell differentiation, and growth during embryogenesis, with Hh ligands functioning as morphogens, mitogens, or cell survival factors to help induce cell fates and pattern the embryo (Hui and Angers, 2011; Ingham et al., 2011) These functions are maintained throughout the life cycle in select tissues and include the regulation of adult neurogenesis (Jacob and Briscoe, 2003; Tannahill et al., 2005). Mutations in Hh signaling underlie a range of human birth defects, including holoprosencephaly and polydactyly (Hui and Angers, 2011). Misregulation of Hh signaling at postembryonic stages and in the adult is associated with a large number of human cancers including medulloblastoma, glioma, basal cell carcinoma (BCC) (Stecca and Ruiz, 2010). Inappropriate activation of Hh signaling is involved in multiple tumorigenic processes including proliferation, metastasis, and relapse (Stecca and Ruiz, 2010). Hh signaling has now been implicated in neural regeneration following spinal cord injury in fish (Reimer et al., 2009; Kuscha et al., 2012), further underscoring the importance of understanding the mechanisms underlying these multiple postembryonic roles for Hh signaling.
A variety of methods have been developed that provide temporal and/or spatial control of gene expression. These experimental tools include the GAL4/UAS transcription regulatory system from yeast (Lewandoski, 2001; Halpern et al., 2008), site specific recombination-based systems for transgene insertion, deletion, or activation (e.g., Feil, 2007; Bischof and Basler, 2008; Hans et al., 2009) and tetracycline-inducible transcriptional activation systems (e.g., Huang et al., 2005). Heat-shock–inducible promoters that allow transgene activation following acute heat shock have been well characterized. The hsp70l gene encodes a chaperonin that functions to prevent the accumulation of misfolded proteins, and the hsp70l promoter has been used to manipulate gene expression throughout the zebrafish life cycle (Shin et al., 2007; Shoji and Sato-Maeda, 2008; Kaslin et al., 2009).
We have taken advantage of the hsp70l promoter to generate four transgenic zebrafish lines that allow the conditional activation or inactivation of Hh signaling at critical steps in the Hh signal transduction pathway. Hh signaling can be activated extracellularly by induction of a transgene encoding the Sonic Hh (Shh) ligand, intracellularly by induction of a transgene encoding a dominant-negative (dn) form of the inhibitory kinase protein kinase A (PKA) (dnPKA) (Hammerschmidt et al., 1996; Ungar and Moon, 1996), or directly at the transcriptional level by activation of a transgene encoding the Gli1 transcription factor (Karlstrom et al., 2003). Conversely, Hh signaling can be efficiently blocked at the transcriptional level by induction of a transgene encoding a truncated Gli2a transcription factor that acts as a potent dominant transcriptional repressor (Gli2DR) (Karlstrom et al., 2003). All four transgenic constructs also encode the green fluorescent protein (GFP) so that transgene activation can be easily visualized. Here we demonstrate that global heat shock can be used to effectively manipulate Hh/Gli signaling in transgenic zebrafish from embryonic to juvenile stages. We also describe two new lines that sensitively report graded Hh signaling in responding cells. These lines provide important new experimental tools for the study of the embryonic and postembryonic roles of Hh/Gli signaling, including the regulation of stem cell proliferation and differentiation, as well as tumorigenesis.
RESULTS AND DISCUSSION
- Top of page
- RESULTS AND DISCUSSION
- EXPERIMENTAL PROCEDURES
Heat-Shock–Regulated Hh/Gli Transgenic Lines
We have generated four transgenic constructs to allow the conditional manipulation of Hh/Gli signaling at different levels of the Shh signaling cascade (Fig. 1A). All transgenic constructs contain the zebrafish hsp70l promoter (Halloran et al., 2000) as well as sequences needed for Tol2-mediated transgenesis (Kawakami and Shima, 1999). For extracellular activation of the pathway, we generated an hsp70l:shha-EGFP transgene construct. We fused EGFP to the C-terminus of the full-length shha coding region. Shh proteins are post-translationally cleaved and modified to create a secreted N-terminal fragment (Shh-N) and a C-terminal fragment that remains in the cell (reviewed in Ingham et al., 2011). This fusion construct is thus designed to allow proper processing and secretion of an unlabeled Shh-N fragment, with the GFP containing C-terminal fragment reporting transgene induction in Shh producing cells. Similar heat-shock–mediated activation of Shh signaling was recently reported using a hsp:GAL4/UAS-Shh system (Balczerski et al., 2012). To cell-autonomously activate the Shh signaling pathway, we generated two additional lines. One uses the dominant-negative form of protein kinase A (dnPKA), which prevents the PKA-dependent processing of Gli transcription factors into transcriptional repressor forms (Epstein et al., 1996). dnPKA is an effective activator of Hh signaling, with overexpression in zebrafish embryos reproducing Shh over-expression defects (Concordet et al., 1996; Barresi et al., 2000). dnPKA could potentially affect other signaling systems, including the Wnt signaling pathway (Hino et al., 2005), although one study showed no effect of dnPKA on Wnt signaling in vitro (Liu and Habener, 2008). The second encodes full-length Gli1, which functions as a transcriptional activator and is similar to the line reported by Huang and Schier (2009). Finally, to inactivate Hh signaling at the transcriptional level, we took advantage of the dominant repressor form of Gli2a encoded by the zebrafish you-too (yot) mutants (Karlstrom et al., 1999, 2003). We used a luciferase reporter assay to verify the repressor activity of the Gli2DR-GFP fusion protein (Fig. 1B). Both the Gli2DR protein and the Gli2DR-GFP fusion protein efficiently blocked Gli1-induced luciferase activity of an 8x Gli Binding Site (GBS) luciferase reporter construct (Sasaki et al., 1997).
