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Role of Rb during Neurogenesis and Axonal Guidance in the Developing Olfactory System

View Article: PubMed Central - PubMed

ABSTRACT

The Retinoblastoma protein, Rb, was shown to regulate distinct aspects of neurogenesis in the embryonic and adult brain besides its primary role in cell cycle control. It is still unknown, however, whether Rb is required for tissue morphogenesis and the establishment of synaptic connections between adjacent tissues during development. We have investigated here the role of Rb during development of the olfactory system (OS), which heavily relies on reciprocal interactions between the olfactory epithelium (OE) and the olfactory bulb (OB). We show that mice carrying a telencephalic-specific deletion of Rb display several neurogenic defects in the OS during late development. In the OE, loss of Rb leads to ectopic proliferation of late-born progenitors (Tuj-1+), abnormal radial migration and terminal maturation of olfactory sensory neurons (OSNs). In the OB, deletion of Rb causes severe lamination defects with loss of clear boundaries between distinct layers. Importantly, starting around E15.5 when OB glomerulogenesis is initiated, many OSNs axons that project along the olfactory nerve layer (ONL) fail to properly innervate the nascent bulb, thus resulting in partial loss of connectivity between OE-OB and gradual neuronal degeneration in both tissues peaking at birth. This deficiency correlates with deregulated expressions of two key chemo-repellant molecules, Robo2/Slit1 and Nrp2/Sema3F that control the formation of dorsal-ventral topographic map of OSNs connections with OB glomeruli. This study highlights a critical requirement for Rb during neurogenesis and the establishment of proper synaptic connections inside the OS during development.

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Massive apoptosis inside the OS during late development in the absence of Rb. (A–B′) Immunostaining against AC-3 (active-caspase 3; apoptotic marker) performed on sagittal sections and showing increased cell death in the OB (A,A′) and the OE (B,B′) in Rb−/− vs. Rb+/− embryos at E18.5 (arrows in A′ and B′). (C) Quantification of AC-3+ cells performed at medial levels in the OE revealed 2.94-, 4.2- and 1.63-fold increase in cell death at E14.5, E18.5, and P0, respectively, and 2.77-, 3.19- and 11.4- increase in cell death inside the OB at the same ages, respectively. Error bars represent SD of measurements from n = 3 per genotype and asterisks indicate a statistical significant difference between genotypes using t-tests, **p < 0.01 and ***p < 0.001. Scale bars = 100 μm.
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Figure 5: Massive apoptosis inside the OS during late development in the absence of Rb. (A–B′) Immunostaining against AC-3 (active-caspase 3; apoptotic marker) performed on sagittal sections and showing increased cell death in the OB (A,A′) and the OE (B,B′) in Rb−/− vs. Rb+/− embryos at E18.5 (arrows in A′ and B′). (C) Quantification of AC-3+ cells performed at medial levels in the OE revealed 2.94-, 4.2- and 1.63-fold increase in cell death at E14.5, E18.5, and P0, respectively, and 2.77-, 3.19- and 11.4- increase in cell death inside the OB at the same ages, respectively. Error bars represent SD of measurements from n = 3 per genotype and asterisks indicate a statistical significant difference between genotypes using t-tests, **p < 0.01 and ***p < 0.001. Scale bars = 100 μm.

Mentions: We next examined whether terminal differentiation and survival of OSNs is affected by the loss of Rb. Following one or more divisions, late neural precursors exit the cell cycle and differentiate into immature OSNs (iOSNs), which are located immediately apical to the GBCs in the intermediate zone (IZ) of the OE. Later on, iOSNs migrate radially to the apical surface as they upregulate the expression of the Olfactory Marker Protein (OMP) and become mature OSNs (mOSNs) with bipolar morphology (Graziadei and Graziadei, 1979; Farbman and Margolis, 1980; Calof and Chikaraishi, 1989). As a result, we found a dramatic increase by 2–4-fold in cell death in both the OE and the OB in Rb−/− vs. Rb+/− embryos between E14.5 and birth as indicated by the number of cells expressing the apoptotic marker active-caspase 3 (AC-3) (Figures 5A–C; arrows in Figures 5A′–B′, AC3+ at E14.5: Rb+/− vs. Rb−/−, 42.9+/−20.9 vs. 126.5+/−11.4, p = 0.00001, 2.95-fold increase in OE, and 125.16+/− 82.76 vs. 346.85+/−74.45, p = 0.0004, 2.77-fold increase in OB). Moreover, co-staining with (AC3/Tuj1) and (Bax (6A7)/OMP, Bax (6A7); active form-apoptotic marker) revealed significant increase in the numbers of apoptotic iOSNs between E14.5 and E18.5 in the absence of Rb (Figures 6A–D′; arrowheads in Figure 6D′, and Figure 6I; (AC+Tuj1+) at E18.5: Rb+/− vs. Rb−/−: 50.94+/−11.81 vs. 229.7+/−54.6, p = 0.0003, 4.5-fold increase)], and mOSNs at E18.5 [(Figures 6E–H′; arrowheads in Figure 6H′, and Figure 6J; Bax (6A7)+, Rb+/− vs. Rb−/−: 111.6+/−16.3 vs. 423.5+/−84.09, p = 0.0009, 3.79-fold increase, and (OMP+Bax (6A7)+): 110+/−16.8 vs. 349.7+/−66.5, p = 0.0002, 3.17-fold increase)]. These data indicate that survival of both iOSNs and mOSNs is severely compromised in the absence of Rb during late development.


