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Collapsin response mediator proteins (CRMPs) are cytosolic phosphoproteins that are functionally important during vertebrate development. We have generated a zebrafish genetrap line that produces fluorescently tagged Crmp1 protein, which can be dynamically tracked in living fish at subcellular resolution. The results show that Crmp1 is expressed in numerous sites in the developing nervous system. Early expression is apparent in the forebrain, epiphysis, optic tectum and the developing spinal cord. In the larval brain, Crmp1 is expressed in several distinct brain regions, such as the telencephalon, habenula and cerebellum. In addition, it is expressed in the spinal cord in a manner that persists in the larva. The results suggest that this Crmp1 protein trap line offers a powerful tool to track selected neuronal populations at high resolution.
Collapsin response mediator proteins (CRMPs) are a family of cytosolic phosphoproteins proteins thought to play important roles during neural development (CRMP 1–5; previously called TUC, Drp, Ulip and TOAD-64; reviewed by (Charrier et al., 2003; Quinn et al., 1999). CRMP-1 and CRMP-2 are key downstream mediators of the Semaphorin-3A signal transduction pathway involved in axon guidance and neuronal migration (Goshima et al., 1995; Uchida et al., 2005; Yamashita et al., 2007). Analysis of mouse CRMP1 suggests a function during maintenance of long term potentiation and spatial learning and memory (Su et al., 2007).
Recent advances in transgenesis have made the zebrafish a powerful model organism for examining expression and function of developmentally important genes. Here, we describe a zebrafish transgenic line for Crmp1. Generated by a Tol2 transposase mediated recombination (Trinh et al, submitted), we report visualization and dynamic tracking of fluorescently tagged endogenous Crmp1 fusion protein expression in living embryos at subcellular resolution. Previous in situ hybridization analysis has described the distribution of Crmp1-5 mRNA at 16 and 24 hours post fertilization (hpf) (Schweitzer et al., 2005). We extend these findings and report the high-resolution expression pattern of Crmp1 protein in live zebrafish during embryonic and larval development.
From a gene and protein trap screen, in which a Tol2 transposase based vector containing Citrine (a variant of the yellow fluorescent protein) was randomly inserted into the genome, we discovered Gt(crmp1:Citrine)ct130a based on its interesting expression pattern in the developing nervous system. Molecular analysis by 3′ Rapid amplification of cDNA ends using Citrine primers determined genomic integration within the locus of Zebrafish crmp1. The crmp1 gene is comprised of 14 exons based on previous published data (Schweitzer et al., 2005). Our 3′ Rapid amplification of cDNA ends (RACE) results matched the previously published sequence downstream from exon 2 (Schweitzer et al., 2005) (data not shown), with the Citrine insertion occurring in the intronic region located between exons 1 and 2 (Fig. 1). Citrine is flanked by splice acceptor and splice donor sites and thus becomes integrated into the Crmp1 protein as an artificial exon (Fig. 1). Gt(crmp1:Citrine)ct130a homozygotes are viable suggesting that the Citrine integration does not impair the function of full length Crmp1.
As Crmps are highly enriched in the nervous system, we focused on its expression and localization between 1 to 4 days of development, as early born neurons were differentiating and eliciting axons and different regions of the brain were forming.
At 24–31 hours post-fertilization (hpf), high levels of expression were noted in the forebrain regions: the telen- and diencephalons (Fig. 2A, ,5C).5C). This protein expression is consistent with previous in situ hybridization data at this time point (Schweitzer, J et al 2005). Crmp1 expression persists and heightens in the forebrain at 31 hpf (Fig. 2A) and also becomes apparent in the epiphysis. Faint signal is also observed in developing spinal cord neurons (asterisks, Fig. 2B).
Tracking protein expression from the embryo to early larval revealed that, once expressed, Crmp1 appears to be maintained in selected sites, including the epiphysis, forebrain regions and spinal cord (Fig. 3A,B) at 42 hpf as well as in the larva (Fig. 3C). As the brain enlarged and compartmentalized, Crmp1 was observed in selected brain regions, including a portion of the telencephalon, the habenula, the optic tectum, and the cerebellum (Fig. 3C–E). Crmp1 expressing cells are observed in closely packed linear arrays in the cerebellum (Fig. 3E).