Transgenesis was done as previously described (Kikuta and Kawakami, 2009; Suster et al., 2009) and founders were identified by heat-shocking progeny and screening for GFP fluorescence. Lines were validated by examining the effects of heat shock on ptch2 expression by in situ hybridization and real-time PCR (Figs. 2, 3). All four lines have been through at least five generations of outcrossing without observable changes in transgene efficacy. For convenience, these four lines will subsequently be referred to as hsp:shh, hsp:dnPKA, hsp:gli1, and hsp:gli2DR.
To characterize the timing and location of transgene activation following heat-shock embryos derived from crosses between identified transgenic carriers were heat shocked (hs) at 20 hours postfertilization (hpf) for 1 hr at 37°C and monitored for GFP fluorescence (Fig. 1C–F). In all lines, GFP fluorescence was visible 2 to 3 hr after heat shock when observed using a fluorescent dissecting microscope, and reached maximum intensity within approximately 4 hr. GFP fluorescence was detectable 30 min to 1 hr earlier using a confocal microscope. Confocal analysis revealed GFP fluorescence predominantly in the cytoplasm of cells in the hsp:shh, hsp:dnPKA, and hsp:gli1 lines (Fig. 1C–E), while GFP labeling was restricted to nuclei in hsp:gli2DR transgenic fish (Fig. 1F), consistent with this truncated gli protein acting as dominant repressor of transcription in the nucleus.
We next tested whether these transgenic lines appropriately alter Hh signaling in vivo by heat-shocking embryos from each transgenic line at the tail bud/one-somite stage (10 hpf), a time when Hh signaling is required for zebrafish embryonic patterning (Bergeron et al., 2011; Huang et al., 2012). Heat shock of hsp:shh, hsp:dnPKA, and hsp:gli1 transgenic embryos at 10 hpf led to variable defects associated with a gain of Hh function, including visible somite, brain, and eye defects (Fig. 1H–J) similar to those observed in embryos injected with shh mRNA (Fig. 1M and Hammerschmidt et al., 1996). Activation of the shh-GFP transgene gave the most prominent defects, with the hsp:gli1 line producing milder defects, similar to a previously described hsp:GFP-gli1 line (Huang and Schier, 2009). These data indicate that heat-shock–induced activation of all three transgenes results in up-regulation of Hh signaling, albeit to different degrees. Differences seen between lines could be due to the variations in levels of Hh signal activation, the differential timing required for each line to activate Hh signaling, the stability and the half-life of the fusion proteins, or the different functions of each protein in the Hh pathway.
Heat-shock–induced expression of the gli2DR-GFP transgene during early development (at 10 hpf) produced defects similar to those seen in the yot(gli2DR) mutant (Karlstrom et al., 1996). Visible defects include U-shaped somites, ventrally misplaced but well-formed eyes, and an ectopic lens at the midline (Fig. 1K,L). These embryos also had posterior tail defects not seen in yot mutants (see Fig. 2). These tail defects may be due to the broad expression of the Gli2DR protein in all cells of the embryo (as opposed to being restricted to the gli2 expression domains in yot), the timing of transgene activation, and/or because the gli2DR protein is expressed at higher levels than in mutant animals. Overall, these results suggest that the Gli2DR-EGFP fusion protein effectively blocks Gli-mediated Hh signaling in vivo as observed in vitro (Fig. 1K).
