Dbx1b defines the dorsal habenular progenitor domain in the zebrafish epithalamus
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The conserved habenular nuclei function as a relay system connecting the forebrain with the brain stem. They play crucial roles in various cognitive behaviors by modulating cholinergic, dopaminergic and serotonergic activities. Despite the renewed interest in this conserved forebrain region because of its importance in regulating aversion and reward behaviors, the formation of the habenular nuclei during embryogenesis is poorly understood due to their small size and deep location in the brain, as well as the lack of known markers for habenular progenitors. In zebrafish, the bilateral habenular nuclei are subdivided into dorsal and ventral compartments, are particularly large and found on the dorsal surface of the brain, which facilitates the study of their development.
Here we examine the expression of a homeodomain transcription factor, dbx1b, and its potential to serve as an early molecular marker of dorsal habenular progenitors. Detailed spatiotemporal expression profiles demonstrate that the expression domain of dbx1b correlates with the presumptive habenular region, and dbx1b-expressing cells are proliferative along the ventricle. A lineage-tracing experiment using the Cre-lox system confirms that all or almost all dorsal habenular neurons are derived from dbx1 b-expressing cells. In addition, mutant analysis and pharmacological treatments demonstrate that both initiation and maintenance of dbx1b expression requires precise regulation by fibroblast growth factor (FGF) signaling.
We provide clear evidence in support of dbx1b marking the progenitor populations that give rise to the dorsal habenulae. In addition, the expression of dbx1b in the dorsal diencephalon is tightly controlled by FGF signaling.
KeywordsAttention Deficit Hyperactivity Disorder Attention Deficit Hyperactivity Disorder Fibroblast Growth Factor Signaling Homeodomain Transcription Factor Habenular Nucleus
actin, beta 2
attention deficit hyperactivity disorder
amine oxidase, copper containing 1
bacterial artificial chromosome
Ca2+-dependent secretion activator 2
chemokine (C-X-C motif), receptor 4b
developing brain homeobox 1b
ELAV like neuron-specific RNA binding protein 3
eomesodermin homolog a
fibroblast growth factor
green fluorescent protein
Institutional Animal Care and Use Committee
potassium channel tetramerization domain containing 12
4-nitro blue tetrazolium
orthodenticle homolog 5
POU domain, class 4, transcription factor 1 (also called Brain-specific homeobox/POU domain protein 3a, brn3a)
wingless-type MMTV integration site family, member 1
zona limitans intrathalamica
The habenular nuclei (habenulae) develop in the dorsal diencephalon of vertebrates. These bilaterally paired nuclei receive inputs from the limbic system and basal ganglia and send outputs to dopaminergic and serotonergic centers. Despite their small size, these nuclei play crucial roles in regulating aversion and reward behaviors . Moreover, the fact that the habenulae are a nexus for monoamine circuits highlights the importance of this brain region for studies of neuromodulation and multi-circuit integration.
The habenular nuclei can be divided into two functionally distinct subnuclei alternately referred to as medial and lateral (mammals) or dorsal and ventral (zebrafish) subnuclei. Recent study of the habenulae has largely focused on the ventral habenulae, but interest in the dorsal habenulae is increasing . Alterations in dorsal habenular structure and function during development have been linked to dopamine receptor expression as well as impulsivity, attention, aversion and spatial memory endophenotypes in rodents [3, 4]. In zebrafish, the dorsal habenulae regulate fear and aversion [5, 6]. Together these studies implicate the dorsal habenulae as a possible mediator of attention deficit hyperactivity disorder (ADHD), depression and anxiety . Interestingly, the dorsal habenulae are also sites of intense acetylcholine receptor and transporter expression and control nicotine intake [7, 8, 9]. This raises the possibility of targeting the habenulae as a therapeutic intervention to nicotine addiction. Beyond translational research, the zebrafish habenulae also serve as an excellent model to study the basic mechanisms underlying the development of left-right brain asymmetry. Unlike the mammalian habenulae, teleost dorsal habenulae are robustly asymmetric in anatomy, gene expression and functional connectivity .
