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Expression of two insm1-like genes in the developing zebrafish nervous system
Gene Expression Patterns 6 (2006) 711–718 www.elsevier.com/locate/modgep
Expression of two insm1-like genes in the developing zebrafish nervous system Chris M. Lukowski, R. Gary Ritzel, Andrew Jan Waskiewicz
*
Department of Biological Sciences, University of Alberta, CW405 Biological Sciences Building, Edmonton, Alta., Canada T6G 2E9 Received 10 November 2005; received in revised form 22 December 2005; accepted 23 December 2005 Available online 17 February 2006
Abstract Insulinoma associated-1 (INSM1, formerly IA-1) is a Cys2-His2 zinc finger transcription factor sharing conserved regions with Caenorhabditis elegans EGL-46 and Drosophila Nerfin-1. INSM, EGL-46, and Nerfin proteins comprise the EIN family of zinc finger transcription factors. egl-46 and nerfin-1 have been implicated in various aspects of neuronal differentiation including cell fate specification, axon guidance decisions and cell migration. Murine Insm1 has a restricted expression pattern in the developing CNS. We have characterized two zebrafish (Danio rerio) Insm1-like genes, insm1a and insm1b, and analyzed their expression patterns during embryonic development. Zebrafish insm1a and insm1b share an embryonic expression pattern comparable to the proneural deltaA as well as overlapping the neuronal marker elavl3. The expression pattern observed for zebrafish insm1a and insm1b is similar to other EIN homologues. Both zebrafish insm1-like transcripts are also present in a region of the embryo where pancreatic progenitors originate. The expression data along with functional characterization of invertebrate homologues suggest a conserved pathway involving the EIN transcription factors in early neurogenesis. 2006 Elsevier B.V. All rights reserved. Keywords: Zebrafish; Danio rerio; Insulinoma associated-1; INSM1; IA-1; insm1a; insm1b; Neuronal differentiation; CNS; Pancreatic precursor; Neurogenesis
1. Results and discussion Insulinoma-associated 1 (INSM1, formerly IA-1) was originally isolated from a cDNA subtraction library between human insulinoma and glucagonoma tumor cells (Goto et al., 1992). Murine Insm1 has a restricted expression pattern in the embryonic CNS (Breslin et al., 2003) and mRNA is undetectable past 2 weeks post-partum (Xie et al., 2002), suggesting a function in both embryonic and neonatal, but not adult tissues. However, Insm1 mRNA has been detected in human, mouse, and rat tumors of neuroendocrine origin including insulinoma, medulloblastoma, pheochromacytoma, retinoblastoma, pituitary tumor, medullary thyroid carcinoma, and small cell lung carcinoma (Goto et al., 1992; Lan et al., 1993). *
Corresponding author. Tel.: +1 780 492 4403; fax: +1 780 492 9234. E-mail address: [email protected] (A.J. Waskiewicz).
1567-133X/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2005.12.008
IA-1 expression in the rat pancreatic AR42J cell line is increased after conversion to an insulin-positive phenotype and decreased following conversion to an acinar-like phenotype (Zhu et al., 2002), indicating a potential role in islet cell differentiation. Mammalian Insm1 expression data suggests a role in differentiation of both neural and pancreatic precursors. INSM1 possesses an amino-terminal SNAIL/GFI (SNAG) repressor domain and five carboxy-terminal Cys2-His2 zinc fingers (Xie et al., 2002). Its protein structure is similar to the SNAIL, SLUG, and GFI superfamily of transcriptional repressors, a group of proteins that have been implicated in tumor progression. In addition, INSM1 contains similar zinc fingers to the Caenorhabditis elegans Egg Laying defective-46 (EGL-46; Wu et al., 2001) and the three zinc fingers of Drosophila Nervous fingers-1 (Nerfin-1; Stivers et al., 2000). The high degree of sequence similarity among the zinc fingers of EGL-46, Nerfin and INSM
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proteins suggests they recognize a common DNA sequence and has been termed the EIN domain for EGL-46, INSM1 and Nerfin-1 (Stivers et al., 2000). The consensus INSM1 DNA-binding target contains a core GGGG, but other EIN domain proteins have yet to be characterized biochemically (Breslin et al., 2002). Caenorhabditis elegans egl-46 mutants display neuronal defects including abnormal cell migration, misguided axonal outgrowth, changes in cell fate, and extra rounds of cellular divisions in presumptive post-mitotic neurons (Wu et al., 2001). EGL-46 has been implicated in specification of the HSN egg laying motor neurons, the FLP neuron (Wu et al., 2001), and the HOB hook neuron, which is necessary for vulva location (Yu et al., 2003). Dynamic expression of egl-46 during larval development is also seen in ventral cord and head neurons, Q-lineage neuroblasts, PVD neurons, touch cells (Wu et al., 2001) and ray neurons (Yu et al., 2003). The Drosophila EIN homologue nerfin-1 is widely detected in the developing CNS including early ventral cord neuroblasts, ganglion mother cells, sensory organ precursors, and precursors of the compound eye (Stivers et al., 2000). Drosophila embryos deficient for nerfin-1 display defects in axon fasciculation and guidance (Kuzin et al., 2005). The activity of the Insm1 promoter has been analyzed in transgenic mice using a lacZ fusion. b-Galactosidase activity was observed in regions of the forebrain, midbrain, hindbrain, spinal cord, retina, olfactory bulb, and cerebellum (Breslin et al., 2003). Here, we report the molecular cloning of two zebrafish insm1-like cDNAs, insm1a and insm1b. A combination of RT-PCR and RNA in situ hybridization demonstrates that both insm1-like transcripts are expressed in a similar pattern to the proneural marker deltaA and overlap expression of the neuronal marker elavl3. The dynamic expression pattern of insm1a and insm1b in the zebrafish central nervous system is comparable to the expression of C. elegans egl-46, Drosophila nerfin-1 and murine Insm1, making zebrafish an attractive vertebrate model organism to determine whether EIN protein functions have been evolutionarily conserved. 1.1. Cloning of zebrafish insm1a and insm1b A zebrafish embryonic cDNA library was differentially screened for genes involved in early embryogenesis. Of the selected clones, BLAST sequence analysis revealed a cDNA with a high degree of sequence similarity to mammalian Insm1. Examination of the Danio rerio genome revealed a second hypothetical insm1-like gene, which we believe to be the product of the teleost lineage genome duplication as both Tetraodon nigroviridis and Takifugu rubripes also have two insm1-like genes and all other complete or nearly complete vertebrate genomes examined contain a single Insm1 gene. We amplified both putative open reading frames (ORFs) with RT-PCR using primers flanking each hypothetical ORF. Sequence analysis revealed predicted ORFs of 383 (Insm1a) and 453 (Insm1b) amino acids that are 71% identical and 81% similar to each other.