Duration and Relative Magnitude of Hh Signal Manipulation
To determine how activation of these transgenes affects Hh signaling, we heat-shocked embryos at 24 hpf and monitored expression of the Hh transcriptional target gene ptch2 (formerly ptc1 in zebrafish) by in situ hybridization (Fig. 2A–J) and real-time PCR (Fig. 2P). For all three activator lines (hsp:shh, hsp:dnPKA, and hsp:gli1), ptch2 expression was noticeably increased 3 hr after the onset of heat shock, with high levels of ptch2 gene expression seen by 5–12 hr post heat shock (Fig. 2B,D,E,P). ptch2 expression levels gradually decreased over the next 12 hr but some ectopic expression remained 19–24 hr post heat shock (data not shown). Robust ptch2 expression was seen throughout the somites of all lines (Fig. 2B,D,E). In the central nervous system (CNS), ptch2 expression was expanded dorsally in the spinal cord and brain, but was not seen in all cells (Fig. 2G–J), similar to what was reported using a hsp:GAL4/ UAS:shha Shh overexpression system (Balczerski et al., 2012). Because all three activator transgenes were expressed robustly throughout the CNS following heat shock at 20 (Fig. 1) and 24 hpf (data not shown), this may reflect either that activation levels are sub-threshold for ptch2 induction or that a subset of CNS cells are not competent to respond to Hh signaling after 24 hpf. Gli1–GFP activation resulted in more punctate labeling in the dorsal brain (Fig. 2J), which may indicate higher levels of Hh signal activation but in fewer cells.
Heat-shock–induced activation of the gli2DR-GFP transgene resulted in near complete loss of ptch2 expression 5 hr after heat shock in the spinal cord, somites, and brain (Fig. 2C). Nine hours after heat-shock treatment, ptch2 expression had recovered in the spinal cord, with ectopic dorsal expression in the spinal cord (Fig. 2F). This dorsal expression in the recovery period is likely due to the loss of the Ptch2 “boundary effect,” in which ventrally expressed Ptch proteins bind Shh and thus restrict its diffusion (reviewed in Goodrich et al., 1997; Ribes and Briscoe, 2009). The loss of Ptch2 protein following heat shock would allow the Shh protein to diffuse dorsally and activate ptch2 gene expression in more dorsal regions until high levels of Ptch2 are re-established ventrally.
To more precisely determine the time between heat-shock–induced transgene activation and changes in functional Hh signaling levels, we took advantage of the clocklike addition of new somites in the trunk during the segmentation period (Kimmel et al., 1995), and the fact that Hh signaling is necessary and sufficient to induce slow muscle fiber differentiation during this period (Karlstrom et al., 1999; Barresi et al., 2000). hsp:gli2DR and hsp:shh embryos were heat-shocked for 1 hr at the 10 somite (S) stage (14 hpf) and slow muscle fibers were examined at 38 hpf using the F59 antibody (Devoto et al., 1996). During this period, somites form at a rate of approximately two somites per hour. In both the hsp:shh and hsp:gli2DR lines, major slow muscle fiber defects were present starting at somite 16, representing a delay of approximately 3 hr from the onset of heat shock (Fig. 2L,O). Overexpression of Shh in the hsp:shh line resulted in increased slow muscle fiber numbers starting at somite 16–17 (Fig. 2L). Ectopic fibers were seen in all somites after somite 17, indicating that high levels of Hh signaling persisted until the end of somitogenesis at approximately 26 hpf, or for over 9 hr of developmental time. More subtle slow fiber and somite boundary defects were seen in both the hsp:gli1 and hsp:dnPKA lines with similar timing, starting at somite 16 and persisting through all remaining somites (Fig. 2M,N). In contrast, loss of Hh signaling was more temporally restricted in the hsp:gli2DR line, with slow muscle fibers being affected in somite 16 but beginning to reappear in somite 22 (Fig. 2O). This reveals that a single heat shock of the hsp:gli2DR line lowers Hh signaling to a level below the threshold needed for slow fiber differentiation for approximately 3 hr.
We next used quantitative real-time polymerase chain reaction (qPCR) to measure the effects of transgene activation on Hh signaling levels. Heat shock of the three activator lines induced ptch2 expression more than three-fold, with distinct kinetics for each line (Fig. 2P). ptch2 expression steadily increased up to 24 hr after the completion of a 1-hr heat shock in the hsp:shh and hsp:dnPKA lines, while in the hsp:gli1 line ptch2 levels peaked ∼5-hr post heat shock and then returned to wild-type levels by 24 hr. In the hsp:gli2DR line, ptch2 levels were reduced to 30% of wild-type levels within 4 hr of heat shock, remained low through 13 hr, and then returned to wild-type levels by 24-hr post heat shock. The more rapid and transient effects seen in the hsp:gli1 and hsp:gli2DR lines may reflect the fact that these transgenes encode the Hh-responsive transcription factors themselves, which can immediately act on target gene transcription. Furthermore, a rapid turnover of the Gli-GFP fusion proteins might be expected for highly regulated transcription factors.
