Crucial role of zebrafish prox1in hypothalamic catecholaminergic neurons development
- Anna Pistocchi1,
- Germano Gaudenzi1,
- Silvia Carra1,
- Erica Bresciani1,
- Luca Del Giacco†1Email author and
- Franco Cotelli†1
© Pistocchi et al; licensee BioMed Central Ltd. 2008
Received: 20 September 2007
Accepted: 10 March 2008
Published: 10 March 2008
Prox1, the vertebrate homolog of prospero in Drosophila melanogaster, is a divergent homeogene that regulates cell proliferation, fate determination and differentiation during vertebrate embryonic development.
Here we report that, in zebrafish, prox1 is widely expressed in several districts of the Central Nervous System (CNS). Specifically, we evidenced prox1 expression in a group of neurons, already positive for otp1, located in the hypothalamus at the level of the posterior tuberculum (PT). Prox1 knock-down determines the severe loss of hypothalamic catecholaminergic (CA) neurons, identified by tyrosine hydroxylase (TH) expression, and the synergistic prox1/otp1 overexpression induces the appearance of hypothalamic supernumerary TH-positive neurons and ectopic TH-positive cells on the yolk epitelium.
Our findings indicate that prox1 activity is crucial for the proper development of the otp1-positive hypothalamic neuronal precursors to their terminal CA phenotype.
The catecholaminergic neurons of the CNS of vertebrates participate in a wide variety of tasks, including motor coordination, mood regulation, and cognitive function, among others. Neurotransmitters catecholamines (CA), namely Dopamine (DA), Adrenaline (AD), and Noradrenaline (NA), are neuroactive molecules that exert strong influence on vertebrates behavior  and serve a variety of central and peripheral functions .
Embryological studies indicate that several extracellular signals, as Hedgehog and FGF, are vital to define the development of the prosencephalic CA neurons [3–7]. The homeodomain transcription factor Orthopedia (Otp), regulated by such signaling pathways , is crucial in restricting the fate of multiple classes of secreting neurons in the neuroendocrine hypothalamus of vertebrates [9, 10]. Specifically, Otp is required for the correct differentiation of the CA neurons positioned in the zebrafish Posterior Tuberculum (PT) and hypothalamus [8, 11]. Despite all these evidences, the role of specific transcription factors leading to the proper differentiation of the hypothalamic CA neurons remains largely unclear .
Prox1 homeobox gene is the vertebrate homologous of prospero in Drosophila melanogaster. During Drosophila embryonic development, prospero is expressed in neuronal precursors and determines the neuronal/glial fate of sibling cells [12, 13]. prospero/Prox1's high level of homology pinpoints possible functional conservation through evolution, suggesting Prox1 involvement in vertebrate cell fate determination. Indeed, also during murine brain development, Prox1 is expressed in most of the locations in which neurogenesis and glial formation occur during middle and late prenatal and postnatal stages, as the subventricular zone, several regions of the prethalamus and hypothalamus, the cerebellum, and the hippocampus .
Here, we demonstrated that, in zebrafish, prox1 is widely expressed in the developing CNS, and one of its expression domains is located in the area corresponding to the ventral part of the PT and the adjacent hypothalamic district, the area hosting the cluster of CA neurons positive for otp1 expression [8, 11]. Moreover, we took advantage of the zebrafish animal model to investigate the in vivo influence of prox1 on hypothalamic CA neuronal development by means of morpholino- and mRNA- loss and gain of function methodologies.
We provide evidence that prox1 is required for the development of hypothalamic neuronal progenitors into mature CA neurons.
Results and discussion
Homeobox genes are expressed in a temporal and spatial restricted manner and play crucial roles for cell type specification [15, 16]. Zebrafish prox1 is a divergent homeodomain transcription factor whose homologues in Drosophila and mice regulate cell proliferation, fate determination and differentiation in CNS and sensory tissues [17–21]. Noteworthingly, during murine brain development, Prox1 is also expressed in the hypothalamus , where several CA neurons differentiate.
Spatio-temporal expression of prox1 during embryogenesis
prox1 is required for the development of a group of hypothalamic CA neurons
The determination of the neurotransmitter phenotype is an important aspect of neuronal differentiation. Degeneration of substantia nigra DA neurons in humans is a hallmark of Parkinson's disease, and the malfunction of CA neurons in other brain regions is implicated in psychiatric disorders and neuroendocrine dysregulation [26–28].
In order to address whether prox1 is involved in neurogenesis processes, we analyzed the expression pattern of the proneural gene ngn1 . ngn1 expression domains resulted unaffected in prox1 MO injected embryos, allowing us to conclude that loss of CA neurons in the hypothalamus of prox1 MO injected embryos is not caused by alteration in neurogenesis (see Additional file 5). Moreover, in order to address this issue, we analyzed the development of other neurotransmitter-producing neurons. Specifically, the neighboring serotonergic neurons appeared only slightly affected by Prox1 ablation in comparison to the most relevant effects we observed in the CA population (see Additional file 6). However, our description of prox1 effects on CA neuron development cannot rule out its potential involvement in the differentiation or fate determination of other neuronal types.
prox1 functions are required for proper otp1 and TH phenotypes in the hypothalamic area
Overexpression of prox1 and otp1 together leads to supernumerary CA neurons in the ventral diencephalon and TH positive cells on the yolk surface
In conclusion, we highlight for the first time the role of prox1 in the proper development of the CA neurons in the ventral diencephalon. Moreover, we provide evidence of regulatory links between prox1 and otp1 genes in defining the terminal TH phenotype in the hypothalamus. The identification of prox1 as a key component in the differentiation of hypothalamic CA neurons will help in clarifying the developmental bases of several human behavioral aspects as well as pathologies such as addictions and Parkinson's disease.
