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Retinoic acid regulates olfactory progenitor cell fate and differentiation.

Paschaki M, Cammas L, Muta Y, Matsuoka Y, Mak SS, Rataj-Baniowska M, Fraulob V, Dollé P, Ladher RK - Neural Dev (2013)

Bottom Line: Using an explant system, we further show that renewal of olfactory neurons is hindered if the olfactory epithelium is unable to synthesize retinoic acid.Our data show that retinoic acid is not a simple placodal inductive signal, but rather controls olfactory neuronal production by regulating the fate of olfactory progenitor cells.Retinaldehyde dehydrogenase 3 (RALDH3) is the key enzyme required to generate retinoic acid within the olfactory epithelium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Sensory Development, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan.

ABSTRACT

Background: In order to fulfill their chemosensory function, olfactory neurons are in direct contact with the external environment and are therefore exposed to environmental aggressive factors. Olfaction is maintained through life because, unlike for other sensory neuroepithelia, olfactory neurons have a unique capacity to regenerate after trauma. The mechanisms that control the ontogenesis and regenerative ability of these neurons are not fully understood. Here, we used various experimental approaches in two model systems (chick and mouse) to assess the contribution of retinoic acid signaling in the induction of the olfactory epithelium, the generation and maintenance of progenitor populations, and the ontogenesis and differentiation of olfactory neurons.

Results: We show that retinoic acid signaling, although dispensable for initial induction of the olfactory placode, plays a key role in neurogenesis within this neuroepithelium. Retinoic acid depletion in the olfactory epithelium, both in chick and mouse models, results in a failure of progenitor cell maintenance and, consequently, differentiation of olfactory neurons is not sustained. Using an explant system, we further show that renewal of olfactory neurons is hindered if the olfactory epithelium is unable to synthesize retinoic acid.

Conclusions: Our data show that retinoic acid is not a simple placodal inductive signal, but rather controls olfactory neuronal production by regulating the fate of olfactory progenitor cells. Retinaldehyde dehydrogenase 3 (RALDH3) is the key enzyme required to generate retinoic acid within the olfactory epithelium.

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Retinoid deficiency affects the olfactory neuronal lineage. (A-C) Treatment of HH14 stage chicken embryos with disulphiram (B) or AT-RA (C) beads impacts positively or negatively on OP mitotic (pH3-labeled) cells. (D,E) pH3 immunodetection in the OE of E12.5 wild-type and Raldh3−/− mouse embryos, showing an increased number of labeled cells in the mutant. (F) Quantification of pH3+ (mitotic) or activated-caspase 3+ (apoptotic) cells in disulphiram or AT-RA-treated HH14 chicken OPs (n = 6 embryos/condition, expressed as percentages with respect to controls; *: p <0.05; **: p <0.01). (G) Quantification of pH3+ cells in the OE of E10.5 or E12.5 wild-type and Raldh3−/− mouse embryos (n = 6 embryos/genotype; three sections of the OE were counted for each embryo; ***: p <0.001). (H) Examples of immunofluorescence detection of Ascl1 (top panels) or Neurog1 (bottom panels), after culturing E10.5 or E12.5 murine olfactory explants for 48 h in defined medium, in the presence (+Vit. A) or absence (−Vit. A) of vitamin A. The corresponding data are quantified in K,L. (I,J) Pax6 immunodetection in the OE of E12.5 wild-type and Raldh3−/− embryos. (K,L) Quantitative analysis of neuronal progenitor populations in murine olfactory explants collected at E10.5 (left-side histograms) or E12.5 (right-side histograms), from wild-type (K) or Raldh3−/−(L) mice, and cultured for 48 h in medium containing (+Vit. A, front row) or devoid of (−Vit. A, back row) vitamin A (n = 3 explants per stage and genotype; Additional file 3: Table S1 for statistics). Explants were analyzed by immunofluorescence using anti-Ascl1, -Neurog1, and/or TuJ1 antibodies. (M-P) TuJ1 immunodetection in the OE of E12.5 wild-type (M,O) and Raldh3−/−(N,P) mice. (O,P) are higher magnifications of the distal OE, taken from specimens labeled with a different fluorochrome.
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Figure 3: Retinoid deficiency affects the olfactory neuronal lineage. (A-C) Treatment of HH14 stage chicken embryos with disulphiram (B) or AT-RA (C) beads impacts positively or negatively on OP mitotic (pH3-labeled) cells. (D,E) pH3 immunodetection in the OE of E12.5 wild-type and Raldh3−/− mouse embryos, showing an increased number of labeled cells in the mutant. (F) Quantification of pH3+ (mitotic) or activated-caspase 3+ (apoptotic) cells in disulphiram or AT-RA-treated HH14 chicken OPs (n = 6 embryos/condition, expressed as percentages with respect to controls; *: p <0.05; **: p <0.01). (G) Quantification of pH3+ cells in the OE of E10.5 or E12.5 wild-type and Raldh3−/− mouse embryos (n = 6 embryos/genotype; three sections of the OE were counted for each embryo; ***: p <0.001). (H) Examples of immunofluorescence detection of Ascl1 (top panels) or Neurog1 (bottom panels), after culturing E10.5 or E12.5 murine olfactory explants for 48 h in defined medium, in the presence (+Vit. A) or absence (−Vit. A) of vitamin A. The corresponding data are quantified in K,L. (I,J) Pax6 immunodetection in the OE of E12.5 wild-type and Raldh3−/− embryos. (K,L) Quantitative analysis of neuronal progenitor populations in murine olfactory explants collected at E10.5 (left-side histograms) or E12.5 (right-side histograms), from wild-type (K) or Raldh3−/−(L) mice, and cultured for 48 h in medium containing (+Vit. A, front row) or devoid of (−Vit. A, back row) vitamin A (n = 3 explants per stage and genotype; Additional file 3: Table S1 for statistics). Explants were analyzed by immunofluorescence using anti-Ascl1, -Neurog1, and/or TuJ1 antibodies. (M-P) TuJ1 immunodetection in the OE of E12.5 wild-type (M,O) and Raldh3−/−(N,P) mice. (O,P) are higher magnifications of the distal OE, taken from specimens labeled with a different fluorochrome.