Conditional Hh Signal Manipulation at Postembryonic Stages
We next assessed the utility of our transgenic lines to manipulate Hh/Gli signaling at larval and juvenile stages. We heat shocked 1-week-old larvae or 1-month-old juvenile transgenic fish at 37°C for 1 hr and examined the expression of GFP and ptch2 using in situ hybridization (ISH) and quantitative real-time PCR (qPCR). As seen at embryonic stages, heat-shock activation of the gli2DR-GFP transgene in 7-dpf larvae resulted in ptch2 down regulation within 6 hr. ISH labeling revealed a clear reduction of expression throughout the fish and qPCR analysis indicated a 70% reduction in ptch2 mRNA (Fig. 3A). In 1-month-old juvenile fish, ptch2 expression was visibly reduced when assayed by ISH and was reduced by 30% when assayed by qPCR (Fig. 3A). GFP mRNA was induced approximately five- and two-fold in 1-week larvae and 1-month juveniles, respectively, relative to a non–heat-shocked transgenic fish.
The hsp:gli2DR repressor line remained effective in reducing ptch2 expression through larval and juvenile stages (Fig. 3A) while the activator lines became less effective in inducing ptch2 expression with increasing age. The hsp:dnPKA and hsp:gli1 lines only weakly affected Hh signaling as determine by ISH analyses starting at approximately 4 dpf (data not shown). The hsp:shh line continued to be effective at increasing Hh signaling through larval and adult stages, with a double heat-shock paradigm further increasing ptch2 expression (Fig. 3B, data not shown). We thus focused our analysis on the hsp:shh activator line at 1-week and later stages (Fig. 3B). Double heat shock of the hsp:shh line led to 1.8- and 1.6-fold increases in ptch2 expression in 7-dpf larvae and 1-month juveniles, respectively (Fig. 3B). ISH revealed increased ptch2 expression mainly in its normal expression domain around the ventricles, with a small region of ectopic ptch2 expression seen in the anterior tectum of 1-week larvae (Fig. 3B, arrows). Because transgene expression is clearly activated throughout the CNS at these ages (Fig. 3B inset), this suggests either that Hh levels are not elevated sufficiently to induce broad ectopic ptch2 expression at these ages, or that only a subset of CNS cells remain competent to express ptch2. For the hsp:shh line, qPCR analysis revealed that EGFP mRNA expression was induced 15- and 25-fold in larvae and juveniles, respectively (Fig 3B).
Several lines of evidence suggest that any “leakiness” of these HSP70l-driven transgenes is not sufficient to disrupt Hh signaling, as has been observed by others (Hans et al., 2009). First, both homozygous and heterozygous transgene carriers are viable and fertile, indicating any leaky transgene expression is insufficient to cause developmental defects associated with altered Hh signaling. Second, in the absence of heat shock, no GFP expression is seen in any tissue, with the exception of the lens in the hsp:dnPKA and hsp:gli2DR lines, a site of normal HSP70l gene activation (Blechinger et al., 2002). Third, ptch2 expression was unaltered in non–heat-shocked transgenic embryos derived from any of the lines at 24 hpf, 7 days, and at 1 month (Fig. 3, data not shown).
New Hh Signaling Reporter Lines
To better visualize cells that respond to Hh signaling we generated Hh reporter lines that express GFP under the control of a 900 bp zebrafish ptch2 promoter fragment containing an additional Gli recognition sequence (Kinzler and Vogelstein, 1990). We generated two independent transgenic lines that express either cytoplasmically localized EGFP (Fig. 4A) or EGFP containing a nuclear localization signal (nlsEGFP) (Fig. 4B–D). Both lines express GFP in all tissues known to express ptch2 mRNA (Fig. 4A–H), including the floor plate of the spinal cord, brain, and somites. This labeling is similar to but more intense than that seen in a line generated using an eight Gli Binding Site-repeat/minimal lens crystallin promoter containing construct (Schwend et al., 2010). The line encoding cytoplasmic GFP (Tg(GBS-ptch2:EGFP)) showed inappropriate expression in the otic placode (Fig. 4A), possibly due to a transgene position effect. The nuclear GFP expressing line (Tg(GBS-ptch2:nlsEGFP)) had no inappropriate expression and was found to be significantly brighter than the EGFP line. This line was more sensitive in reporting Hh signaling levels in vivo and accurately reported the ventral to dorsal gradient of Shh signaling levels (Fig. 4D). Transgene expression from this promoter was drastically reduced when Hh signaling was blocked using the small molecule inhibitor cyclopamine (Fig. 4J–L) and was up-regulated when Hh signaling levels were artificially increased by the injection of shh mRNA (Fig. 4M), indicating these transgenes are appropriately regulated by changes in Hh signaling levels. Both lines continue to express GFP in known Hh responsive regions through larval (Fig. 4N) and adult stages (Fig. 4O).
