BACKGROUND
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  <cit> .
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  <cit> .
the hair cells are surrounded by a group of accessory cells of at least two types: mantle cells and supporting cells  <cit> .
the hair cells can be evidenced easily in live fish because they incorporate fluorescent styryl dyes  <cit>  or by labeling with anti-acetylated tubulin antibody  <cit> .
the posterior lateral line  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 , from  <dig> until  <dig> hours post fertilization   <cit> .
by  <dig> 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 prox <dig> 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  <cit> .
prospero/prox <dig> protein can act as transcriptional activator or repressor, depending on the target gene and subcellular distribution  <cit> .
the protein structure is highly conserved in insects and vertebrates and contains both a nuclear localization signal  and a nuclear export signal , regulated by a prospero domain  <cit> .
several studies demonstrated that prospero/prox <dig> subcellular distribution can be either cytoplasmatic or nuclear, depending on the cell fate  <cit> .
in fact, there is a direct correlation between prox <dig>  cell cycle regulation and cell fate specification during the development of several vertebrate organs such as the inner ear  <cit> , liver  <cit> , lens  <cit> , lymphatic system  <cit> , gustatory system  <cit> , and central nervous system  <cit> .
in the chick inner ear, prox <dig> labels dividing progenitor supporting cells that are fated to become hair cells  <cit> .
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 prox <dig> mrna is expressed only in the pllp and recently deposited neuromasts.
furthermore, we characterize prox <dig> protein expression in  <dig> and  <dig> hpf fish using immunohistochemistry with an anti-prox <dig> 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 prox <dig> in pll development by means of morpholino- and mrna- microinjection to achieve loss- and gain-of-function, respectively.
we show that prox <dig> 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 prox <dig> function results in defects in hair cell differentiation, suggesting that it is a critical transcription factor for sensory function.
RESULTS
prox <dig> expression in the lateral line primordium and neuromasts
a previous description of the prox <dig> mrna expression pattern in zebrafish revealed that the gene is expressed, among other tissues, in the pll system  <cit> .
in zebrafish, the embryonic pllp begins its migration at  <dig> hpf and reaches the tip of the tail at about  <dig> hpf.
prox <dig> mrna is detected during the entire journey of the migrating pllp and shortly after deposition of the neuromasts .
proneuromasts  also express prox <dig> mrna, specifically in a group of cells at the center of the cell cluster, including the location where the hair cells will eventually arise .
prox <dig> mrna was not detected after  <dig> hpf, indicating a strong reduction in mrna levels beginning at this time point.
to more precisely analyze the expression of the prox <dig> product in the lateral line system, we used an antibody against prox <dig>  <cit>  to carry out immunohistochemistry in zebrafish embryos and larvae.
prox <dig> protein expression had been described in cavefish lateral line hair cells  <cit>  and in the lateral line primordium in zebrafish  <cit> .
as previously shown by roy and collegues  <cit> , our initial immunostaining experiments confirmed that expression of prox <dig> is detected extensively in muscle cells , 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  without labeling the muscle cells .
prox <dig> expression was detected in few cells in each deposited neuromasts at  <dig> hpf  and  <dig> hpf , with the number of labeled cells increasing at the later developmental timepoint.
at  <dig> hpf, immunolabel is seen in a small group of centrally located cells  suggesting that expression occurs predominantly in mechanosensory hair cells and/or their precursors .
at  <dig> hpf, the cluster of labeled cells is larger  and we often observed labeling in more peripheral cells .
since the number of hair cells at this timepotint is, on average, around 10- <dig>  <cit> , expression of prox <dig> is likely to occur predominantly in hair cells.
to confirm expression of prox <dig> in hair cells, we perfomed immunostains against prox <dig> in pou4f1::gfp transgenic larvae.
this transgenic line carries a dna construct that directs cytoplasmatic green fluorescent protein  to hair cells, at different stages of their differentiation process  <cit> .
comparison of immunostaining  and gfp expression  at  <dig> hpf shows that prox <dig> positive cells coincide, for the most part, with gfp-labeled cells .
most peripheral cells of the neuromast  do not show staining.
prox <dig> label is seen in mature hair cells  as well as in immature hair cells .
we conclude that prox <dig> is predominantly expressed in cells that are committed to the hair cell lineage and in differentiating hair cells.
our results show that prox <dig> 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-prox <dig> 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 prox <dig> 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.
to more accurately localize prox <dig> protein expression to specific neuromast cells, we carried out immunostaining using additional transgenic zebrafish lines, which express gfp in the different cell types in the lateral line system .
