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
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 (). Citrine is flanked by splice acceptor and splice donor sites and thus becomes integrated into the Crmp1 protein as an artificial exon (). Gt(crmp1:Citrine
homozygotes are viable suggesting that the Citrine integration does not impair the function of full length Crmp1.
Figure 1 The Citrine insertion (green) occurred within the first intron (I1) in crmp1 in the Gt(crmp1:citrine)ct130a line and produces a fluorescently tagged full-length Crmp1 protein. The exons (blue boxes) and introns are not drawn to scale. Pink, 5′ (more ...)
1.1. Live Citrine expression in the Gt(crmp1:Citrine)ct130a line
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 (, ). 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 () and also becomes apparent in the epiphysis. Faint signal is also observed in developing spinal cord neurons (asterisks, ).
Figure 2 Crmp1 (green) is expressed in the forebrain at 31 hpf. All embryos were treated with the vital dye Bodipy-TR (red) to act as a contrast against the citrine signal. (A–B) Live confocal images. (A) Confocal z-stack showing Citrine/ Crmp1 (green) (more ...)
Figure 5 In situ hybridization results showing expression of crmp1 (A–B) and citrine (D–E). In wildtype fish crmp1 expression is localized to the nervous system at 24 hpf (A) and 48 hpf (B). (B) Lateral and frontal views at 48 hpf. (C) Confocal (more ...)
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 () at 42 hpf as well as in the larva (). 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 (). Crmp1 expressing cells are observed in closely packed linear arrays in the cerebellum ().
Figure 3 Crmp1 expression in the developing brain in live 42 hpf- 4 day fish. (A) Lateral view at 42 hpf. Crmp1 is expressed in the epiphysis (e) and forebrain regions: telencephalon (t) and diencephalons (d). Expression is also detected in the retina (r). Arrows (more ...)
1.2. Further analysis of Crmp1 expression during zebrafish development compared to neuronal markers
We examined Crmp1 expression and its relationship to neuronal markers HuC/D and Neurofilament (NFM) (). 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 (). One population of Crmp1 expressing cells co-localizes with HuC/D (asterisk, ) whereas the second population of cells is HuC/D negative (arrowheads, and ). For example, in the developing epiphysis, a proportion of Crmp1 positive cells do not express HuC/D (). 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+
(). By 42 hpf, the population of cells co-expressing HuC/D and Crmp1 has expanded in the telencephalon and the epiphysis (). 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 ( and inset). Within the diencephalon, cell tracts or cell extensions can be visualized (arrows, ).
Figure 4 Crmp1 expression (green) compared to neuronal markers HuC/D (A–C) and NFM (D–E) (red). Gt(crmp1:citrine)ct130a heterozygotes were stained with anti-green fluorescent protein to enhance the citrine signal and the nuclear stain (DAPI; blue) (more ...)
By 48 hpf, NFM positive axon fascicles expressing Crmp1 can be seen emanating from the cerebellum (arrowheads, ), although the Crmp1 expressing domain is greater than the NFM expression within the cerebellum (). 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 (). 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, inset).
1.3. In situ expression of citrine in Gt(crmp1:Citrine)ct130a line during development
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
line (). At 48hpf, crmp1
expression correlates well with Crmp1 protein expression observed in the genetrap line (). 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 ().
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.