The zebrafish Insm1 proteins also share amino acid sequence conservation with two characterized invertebrate zinc finger transcription factors, Nerfin-1 and EGL-46. Insm1a is 34% identical to Nerfin-1 and 35% identical to EGL-46. Insm1b shares 30% identity with Nerfin-1 and 36% identity with EGL-46. From current genomic sequences, zebrafish insm1a is in a region of conserved synteny to mammalian Insm1 while insm1b is unmapped. We confirmed that zebrafish insm1a and insm1b are intronless, resembling their murine (Xie et al., 2002) and Drosophila (Kuzin et al., 2005) homologues (data not shown). Amino acid alignment of zebrafish Insm1a and Insm1b predicted ORFs to mammalian homologues reveals several highly conserved regions, including an amino-terminal SNAG motif (Grimes et al., 1996), five carboxy-terminal Cys2-His2 zinc fingers and a putative nuclear localization signal (Fig. 1A). A conserved arginine residue has replaced the second histidine of zinc finger 1. The carboxy terminus is also highly conserved amongst vertebrate EIN proteins, indicating a potential functional domain. The putative nuclear localization signal (NLS) we have identified is upstream of zinc finger 1 in a region that is conserved in both vertebrate and invertebrate EIN homologues. For vertebrate Insm1 proteins, the sequences of zinc fingers 1, 2, 3, and 5 are very well conserved while the majority of conservation in zinc finger four surrounds the zinc coordinating cysteine and histidine residues (Fig. 1A). Zebrafish Insm1 proteins have a similar structure to mammalian homologues, which all share a highly conserved EIN domain encompassing the first two zinc fingers (Fig. 1B). The EIN domain is 59% identical and 73% similar in species from nematodes to humans in addition to being 82% identical and 92% similar in identified vertebrate homologues. A common feature of mammalian INSM1 is zinc finger spacing; two pairs are symmetrically positioned around a central zinc finger. Although this feature is more evident in Insm1b than Insm1a, zinc finger spacing in a linear schematic may not accurately reflect the implications on its tertiary structure. Together with EGL-46 and Nerfin-1, vertebrate Insm1 proteins clearly share a conserved domain architecture and considerable sequence conservation. Vertebrates possess a second INSM1-like protein named MLT1 (Tateno et al., 2001), which is fairly similar in amino acid sequence to INSM1, but has a different zinc finger distribution. Phylogenetic analysis of predicted amino acid sequences demonstrate that Insm1a (79% similar) and Insm1b (73% similar) are more closely related to human INSM1 (Fig. 1C) than to human MLT1 (Tateno et al., 2001). Human MLT1 amino acid sequences are 52% similar to INSM1, but the zinc finger distribution is distinct from INSM1, and several unique conserved sequences in mammalian MLT1 are not present in either Insm1a or Insm1b. Analysis of the zebrafish genome revealed a putative mlt1 gene encoding an ORF of 508 amino acids with 63% similarity to human MLT1 and 51% similarity to human INSM1. The hypothetical zebrafish Mlt1 shares
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Fig. 1. Comparison of zebrafish Insm1a and Insm1b to mammalian and invertebrate homologues. (A) Amino acid alignment of predicted zebrafish Insm1a (AAZ94623) and Insm1b (AAZ94624) to human (NP_002187) and mouse (NP_058585) INSM1. Amino acid identity (asterisks) and similarity (periods) are indicated below the aligned sequences. Insm1 is predicted to contain an amino-terminal SNAG motif (blue), a putative nuclear localization sequence (green), and five (numbered) carboxy-terminal Cys2-His2 zinc fingers (red). Zinc coordination residues of each zinc finger are highlighted in red. (B) Protein schematic showing the predicted domain structure of Insm1, Nerfin-1, and EGL-46 proteins. Shown are the amino-terminal SNAG repressor domains (blue), putative nuclear localization signals (green) and carboxy-terminal Cys2-His2 zinc fingers (red). Proteins were drawn to scale and aligned about the highly conserved EIN domain. Zinc fingers are numbered based on homology as in (A) and indicated in invertebrate homologues. Zinc finger 3 of EGL-46 (asterisk) does not share the conservation seen in other EIN homologues. Numbers in brackets indicate the amino acid length. (C) Phylogenetic analysis showing zebrafish Insm1a and Insm1b (highlighted in orange) are more similar to mammalian INSM1 than to another INSM1-like protein, MLT1. We have identified a zebrafish MLT1-like gene from genomic sequences that is more similar to mammalian MLT1 than to INSM1. z, zebrafish; h, human; and m, mouse.
the distinct zinc finger distribution seen in mammalian MLT1 and contains several of the unique sequences mentioned previously. These data further support our hypothesis that zebrafish Insm1a and Insm1b are co-orthologues of mammalian INSM1. 1.2. Developmental expression of insm1a and insm1b Mouse Insm1 expression has previously been determined using a promoter-lacZ fusion transgene (Breslin et al., 2003). Since the visualization of b-galactosidase activity is necessarily an accumulation assay, there is no data precisely measuring the spatiotemporal expression pattern of vertebrate insm1 mRNA during development. Furthermore, the Drosophila EIN homologue has been shown to be regulated post-transcriptionally, consequently a promoter-fusion may not be the most accurate method to ascertain the expression of vertebrate INSM1. Therefore, we have conducted a detailed analysis of vertebrate insm1 spatiotemporal gene regulation in zebrafish. To obtain a measure of insm1a and insm1b transcripts during zebrafish development, we performed semi-quantitative RT-PCR with RNA from a series of embryonic stages (Fig. 2). insm1a
Fig. 2. Reverse transcriptase-PCR analysis of zebrafish insm1a and insm1b during development. Two hundred and fifty nanograms total RNA was used per reaction. Time listed above each lane indicates embryonic age post-fertilization at which the RNA was extracted. Zebrafish neurogenesis begins at tailbud stages (9 hpf) and reaches maximal levels between 24 and 72 hpf, a pattern that is precisely followed by both insm1 transcripts. Neither transcript is expressed in adult tissues, which is consistent with mammalian expression data. The final lane is a no RNA control. Note that insm1a transcripts appear to be maternally inherited as zygotic transcription does not begin until 3 hpf, while insm1b is not.
transcripts appear to be maternally contributed while insm1b transcripts are not, as zygotic transcription in zebrafish does not begin until 3 h post-fertilization (hpf; Mathavan et al., 2005). Both transcripts are upregulated at
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10 hpf, reach peak levels at 48 hpf and are not detectable in adult tissues. Zebrafish neurogenesis begins at early tailbud stages (9 hpf), reaches high levels between 24 and 48 hpf, and progresses past 72 hpf. The timing of peak insm1a and insm1b expression coincides precisely with periods of zebrafish neurogenesis. Mouse insm1 transcripts are seen from embryonic day 10.5 until 2 weeks post-partum (Xie et al., 2002), a time course similar to our data in zebrafish. To determine if transcript levels from RT-PCR analysis are consistent with a hypothesized expression during neurogenesis, we performed RNA in situ hybridization on staged zebrafish embryos ranging from 10 to 72 hpf. To avoid potential cross hybridization of insm1a and insm1b ORF probes, antisense probes corresponding to the 3 0 -untranslated regions (UTRs) of both insm1a and insm1b were used. We also used a probe for the proneural Notch ligand deltaA to indicate where and when neurogenesis is initiated during embryonic development. During the formation of proneural fields, cells express both notch and deltaA. Through the mechanism of lateral inhibition, only subsets of these cells are fated to become neurons, therefore deltaA-expression correlates with proneural domains rather than early born neurons. Sense control probes for insm1a or insm1b 3 0 -UTRs showed no staining after an extended coloring reaction time (data not shown). Expression of insm1a and insm1b is first detected prior to somitogenesis, at late tailbud stages (9.5 hpf) with a pattern overlapping the proneural marker deltaA. At the two somite stage (10 hpf), insm1a is expressed in the presumptive trigeminal ganglia, telencephalon and lateral trunk neurons (Figs. 3A and A 0 ). insm1b is expressed in the presumptive trigeminal ganglia, midbrain, and several types of trunk neurons (Figs. 3B and B 0 ). These regions of insm1 expression are similar to that of deltaA (Figs. 3C and C 0 ), albeit with weaker staining intensity. We noticed a short delay between the regions of deltaA expression and insm1 expression at a slightly later stage, suggesting insm1 may be a downstream target of deltaA or other neurogenic factors. At the 10 somite stage (14 hpf), insm1a and insm1b are expressed in the telencephalon, ventral diencephalon, midbrain, and hindbrain (Figs. 3D and E). Strong insm1b expression is visible in the presumptive trigeminal and cranial ganglia at the 10 somite stage (14 hpf; Fig. 3E). Both insm1 transcripts are present in a group of cells in the ventral endoderm (Fig. 3D 0 ; insm1b not shown), a region that is known to give rise to pancreatic precursors. The disorganized pattern of cells expressing insm1a and insm1b converge as development continues, a pattern similar to insulin expression and the pancreatic markers neuroD, nkx2.2, islet1 and pax6.2 (Fig. 3F 0 ; Biemar et al., 2001; Mavropoulos et al., 2005). insm1a expression in the ventral endoderm is seen very early while insm1b appears later. Together with the fact that human INSM1 was isolated from an insulinoma cDNA library, presumptive pancreatic expression of zebrafish insm1 genes suggests a potential role for vertebrate Insm1 proteins in pancreatic cell differentiation.