In conclusion, we have characterized a new set of zebrafish transgenic lines that allow the temporal manipulation and visualization of Hh signaling at embryonic, larval, and juvenile stages. New reporter lines accurately and sensitively mark Hh responding cells at all stages of the life cycle. Activation of Hh pathway components by heat-shock recapitulates known loss or gain of function Hh/Gli signaling phenotypes and remains effective into early adult stages, but becomes less robust with age, perhaps due to changes in heat-shock promoter activation and/or changes in tissue responsiveness (Murtha and Keller, 2003). Region-specific manipulation of Hh signaling using these lines is now possible using a micro-scale heating device (Hardy et al., 2007; Placinta et al., 2009). Two new Hh reporter lines provide a highly sensitive read-out of Hh signal activity throughout the life cycle. Together, these transgenic lines provide important new tools for the study of Hh/Gli signaling throughout embryonic and postembryonic stages, periods associated with major tissue growth as well as continued cell differentiation and tissue patterning. Detailed analyses of the mechanisms by which Hh/Gli regulates postembryonic cell proliferation, differentiation, and survival are particularly important given the role this signaling system plays in somatic stem cell regulation, tissue regeneration, and cancer.
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- RESULTS AND DISCUSSION
- EXPERIMENTAL PROCEDURES
The hsp70l:shha-EGFP and hsp70l:gli1-EGFP transgenes was constructed using multisite Gateway technology (Invitrogen) and Gateway compatible tol2 kits generously supplied by the Chien and Lawson Labs (Kwan et al., 2007; Villefranc et al., 2007). Full-length shha or gli1 encoding DNA was amplified with attB1 and attB2 site containing PCR primers (Table 1) and a middle entry clone (pMEshha or pMEgli1) was created by BP recombination of the PCR product with the pDONR221 vector. To make the expression vectors, three fragment LR recombination reactions were performed using the pMEshh or pMEgli1, p5Ehsp70l, p3EEGFP, and pDestTol2pA fragments. To generate the hedgehog signaling reporter lines, a 0.9 kb ptch2 promoter region was amplified from zebrafish genomic DNA using primers that included an additional Gli Binding Site (GBS, GACCACCCA): attB4.GBS.ptch2.Fw and attB1.ptch2.Rv. This amplicon was then used in a BP reaction with the pDONRP4-P1R plasmid to create the ptch2 entry clone. The final expression clone was made using the LR reaction with the ptch2 entry clone and either the pME-EGFP (cytoplasmic EGFP) or pME-nlsEGFP (nuclear EGFP) plasmids (Kwan et al., 2007) and the pTolDestR4R2pA plasmid (Villefranc et al., 2007). The hsp70l:gli2DR-EGFP and hsp70l:dnPKA-BGFP transgenes were constructed using restriction fragment-based sub-cloning methods starting with the truncated Gli2DR transcription factor sequences encoded by the yotty119 mutant allele (Karlstrom et al., 1999) or the dnPKA sequence from (Hammerschmidt et al., 1996). These sequences were then combined with hsp70l-EGFP (Halloran et al., 2000) into the pTol2000 vector (Kawakami, 2007; Kwan et al., 2007). Gli activator and repressor activity was assayed using a luciferase reporter assay as in Tyurina et al. (2005) using the C17 neural cell line (Snyder et al., 1992).