Mentions: Since modulation of the RA pathway appears to regulate the pool of Pax6+ progenitor cells, and considering that olfactory neurogenesis is inhibited by RA in vitro, we wondered at which step of the ORN lineage RA may act. We first assessed if modulation of RA signaling affects apoptotic cell death or progenitor cell proliferation in the OE. Apoptotic cells, identified by activated caspase-3 immunolabeling, were not significantly changed in the OE of chicken embryos, 48 h after disulphiram or AT-RA bead implantation (Figure 3F). However, disulphiram administration increased phospho-histone H3 (pH3)-labeled proliferative cells (Figure 3A, B, F), and AT-RA inhibited cell proliferation (Figure 3C, F). An effect of RA deficiency on cell proliferation was also observed in Raldh3−/− mouse mutants, with a slight increase of pH3-labeled mitotic progenitors in the OP at E10.5 (Figure 3G), and a more pronounced increase in the OE of E12.5 mutant embryos (Figure 3D, E, G).


Retinoic acid regulates olfactory progenitor cell fate and differentiation.

Paschaki M, Cammas L, Muta Y, Matsuoka Y, Mak SS, Rataj-Baniowska M, Fraulob V, Dollé P, Ladher RK - Neural Dev (2013)

Retinoid deficiency affects the olfactory neuronal lineage. (A-C) Treatment of HH14 stage chicken embryos with disulphiram (B) or AT-RA (C) beads impacts positively or negatively on OP mitotic (pH3-labeled) cells. (D,E) pH3 immunodetection in the OE of E12.5 wild-type and Raldh3−/− mouse embryos, showing an increased number of labeled cells in the mutant. (F) Quantification of pH3+ (mitotic) or activated-caspase 3+ (apoptotic) cells in disulphiram or AT-RA-treated HH14 chicken OPs (n = 6 embryos/condition, expressed as percentages with respect to controls; *: p <0.05; **: p <0.01). (G) Quantification of pH3+ cells in the OE of E10.5 or E12.5 wild-type and Raldh3−/− mouse embryos (n = 6 embryos/genotype; three sections of the OE were counted for each embryo; ***: p <0.001). (H) Examples of immunofluorescence detection of Ascl1 (top panels) or Neurog1 (bottom panels), after culturing E10.5 or E12.5 murine olfactory explants for 48 h in defined medium, in the presence (+Vit. A) or absence (−Vit. A) of vitamin A. The corresponding data are quantified in K,L. (I,J) Pax6 immunodetection in the OE of E12.5 wild-type and Raldh3−/− embryos. (K,L) Quantitative analysis of neuronal progenitor populations in murine olfactory explants collected at E10.5 (left-side histograms) or E12.5 (right-side histograms), from wild-type (K) or Raldh3−/−(L) mice, and cultured for 48 h in medium containing (+Vit. A, front row) or devoid of (−Vit. A, back row) vitamin A (n = 3 explants per stage and genotype; Additional file 3: Table S1 for statistics). Explants were analyzed by immunofluorescence using anti-Ascl1, -Neurog1, and/or TuJ1 antibodies. (M-P) TuJ1 immunodetection in the OE of E12.5 wild-type (M,O) and Raldh3−/−(N,P) mice. (O,P) are higher magnifications of the distal OE, taken from specimens labeled with a different fluorochrome.
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Figure 3: Retinoid deficiency affects the olfactory neuronal lineage. (A-C) Treatment of HH14 stage chicken embryos with disulphiram (B) or AT-RA (C) beads impacts positively or negatively on OP mitotic (pH3-labeled) cells. (D,E) pH3 immunodetection in the OE of E12.5 wild-type and Raldh3−/− mouse embryos, showing an increased number of labeled cells in the mutant. (F) Quantification of pH3+ (mitotic) or activated-caspase 3+ (apoptotic) cells in disulphiram or AT-RA-treated HH14 chicken OPs (n = 6 embryos/condition, expressed as percentages with respect to controls; *: p <0.05; **: p <0.01). (G) Quantification of pH3+ cells in the OE of E10.5 or E12.5 wild-type and Raldh3−/− mouse embryos (n = 6 embryos/genotype; three sections of the OE were counted for each embryo; ***: p <0.001). (H) Examples of immunofluorescence detection of Ascl1 (top panels) or Neurog1 (bottom panels), after culturing E10.5 or E12.5 murine olfactory explants for 48 h in defined medium, in the presence (+Vit. A) or absence (−Vit. A) of vitamin A. The corresponding data are quantified in K,L. (I,J) Pax6 immunodetection in the OE of E12.5 wild-type and Raldh3−/− embryos. (K,L) Quantitative analysis of neuronal progenitor populations in murine olfactory explants collected at E10.5 (left-side histograms) or E12.5 (right-side histograms), from wild-type (K) or Raldh3−/−(L) mice, and cultured for 48 h in medium containing (+Vit. A, front row) or devoid of (−Vit. A, back row) vitamin A (n = 3 explants per stage and genotype; Additional file 3: Table S1 for statistics). Explants were analyzed by immunofluorescence using anti-Ascl1, -Neurog1, and/or TuJ1 antibodies. (M-P) TuJ1 immunodetection in the OE of E12.5 wild-type (M,O) and Raldh3−/−(N,P) mice. (O,P) are higher magnifications of the distal OE, taken from specimens labeled with a different fluorochrome.
Mentions: Since modulation of the RA pathway appears to regulate the pool of Pax6+ progenitor cells, and considering that olfactory neurogenesis is inhibited by RA in vitro, we wondered at which step of the ORN lineage RA may act. We first assessed if modulation of RA signaling affects apoptotic cell death or progenitor cell proliferation in the OE. Apoptotic cells, identified by activated caspase-3 immunolabeling, were not significantly changed in the OE of chicken embryos, 48 h after disulphiram or AT-RA bead implantation (Figure 3F). However, disulphiram administration increased phospho-histone H3 (pH3)-labeled proliferative cells (Figure 3A, B, F), and AT-RA inhibited cell proliferation (Figure 3C, F). An effect of RA deficiency on cell proliferation was also observed in Raldh3−/− mouse mutants, with a slight increase of pH3-labeled mitotic progenitors in the OP at E10.5 (Figure 3G), and a more pronounced increase in the OE of E12.5 mutant embryos (Figure 3D, E, G).

Bottom Line: Using an explant system, we further show that renewal of olfactory neurons is hindered if the olfactory epithelium is unable to synthesize retinoic acid.Our data show that retinoic acid is not a simple placodal inductive signal, but rather controls olfactory neuronal production by regulating the fate of olfactory progenitor cells.Retinaldehyde dehydrogenase 3 (RALDH3) is the key enzyme required to generate retinoic acid within the olfactory epithelium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Sensory Development, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan.

ABSTRACT

Background: In order to fulfill their chemosensory function, olfactory neurons are in direct contact with the external environment and are therefore exposed to environmental aggressive factors. Olfaction is maintained through life because, unlike for other sensory neuroepithelia, olfactory neurons have a unique capacity to regenerate after trauma. The mechanisms that control the ontogenesis and regenerative ability of these neurons are not fully understood. Here, we used various experimental approaches in two model systems (chick and mouse) to assess the contribution of retinoic acid signaling in the induction of the olfactory epithelium, the generation and maintenance of progenitor populations, and the ontogenesis and differentiation of olfactory neurons.

Results: We show that retinoic acid signaling, although dispensable for initial induction of the olfactory placode, plays a key role in neurogenesis within this neuroepithelium. Retinoic acid depletion in the olfactory epithelium, both in chick and mouse models, results in a failure of progenitor cell maintenance and, consequently, differentiation of olfactory neurons is not sustained. Using an explant system, we further show that renewal of olfactory neurons is hindered if the olfactory epithelium is unable to synthesize retinoic acid.

Conclusions: Our data show that retinoic acid is not a simple placodal inductive signal, but rather controls olfactory neuronal production by regulating the fate of olfactory progenitor cells. Retinaldehyde dehydrogenase 3 (RALDH3) is the key enzyme required to generate retinoic acid within the olfactory epithelium.

Show MeSH
Related in: MedlinePlus