At late somitogenesis (24 hpf) both insm1a and insm1b are expressed in the olfactory placode, ventral diencephalon, tegmentum, hindbrain, presumptive pancreas, and a subset of spinal cord neurons (Figs. 3F, F 0 , and I). insm1b expression occupies the most posterior region of the epiphysis, while insm1a is seen throughout this region. Spinal cord expression of insm1b occupies three planes along the dorsoventral axis, presumably the motoneurons, interneurons, and Rohon-beard (Rb) neurons. Spinal cord expression of insm1a is absent from rostral Rb neurons and is restricted to the ventral motoneurons. Expression of deltaA at 24 hpf illustrates the neural fields in the developing embryo (Fig. 3L), and insm1a and insm1b are expressed in a more refined but markedly similar fashion. By late pharyngula stages (48 hpf), the expression of insm1a (Fig. 3G) and insm1b (Fig. 3J) is widespread throughout the CNS, and deltaA (Fig. 3M) also exhibits a similar overall expression pattern. insm1a expression is visible in the otic vesicle and has expanded throughout the retina, whereas insm1b expression was not detectable in the retina at any stage examined. Stripes of insm1 expression are seen along the dorsoventral axis in the hindbrain at 48 hpf, which correspond to a region directly adjacent to rhombomere boundaries. This expression creates a pattern of alternating large and small gaps in insm1 expression that is clearly visible in a lateral view of insm1b expression (Fig. 3J) but less so for insm1a. deltaA expression also exhibits this pattern within the hindbrain. At 48 hpf there is faint staining for insm1a and insm1b along the lateral edge of the hindbrain and this expression is visible starting at 18 hpf (data not shown). At early larval stages (72 hpf) insm1a expression is confined to a specific cell layer in the retina (Fig. 3H), however we observed a moderate degree of variability at this stage, indicating a transient nature of insm1a in the retina from 48 to 72 hpf. This variability may be due to slight inconsistencies in timing of neurogenic events such as neuronal maturation. insm1a is also seen in regions of the forebrain, optic tectum, epiphysis, and cerebellum. Hindbrain expression of insm1a becomes restricted to the midline and does not exhibit the striped pattern seen at 48 hpf. insm1b at 72 hpf is seen in the forebrain and cerebellum, barely visible in the optic tectum and absent from the hindbrain (Fig. 3K). Again, both insm1a and insm1b patterns resemble deltaA expression (Fig. 3N). Neuronal expression of insm1a and insm1b was shown by two-color in situ hybridization with the neuronal marker elavl3 (Park et al., 2000). During early neurogenic stages both insm1 transcripts overlap elavl3 expression (Figs. 4A and B) in the trigeminal ganglion and Rohon-beard sensory neurons. insm1b also overlaps elavl3 in the primary motoneurons and interneurons of the trunk, and in the midbrain. Expression of insm1 in the telencephalon precedes elavl3 and also in the presumptive cranial ganglia (data not shown). At 24 hpf insm1a and insm1b are clearly expressed in the majority of elavl3-positive cells with insm1b being more strongly expressed in the cranial ganglia
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Fig. 3. Embryonic expression patterns of insm1a (A, A 0 , D, D 0 , F, F 0 , G, and H) and insm1b (B, B 0 , E, I, J, and K). At 10 hpf (A, A 0 , B, B 0 , C, and C 0 ) shortly after neurogenesis has been initiated, insm1a is barely detectable in the lateral trunk while insm1b is expressed in a broader range of presumptive trunk neurons, as shown by similarities to expression of the proneural gene deltaA (C, C 0 , L, M, and N). (A–C) Anterior expression while A 0 , B 0 , and C 0 show trunk expression of the same embryo. At 14 hpf (D, D 0 , and E) insm1a is visible in a disorganized manner in the rostral trunk region (inset D 0 ), presumably pancreatic progenitors. insm1b is clearly visible in the presumptive trigeminal ganglia (E). By 24 hpf (F, F 0 , I, and L) it is evident that both insm1a and insm1b are expressed in a subset of proneural, deltaA-positive cells. Ventral endoderm in the rostral trunk shows a clustering of cells expressing insm1a (inset F 0 ). At 48 hpf (G, J, and M) both insm1a and insm1b are visible throughout the CNS and expression in the spinal cord is no longer detectable. Expression is also seen as dorsoventral stripes in the hindbrain. Retinal expression of insm1a is prevalent at 48 hpf, however by 72 hpf (H, K, and N) it is restricted to a subset of cells, consistent with a transient process such as neuronal differentiation or maturation. Scale bars represent 100 lm. Inset images were taken of flat mounts of the same embryo shown in its respective panel. cb, cerebellum; cg, cranial ganglia; d, diencephalon; ep, epiphysis; hb, hindbrain; mb, midbrain; olf, olfactory placode; ot, otic vesicle; r, rhombomere; ret, retina; scn, spinal cord neurons; tec, optic tectum; teg, tegmentum; tel, telencephalon; tg, trigeminal ganglia; and tn, trunk neurons.
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Fig. 4. Neuronal and pancreatic expression of insm1a (A, C, E, G, and I) and insm1b (B, D, F, H, and J) in zebrafish. All panels show anterior to the left with complete (A–D), lateral (E and F), and ventral focal planes (G–J). Scale bars represent 50 lm. Fluorescein labeled elavl3 (A–F) or neuroD (G–J) antisense probe was used to indicate specified neurons or pancreatic cells (red), respectively. insm1 is shown in purple and the resulting overlap appears brown with varying intensities. At 11 hpf (A and B) elavl3 is expressed in neurons of the early CNS and insm1 overlap this expression precisely in several types of neurons. At 24 hpf (C and D) there is a large degree of overlap between insm1 and elavl3. By 48 hpf (E and F) elavl3 labels the entire CNS and darker regions indicate where insm1 is expressed, notably the cranial ganglia for insm1b and retina for insm1a. As a reference for elavl3-only (red) neurons, insm1a is not present in pmn at 11 hpf and barely detectable in the cranial ganglia at 24 hpf; otic vesicles are indicated for positional reference. Pancreatic expression of insm1a and insm1b coincide with neuroD in a disorganized manner at 18 hpf (G and H) and later come together to a continuous expression domain at 24 hpf (I and J). cb, cerebellum; cg, cranial ganglia; in, interneurons; mb, midbrain; nc, notochord; olf, olfactory placode; ot, otic vesicle; pmn, primary motoneurons; Rb, Rohon-beard neurons; ret, retina; tel, telencephalon; and tg, trigeminal ganglia.
than insm1a (Figs. 4C and D). insm1a is seen in the ventronasal retina prior to elavl3, the site where retinal differentiation is initiated and that insm1a follows the expression wave seen by ath5 during retinal differentiation (Kay et al., 2005). Retinal expression is unique to insm1a and occurs transiently from 24 to 72 hpf. By 48 hpf expression of elavl3 is present throughout the CNS and also overlaps insm1b in the cranial ganglia (Figs. 4E and F). To demonstrate that the ventral endoderm expression of insm1 is indeed in the pancreas we carried out two-color
in situ hybridization with the pancreatic marker neuroD (Mavropoulos et al., 2005). At the 18 somite stage (18 hpf) strong insm1a and weak insm1b expression overlap that of neuroD (Figs. 4G and H) forming a disorganized cluster of cells ventral to the notochord. By 24 hpf these cells have coalesced to form a more organized group of cells expressing both insm1 and neuroD (Figs. 4I and J). Two-color in situ analysis was not sensitive enough to show insm1 overlaps neuroD at very early stages. However, from the overlapping expression at later stages combined with separate detection of DIG-labeled neuroD and insm1 probes (data not shown) we conclude that the early ventral endoderm expression of insm1a and later expression of insm1b is in pancreatic progenitors. The expression seen in the hindbrain, primarily from 18 to 48 hpf, follows a segmental pattern. In order to determine where insm1 expression is in relation to rhombomere boundaries we performed two-color in situ hybridization with egr2b (formerly krox20) or ephA4a (formerly ephA4) probes. At 10 somites (14 hpf) insm1 expression is seen primarily in rhombomeres 2 and 4 of the hindbrain (Figs. 5A and B) and by 18 somites (18 hpf) the segmental expression of insm1a is visible in the ventral hindbrain (Figs. 5C, D). By 32 hpf insm1a is more prevalent adjacent to the midline and faint expression extends laterally (Fig. 5E). insm1b does not share this concentrated expression adjacent to the midline but instead is predominantly found in the cranial ganglia (Fig. 5F). Lateral views of 32 hpf embryos demonstrate that insm1a is primarily found in the ventral-most hindbrain while insm1b expression is present in the ventral half of the hindbrain. Both transcripts are expressed in cells that occupy a thin plane adjacent to rhombomere boundaries (Figs. 5G and H). This plane becomes more refined by 48 hpf to a single cell width that forms a funnel-shape at the lateral edges of rhombomeres 2–6 (Figs. 5I and J). 1.3. Conclusion We have identified two zebrafish insm1-like genes with an embryonic expression pattern that is comparable to the proneural marker deltaA as well as coinciding specifically with the neuronal marker elavl3 (Park et al., 2000) and the pancreatic marker neuroD (Mavropoulos et al., 2005). This pattern is similar to the previously identified neurogenic basic-helix–loop–helix transcription factors neurogenin1 (Blader et al., 1997) and atonal homolog 3 (Park et al., 2003; Wang et al., 2003). Proteins containing Insm-like Cys2-His2 zinc fingers, including EGL-46 from C. elegans and Nerfin-1 from Drosophila melanogaster belong to a conserved family of transcription factors that have been shown to be important in neural development (Kuzin et al., 2005; Yu et al., 2003). The spatial and temporal expression of zebrafish insm1 genes is consistent with the earliest stages of neuronal differentiation and provides us with a system to assess the evolutionary conservation of EIN transcription factors.