Breeding wild type fish of the AB strain were maintained at 28°C on a 14 h light/10 h dark cycle. Embryos were collected by natural spawning, staged according to Kimmel and colleagues , and raised at 28°C in fish water (Instant Ocean, 0,1% Methylene Blue) in Petri dishes. We express the embryonic ages in somites (s), hours post fertilization (hpf) and days post fertilization (dpf).
Total RNA from 17 samples (an average of 30 embryos per sample) corresponding to 10 different developmental stage embryos (1–2 cells, 30% epiboly, 50% epiboly, 80% epiboly, tailbud, 8 s, 15 s, 24 hpf, 72 hpf, and 5 dpf) and 7 adult organs (testis, ovary, gills, gut, eyes, brain, and liver) was extracted with the TOTALLY RNA isolation kit (Ambion), treated with RQ1 RNase-Free DNase (Promega) and oligo(dT)-reverse transcribed using SuperScript II RT (Invitrogen), according to manufacturers' instructions. The following primers were used for PCR reactions: prox1_sense 5'-ACCTCAGCCACCATCGTTCCATC-3' and prox1_antisense 5'-CACTATTCATGCAGAAGCTCCTGC-3'. PCR products were loaded and resolved onto 2% agarose gels.
In situ hybridization and immunohistochemistry
Whole mount in situ hybridization (WISH), was carried out as described  on embryos fixed for 2 h in 4% paraformaldehyde/phosphate buffered saline, then rinsed with PBS-Tween, dehydrated in 100% methanol and stored at -20°C until processed for WISH . Antisense riboprobes were previously in vitro labelled with modified nucleotides (i.e. digoxigenin, fluorescin, Roche). For histological sections, stained embryos were re-fixed in 4% PFA, dehydrated and stored in methanol, wax embedded and sectioned (5 μm). For immunohistochemistry, embryos were exposed to rabbit anti-Tyrosine Hydroxilase (anti-TH) (Chemicon), or rabbit anti-Serotonin (anti-5HT) (Chemicon), then treated with biotinylated or fluorescent secondary antibody (Vector Laboratories).
Quantitative real time RT-PCR
Reverse transcriptions (RTs) were performed using 2 μg of DNase treated (DNA-free™, Ambion Inc) total RNA in presence of random hexamers (Invitrogen™) and SuperScript II reverse transcriptase (Invitrogen™). Real-time PCRs were carried out in a total volume of 15 μl containing 1× iQ SYBR Green Super Mix (BioRad), using 1 μl of the RT reaction. PCRs were performed using the BioRad iCycler iQ Real Time Detection System (BioRad Laboratories). For normalization purposes, 18S ribosomal RNA level was tested in parallel with the gene of interest. The following primers were used:
For TUNEL assay, 24 and 36 hpf embryos were fixed with 4% PFA for 2 h at room temperature. Embryos were permeabilized with methanol at -20°C and washed twice with PBC (0.001% Triton ×-100, 0.1% sodium citrate in PBS) for 10 minutes. Labeling for apoptotic cells was performed using In situ Cell Death Detection Kit (Roche). The embryos were incubated at 37°C for 1 h, washed and mounted for fluorescent microscopic imaging.
Injections were carried out on 1- to 2-cell stage embryos; the dye tracer rhodamine dextran was also coinjected. Synthetic capped prox1 or otp1 mRNA were injected repeatedly (n > 3) at concentrations of 450 pg and 300 pg per embryo, respectively. Double mRNA injection was performed with 450 pg prox1 mRNA and 300 pg otp1 mRNA per embryo; 450 pg of synthetic capped GFP mRNA was injected as control. To repress prox1 mRNA translation, an ATG-targeting morpholino was synthesized (Gene Tools, LLC): 5'-ATGTGCTGTCATGGTCAGGCATCAC-3' [32, 33]. prox1 MO was used at the concentration of 1 pmole in 1× Danieau buffer (pH 7,6) as previously reported . To repress otp1 mRNA translation, an ATG-targeting morpholino was designed (Gene Tools, LLC): 5'-CCAAGAGGTCGGCATGAGAGAGCAT-3' . otp1 MO was used at the concentration of 0,7 pmole. As control we injected a standard control morpholino oligonucleotide (stdr MO). Double prox1 MO/otp1 mRNA was performed with 1 pmole of MO and 300 pg of synthetic mRNA per embryo.
We thank P. Sordino and M. Beltrame for the reading and critical comments on the manuscript. AP thanks S. Cimbro and C. Fognani for their priceless support. This work was supported by grants from "CARIPLO N.O.B.E.L." (FC).
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