We examined Crmp1 expression and its relationship to neuronal markers HuC/D and Neurofilament (NFM) (Fig. 4). HuC/D is expressed by newly born post-mitotic neurons and marks RNA binding proteins of the Elav family (Kim et al., 1996). There are clearly two populations of cells within the telencephalon that express Crmp1 (Fig. 4A–B). One population of Crmp1 expressing cells co-localizes with HuC/D (asterisk, Fig. 4A–B) whereas the second population of cells is HuC/D negative (arrowheads, Fig. 2D and Fig. 4A). For example, in the developing epiphysis, a proportion of Crmp1 positive cells do not express HuC/D (Fig. 4A–B). In addition, a substantial proportion of HuC/D positive neurons fail to express Crmp1 in the optic tectum at 31 hpf, whereas the majority of neurons in the telencephalon are Crmp1+ (Fig. 4A). By 42 hpf, the population of cells co-expressing HuC/D and Crmp1 has expanded in the telencephalon and the epiphysis (Fig. 4B). A ventral view at 42 hpf reveals that unlike in the telencephalon, in the diencephalon there is little to no overlap in Crmp1 and HuC/D expression (Fig. 4C and inset). Within the diencephalon, cell tracts or cell extensions can be visualized (arrows, Fig. 4C).
By 48 hpf, NFM positive axon fascicles expressing Crmp1 can be seen emanating from the cerebellum (arrowheads, Fig. 4D), although the Crmp1 expressing domain is greater than the NFM expression within the cerebellum (Fig. 4D). This is consistent with the proposed role for Sema3a signaling in axon guidance and fasciculation (Kawasaki et al., 2002; Yamashita et al., 2007). In contrast, within the forebrain (asterisk) and optic tectum region (arrow), Crmp1 positive cells do not co-express NFM (Fig. 4E). At 48hpf, Crmp1 expression in the forebrain is remarkably similar to the radial glial marker Glial Fibrillary Acidic Protein (GFAP) (Barresi et al., 2005). It is known that glial bridge formation mediated by Slit-Robo signaling facilitates the correct formation of commissures in the zebrafish forebrain (Barresi et al., 2005). Intriguingly, there is evidence to suggest that Robo signaling modulates Semaphorin signaling during interneuron migration in the forebrain (Hernandez-Miranda et al., 2011). Thus, in a similar manner, Robo-expressing glia in the forebrain may also integrate Semaphorin signaling during commissure formation.
A significant advantage of the Crmp1 transgenic line is that it reflects expression of the endogenous protein that can be visualized at high magnification, revealing the subcellular distribution of the protein. The results reveal that Crmp1 expression is clearly restricted to cell bodies and excluded from the nucleus (arrowheads, Fig. 4B′ inset).
The above results demonstrate that Crmp1 protein is expressed as early as 24 hpf and maintained at later stages. To compare this distribution with that of the crmp1 transcript and of the citrine fusion transcript, we analyzed the expression of crmp1 in wildtype fish at 24 and 48 hpf. The 24 hpf crmp1 expression is consistent with previously published data (Schweitzer, J et al 2005) and overlaps with that of both Citrine protein and transcript expression in the Gt(crmp1:Citrine)ct130a line (Fig. 5). At 48hpf, crmp1 and citrine expression correlates well with Crmp1 protein expression observed in the genetrap line (Fig. 5B and 5D). However, crmp1 expression in the retina in wildtype fish is not as obvious as citrine expression in the genetrap line. At 72 hpf, Citrine mRNA is expressed and matches well with Citrine protein expression (Fig. 5E).
Gt(crmp1:Citrine)ct130a is a useful tool to study the nervous system. This transgenic line reflects the endogenous pattern of Crmp1 expression and the tagged version of the Crmp1 can be used in proteomic studies to identify binding partners in an in vivo relevant manner. By revealing expression of the endogenous protein at subcellular resolution, we show that Crmp1 is present throughout the cytoplasm and axon projections, consistent with its proposed role as a mediator of signal transduction downstream of Semaphorins and possibly other signaling pathways (Goshima et al., 1995; Uchida et al., 2005; Yamashita et al., 2007). Furthermore, the protein distribution largely correlates with that of its mRNA transcripts. The distribution of Crmp1 in subpopulations of neurons in the developing central nervous system of zebrafish is consistent with important functions in neuronal development, synaptic plasticity, and behavior.