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2.2. Cloning and sequencing Purified poly-A RNA from 18 hpf embryos was used to construct a cDNA library in k Uni-ZAP XR according to manufacturers specifications (Stratagene). Plaques were lifted in duplicate onto Hybond-N (Amersham) nylon membranes and differentially screened with radiolabeled 18 hpf or adult cDNAs to determine potential embryo-specific cDNAs. Inserts >1 kb were sequenced using a DYEnamic ET sequencing kit (Amersham). BLAST searches of sequenced clones revealed a cDNA that corresponded to a gene having a predicted open reading frame (ORF) similar to human INSM1. An additional INSM1-like gene was identified from BLAST searches with a high degree of similarity to mammalian INSM1. To confirm their coding sequences, both putative ORFs were amplified using a Superscript III One-step RT-PCR kit (Invitrogen) and cloned into pCR 4-TOPO (Invitrogen). Sequencing revealed two distinct ORFs that were designated insm1a (383 amino acids) and insm1b (453 amino acids) after we examined their expression patterns; their coding sequences are deposited in GenBank under Accession Nos. DQ164184 and DQ164185, respectively. BLAST searches revealed a conserved EIN domain encompassing two Cys2-His2 zinc fingers that are present in C. elegans EGL-46, Drosophila Nerfin-1 and -2, and mammalian MLT1. To date only egl-46 and nerfin-1 have been functionally characterized and these two possess the highest degree of similarity at the level of protein sequence and structure, to vertebrate Insm1.
2.3. Reverse transcriptase-PCR
Fig. 5. Hindbrain expression of zebrafish insm1a (A, C, E, G, and I) and insm1b (B, D, F, H, and J). Rhombomeres 3 and 5 are marked by egr2b (A– D) or ephA4a (E–J) expression (red). All panels are shown with anterior to the left in ventral (A, B, E, F, I, and J) or mediolateral (C, D, G, and H) focal planes. Scale bars represent 50 lm. At 14 hpf (A and B) insm1a and insm1b are seen in rhombomeres 2 and 4. At 18 hpf (C and D), insm1a becomes more prevalent in the ventral hindbrain while insm1b is fairly dispersed. During pharyngula stages (32 hpf; E–H) insm1a expression is dominant adjacent to the midline and insm1b in the cranial ganglia. Alternating large and small gaps representing rhombomeres and rhombomere boundaries, respectively, are seen in r1 through r6. By 48 hpf (I and J) both insm1a and insm1b occupy a dorsoventral plane that extends laterally, directly adjacent to rhombomere boundaries (arrows). However, this plane does not extend entirely to the edge of the rhombomere, instead it converges within a rhombomere to form a funnel-shape (asterisk) between posterior and anterior segments of adjacent rhombomeres. There is considerable expression of insm1a adjacent to the midline (arrowhead). For lateral views tissues surrounding the hindbrain were removed for clarity. Note that the majority of egr2b or ephA4a staining is dorsal (seen in C and D) which results in a blurred boundary in the ventral focal planes shown. cb, cerebellum; ot, otic vesicle; and r, rhombomere.
2. Experimental procedures 2.1. Organisms Zebrafish AB strain embryos were collected and staged as previously described (Kimmel et al., 1995; Westerfield, 1995). To block pigment formation in embryos to be fixed at later stages, 0.003% phenylthiourea (PTU) was added to the media (Westerfield, 1995) at 18 h post-fertilization (hpf) and replenished every 24 h thereafter. Embryos were fixed in 4% paraformaldehyde-phosphate buffered saline (PBS) at 4 C and stored in 100% methanol at 20 C prior to in situ hybridization.
Total RNA was isolated from staged zebrafish embryos with TriZol (Invitrogen) following the manufacturer’s protocol and purified with an RNeasy kit (Qiagen). PCR conditions were optimized for each transcript individually; we used 33 cycles for insm1a and 36 cycles for insm1b. Using 250 ng total RNA as template, RT-PCR was performed with primers producing a 189-bp product representing insm1a and a 661-bp product representing insm1b. As a control, primers producing a 463-bp product corresponding to elongation factor 1 alpha (ef1a) were included in each reaction. Multiplex reactions with all six primers were not possible due to insm1a–insm1b primer incompatibility.
2.4. In situ hybridization To eliminate the chances of cross reactivity of ORF probes during in situ hybridization, we used probes against the 3 0 -UTRs of insm1a and insm1b as they have little sequence homology. RNA in situ hybridization was carried out as previously described (Oxtoby and Jowett, 1993) with the following modifications. DIG-labeled RNA probes were synthesized from cloned 3 0 -UTRs (1 kb). The coloring reactions proceeded for 2–3 h at 28.5 C and embryos were stored in 100% methanol, 0.1% Tween 20 at 4 C. For two-color in situ hybridization, embryos were probed with a DIG-labeled (Roche) insm1a or insm1b RNA probe and a fluorescein-labeled (Roche) egr2b, ephA4a, elavl3, and neuroD RNA probes. Following the NBT/BCIP coloring reaction used to detect the DIG-labeled probe, embryos were rinsed twice in water and incubated for 10 min in 0.1 M glycine, pH 2.2 at room temperature to inactivate the alkaline phosphatase (AP); washed in phosphate buffered saline 0.1% Tween 20 (PBTw) several times; re-blocked for 1 h at room temperature; and incubated with a 1:10,000 dilution of AP-conjugated anti-fluorescein antibody (Roche) overnight at 4 C. The second color reaction was carried out with iodonitrotetrazolium chloride (Sigma) to produce a red precipitate, which went for between 1 and 5 h at 28.5 C. Reactions were stopped with two water rinses and embryos were stored in 4% paraformaldehyde in 1· PBS at 4 C. Whole embryos were photographed in 1% methylcellulose using an Olympus SZX12 stereomicroscope. Flat mounts were prepared by deyolking embryos, transferring them through 30%, 50%, and 70% glycerol solutions and photographed with a Zeiss Imager.Z1 microscope. All figures were prepared with Adobe Photoshop. Panel 4F was modified to remove a bubble adjacent to the embryo.
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Acknowledgements We thank members of the Waskiewicz lab for comments and suggestions on the manuscript. This work was supported by the Department of Biological Sciences at the University of Alberta as part of an undergraduate course, the Natural Science and Engineering Research Council (A.W.), the Canadian Institutes for Health Research (A.W.), and Alberta Ingenuity Fund (A.W.). A.W. is a recipient of a Canada Research Chair award. References Biemar, F., Argenton, F., Schmidtke, R., Epperlein, S., Peers, B., Driever, W., 2001. Pancreas development in zebrafish: early dispersed appearance of endocrine hormone expressing cells and their convergence to form the definitive islet. Dev. Biol. 230, 189–203. Blader, P., Fischer, N., Gradwohl, G., Guillemot, F., Stra¨hle, U., 1997. The activity of neurogenin1 is controlled by local cues in the zebrafish embryo. Development 124, 4557–4569. Breslin, M.B., Zhu, M., Notkins, A.L., Lan, M.S., 2002. Neuroendocrine differentiation factor, IA-1 is a transcriptional repressor and contains a specific DNA-binding domain: identification of a consensus IA-1 binding sequence. Nucleic Acids Res. 30, 1038–1045. Breslin, M.B., Zhu, M., Lan, M.S., 2003. NeuroD1/E47 regulates the Ebox element of a novel zinc finger transcription factor, IA-1, in the developing nervous system. J. Biol. Chem. 278, 38991–38997. Goto, Y., De Silva, M.G., Toscani, A., Prabhaker, B.S., Notkins, A.L., Lan, M.S., 1992. A novel human insulinoma-associated cDNA, IA-1, encodes a protein with ‘‘zinc-finger’’ DNA-binding motifs. J. Biol. Chem. 267, 15252–15257. Grimes, H.L., Chan, T.O., Zweidler-McKay, P.A., Tong, B., Tsichlis, P.N., 1996. The gfi-1 proto-oncoprotein contains a novel transcriptional repressor domain, SNAG, and inhibits G1 arrest induced by interleukin-2 withdrawal. Mol. Cell. Biol. 16, 6263–6272. Kay, J.N., Link, B.A., Baier, H., 2005. Staggered cell-intrinsic timing of ath5 expession underlies the wave of ganglion cell neurogenesis in the zebrafish retina. Development 132, 2573–2585. Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B., Schilling, T.F., 1995. Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253–310. Kuzin, A., Brody, T., Moore, A.W., Odenwald, W.F., 2005. Nerfin-1 is required for early axon guidance decisions in the developing Drosophila CNS. Dev. Biol. 277, 347–365. Lan, M.S., Russell, E.K., Lu, J., Johnson, B.E., Notkins, A.L., 1993. IA-1, a new marker for neuroendocrine differentiation in human lung cancer cell lines. Cancer Res. 53, 4169–4171.
