The zebrafish prospero homolog prox1 is required for mechanosensory hair cell differentiation and functionality in the lateral line
- Anna Pistocchi†1,
- Carmen G Feijóo†2, 3,
- Pablo Cabrera2,
- Eduardo J Villablanca4,
- Miguel L Allende2Email author and
- Franco Cotelli1Email author
© Pistocchi et al; licensee BioMed Central Ltd. 2009
Received: 14 May 2009
Accepted: 30 November 2009
Published: 30 November 2009
The lateral line system in zebrafish is composed of a series of organs called neuromasts, which are distributed over the body surface. Neuromasts contain clusters of hair cells, surrounded by accessory cells.
In this report we describe zebrafish prox1 mRNA expression in the migrating primordium and in the neuromasts of the posterior lateral line. Furthermore, using an antibody against Prox1 we characterize expression of the protein in different cell types within neuromasts, and we show distribution among the supporting cells and hair cells.
Functional analysis using antisense morpholinos indicates that prox1 activity is crucial for the hair cells to differentiate properly and acquire functionality, while having no role in development of other cell types in neuromasts.
The lateral line of fish and amphibians comprises a set of sensory organs, the neuromasts, arranged on the head and body surface in a species-specific pattern [1, 2]. Within each neuromast there is a centrally located cluster of mechanosensory cells, the hair cells, which are functionally and morphologically equivalent to the mechanosensory hair cells of the vertebrate inner ear . The hair cells are surrounded by a group of accessory cells of at least two types: mantle cells and supporting cells [4, 5]. The hair cells can be evidenced easily in live fish because they incorporate fluorescent styryl dyes [6, 7] or by labeling with anti-acetylated tubulin antibody .
The posterior lateral line (PLL) in the zebrafish larva consists of a single line of neuromasts running along the horizontal myoseptum of the trunk and tail; the neuromasts are innervated by afferences from the PLL ganglion located behind the otic vesicle. The neuromasts are deposited by the migration of a posterior lateral line placodal primordium (PLLP), from 20 until 42 hours post fertilization (hpf) . By 72 hpf the pattern of neuromasts is complete: five to six neuromasts along each side of the body plus an additional cluster of two to three neuromasts at the end of the tail.
The prox1 homeobox gene is the vertebrate homolog of prospero in Drosophila melanogaster that is responsible for neuronal/glial fate of sibling cells during Drosophila embryonic development [10, 11]. Prospero/Prox1 protein can act as transcriptional activator or repressor, depending on the target gene and subcellular distribution [12–14]. The protein structure is highly conserved in insects and vertebrates and contains both a nuclear localization signal (NLS) and a nuclear export signal (NES), regulated by a Prospero domain [15, 16]. Several studies demonstrated that Prospero/Prox1 subcellular distribution can be either cytoplasmatic or nuclear, depending on the cell fate [11, 15, 16]. In fact, there is a direct correlation between Prox1, cell cycle regulation and cell fate specification during the development of several vertebrate organs such as the inner ear , liver , lens , lymphatic system [20, 21], gustatory system , and central nervous system [23–25]. In the chick inner ear, Prox1 labels dividing progenitor supporting cells that are fated to become hair cells . Thus, it is of interest to determine whether this gene is also expressed in the mechanosensory cells of the fish lateral line system.
Here, using in situ hybridization techniques in zebrafish embryos and larvae, we demonstrate that prox1 mRNA is expressed only in the PLLP and recently deposited neuromasts. Furthermore, we characterize Prox1 protein expression in 48 and 96 hpf fish using immunohistochemistry with an anti-Prox1 antibody in combination with other markers or transgenic lines expressing GFP in the diverse cell types of the PLL. Finally, we investigate the functional role of prox1 in PLL development by means of morpholino- and mRNA- microinjection to achieve loss- and gain-of-function, respectively. We show that prox1 does not participate in development of accessory cell types in the lateral line system, nor is it involved in the first stages of hair cell specification. However, we provide evidence that loss of prox1 function results in defects in hair cell differentiation, suggesting that it is a critical transcription factor for sensory function.