Wildtype and transgenic zebrafish lines were maintained according to Institutional Animal Care and Use protocols. Embryos were collected, maintained and staged according to (Kimmel et al., 1995). Gt(crmp1:citrine)ct130a AB/TL F1 were generated by crossing AB founder to TL wildtype.
RNA was extracted from 3 dpf citrine positive fish and a complementary DNA (cDNA) library was generated using random hexamers. 3′ RACE was performed with the following primers: Citrine primer: 5′-GACAACCACTACCTGAGCTACC-3′ and dT15 primer: 5′-GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGTTTTTTTTTTTTTTT-3′. A second round of amplification was performed using dT15 primer and nested Citrine primer-5′: ACATGGTCCTGCTGGAGTTC-3′. A 2.1 kb product was amplified, cloned into pGEMTeasy (Promega) and sequenced.
To generate crmp1 probe the following primers were used: crmp1 forward primer-5′-TGCATGCAGAGAATGGAGACCTGA-3′ and reverse primer containing a T7 promoter site T7-crmp1-R-5′-TAATACGACTCACTATAGGGACTGGACCATCATACATGCCACGA-3′. The 3′ RACE product was used to generate crmp1 probe. To generate citrine anti-sense probe the following primers were used with the reverse primer containing a T7 promoter site; Sp6-citrine-F-5′-GATTTAGGTGACACTATAGATGGTGAGCAAGGGCGAGGA-3′ and T7-citrine-R-5′-TAATACGACTCACTATAGGGCTTGTACAGCTCGTCCATGC-3′. In situ hybridization was carried out according to standard protocols (Jayasena et al., 2008) and detailed protocols are available upon request.
Live Gt (crmp1:citrine)ct130a homozygous fish were soaked in BODIPY® TR methyl ester vital dye (Invitrogen) for 30–60 min at 28 °C to label all cells. Fish of appropriate stage were anesthetized using 0.1% Tricaine (pH 7.2) in egg water (Sigma) and mounted laterally onto imaging moulds (1% agarose in egg water). Confocal z-stack images ranging from 40–100 μM were collected using Zeiss LSM 510 microscope and Zeiss 25x LD LCI Plan-Apochromat NA 0.80 DIC 1mm Korr or Zeiss LD C-Apochromat 40X/ 1.1W Korr UV-VIS-IR objectives. Wholemount live images were collected using an Olympus MVX10 microscope and Zeiss software LSM 510 version 4. Images were processed using ImageJ 1.38x (NIH) and Adobe Photoshop 7. Z-stack projections were generated using LSM Image Examiner version 4.
Immunofluorescent staining was performed as follows. Embryos were manually deyolked with a pulled glass Pasteur pipette and fixed in 4% paraformaldehyde in 0.1M Phosphate buffer at room temperature for 1–2 hours. After several rinses in Phosphate buffer saline containing 0.1% Tween-20 (PBSTw), the embryos/ larvae were Trypsin treated (1mg/ml) at room temperature. Following a 20 minute post-fix in 4% paraformaldehyde in 0.1M Phosphate buffer at room temperature, the embryos/ larvae were blocked in 5% goat serum containing 0.5% Triton-X in PBS (5% GS+ PBSTx). Primary and secondary antibody incubations were performed at 4 °C overnight in 5% GS+ PBSTx. Post antibody washes were performed in PBS containing 0.1% Triton-X. The embryos were mounted as stated above for live confocal imaging. The following antibodies were used in this study: mouse anti-HuC/D (Molecular probes), mouse anti-NFM (Zymed), rabbit-anti-GFP (Molecular probes), goat anti-rabbit Alexa-488 (Molecular probes) and donkey anti-mouse Alexa-594 (Molecular probes).
We wish to thank the following people: Dr. Le. A. Trinh for helpful discussions and technical help, Ho-yin Leung for performing 3′ RACE and Leigh-Anne Fletcher for technical help in the Caltech Centers of Excellence in Genomic Science fish facility. This work is supported by USPHS P50HG004071.
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