Mathavan, S., Lee, S.G.P., Mak, A., Miller, L.D., Radha, K., Murthy, K., Govindarajan, K.R., Tong, Y., Wu, Y.L., Lam, S.H., Yang, H., Ruan, Y., Korzh, V., Gong, Z., Liu, E.T., Lufkin, T., 2005. Transcriptome analysis of zebrafish embryogenesis using microarrays. PLoS Genet. 1, e29. Mavropoulos, A., Devos, N., Biemar, F., Zecchin, E., Argenton, F., Edlund, H., Motte, P., Martial, J.A., Peers, B., 2005. sox4b is a key player of pancreatic a cell differentiation in zebrafish. Dev. Biol. 285, 211–223. Oxtoby, E., Jowett, T., 1993. Cloning of the zebrafish krox-20 gene (krx20) and its expression during hindbrain development. Nucleic Acids Res. 21, 1087–1095. Park, H., Hong, S., Kim, H., Kim, S., Yoon, E., Kim, C., Miki, N., Huh, T., 2000. Structural comparison of zebrafish elav/hu and their differential expressions during neurogenesis. Neurosci. Lett. 279, 81– 84. Park, S., Yeo, S., Yoo, K., Hong, S., Lee, S., Rhee, M., Chitnis, A.B., Kim, C., 2003. Zath3, a neural basic helix–loop–helix gene, regulates early neurogenesis in the zebrafish. Biochem. Biophys. Res. Commun. 308, 184–190. Stivers, C., Brody, T., Kuzin, A., Odenwald, W.F., 2000. Nerfin-1 and -2, novel Drosophila Zn-finger transcription factor genes expressed in the developing nervous system. Mech. Dev. 97, 205–210. Tateno, M., Fukunishi, Y., Komatsu, S., Okazaki, Y., Kwai, J., Shibata, K., Itoh, M., Muramatsu, M., Held, W.A., Hayashizaki, Y., 2001. Identification of a novel member of the snail/gfi-1 repressor family, mlt1, which is methylated and silenced in liver tumors of SV40 T antigen transgenic mice. Cancer Res. 61, 1144– 1153. Wang, X., Emelyanov, A., Korzh, V., Gong, Z., 2003. Zebrafish atonal homologue zath3 is expressed during neurogenesis in embryonic development. Dev. Dyn. 227, 587–592. Westerfield, M., 1995. The Zebrafish Book: A Guide for the Laboratory use of Zebrafish (Danio rerio). University of Oregon Press, Eugene, OR. Wu, J., Duggan, A., Calfie, M., 2001. Inhibition of touch cell fate by egl44 and egl-46 in C. elegans. Genes Dev. 15, 789–802. Xie, J., Cai, T., Zhang, H., Lan, M.S., Notkins, A.L., 2002. The zincfinger transcription factor INSM1 is expressed during embryo development and interacts with the Cb1-associated protein. Genomics 80, 54–61. Yu, H., Pre´toˆt, R.F., Bu¨rglin, T.R., Sternberg, P.W., 2003. Distinct roles of transcription factors EGL-46 and DAF-19 in specifying the functionality of a polycystin-expressing sensory neuron necessary for C. elegans male vulva location behavior. Development 130, 5127–5217. Zhu, M., Breslin, M.B., Lan, M.S., 2002. Expression of a novel zinc-finger cDNA, IA-1, is associated with rat AR42J cells differentiation into insulin-positive cells. Pancreas 24, 139–145.
Expression of two insm1-like genes in the developing zebrafish nervous system Chris M. Lukowski, R. Gary Ritzel, Andrew Jan Waskiewicz
*
Department of Biological Sciences, University of Alberta, CW405 Biological Sciences Building, Edmonton, Alta., Canada T6G 2E9 Received 10 November 2005; received in revised form 22 December 2005; accepted 23 December 2005 Available online 17 February 2006
Abstract Insulinoma associated-1 (INSM1, formerly IA-1) is a Cys2-His2 zinc finger transcription factor sharing conserved regions with Caenorhabditis elegans EGL-46 and Drosophila Nerfin-1. INSM, EGL-46, and Nerfin proteins comprise the EIN family of zinc finger transcription factors. egl-46 and nerfin-1 have been implicated in various aspects of neuronal differentiation including cell fate specification, axon guidance decisions and cell migration. Murine Insm1 has a restricted expression pattern in the developing CNS. We have characterized two zebrafish (Danio rerio) Insm1-like genes, insm1a and insm1b, and analyzed their expression patterns during embryonic development. Zebrafish insm1a and insm1b share an embryonic expression pattern comparable to the proneural deltaA as well as overlapping the neuronal marker elavl3. The expression pattern observed for zebrafish insm1a and insm1b is similar to other EIN homologues. Both zebrafish insm1-like transcripts are also present in a region of the embryo where pancreatic progenitors originate. The expression data along with functional characterization of invertebrate homologues suggest a conserved pathway involving the EIN transcription factors in early neurogenesis. 2006 Elsevier B.V. All rights reserved. Keywords: Zebrafish; Danio rerio; Insulinoma associated-1; INSM1; IA-1; insm1a; insm1b; Neuronal differentiation; CNS; Pancreatic precursor; Neurogenesis
1. Results and discussion Insulinoma-associated 1 (INSM1, formerly IA-1) was originally isolated from a cDNA subtraction library between human insulinoma and glucagonoma tumor cells (Goto et al., 1992). Murine Insm1 has a restricted expression pattern in the embryonic CNS (Breslin et al., 2003) and mRNA is undetectable past 2 weeks post-partum (Xie et al., 2002), suggesting a function in both embryonic and neonatal, but not adult tissues. However, Insm1 mRNA has been detected in human, mouse, and rat tumors of neuroendocrine origin including insulinoma, medulloblastoma, pheochromacytoma, retinoblastoma, pituitary tumor, medullary thyroid carcinoma, and small cell lung carcinoma (Goto et al., 1992; Lan et al., 1993). *
Corresponding author. Tel.: +1 780 492 4403; fax: +1 780 492 9234. E-mail address: [email protected] (A.J. Waskiewicz).
1567-133X/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.modgep.2005.12.008
IA-1 expression in the rat pancreatic AR42J cell line is increased after conversion to an insulin-positive phenotype and decreased following conversion to an acinar-like phenotype (Zhu et al., 2002), indicating a potential role in islet cell differentiation. Mammalian Insm1 expression data suggests a role in differentiation of both neural and pancreatic precursors. INSM1 possesses an amino-terminal SNAIL/GFI (SNAG) repressor domain and five carboxy-terminal Cys2-His2 zinc fingers (Xie et al., 2002). Its protein structure is similar to the SNAIL, SLUG, and GFI superfamily of transcriptional repressors, a group of proteins that have been implicated in tumor progression. In addition, INSM1 contains similar zinc fingers to the Caenorhabditis elegans Egg Laying defective-46 (EGL-46; Wu et al., 2001) and the three zinc fingers of Drosophila Nervous fingers-1 (Nerfin-1; Stivers et al., 2000). The high degree of sequence similarity among the zinc fingers of EGL-46, Nerfin and INSM
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proteins suggests they recognize a common DNA sequence and has been termed the EIN domain for EGL-46, INSM1 and Nerfin-1 (Stivers et al., 2000). The consensus INSM1 DNA-binding target contains a core GGGG, but other EIN domain proteins have yet to be characterized biochemically (Breslin et al., 2002). Caenorhabditis elegans egl-46 mutants display neuronal defects including abnormal cell migration, misguided axonal outgrowth, changes in cell fate, and extra rounds of cellular divisions in presumptive post-mitotic neurons (Wu et al., 2001). EGL-46 has been implicated in specification of the HSN egg laying motor neurons, the FLP neuron (Wu et al., 2001), and the HOB hook neuron, which is necessary for vulva location (Yu et al., 2003). Dynamic expression of egl-46 during larval development is also seen in ventral cord and head neurons, Q-lineage neuroblasts, PVD neurons, touch cells (Wu et al., 2001) and ray neurons (Yu et al., 2003). The Drosophila EIN homologue nerfin-1 is widely detected in the developing CNS including early ventral cord neuroblasts, ganglion mother cells, sensory organ precursors, and precursors of the compound eye (Stivers et al., 2000). Drosophila embryos deficient for nerfin-1 display defects in axon fasciculation and guidance (Kuzin et al., 2005). The activity of the Insm1 promoter has been analyzed in transgenic mice using a lacZ fusion. b-Galactosidase activity was observed in regions of the forebrain, midbrain, hindbrain, spinal cord, retina, olfactory bulb, and cerebellum (Breslin et al., 2003). Here, we report the molecular cloning of two zebrafish insm1-like cDNAs, insm1a and insm1b. A combination of RT-PCR and RNA in situ hybridization demonstrates that both insm1-like transcripts are expressed in a similar pattern to the proneural marker deltaA and overlap expression of the neuronal marker elavl3. The dynamic expression pattern of insm1a and insm1b in the zebrafish central nervous system is comparable to the expression of C. elegans egl-46, Drosophila nerfin-1 and murine Insm1, making zebrafish an attractive vertebrate model organism to determine whether EIN protein functions have been evolutionarily conserved. 1.1. Cloning of zebrafish insm1a and insm1b A zebrafish embryonic cDNA library was differentially screened for genes involved in early embryogenesis. Of the selected clones, BLAST sequence analysis revealed a cDNA with a high degree of sequence similarity to mammalian Insm1. Examination of the Danio rerio genome revealed a second hypothetical insm1-like gene, which we believe to be the product of the teleost lineage genome duplication as both Tetraodon nigroviridis and Takifugu rubripes also have two insm1-like genes and all other complete or nearly complete vertebrate genomes examined contain a single Insm1 gene. We amplified both putative open reading frames (ORFs) with RT-PCR using primers flanking each hypothetical ORF. Sequence analysis revealed predicted ORFs of 383 (Insm1a) and 453 (Insm1b) amino acids that are 71% identical and 81% similar to each other.