Results and discussion
prox1expression in the lateral line primordium and neuromasts
To more precisely analyze the expression of the prox1 product in the lateral line system, we used an antibody against Prox1  to carry out immunohistochemistry in zebrafish embryos and larvae. Prox1 protein expression had been described in cavefish lateral line hair cells  and in the lateral line primordium in zebrafish . As previously shown by Roy and collegues , our initial immunostaining experiments confirmed that expression of Prox1 is detected extensively in muscle cells (not shown), which prevented us from clearly distinguishing the label in the overlying lateral line. Thus, in order to visualize expression in neuromasts, we used reduced amounts of detergent during immunolabeling to preclude penetrance of the antibody; in this fashion, we were able to obtain specific staining of superficially located cells (such as neuromast cells) without labeling the muscle cells (Fig. 1C and 1E). Prox1 expression was detected in few cells in each deposited neuromasts at 48 hpf (Fig. 1C-D) and 96 hpf (Fig. 1E-F), with the number of labeled cells increasing at the later developmental timepoint. At 48 hpf, immunolabel is seen in a small group of centrally located cells (4-8 cells) suggesting that expression occurs predominantly in mechanosensory hair cells and/or their precursors (Fig. 1D). At 96 hpf, the cluster of labeled cells is larger (6-12 cells) and we often observed labeling in more peripheral cells (arrows in Fig. 1F). Since the number of hair cells at this timepotint is, on average, around 10-12 , expression of Prox1 is likely to occur predominantly in hair cells. To confirm expression of Prox1 in hair cells, we perfomed immunostains against Prox1 in pou4f1::GFP transgenic larvae. This transgenic line carries a DNA construct that directs cytoplasmatic Green Fluorescent Protein (GFP) to hair cells, at different stages of their differentiation process . Comparison of immunostaining (red label) and GFP expression (green label) at 96 hpf shows that Prox1 positive cells coincide, for the most part, with GFP-labeled cells (Fig. 1G-I). Most peripheral cells of the neuromast (labeled with DAPI in Fig. 1I) do not show staining. Prox1 label is seen in mature hair cells (strong GFP expressing cells in the center of the cluster) as well as in immature hair cells (weak GFP labeled cells). We conclude that Prox1 is predominantly expressed in cells that are committed to the hair cell lineage and in differentiating hair cells.
Our results show that prox1 mRNA is expressed at high levels during development of the lateral line system, but then diminishes as the system matures. Despite this reduction in mRNA expression, we observe strong protein label when using the anti-Prox1 antibody after neuromast deposition and in a group of centrally located cells as the neuromast matures. Therefore, high levels of protein expression follow a temporally distinct pattern to mRNA expression and could indicate that prox1 mRNA is short lasting while the protein is stable, at least in hair cells. More work will be required to determine whether this is indeed the case.
Our interpretation of the expression pattern of Prox1 protein is that it is likely to be expressed in a group of precursor cells, supporting cells, and in differentiating hair cells (Fig. 2F). After differentiation, Prox1 becomes down-regulated as it is not observed in all mature hair cells (Fig. 2E). Whether Prox1 expression is a marker for immediate hair cell progenitors that are fated to become hair cells , as occurs in the chick inner ear , will require further analysis. Studies performed in other species have demonstrated that Prox1 promotes terminal mitoses. For example in the ganglion mother cell (GMC) of Drosophila, the prox1 homolog Prospero, represses positive regulators of the cell cycle and diminishes mitotic activity [38, 39]. Moreover, in the lens of Prox1 null mice, cells fail to correctly exit the cell cycle because of the delayed expression of negative regulators such as p27kip1, and their differentiation is altered .
prox1loss- and gain-of-function experiments
We sought to learn whether prox1 is important for PLL development in the zebrafish. We prevented translation of the gene by injecting, into one-cell stage embryos, 8 ng of a specific ATG-targeted antisense morpholino oligonucleotide (prox1 MO) that has been previously described [25, 40, 41]. The efficacy of the morpholino was tested with Prox1 immunostain experiments that show reduction of the protein levels in morphants compared to contro injected fish [see Additional file 1]. Identical results were obtained by injecting the ATG-targeted morpholino and a splice site morpholino, splice prox1 MO, indicating that the effect is specific to prox1 loss of function (data not shown). Control fish were injected with 8 ng of a non-specific morpholino which did not elicit a phenotype. As an additional functional assay, we microinjected prox1 mRNA in the same fashion to determine whether a gain-of-function experiment would be indicative of the role of this gene in zebrafish.