the sqet <dig> transgenic line  <cit>  labels the mantle cells in neuromasts, which surround the hair cells and provide a central opening for protrusion of kinocilia into the environment  <cit> .
visualization of both gfp and anti-prox <dig> label  shows that prox1-positive cells are contained within the ring of mantle cells, with little or no overlap between them .
therefore, prox <dig> is likely to be expressed in hair cells and possibly in underlying progenitors and/or in supporting cells, but not in mantle cells.
we next used two transgenic lines that label accessory cells in neuromasts: the claudinb::gfp line  <cit>  that labels all accessory cells  and the scm <dig> line  <cit>  that labels all supporting cells .
in these larvae, localization of the prox <dig> signal in a subset of the gfp-labeled cells is observed but, clearly, not all supporting cells express prox <dig>  to determine the extent of overlap between prox <dig> protein expression and lateral line hair cells, we used the pou4f3::mgfp line  <cit> , in which gfp is directed to the membrane of differentiated hair cells .
in the developmental stages analyzed  we observed a partial coincidence between the expression of both markers, indicating that prox <dig> protein is present in some, but not all, differentiated hair cells and is also found in other  cells.
these findings suggests that prox <dig> may be expressed in a specific progenitor cell population or during early stages of maturation of hair cells, prior to the appearance of differentiation markers.
this is consistent with the situation in the chick embryonic otocyst where cprox <dig> protein levels remain elevated in dividing sensory progenitor cells and in newly formed hair cells and supporting cells, and expression becomes down-regulated as these cells mature  <cit> .
our interpretation of the expression pattern of prox <dig> protein is that it is likely to be expressed in a group of precursor cells, supporting cells, and in differentiating hair cells .
after differentiation, prox <dig> becomes down-regulated as it is not observed in all mature hair cells .
whether prox <dig> expression is a marker for immediate hair cell progenitors that are fated to become hair cells  <cit> , as occurs in the chick inner ear  <cit> , will require further analysis.
studies performed in other species have demonstrated that prox <dig> promotes terminal mitoses.
for example in the ganglion mother cell  of drosophila, the prox <dig> homolog prospero, represses positive regulators of the cell cycle and diminishes mitotic activity  <cit> .
moreover, in the lens of prox <dig> null mice, cells fail to correctly exit the cell cycle because of the delayed expression of negative regulators such as p27kip <dig>  and their differentiation is altered  <cit> .
prox <dig> loss- and gain-of-function experiments
we sought to learn whether prox <dig> is important for pll development in the zebrafish.
we prevented translation of the gene by injecting, into one-cell stage embryos,  <dig> ng of a specific atg-targeted antisense morpholino oligonucleotide  that has been previously described  <cit> .
the efficacy of the morpholino was tested with prox <dig> immunostain experiments that show reduction of the protein levels in morphants compared to contro injected fish .
identical results were obtained by injecting the atg-targeted morpholino and a splice site morpholino, splice prox <dig> mo, indicating that the effect is specific to prox <dig> loss of function .
control fish were injected with  <dig> ng of a non-specific morpholino which did not elicit a phenotype.
as an additional functional assay, we microinjected prox <dig> mrna in the same fashion to determine whether a gain-of-function experiment would be indicative of the role of this gene in zebrafish.
analysis of lateral line development in prox <dig> loss- and gain-of-function animals was first carried out by staining the larvae with diasp, a vital stain for mature and functional hair cells  <cit> .
at  <dig> hpf, neuromasts of the primary lateral line system have been deposited and functional hair cells incorporate diasp in neuromasts of control fish.
90% of the control mo injected embryos , presented between  <dig> and  <dig> labeled neuromasts per side at this stage .
in contrast, only 15% of prox <dig> mo injected embryos showed a nearly normal number of labeled neuromasts , 65% presented a reduced number of labeled neuromasts  and 20% a complete absence of labeling  .
the effect of prox <dig> loss of function was not due to developmental delay: at  <dig> hpf, mo injected embryos still presented less neuromasts than control mo injected embryos .
gain of function experiments by means of prox <dig> mrna injection did not significantly affect diasp labeling of neuromasts .
while our results indicated that hair cell development is impaired when prox <dig> expression is reduced, it did not clarify whether the phenotype was due to defective migration of the pll primordium, aberrant deposition of neuromasts or a failure of hair cell differentiation within neuromasts.
to discriminate between these possibilities, we took advantage of the claudinb::gfp transgenic line, that expresses gfp in the migrating primordium, and we injected the control and prox <dig> morpholinos in these fish.