Fish Strains and Generation of Transgenic Lines
Wild-type and transgenic fish are maintained in the Karlstrom Lab Zebrafish Facility at the University of Massachusetts, Amherst. The embryos are incubated at 28.5°C and staged as described (Kimmel et al., 1995). The wild-type lines used are TL/GOL and WIK. Transgenic lines were generated as in (Kikuta and Kawakami, 2009; Suster et al., 2009). Briefly, 25 pg of purified plasmid DNA was injected to one- to two-cell zebrafish embryos with 25 pg of transposase RNA. Injected fish (potentially mosaic carriers) were raised to adulthood and out-crossed to wild-type individuals to identify potential founder fish by examining 24 hpf embryos for GFP fluorescence 4 hr after a 1 hr heat shock at 37°C. Founders were then outcrossed again and the progeny were raised to adulthood. These adults (potential stable transgenic fish) were screened for the presence of the GFP(EGFP or BGFP) transgene using PCR-based genotyping and DNA isolated from fin clips (see Table 1 for primer sequences). Three founder fish (F0) were identified for the Tg(hsp70l:shha-EGFP) line, two for the Tg(hsp70l:gli2DR-EGFP) and Tg(hsp70l:dnPKA-BGFP) lines, and one for the Tg(hsp70l:gli1-EGFP) line. For convenience, these lines are abbreviated as hsp:shh, hsp:gli2DR, hsp:dnPKA, and hsp:gli1, respectively (see Fig. 1). Transgenic lines were compared for GFP fluorescence following heat shock. While the different founder lines behaved similarly, one founder from the hsp:shh line showed significantly brighter GFP fluorescence, and this line was used for larval and juvenile experiments. Hh reporter lines were generated similarly, with plasmid injected fish being grown to adulthood and out crossed to wild-type fish. The offspring were then screened under a fluorescent dissection microscope for GFP expression between 1 and 4 dpf. GFP expressing larvae were raised to establish stable transgenic lines. One founder was identified for the GBS-ptch2:EGFP line, and four founders were found for the GBS-ptch2:nlsEGFP line. The four different GBS-ptch2:nlsEGFP lines show same expression pattern, but with different GFP intensities. The brightest line was chosen for all further studies. Lines/alleles have been registered with zfin.org as follows: Tg(GBS-ptch2:EGFP)umz23, Tg(GBS-ptch2:nlsEGFP)umz24, Tg(hsp70l:shha-EGFP)umz30, Tg(hsp70l:gli1-EGFP)umz31, Tg(hsp70l:dnPKA-BGFP)umz32, Tg(hsp70l:gli2aDR-EGFP)umz33.
Heat Shock and Drug Treatment
Staged embryos and larvae (up to 7 dpf) were placed into a 50-ml conical tube containing 25 ml of embryo raising medium (Westerfield, 2000) and the tube was then placed in a 37°C water bath for 1 hr. After heat shock, fish were returned to Petri dishes and placed at 28.5°C. For 1-month juveniles, a beaker containing 500 ml of system water was preheated to 37°C in a water bath. Fish were netted and transferred directly into the beaker for 1 hr. Double heat shocks were for 1 hr (37°) at t = 0 and t = 13 hr. After heat shock, 100 ml of room temperature system water was added to the beaker and fish were placed at 28.5°C. GFP fluorescence was visualized using a Leica MZ 16FA fluorescence dissecting scope. To validate the appropriate response to Hh signal manipulation, Tg(GBS-ptch2:EGFP) or Tg(GBS-ptch2:nlsEGFP) embryos were (1) incubated in 100 μM cyclopamine (LC Labs) from 6 to 24 hpf to block Hh signaling as in (Sbrogna et al., 2003) or (2) injected with 100 pg shha mRNA at the one- to two-cell stage to activate Hh signaling. Embryos were assayed either for GFP fluorescence or for ptch2 expression by ISH at 24 hpf. Confocal imaging was done on a Zeiss LSM-700 confocal microscope.
In Situ Hybridization and Immunohistochemistry
Fish were fixed in 4% paraformaldehyde overnight at 4°C, dehydrated in 100% methanol, and stored at −20°C. Whole mount ISH was performed on fish up to 4 dpf as previously described (Karlstrom et al., 1999). For larval and juvenile stages, 2–10 fixed fish per experimental condition were rinsed with PBS, placed in embedding media (1.5% agar, 5% sucrose), and then immersed in 30% sucrose solution overnight at 4°C. Embedded samples were sectioned (20 μm) using a Leica CM 1950 cryostat. Immunohistochemistry was performed as previously described (Karlstrom et al., 1999). Primary antibodies used were rabbit anti-GFP (1:400, Invitrogen) and the F59 monoclonal antibody that recognizes the myosin heavy chain to label slow adaxial muscle cells (1:10, Developmental Studies Hybridoma Bank), and mouse IgG2b anti-HuC/D (1:200, Invitrogen). Secondary antibodies used were goat anti-rabbit alexa 488, goat anti-mouse alexa 546, and goat anti-mouse IgG2b Alexa 546 (1:800, Invitrogen).