Overall, our studies reveal that prox1 mRNA and protein are expressed in the migrating PLLP and in deposited neuromasts, in particular in the progenitor/supporting cell layer and in hair cells. It is noteworthy that Prox1 protein levels and distribution were distinct from mRNA distribution suggesting that regulation of this gene at the transcriptional and posttranscriptional levels may be highly dynamic. We relied on gene inactivation and overexpression to analyze the role of prox1 during PLLP migration, neuromast deposition and differentiation. Interestingly, primordium migration and deposition, and differentiation of most cells types are not controlled by this gene. In other model systems, the presence and activity of Prox1 in progenitor cells directs cell fate selection: while cells with high Prox1 levels become hair cells, cells with low Prox1 levels acquire supporting cell or other fates. Interestingly, Prox1 protein localization in supporting cells may play a role in the switch from proliferation to differentiation that leads to the development of functional hair cells. In fact, in other organisms, nuclear accumulation of Prospero/Prox1 protein has been argued to regulate genes specific for the differentiated state, while in proliferating cells the protein remains in the cytoplasm [11, 15, 16]. In our study, prox1 loss-of-function causes defects in the functionality of hair cells in the neuromasts, as assayed by incorporation of DiAsp, a vital dye that is likely to enter hair cells through the mechanotransduction channel [6, 42–44]. However, GFP expression driven by a regulatory element active during initial stages of hair cell differentiation is not affected by absence of Prox1. Therefore, initial hair cell specification does not appear to require prox1, but only terminal differentiation. It will be of interest to dissect the exact molecular players that are regulated by this gene in the zebrafish.
Fish and embryo maintenance
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. Embryos used in whole-mount in situ hybridization were raised in 0,003% PTU (Sigma) to prevent pigmentation. We express the embryonic ages in hours post fertilization (hpf) or days post fertilization (dpf). The transgenic lines used in this study are SqET20 ; claudinB::GFP , SCM1 , pou4f1::GFP  and pou4f3::GFP . Zebrafish (Danio rerio) were raised and maintained in agreement with local and national sanitary regulations.
Whole-mount in situhybridization 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 (PBS), then rinsed with PBS-Tween, dehydrated in 100% methanol and stored at -20°C until processed for WISH . Antisense riboprobes were previously in vitro labeled with modified nucleotides (i.e. digoxigenin, fluorescein, Roche). For immunohistochemistry, the following antibodies were used: rabbit anti-Prox1 (Chemicon AB5475) at a dilution of 1:250; mouse anti-GFP (Chemicon MAB3580) at a dilution of 1:200, Alexa Fluor 594 rabbit (Invitrogen A31632) at a dilution of 1:200; Alexa Fluor 488 mouse (Invitrogen A11029) at a dilution of 1:200.
Loss- and gain-of-function analysis
For loss- and gain-of-function experiments, specific prox1 morpholino (prox1 MO) and capped RNA were injected as previously described . Two prox1 morpholinos were designed to knockdown translation of the Prox1 protein. prox1 MO was directed against the translation initiation region of the mRNA (5'-ATGTGCTGTCATGGTCAGGCATCAC-'3) while prox1 MO splice was designed to bind to the donor splice site between exon 2 and intron 3 (5'-GGAACCTAGCCAGAAAGAAAGGACT-'3). Both were injected at a concentration of 8 ng into one-cell stage embryos.
The neuromast hair cells were labeled in live embryos or larvae with 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (Di-Asp; Sigma D3418, USA) as described in Collazo et al. . For live staining, 48-72-hpf larvae were incubated in 5 mM Di-Asp in embryo medium for 5 min and then rinsed with fresh medium and visualized under fluorescent light in a dissection microscope. For carrying out statistical tests we counted Di-Asp-stained hair cells in the P1 neuromast (see neuromast nomenclature in Harris and collegues  on one side of each larva. To determine significance of differences, we used the Student's t test (SigmaStat 3.1).
We thank Catalina Lafourcade and Florencio Espinoza for technical help; Herwig Baier and Hitoshi Okamoto for the pou4f3::GFP and pou4f1::mGFP transgenic fish, respectively; Kenna Shaw and Brant Weinstein for the SCM1 line; Vladimir Korzh for SqET20; Darren Gilmour for ClaudinB::GFP. MA and CGF were supported by grants from FONDECYT (1070867), ICM (P06-039F) and UNAB (DI23-08/R). AP and FC were supported by grants from CARIPLO N.O.B.E.L. Molecular and cellular biology of tumor stem cell.
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