analysis of gfp expression in both groups of animals showed that the number of cells, shape of the primordia, and neuromast deposition were indistinguishable between them .
we fixed these fish and carried out immunostaining with an antibody against acetylated tubulin, which labels neural processes  and the differentiated hair cells, identified by means of the label in their kinocilia  <cit> .
the pll nerve, which innervates the neuromasts, is intact in morphant larvae  indicating that the pll neurons and neural process formation are unaffected by prox <dig> loss-of-function.
however, tubulin staining in differentiated hair cells was absent in morphant fish .
we were certain of the position of the neuromasts in these fish as the gfp label was still visible after immunostaining .
importantly, tunel assays  showed no differences in cell death levels, in the lateral line or elsewhere, between control and prox <dig> mo injected fish, indicating that the absence of hair cell kinociliae in morphants was not likely due to death of the hair cells after neuromast deposition.
to examine in more detail the prox <dig> morphant phenotype, we carried out injection of control and prox <dig> antisense morpholinos in the sqet <dig> and scm <dig> transgenic lines.
comparison of control and morphant neuromasts in these fish at  <dig> hpf did not show any significant differences in number and appearance of labeled cell types , indicating no essential role for prox <dig> in their development .
since we had observed loss of acetylated tubulin and diasp staining in morphant neuromasts, we examined gfp expression in pou4f3::mgfp transgenic fish injected with control and prox <dig> morpholinos.
again, in this line, the number of gfp labeled cells was not significantly altered by prox <dig> loss of function .
the pou4f <dig> promoter-enhancer directs gfp expression to developing and mature hair cells.
to distinguish fully differentiated  from immature hair cells, diasp staining should be used.
when we labeled pou4f3::mgfp transgenic control and morphant fish with diasp, a clear difference in the number of diasp labeled hair cells was observed between both conditions .
while control embryos presented 75% of the pou4f3::mgfp positive cells also positive for diasp staining, in prox <dig> mo injected embryos the percentage was only 38%, indicating that these hair cells are unable to reach full functionality in the absence of prox <dig> .
as only a fraction of pou4f3::mgfp positive cells were stained with diasp in morphant fish, we conclude that development of hair cells in morphants is arrested prior to their final differentiation and are thus unable to reach full functionality in the absence of prox <dig>  our results suggest that prox <dig> has a role in the late stages of hair cell differentiation, when they acquire the mechanotransduction capacity.
CONCLUSIONS
overall, our studies reveal that prox <dig> 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 prox <dig> 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 prox <dig> 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 prox <dig> in progenitor cells directs cell fate selection: while cells with high prox <dig> levels become hair cells, cells with low prox <dig> levels acquire supporting cell or other fates.
interestingly, prox <dig> 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/prox <dig> protein has been argued to regulate genes specific for the differentiated state, while in proliferating cells the protein remains in the cytoplasm  <cit> .
in our study, prox <dig> 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  <cit> .
however, gfp expression driven by a regulatory element active during initial stages of hair cell differentiation is not affected by absence of prox <dig>  therefore, initial hair cell specification does not appear to require prox <dig>  but only terminal differentiation.
it will be of interest to dissect the exact molecular players that are regulated by this gene in the zebrafish.