The zebrafish Insm1 proteins also share amino acid sequence conservation with two characterized invertebrate zinc finger transcription factors, Nerfin-1 and EGL-46. Insm1a is 34% identical to Nerfin-1 and 35% identical to EGL-46. Insm1b shares 30% identity with Nerfin-1 and 36% identity with EGL-46. From current genomic sequences, zebrafish insm1a is in a region of conserved synteny to mammalian Insm1 while insm1b is unmapped. We confirmed that zebrafish insm1a and insm1b are intronless, resembling their murine (Xie et al., 2002) and Drosophila (Kuzin et al., 2005) homologues (data not shown). Amino acid alignment of zebrafish Insm1a and Insm1b predicted ORFs to mammalian homologues reveals several highly conserved regions, including an amino-terminal SNAG motif (Grimes et al., 1996), five carboxy-terminal Cys2-His2 zinc fingers and a putative nuclear localization signal (Fig. 1A). A conserved arginine residue has replaced the second histidine of zinc finger 1. The carboxy terminus is also highly conserved amongst vertebrate EIN proteins, indicating a potential functional domain. The putative nuclear localization signal (NLS) we have identified is upstream of zinc finger 1 in a region that is conserved in both vertebrate and invertebrate EIN homologues. For vertebrate Insm1 proteins, the sequences of zinc fingers 1, 2, 3, and 5 are very well conserved while the majority of conservation in zinc finger four surrounds the zinc coordinating cysteine and histidine residues (Fig. 1A). Zebrafish Insm1 proteins have a similar structure to mammalian homologues, which all share a highly conserved EIN domain encompassing the first two zinc fingers (Fig. 1B). The EIN domain is 59% identical and 73% similar in species from nematodes to humans in addition to being 82% identical and 92% similar in identified vertebrate homologues. A common feature of mammalian INSM1 is zinc finger spacing; two pairs are symmetrically positioned around a central zinc finger. Although this feature is more evident in Insm1b than Insm1a, zinc finger spacing in a linear schematic may not accurately reflect the implications on its tertiary structure. Together with EGL-46 and Nerfin-1, vertebrate Insm1 proteins clearly share a conserved domain architecture and considerable sequence conservation. Vertebrates possess a second INSM1-like protein named MLT1 (Tateno et al., 2001), which is fairly similar in amino acid sequence to INSM1, but has a different zinc finger distribution. Phylogenetic analysis of predicted amino acid sequences demonstrate that Insm1a (79% similar) and Insm1b (73% similar) are more closely related to human INSM1 (Fig. 1C) than to human MLT1 (Tateno et al., 2001). Human MLT1 amino acid sequences are 52% similar to INSM1, but the zinc finger distribution is distinct from INSM1, and several unique conserved sequences in mammalian MLT1 are not present in either Insm1a or Insm1b. Analysis of the zebrafish genome revealed a putative mlt1 gene encoding an ORF of 508 amino acids with 63% similarity to human MLT1 and 51% similarity to human INSM1. The hypothetical zebrafish Mlt1 shares
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Fig. 1. Comparison of zebrafish Insm1a and Insm1b to mammalian and invertebrate homologues. (A) Amino acid alignment of predicted zebrafish Insm1a (AAZ94623) and Insm1b (AAZ94624) to human (NP_002187) and mouse (NP_058585) INSM1. Amino acid identity (asterisks) and similarity (periods) are indicated below the aligned sequences. Insm1 is predicted to contain an amino-terminal SNAG motif (blue), a putative nuclear localization sequence (green), and five (numbered) carboxy-terminal Cys2-His2 zinc fingers (red). Zinc coordination residues of each zinc finger are highlighted in red. (B) Protein schematic showing the predicted domain structure of Insm1, Nerfin-1, and EGL-46 proteins. Shown are the amino-terminal SNAG repressor domains (blue), putative nuclear localization signals (green) and carboxy-terminal Cys2-His2 zinc fingers (red). Proteins were drawn to scale and aligned about the highly conserved EIN domain. Zinc fingers are numbered based on homology as in (A) and indicated in invertebrate homologues. Zinc finger 3 of EGL-46 (asterisk) does not share the conservation seen in other EIN homologues. Numbers in brackets indicate the amino acid length. (C) Phylogenetic analysis showing zebrafish Insm1a and Insm1b (highlighted in orange) are more similar to mammalian INSM1 than to another INSM1-like protein, MLT1. We have identified a zebrafish MLT1-like gene from genomic sequences that is more similar to mammalian MLT1 than to INSM1. z, zebrafish; h, human; and m, mouse.
the distinct zinc finger distribution seen in mammalian MLT1 and contains several of the unique sequences mentioned previously. These data further support our hypothesis that zebrafish Insm1a and Insm1b are co-orthologues of mammalian INSM1. 1.2. Developmental expression of insm1a and insm1b Mouse Insm1 expression has previously been determined using a promoter-lacZ fusion transgene (Breslin et al., 2003). Since the visualization of b-galactosidase activity is necessarily an accumulation assay, there is no data precisely measuring the spatiotemporal expression pattern of vertebrate insm1 mRNA during development. Furthermore, the Drosophila EIN homologue has been shown to be regulated post-transcriptionally, consequently a promoter-fusion may not be the most accurate method to ascertain the expression of vertebrate INSM1. Therefore, we have conducted a detailed analysis of vertebrate insm1 spatiotemporal gene regulation in zebrafish. To obtain a measure of insm1a and insm1b transcripts during zebrafish development, we performed semi-quantitative RT-PCR with RNA from a series of embryonic stages (Fig. 2). insm1a
Fig. 2. Reverse transcriptase-PCR analysis of zebrafish insm1a and insm1b during development. Two hundred and fifty nanograms total RNA was used per reaction. Time listed above each lane indicates embryonic age post-fertilization at which the RNA was extracted. Zebrafish neurogenesis begins at tailbud stages (9 hpf) and reaches maximal levels between 24 and 72 hpf, a pattern that is precisely followed by both insm1 transcripts. Neither transcript is expressed in adult tissues, which is consistent with mammalian expression data. The final lane is a no RNA control. Note that insm1a transcripts appear to be maternally inherited as zygotic transcription does not begin until 3 hpf, while insm1b is not.