Interest in how the dorsal habenulae integrate into cholinergic and monoaminergic circuitry has put pressure on researchers to understand dorsal habenular development. Indeed, Beretta et al.  have shown that dorsal and ventral habenular neurons arise from distinct progenitor populations. While progress has been made in describing habenular neurogenesis, differentiation and elaboration of processes, there are no known markers for habenular progenitors [12, 13, 14, 15, 16]. Therefore, finding marker genes that label dorsal habenular progenitors will be fundamental to studying how the diverse set of dorsal habenular neurons are generated and integrated into neural circuits underpinning aversive behavior as well as pathological addictive and depressive behaviors.
The dbx homeodomain transcription factors play a central role in regulating progenitor status in several regions in the developing central nervous system, including the spinal cord [17, 18]. However, the upstream regulatory pathways that regulate dbx gene-family expression are not known. Here we report that in zebrafish, dbx1b is expressed in the dorsal diencephalon where it marks dorsal habenular progenitors, and further, that dorsal diencephalic expression of dbx1b is controlled by fibroblast growth factor (FGF) signaling.
Results and discussion
Dbx1b is expressed in the presumptive habenulae
During our ongoing efforts to characterize transcription factors (TFs) that are expressed in the dorsal diencephalic region between 24 and 48 hours post-fertilization (hpf), we focused on a family of homeodomain-containing TFs encoded by the dbx genes because of their known roles in neural progenitors. There are three dbx genes in the zebrafish genome, and we carefully examined the expression pattern of the two dbx1 paralogs, dbx1a and dbx1b. We excluded dbx2 from our study because its expression has been detailed previously . At 28 hpf, dbx1a and dbx1b showed similar yet distinct expression patterns (Additional file 1). As shown previously , dbx1a was expressed in sharply restricted domains in the diencephalon with prominent expression in the prethalamus and thalamus (Additional file 1: panel A). Expression of dbx1a and dbx1b was similar in the prethalamic region, but in the thalamic region dbx1b was expressed at a much lower level than dbx1a (Additional file 1; panel C). A more striking difference between the patterns of these two paralogs was the expression of dbx1b in the dorsal diencephalon, where dbx1a was completely absent (compare arrowheads in Additional file 1). Expression of dbx1b was excluded from the otx5-positive pineal complex, the other major structure of the dorsal diencephalon (Additional file 1, panel C). These data suggested that dbx1b could be an early molecular marker for the presumptive habenulae.
Dbx1b labels dorsal habenular progenitors
FGF signaling is required for proper development of the dorsal habenulae
This report describes the expression of dbx1b, which we believe is the first reported marker of the neuronal progenitors that give rise to the dorsal habenulae. In addition, we found that FGF signaling controls the expression of dbx1b in the dorsal diencephalon. Together with other existing genetic tools, including various dbx1b BAC transgenic lines, our discovery of dbx1b as a habenular progenitor marker will not only allow for more detailed and nuanced investigation of dorsal habenular development, but also provide an exciting way forward to study proliferation, specification and circuit formation of the diverse neuronal populations in the habenular nuclei, and how these processes influence developmental and adult habenulae-mediated behaviors.
Zebrafish maintenance and strains
Zebrafish were raised at 28.5°C on a 14/10 hour light/dark cycle and staged according to hours post-fertilization. The following fish lines were used: the wildtype strain AB*, TgBAC[dbx1b:Cre-mCherry] nns13a  and Tg[−10actb2:LOXP-mCherry-LOXP-nlsEGFP] pd31 . All experiments were approved by the Vanderbilt University’s Institutional Animal Care and Use Committee (IACUC) and Office of Animal Welfare, and performed according to national regulatory standards.
Whole-mount in situ hybridization
Whole-mount RNA in situ hybridization was performed as described previously , with one change: 5% dextran sulfate was added to the hybridization buffer to enhance hybridization specificity . Hybridized probes were detected using alkaline phosphatase conjugated antibodies (Roche, Indianapolis, IN, USA) and visualized by 4-nitro blue tetrazolium (NBT; Roche) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP; Roche) staining for single colorometric labeling, or by NBT/BCIP followed by iodonitrotetrazolium (INT) and BCIP staining for double colorometric labeling. dbx1a probe  was produced from pCRII-dbx1a plasmid linearized by EcoRV and transcribed by SP6 RNA polymerase. dbx1b probe  using pCRII-dbx1b, BamHI and T7 RNA polymerase, and otx5 using pBS-otx5, Not1 and T7 RNA polymerase.