Quantitative Real-Time PCR (qPCR) and Statistical Analysis
Six replicates were collected for each experimental condition. RNA was purified from homogenized whole fish using Trizol (Invitrogen). Five to 10 embryos, 3–6 larvae, or 1 juvenile were used per replicate. cDNA was synthesized from 50–100 ng total RNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The MxPro3000P RT-PCR system (Stratagene) was used to determine relative gene expression levels. A total reaction volume of 10 μl contained 1 μl cDNA template, 5 μl FastStart Universal SYBR Green Master Kit (Roche), 400 nM forward and reverse primers (Table 2). RT-PCR conditions were as follows: 1 cycle of 95°C for 15 min; 40 cycles each of 95°C for 15 sec, 60°C for 30 sec, 72°C for 30 sec. All values were normalized to ef1a mRNA levels. qPCR data were analyzed using the ΔΔCt method (Livak and Schmittgen, 2001; Pfaffl, 2001). Relative gene expression levels are compared with heat-shocked wild-type (Fig. 2) or non–heat-shocked transgenic controls (Fig. 3). All analyses were conducted using GraphPad Prism software (San Diego, CA). Comparisons between non–heat-shocked and heat-shocked groups were analyzed by Student's t test.
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- RESULTS AND DISCUSSION
- EXPERIMENTAL PROCEDURES
The authors thank Judy Bennett for fish care and the members of the Karlstrom lab for help proofing the manuscript. Special thanks go to Nathan Lawson and the late Chi-Bin Chien for their generosity in sharing tol2Kit cloning constructs and to Mary Halloran for sharing hsp70l promoter constructs. R.O.K. was funded by the NIH.
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- RESULTS AND DISCUSSION
- EXPERIMENTAL PROCEDURES
- 2012. Analysis of sphingosine-1-phosphate signaling mutants reveals endodermal requirements for the growth but not dorsoventral patterning of jaw skeletal precursors. Dev Biol 362:230–241. , , , , , .
- 2000. The zebrafish slow-muscle-omitted gene product is required for Hedgehog signal transduction and the development of slow muscle identity. Development 127:2189–2199. , , .
- 2011. Brother of cdo (umleitung) is cell-autonomously required for Hedgehog-mediated ventral CNS patterning in the zebrafish. Development 138:75–85. , , , , .
- 2008. Recombinases and their use in gene activation, gene inactivation, and transgenesis. Methods Mol Biol 420:175–195. , .
- 2002. The heat-inducible zebrafish hsp70 gene is expressed during normal lens development under non-stress conditions. Mech Dev 112:213–215. , , , , , .
- 1996. Spatial regulation of a zebrafish patched homologue reflects the roles of sonic hedgehog and protein kinase A in neural tube and somite patterning. Development 122:2835–2846. , , , , , , .
- 1996. Identification of separate slow and fast muscle precursor cells in vivo, prior to somite formation. Development 122:3371–3380. , , , .
- 1996. Antagonizing cAMP-dependent protein kinase A in the dorsal CNS activates a conserved Sonic hedgehog signaling pathway. Development 122:2885–2894. , , , .
- 2007. Conditional somatic mutagenesis in the mouse using site-specific recombinases. Handb Exp Pharmacol 3–28. .
- 1997. Altered neural cell fates and medulloblastoma in mouse patched mutants. Science 277:1109–1113. , , , .
- 2000. Laser-induced gene expression in specific cells of transgenic zebrafish. Development 127:1953–1960. , , , , , , , .
- 2008. Gal4/UAS transgenic tools and their application to zebrafish. Zebrafish 5:97–110. , , , , , .
- 1996. Protein kinase A is a common negative regulator of Hedgehog signaling in the vertebrate embryo. Genes Dev 10:647–658. , , .
- 2009. Temporally-controlled site-specific recombination in zebrafish. PLoS ONE 4:e4640. , , , .
- 2007. Focal gene misexpression in zebrafish embryos induced by local heat shock using a modified soldering iron. Dev Dyn 236:3071–3076. , , .
- 2005. Phosphorylation of beta-catenin by cyclic AMP-dependent protein kinase stabilizes beta-catenin through inhibition of its ubiquitination. Mol Cell Biol 25:9063–9072. , , , .
- 2005. Conditional expression of a myocardium-specific transgene in zebrafish transgenic lines. Dev Dyn 233:1294–1303. , , , , , , .
- 2009. Dampened Hedgehog signaling but normal Wnt signaling in zebrafish without cilia. Development 136:3089–3098. , .
- 2012. Attenuation of notch and hedgehog signaling is required for fate specification in the spinal cord. PLoS Genet 8:e1002762. , , , .
- 2011. Gli proteins in development and disease. Annu Rev Cell Dev Biol 27:513–537. , .
- 1998. The teratogenic Veratrum alkaloid cyclopamine inhibits sonic hedgehog signal transduction. Development 125:3553–3562. , , , .
- 2011. Mechanisms and functions of Hedgehog signalling across the metazoa. Nat Rev Genet 12:393–406. , , .