 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. 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. the hair cells can be evidenced easily in live fish because they incorporate fluorescent styryl dyes or by labeling with anti-acetylated tubulin antibody. the posterior lateral line 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, from until hours post fertilization. by 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 prox 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. prospero/prox protein can act as transcriptional activator or repressor, depending on the target gene and subcellular distribution. the protein structure is highly conserved in insects and vertebrates and contains both a nuclear localization signal and a nuclear export signal, regulated by a prospero domain. several studies demonstrated that prospero/prox subcellular distribution can be either cytoplasmatic or nuclear, depending on the cell fate. in fact, there is a direct correlation between prox cell cycle regulation and cell fate specification during the development of several vertebrate organs such as the inner ear, liver, lens, lymphatic system, gustatory system, and central nervous system. in the chick inner ear, prox 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 prox mrna is expressed only in the pllp and recently deposited neuromasts. furthermore, we characterize prox protein expression in and hpf fish using immunohistochemistry with an anti-prox 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 prox in pll development by means of morpholino- and mrna- microinjection to achieve loss- and gain-of-function, respectively. we show that prox 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 prox function results in defects in hair cell differentiation, suggesting that it is a critical transcription factor for sensory function.  prox expression in the lateral line primordium and neuromasts a previous description of the prox mrna expression pattern in zebrafish revealed that the gene is expressed, among other tissues, in the pll system. in zebrafish, the embryonic pllp begins its migration at hpf and reaches the tip of the tail at about hpf. prox mrna is detected during the entire journey of the migrating pllp and shortly after deposition of the neuromasts. proneuromasts also express prox mrna, specifically in a group of cells at the center of the cell cluster, including the location where the hair cells will eventually arise. prox mrna was not detected after hpf, indicating a strong reduction in mrna levels beginning at this time point. to more precisely analyze the expression of the prox product in the lateral line system, we used an antibody against prox to carry out immunohistochemistry in zebrafish embryos and larvae. prox 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 prox is detected extensively in muscle cells, 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 without labeling the muscle cells. prox expression was detected in few cells in each deposited neuromasts at hpf and hpf, with the number of labeled cells increasing at the later developmental timepoint. at hpf, immunolabel is seen in a small group of centrally located cells suggesting that expression occurs predominantly in mechanosensory hair cells and/or their precursors. at hpf, the cluster of labeled cells is larger and we often observed labeling in more peripheral cells. since the number of hair cells at this timepotint is, on average, around 10-, expression of prox is likely to occur predominantly in hair cells. to confirm expression of prox in hair cells, we perfomed immunostains against prox in pou4f1::gfp transgenic larvae. this transgenic line carries a dna construct that directs cytoplasmatic green fluorescent protein to hair cells, at different stages of their differentiation process. comparison of immunostaining and gfp expression at hpf shows that prox positive cells coincide, for the most part, with gfp-labeled cells. most peripheral cells of the neuromast do not show staining. prox label is seen in mature hair cells as well as in immature hair cells. we conclude that prox is predominantly expressed in cells that are committed to the hair cell lineage and in differentiating hair cells. our results show that prox 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-prox 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 prox 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. to more accurately localize prox protein expression to specific neuromast cells, we carried out immunostaining using additional transgenic zebrafish lines, which express gfp in the different cell types in the lateral line system. the sqet transgenic line labels the mantle cells in neuromasts, which surround the hair cells and provide a central opening for protrusion of kinocilia into the environment. visualization of both gfp and anti-prox label shows that prox1-positive cells are contained within the ring of mantle cells, with little or no overlap between them. therefore, prox is likely to be expressed in hair cells and possibly in underlying progenitors and/or in supporting cells, but not in mantle cells. we next used two transgenic lines that label accessory cells in neuromasts: the claudinb::gfp line that labels all accessory cells and the scm line that labels all supporting cells. in these larvae, localization of the prox signal in a subset of the gfp-labeled cells is observed but, clearly, not all supporting cells express prox to determine the extent of overlap between prox protein expression and lateral line hair cells, we used the pou4f3::mgfp line, in which gfp is directed to the membrane of differentiated hair cells. in the developmental stages analyzed we observed a partial coincidence between the expression of both markers, indicating that prox protein is present in some, but not all, differentiated hair cells and is also found in other cells. these findings suggests that prox may be expressed in a specific progenitor cell population or during early stages of maturation of hair cells, prior to the appearance of differentiation markers. this is consistent with the situation in the chick embryonic otocyst where cprox protein levels remain elevated in dividing sensory progenitor cells and in newly formed hair cells and supporting cells, and expression becomes down-regulated as these cells mature. our interpretation of the expression pattern of prox protein is that it is likely to be expressed in a group of precursor cells, supporting cells, and in differentiating hair cells. after differentiation, prox becomes down-regulated as it is not observed in all mature hair cells. whether prox 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 prox promotes terminal mitoses. for example in the ganglion mother cell of drosophila, the prox homolog prospero, represses positive regulators of the cell cycle and diminishes mitotic activity. moreover, in the lens of prox null mice, cells fail to correctly exit the cell cycle because of the delayed expression of