Whole-mount fluorescent in situ hybridization and immunohistochemistry
Whole-mount fluorescent in situ hybridization and immunohistochemical co-labeling was performed as described previously , with the following additional reagents: in addition to Fast Red substrate (F4648; Sigma-Aldrich, St Louis, MO, USA), some experiments used 3 × 5 minute washes in Fast Blue Buffer  and were developed in Fast Blue Substrate (0.25 mg/mL Fast Blue Substrate and 0.25 mg/mL nAMP in Fast Blue Buffer) diluted in Fast Blue Buffer. In addition to the anti-digoxigenin (DIG) antibody, the primary antibodies used were rabbit anti-pHH3 (1:500, EMD Millipore, Billerica, MA, USA), mouse anti-HuC (1:400, Life Technologies, Carlsbad, CA, USA), rabbit anti-GFP (1:500, Torrey Pines Biolab, Secaucus, NJ, USA), rabbit anti-Kctd12.1 and rabbit anti-Kctd12.2 (1:300, see ). Primary antibody was detected using goat-anti-rabbit or goat-anti-mouse antibodies conjugated to Alexa 488, Alexa 568 or Alexa 633 fluorophores (1:300, Molecular Probes, Eugene, OR, USA).
Double fluorescent in situ hybridization was performed with the following modifications to the above colorometric in situ protocol: after hybridization of DIG and fluorescein-labeled probes, anti-DIG antibody was applied (1:5,000, Roche) overnight at 4°C. The following day, embryos were washed 4 × 20 minutes in PBS with Triton (PBSTr) and 3 × 5 minutes in Fast Blue Buffer and developed in Fast Blue Substrate diluted in Fast Blue Buffer. After color development, embryos were washed 2 × 10 minutes in PBSTr. The alkaline phosphatase was acid inactivated by a 10-minute wash in 0.1 M glycine HCl, pH 2.0. After 2 × 10 minute PBSTr washes, embryos were incubated in anti-fluorescein antibody (1:1,000, Roche) overnight at 4°C. The following day, color was developed in Fast Red substrate as in Doll et al. . cxcr4b probe was generated with EcoRV and SP6 RNA polymerase.
For whole-mount in situ hybridizations and antibody labeling, embryos were incubated in their chorions in 12 μM (for complete receptor inhibition; ) of SU5402 (Tocris Bioscience, Bristol, UK) dissolved in 0.3% dimethyl sulfoxide (DMSO) in egg water supplemented with 0.003% N-phenylthiourea (PTU; Sigma-Aldrich) to prevent melanin formation. Control embryos were treated with 0.3% DMSO in parallel with their SU5402-treated siblings. Embryos were either fixed immediately following treatment or SU5402/DMSO was washed off with 5 × 5 minute egg water before being returned to egg water with PTU to develop to the desired stage for fixation.
All samples were cleared in a glycerol series (50%, 100%). Colorometric in situ images were captured on a Leica DM6000 B compound microscope (Leica Microsystems, Buffalo Grove, IL, USA) under a 20× air objective in bright field conditions. Fluorescent images were captured on a PerkinElmer spinning disk confocal microscope (PerkinElmer, Waltham, MA, USA) or a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss Microscopy, Oberkochen, Germany) with a 40× oil-immersion objective and analyzed with Volocity software (Improvision/PerkinElmer, Waltham, MA, USA).
We would like to thank Gamse lab members for helpful discussions. We acknowledge the SC Vanderbilt University Fish Facility Research Assistants for excellent fish care. Experiments were performed in part through the use of the VUMC Cell Imaging Shared Resource (supported by NIH grants CA68485, DK20593, DK58404, HD15052, DK59637 and EY08126). This work was supported by Public Health Service award T32 GM07347 from the National Institute of General Medical Studies for the Vanderbilt Medical-Scientist Training Program to BJD, NIH grants F32HD069148 to SW, and R01HD054534 to JTG.
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