- 2003. Gli proteins and the control of spinal-cord patterning. EMBO Rep 4:761–765. , .
- 1999. Comparative synteny cloning of zebrafish you-too: mutations in the Hedgehog target gli2 affect ventral forebrain patterning. Genes Dev 13:388–393. , , .
- 1996. Zebrafish mutations affecting retinotectal axon pathfinding. Development 123:427–438. , , , , , , , , , , , , , , .
- 2003. Genetic analysis of zebrafish gli1 and gli2 reveals divergent requirements for gli genes in vertebrate development. Development 130:1549–1564. , , , , , , .
- 2009. Stem cells in the adult zebrafish cerebellum: initiation and maintenance of a novel stem cell niche. J Neurosci 29:6142–6153. , , , , , .
- 2007. Tol2: a versatile gene transfer vector in vertebrates. Genome Biol 8(suppl 1):S7. .
- 1999. Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio. Gene 240:239–244. , .
- 2009. Transient and stable transgenesis using tol2 transposon vectors. Methods Mol Biol 546:69–84. , .
- 1995. Stages of embryonic development of the Zebrafish. Dev Dyn 203:253–310. , , , , .
- 1990. The GLI gene encodes a nuclear protein which binds specific sequences in the human genome. Mol Cell Biol 10:634–642. , .
- 2008. Genetic analysis of the two zebrafish patched homologues identifies novel roles for the hedgehog signaling pathway. BMC Dev Biol 8:15. , , , .
- 2012. Plasticity of tyrosine hydroxylase and serotonergic systems in the regenerating spinal cord of adult zebrafish. J Comp Neurol 520:933–951. , , , .
- 2007. The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev Dyn 236:3088–3099. , , , , , , , , , .
- 2001. Conditional control of gene expression in the mouse. Nat Rev Genet 2:743–755. .
- 2008. Glucagon-like peptide-1 activation of TCF7L2-dependent Wnt signaling enhances pancreatic beta cell proliferation. J Biol Chem 283:8723–8735. , .
- 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408. , .
- 2003. Characterization of the heat shock response in mature zebrafish (Danio rerio). Exp Gerontol 38:683–691. , .
- 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45. .
- 2009. A laser pointer driven microheater for precise local heating and conditional gene regulation in vivo. Microheater driven gene regulation in zebrafish. BMC Dev Biol 9:73. , , , .
- 2009. Sonic hedgehog is a polarized signal for motor neuron regeneration in adult zebrafish. J Neurosci 29:15073–15082. , , , , , , , .
- 2009. Establishing and interpreting graded Sonic Hedgehog signaling during vertebrate neural tube patterning: the role of negative feedback. Cold Spring Harb Perspect Biol 1:a002014. , .
- 1997. A binding site for Gli proteins is essential for HNF-3beta floor plate enhancer activity in transgenics and can respond to Shh in vitro. Development 124:1313–1322. , , , .
- 2003. Multiple roles for Hedgehog signaling in zebrafish pituitary development. Dev Biol 254:19–35. , , .
- 2010. Visualization of Gli activity in craniofacial tissues of hedgehog-pathway reporter transgenic zebrafish. PLoS One 5:e14396. , , .
- 2007. Bmp and Fgf signaling are essential for liver specification in zebrafish. Development 134:2041–2050. , , , , , , , , .
- 2008. Application of heat shock promoter in transgenic zebrafish. Dev Growth Differ 50:401–406. , .
- 1992. Multipotent neural cell lines can engraft and participate in development of mouse cerebellum. Cell 68:33–51. , , , , , .
- 2005. A gradient of Gli activity mediates graded Sonic Hedgehog signaling in the neural tube. Genes Dev 19:626–641. , , , , .
- 2010. Context-dependent regulation of the GLI code in cancer by HEDGEHOG and non-HEDGEHOG signals. J Mol Cell Biol 2:84–95. , .
- 2009. Transgenesis in zebrafish with the tol2 transposon system. Methods Mol Biol 561:41–63. , , , , .
- 2005. Role of morphogens in brain growth. J Neurobiol 64:367–375. , , .
- 2005. Zebrafish Gli3 functions as both an activator and a repressor in Hedgehog signaling. Dev Biol 277:537–556. , , , , , , .
- 1996. Inhibition of protein kinase A phenocopies ectopic expression of hedgehog in the CNS of wild-type and cyclops mutant embryos. Dev Biol 178:186–191. , .
- 2007. Gateway compatible vectors for analysis of gene function in the zebrafish. Dev Dyn 236:3077–3087. , , .
- 2000. The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). Eugene: University of Oregon Press. .