negative regulators such as p27kip and their differentiation is altered. prox loss- and gain-of-function experiments we sought to learn whether prox is important for pll development in the zebrafish. we prevented translation of the gene by injecting, into one-cell stage embryos, ng of a specific atg-targeted antisense morpholino oligonucleotide that has been previously described. the efficacy of the morpholino was tested with prox immunostain experiments that show reduction of the protein levels in morphants compared to contro injected fish. identical results were obtained by injecting the atg-targeted morpholino and a splice site morpholino, splice prox mo, indicating that the effect is specific to prox loss of function. control fish were injected with ng of a non-specific morpholino which did not elicit a phenotype. as an additional functional assay, we microinjected prox mrna in the same fashion to determine whether a gain-of-function experiment would be indicative of the role of this gene in zebrafish. analysis of lateral line development in prox loss- and gain-of-function animals was first carried out by staining the larvae with diasp, a vital stain for mature and functional hair cells. at hpf, neuromasts of the primary lateral line system have been deposited and functional hair cells incorporate diasp in neuromasts of control fish. 90% of the control mo injected embryos, presented between and labeled neuromasts per side at this stage. in contrast, only 15% of prox mo injected embryos showed a nearly normal number of labeled neuromasts, 65% presented a reduced number of labeled neuromasts and 20% a complete absence of labeling. the effect of prox loss of function was not due to developmental delay: at hpf, mo injected embryos still presented less neuromasts than control mo injected embryos. gain of function experiments by means of prox mrna injection did not significantly affect diasp labeling of neuromasts. while our results indicated that hair cell development is impaired when prox expression is reduced, it did not clarify whether the phenotype was due to defective migration of the pll primordium, aberrant deposition of neuromasts or a failure of hair cell differentiation within neuromasts. to discriminate between these possibilities, we took advantage of the claudinb::gfp transgenic line, that expresses gfp in the migrating primordium, and we injected the control and prox morpholinos in these fish. analysis of gfp expression in both groups of animals showed that the number of cells, shape of the primordia, and neuromast deposition were indistinguishable between them. we fixed these fish and carried out immunostaining with an antibody against acetylated tubulin, which labels neural processes and the differentiated hair cells, identified by means of the label in their kinocilia. the pll nerve, which innervates the neuromasts, is intact in morphant larvae indicating that the pll neurons and neural process formation are unaffected by prox loss-of-function. however, tubulin staining in differentiated hair cells was absent in morphant fish. we were certain of the position of the neuromasts in these fish as the gfp label was still visible after immunostaining. importantly, tunel assays showed no differences in cell death levels, in the lateral line or elsewhere, between control and prox mo injected fish, indicating that the absence of hair cell kinociliae in morphants was not likely due to death of the hair cells after neuromast deposition. to examine in more detail the prox morphant phenotype, we carried out injection of control and prox antisense morpholinos in the sqet and scm transgenic lines. comparison of control and morphant neuromasts in these fish at hpf did not show any significant differences in number and appearance of labeled cell types, indicating no essential role for prox in their development. since we had observed loss of acetylated tubulin and diasp staining in morphant neuromasts, we examined gfp expression in pou4f3::mgfp transgenic fish injected with control and prox morpholinos. again, in this line, the number of gfp labeled cells was not significantly altered by prox loss of function. the pou4f promoter-enhancer directs gfp expression to developing and mature hair cells. to distinguish fully differentiated from immature hair cells, diasp staining should be used. when we labeled pou4f3::mgfp transgenic control and morphant fish with diasp, a clear difference in the number of diasp labeled hair cells was observed between both conditions. while control embryos presented 75% of the pou4f3::mgfp positive cells also positive for diasp staining, in prox mo injected embryos the percentage was only 38%, indicating that these hair cells are unable to reach full functionality in the absence of prox. as only a fraction of pou4f3::mgfp positive cells were stained with diasp in morphant fish, we conclude that development of hair cells in morphants is arrested prior to their final differentiation and are thus unable to reach full functionality in the absence of prox our results suggest that prox has a role in the late stages of hair cell differentiation, when they acquire the mechanotransduction capacity.  overall, our studies reveal that prox 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 prox 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 prox 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 prox in progenitor cells directs cell fate selection: while cells with high prox levels become hair cells, cells with low prox levels acquire supporting cell or other fates. interestingly, prox 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/prox protein has been argued to regulate genes specific for the differentiated state, while in proliferating cells the protein remains in the cytoplasm. in our study, prox 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. however, gfp expression driven by a regulatory element active during initial stages of hair cell differentiation is not affected by absence of prox therefore, initial hair cell specification does not appear to require prox but only terminal differentiation. it will be of interest to dissect the exact molecular players that are regulated by this gene in the zebrafish. 
neuromasts contain clusters of hair cells, surrounded by accessory cells. furthermore, using an antibody against prox 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 prox 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 system in zebrafish is composed of a series of organs called neuromasts, which are distributed over the body surface. in this report we describe zebrafish prox mrna expression in the migrating primordium and in the neuromasts of the posterior lateral line.
 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 prox mrna expression in the migrating primordium and in the neuromasts of the posterior lateral line. furthermore, using an antibody against prox 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 prox 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. 