transcripts appear to be maternally contributed while insm1b transcripts are not, as zygotic transcription in zebrafish does not begin until 3 h post-fertilization (hpf; Mathavan et al., 2005). Both transcripts are upregulated at
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10 hpf, reach peak levels at 48 hpf and are not detectable in adult tissues. Zebrafish neurogenesis begins at early tailbud stages (9 hpf), reaches high levels between 24 and 48 hpf, and progresses past 72 hpf. The timing of peak insm1a and insm1b expression coincides precisely with periods of zebrafish neurogenesis. Mouse insm1 transcripts are seen from embryonic day 10.5 until 2 weeks post-partum (Xie et al., 2002), a time course similar to our data in zebrafish. To determine if transcript levels from RT-PCR analysis are consistent with a hypothesized expression during neurogenesis, we performed RNA in situ hybridization on staged zebrafish embryos ranging from 10 to 72 hpf. To avoid potential cross hybridization of insm1a and insm1b ORF probes, antisense probes corresponding to the 3 0 -untranslated regions (UTRs) of both insm1a and insm1b were used. We also used a probe for the proneural Notch ligand deltaA to indicate where and when neurogenesis is initiated during embryonic development. During the formation of proneural fields, cells express both notch and deltaA. Through the mechanism of lateral inhibition, only subsets of these cells are fated to become neurons, therefore deltaA-expression correlates with proneural domains rather than early born neurons. Sense control probes for insm1a or insm1b 3 0 -UTRs showed no staining after an extended coloring reaction time (data not shown). Expression of insm1a and insm1b is first detected prior to somitogenesis, at late tailbud stages (9.5 hpf) with a pattern overlapping the proneural marker deltaA. At the two somite stage (10 hpf), insm1a is expressed in the presumptive trigeminal ganglia, telencephalon and lateral trunk neurons (Figs. 3A and A 0 ). insm1b is expressed in the presumptive trigeminal ganglia, midbrain, and several types of trunk neurons (Figs. 3B and B 0 ). These regions of insm1 expression are similar to that of deltaA (Figs. 3C and C 0 ), albeit with weaker staining intensity. We noticed a short delay between the regions of deltaA expression and insm1 expression at a slightly later stage, suggesting insm1 may be a downstream target of deltaA or other neurogenic factors. At the 10 somite stage (14 hpf), insm1a and insm1b are expressed in the telencephalon, ventral diencephalon, midbrain, and hindbrain (Figs. 3D and E). Strong insm1b expression is visible in the presumptive trigeminal and cranial ganglia at the 10 somite stage (14 hpf; Fig. 3E). Both insm1 transcripts are present in a group of cells in the ventral endoderm (Fig. 3D 0 ; insm1b not shown), a region that is known to give rise to pancreatic precursors. The disorganized pattern of cells expressing insm1a and insm1b converge as development continues, a pattern similar to insulin expression and the pancreatic markers neuroD, nkx2.2, islet1 and pax6.2 (Fig. 3F 0 ; Biemar et al., 2001; Mavropoulos et al., 2005). insm1a expression in the ventral endoderm is seen very early while insm1b appears later. Together with the fact that human INSM1 was isolated from an insulinoma cDNA library, presumptive pancreatic expression of zebrafish insm1 genes suggests a potential role for vertebrate Insm1 proteins in pancreatic cell differentiation.
At late somitogenesis (24 hpf) both insm1a and insm1b are expressed in the olfactory placode, ventral diencephalon, tegmentum, hindbrain, presumptive pancreas, and a subset of spinal cord neurons (Figs. 3F, F 0 , and I). insm1b expression occupies the most posterior region of the epiphysis, while insm1a is seen throughout this region. Spinal cord expression of insm1b occupies three planes along the dorsoventral axis, presumably the motoneurons, interneurons, and Rohon-beard (Rb) neurons. Spinal cord expression of insm1a is absent from rostral Rb neurons and is restricted to the ventral motoneurons. Expression of deltaA at 24 hpf illustrates the neural fields in the developing embryo (Fig. 3L), and insm1a and insm1b are expressed in a more refined but markedly similar fashion. By late pharyngula stages (48 hpf), the expression of insm1a (Fig. 3G) and insm1b (Fig. 3J) is widespread throughout the CNS, and deltaA (Fig. 3M) also exhibits a similar overall expression pattern. insm1a expression is visible in the otic vesicle and has expanded throughout the retina, whereas insm1b expression was not detectable in the retina at any stage examined. Stripes of insm1 expression are seen along the dorsoventral axis in the hindbrain at 48 hpf, which correspond to a region directly adjacent to rhombomere boundaries. This expression creates a pattern of alternating large and small gaps in insm1 expression that is clearly visible in a lateral view of insm1b expression (Fig. 3J) but less so for insm1a. deltaA expression also exhibits this pattern within the hindbrain. At 48 hpf there is faint staining for insm1a and insm1b along the lateral edge of the hindbrain and this expression is visible starting at 18 hpf (data not shown). At early larval stages (72 hpf) insm1a expression is confined to a specific cell layer in the retina (Fig. 3H), however we observed a moderate degree of variability at this stage, indicating a transient nature of insm1a in the retina from 48 to 72 hpf. This variability may be due to slight inconsistencies in timing of neurogenic events such as neuronal maturation. insm1a is also seen in regions of the forebrain, optic tectum, epiphysis, and cerebellum. Hindbrain expression of insm1a becomes restricted to the midline and does not exhibit the striped pattern seen at 48 hpf. insm1b at 72 hpf is seen in the forebrain and cerebellum, barely visible in the optic tectum and absent from the hindbrain (Fig. 3K). Again, both insm1a and insm1b patterns resemble deltaA expression (Fig. 3N). Neuronal expression of insm1a and insm1b was shown by two-color in situ hybridization with the neuronal marker elavl3 (Park et al., 2000). During early neurogenic stages both insm1 transcripts overlap elavl3 expression (Figs. 4A and B) in the trigeminal ganglion and Rohon-beard sensory neurons. insm1b also overlaps elavl3 in the primary motoneurons and interneurons of the trunk, and in the midbrain. Expression of insm1 in the telencephalon precedes elavl3 and also in the presumptive cranial ganglia (data not shown). At 24 hpf insm1a and insm1b are clearly expressed in the majority of elavl3-positive cells with insm1b being more strongly expressed in the cranial ganglia
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Fig. 3. Embryonic expression patterns of insm1a (A, A 0 , D, D 0 , F, F 0 , G, and H) and insm1b (B, B 0 , E, I, J, and K). At 10 hpf (A, A 0 , B, B 0 , C, and C 0 ) shortly after neurogenesis has been initiated, insm1a is barely detectable in the lateral trunk while insm1b is expressed in a broader range of presumptive trunk neurons, as shown by similarities to expression of the proneural gene deltaA (C, C 0 , L, M, and N). (A–C) Anterior expression while A 0 , B 0 , and C 0 show trunk expression of the same embryo. At 14 hpf (D, D 0 , and E) insm1a is visible in a disorganized manner in the rostral trunk region (inset D 0 ), presumably pancreatic progenitors. insm1b is clearly visible in the presumptive trigeminal ganglia (E). By 24 hpf (F, F 0 , I, and L) it is evident that both insm1a and insm1b are expressed in a subset of proneural, deltaA-positive cells. Ventral endoderm in the rostral trunk shows a clustering of cells expressing insm1a (inset F 0 ). At 48 hpf (G, J, and M) both insm1a and insm1b are visible throughout the CNS and expression in the spinal cord is no longer detectable. Expression is also seen as dorsoventral stripes in the hindbrain. Retinal expression of insm1a is prevalent at 48 hpf, however by 72 hpf (H, K, and N) it is restricted to a subset of cells, consistent with a transient process such as neuronal differentiation or maturation. Scale bars represent 100 lm. Inset images were taken of flat mounts of the same embryo shown in its respective panel. cb, cerebellum; cg, cranial ganglia; d, diencephalon; ep, epiphysis; hb, hindbrain; mb, midbrain; olf, olfactory placode; ot, otic vesicle; r, rhombomere; ret, retina; scn, spinal cord neurons; tec, optic tectum; teg, tegmentum; tel, telencephalon; tg, trigeminal ganglia; and tn, trunk neurons.
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Fig. 4. Neuronal and pancreatic expression of insm1a (A, C, E, G, and I) and insm1b (B, D, F, H, and J) in zebrafish. All panels show anterior to the left with complete (A–D), lateral (E and F), and ventral focal planes (G–J). Scale bars represent 50 lm. Fluorescein labeled elavl3 (A–F) or neuroD (G–J) antisense probe was used to indicate specified neurons or pancreatic cells (red), respectively. insm1 is shown in purple and the resulting overlap appears brown with varying intensities. At 11 hpf (A and B) elavl3 is expressed in neurons of the early CNS and insm1 overlap this expression precisely in several types of neurons. At 24 hpf (C and D) there is a large degree of overlap between insm1 and elavl3. By 48 hpf (E and F) elavl3 labels the entire CNS and darker regions indicate where insm1 is expressed, notably the cranial ganglia for insm1b and retina for insm1a. As a reference for elavl3-only (red) neurons, insm1a is not present in pmn at 11 hpf and barely detectable in the cranial ganglia at 24 hpf; otic vesicles are indicated for positional reference. Pancreatic expression of insm1a and insm1b coincide with neuroD in a disorganized manner at 18 hpf (G and H) and later come together to a continuous expression domain at 24 hpf (I and J). cb, cerebellum; cg, cranial ganglia; in, interneurons; mb, midbrain; nc, notochord; olf, olfactory placode; ot, otic vesicle; pmn, primary motoneurons; Rb, Rohon-beard neurons; ret, retina; tel, telencephalon; and tg, trigeminal ganglia.
than insm1a (Figs. 4C and D). insm1a is seen in the ventronasal retina prior to elavl3, the site where retinal differentiation is initiated and that insm1a follows the expression wave seen by ath5 during retinal differentiation (Kay et al., 2005). Retinal expression is unique to insm1a and occurs transiently from 24 to 72 hpf. By 48 hpf expression of elavl3 is present throughout the CNS and also overlaps insm1b in the cranial ganglia (Figs. 4E and F). To demonstrate that the ventral endoderm expression of insm1 is indeed in the pancreas we carried out two-color
in situ hybridization with the pancreatic marker neuroD (Mavropoulos et al., 2005). At the 18 somite stage (18 hpf) strong insm1a and weak insm1b expression overlap that of neuroD (Figs. 4G and H) forming a disorganized cluster of cells ventral to the notochord. By 24 hpf these cells have coalesced to form a more organized group of cells expressing both insm1 and neuroD (Figs. 4I and J). Two-color in situ analysis was not sensitive enough to show insm1 overlaps neuroD at very early stages. However, from the overlapping expression at later stages combined with separate detection of DIG-labeled neuroD and insm1 probes (data not shown) we conclude that the early ventral endoderm expression of insm1a and later expression of insm1b is in pancreatic progenitors. The expression seen in the hindbrain, primarily from 18 to 48 hpf, follows a segmental pattern. In order to determine where insm1 expression is in relation to rhombomere boundaries we performed two-color in situ hybridization with egr2b (formerly krox20) or ephA4a (formerly ephA4) probes. At 10 somites (14 hpf) insm1 expression is seen primarily in rhombomeres 2 and 4 of the hindbrain (Figs. 5A and B) and by 18 somites (18 hpf) the segmental expression of insm1a is visible in the ventral hindbrain (Figs. 5C, D). By 32 hpf insm1a is more prevalent adjacent to the midline and faint expression extends laterally (Fig. 5E). insm1b does not share this concentrated expression adjacent to the midline but instead is predominantly found in the cranial ganglia (Fig. 5F). Lateral views of 32 hpf embryos demonstrate that insm1a is primarily found in the ventral-most hindbrain while insm1b expression is present in the ventral half of the hindbrain. Both transcripts are expressed in cells that occupy a thin plane adjacent to rhombomere boundaries (Figs. 5G and H). This plane becomes more refined by 48 hpf to a single cell width that forms a funnel-shape at the lateral edges of rhombomeres 2–6 (Figs. 5I and J). 1.3. Conclusion We have identified two zebrafish insm1-like genes with an embryonic expression pattern that is comparable to the proneural marker deltaA as well as coinciding specifically with the neuronal marker elavl3 (Park et al., 2000) and the pancreatic marker neuroD (Mavropoulos et al., 2005). This pattern is similar to the previously identified neurogenic basic-helix–loop–helix transcription factors neurogenin1 (Blader et al., 1997) and atonal homolog 3 (Park et al., 2003; Wang et al., 2003). Proteins containing Insm-like Cys2-His2 zinc fingers, including EGL-46 from C. elegans and Nerfin-1 from Drosophila melanogaster belong to a conserved family of transcription factors that have been shown to be important in neural development (Kuzin et al., 2005; Yu et al., 2003). The spatial and temporal expression of zebrafish insm1 genes is consistent with the earliest stages of neuronal differentiation and provides us with a system to assess the evolutionary conservation of EIN transcription factors.
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2.2. Cloning and sequencing Purified poly-A RNA from 18 hpf embryos was used to construct a cDNA library in k Uni-ZAP XR according to manufacturers specifications (Stratagene). Plaques were lifted in duplicate onto Hybond-N (Amersham) nylon membranes and differentially screened with radiolabeled 18 hpf or adult cDNAs to determine potential embryo-specific cDNAs. Inserts >1 kb were sequenced using a DYEnamic ET sequencing kit (Amersham). BLAST searches of sequenced clones revealed a cDNA that corresponded to a gene having a predicted open reading frame (ORF) similar to human INSM1. An additional INSM1-like gene was identified from BLAST searches with a high degree of similarity to mammalian INSM1. To confirm their coding sequences, both putative ORFs were amplified using a Superscript III One-step RT-PCR kit (Invitrogen) and cloned into pCR 4-TOPO (Invitrogen). Sequencing revealed two distinct ORFs that were designated insm1a (383 amino acids) and insm1b (453 amino acids) after we examined their expression patterns; their coding sequences are deposited in GenBank under Accession Nos. DQ164184 and DQ164185, respectively. BLAST searches revealed a conserved EIN domain encompassing two Cys2-His2 zinc fingers that are present in C. elegans EGL-46, Drosophila Nerfin-1 and -2, and mammalian MLT1. To date only egl-46 and nerfin-1 have been functionally characterized and these two possess the highest degree of similarity at the level of protein sequence and structure, to vertebrate Insm1.
2.3. Reverse transcriptase-PCR
Fig. 5. Hindbrain expression of zebrafish insm1a (A, C, E, G, and I) and insm1b (B, D, F, H, and J). Rhombomeres 3 and 5 are marked by egr2b (A– D) or ephA4a (E–J) expression (red). All panels are shown with anterior to the left in ventral (A, B, E, F, I, and J) or mediolateral (C, D, G, and H) focal planes. Scale bars represent 50 lm. At 14 hpf (A and B) insm1a and insm1b are seen in rhombomeres 2 and 4. At 18 hpf (C and D), insm1a becomes more prevalent in the ventral hindbrain while insm1b is fairly dispersed. During pharyngula stages (32 hpf; E–H) insm1a expression is dominant adjacent to the midline and insm1b in the cranial ganglia. Alternating large and small gaps representing rhombomeres and rhombomere boundaries, respectively, are seen in r1 through r6. By 48 hpf (I and J) both insm1a and insm1b occupy a dorsoventral plane that extends laterally, directly adjacent to rhombomere boundaries (arrows). However, this plane does not extend entirely to the edge of the rhombomere, instead it converges within a rhombomere to form a funnel-shape (asterisk) between posterior and anterior segments of adjacent rhombomeres. There is considerable expression of insm1a adjacent to the midline (arrowhead). For lateral views tissues surrounding the hindbrain were removed for clarity. Note that the majority of egr2b or ephA4a staining is dorsal (seen in C and D) which results in a blurred boundary in the ventral focal planes shown. cb, cerebellum; ot, otic vesicle; and r, rhombomere.
2. Experimental procedures 2.1. Organisms Zebrafish AB strain embryos were collected and staged as previously described (Kimmel et al., 1995; Westerfield, 1995). To block pigment formation in embryos to be fixed at later stages, 0.003% phenylthiourea (PTU) was added to the media (Westerfield, 1995) at 18 h post-fertilization (hpf) and replenished every 24 h thereafter. Embryos were fixed in 4% paraformaldehyde-phosphate buffered saline (PBS) at 4 C and stored in 100% methanol at 20 C prior to in situ hybridization.
Total RNA was isolated from staged zebrafish embryos with TriZol (Invitrogen) following the manufacturer’s protocol and purified with an RNeasy kit (Qiagen). PCR conditions were optimized for each transcript individually; we used 33 cycles for insm1a and 36 cycles for insm1b. Using 250 ng total RNA as template, RT-PCR was performed with primers producing a 189-bp product representing insm1a and a 661-bp product representing insm1b. As a control, primers producing a 463-bp product corresponding to elongation factor 1 alpha (ef1a) were included in each reaction. Multiplex reactions with all six primers were not possible due to insm1a–insm1b primer incompatibility.
2.4. In situ hybridization To eliminate the chances of cross reactivity of ORF probes during in situ hybridization, we used probes against the 3 0 -UTRs of insm1a and insm1b as they have little sequence homology. RNA in situ hybridization was carried out as previously described (Oxtoby and Jowett, 1993) with the following modifications. DIG-labeled RNA probes were synthesized from cloned 3 0 -UTRs (1 kb). The coloring reactions proceeded for 2–3 h at 28.5 C and embryos were stored in 100% methanol, 0.1% Tween 20 at 4 C. For two-color in situ hybridization, embryos were probed with a DIG-labeled (Roche) insm1a or insm1b RNA probe and a fluorescein-labeled (Roche) egr2b, ephA4a, elavl3, and neuroD RNA probes. Following the NBT/BCIP coloring reaction used to detect the DIG-labeled probe, embryos were rinsed twice in water and incubated for 10 min in 0.1 M glycine, pH 2.2 at room temperature to inactivate the alkaline phosphatase (AP); washed in phosphate buffered saline 0.1% Tween 20 (PBTw) several times; re-blocked for 1 h at room temperature; and incubated with a 1:10,000 dilution of AP-conjugated anti-fluorescein antibody (Roche) overnight at 4 C. The second color reaction was carried out with iodonitrotetrazolium chloride (Sigma) to produce a red precipitate, which went for between 1 and 5 h at 28.5 C. Reactions were stopped with two water rinses and embryos were stored in 4% paraformaldehyde in 1· PBS at 4 C. Whole embryos were photographed in 1% methylcellulose using an Olympus SZX12 stereomicroscope. Flat mounts were prepared by deyolking embryos, transferring them through 30%, 50%, and 70% glycerol solutions and photographed with a Zeiss Imager.Z1 microscope. All figures were prepared with Adobe Photoshop. Panel 4F was modified to remove a bubble adjacent to the embryo.
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