Role of Rb during Neurogenesis and Axonal Guidance in the Developing Olfactory System
Massive apoptosis inside the OS during late development in the absence of Rb. (A–B′) Immunostaining against AC-3 (active-caspase 3; apoptotic marker) performed on sagittal sections and showing increased cell death in the OB (A,A′) and the OE (B,B′) in Rb−/− vs. Rb+/− embryos at E18.5 (arrows in A′ and B′). (C) Quantification of AC-3+ cells performed at medial levels in the OE revealed 2.94-, 4.2- and 1.63-fold increase in cell death at E14.5, E18.5, and P0, respectively, and 2.77-, 3.19- and 11.4- increase in cell death inside the OB at the same ages, respectively. Error bars represent SD of measurements from n = 3 per genotype and asterisks indicate a statistical significant difference between genotypes using t-tests, **p < 0.01 and ***p < 0.001. Scale bars = 100 μm.
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Related In: Results  -  Collection

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Figure 5: Massive apoptosis inside the OS during late development in the absence of Rb. (A–B′) Immunostaining against AC-3 (active-caspase 3; apoptotic marker) performed on sagittal sections and showing increased cell death in the OB (A,A′) and the OE (B,B′) in Rb−/− vs. Rb+/− embryos at E18.5 (arrows in A′ and B′). (C) Quantification of AC-3+ cells performed at medial levels in the OE revealed 2.94-, 4.2- and 1.63-fold increase in cell death at E14.5, E18.5, and P0, respectively, and 2.77-, 3.19- and 11.4- increase in cell death inside the OB at the same ages, respectively. Error bars represent SD of measurements from n = 3 per genotype and asterisks indicate a statistical significant difference between genotypes using t-tests, **p < 0.01 and ***p < 0.001. Scale bars = 100 μm.
Mentions: We next examined whether terminal differentiation and survival of OSNs is affected by the loss of Rb. Following one or more divisions, late neural precursors exit the cell cycle and differentiate into immature OSNs (iOSNs), which are located immediately apical to the GBCs in the intermediate zone (IZ) of the OE. Later on, iOSNs migrate radially to the apical surface as they upregulate the expression of the Olfactory Marker Protein (OMP) and become mature OSNs (mOSNs) with bipolar morphology (Graziadei and Graziadei, 1979; Farbman and Margolis, 1980; Calof and Chikaraishi, 1989). As a result, we found a dramatic increase by 2–4-fold in cell death in both the OE and the OB in Rb−/− vs. Rb+/− embryos between E14.5 and birth as indicated by the number of cells expressing the apoptotic marker active-caspase 3 (AC-3) (Figures 5A–C; arrows in Figures 5A′–B′, AC3+ at E14.5: Rb+/− vs. Rb−/−, 42.9+/−20.9 vs. 126.5+/−11.4, p = 0.00001, 2.95-fold increase in OE, and 125.16+/− 82.76 vs. 346.85+/−74.45, p = 0.0004, 2.77-fold increase in OB). Moreover, co-staining with (AC3/Tuj1) and (Bax (6A7)/OMP, Bax (6A7); active form-apoptotic marker) revealed significant increase in the numbers of apoptotic iOSNs between E14.5 and E18.5 in the absence of Rb (Figures 6A–D′; arrowheads in Figure 6D′, and Figure 6I; (AC+Tuj1+) at E18.5: Rb+/− vs. Rb−/−: 50.94+/−11.81 vs. 229.7+/−54.6, p = 0.0003, 4.5-fold increase)], and mOSNs at E18.5 [(Figures 6E–H′; arrowheads in Figure 6H′, and Figure 6J; Bax (6A7)+, Rb+/− vs. Rb−/−: 111.6+/−16.3 vs. 423.5+/−84.09, p = 0.0009, 3.79-fold increase, and (OMP+Bax (6A7)+): 110+/−16.8 vs. 349.7+/−66.5, p = 0.0002, 3.17-fold increase)]. These data indicate that survival of both iOSNs and mOSNs is severely compromised in the absence of Rb during late development.

View Article: PubMed Central - PubMed

ABSTRACT

The Retinoblastoma protein, Rb, was shown to regulate distinct aspects of neurogenesis in the embryonic and adult brain besides its primary role in cell cycle control. It is still unknown, however, whether Rb is required for tissue morphogenesis and the establishment of synaptic connections between adjacent tissues during development. We have investigated here the role of Rb during development of the olfactory system (OS), which heavily relies on reciprocal interactions between the olfactory epithelium (OE) and the olfactory bulb (OB). We show that mice carrying a telencephalic-specific deletion of Rb display several neurogenic defects in the OS during late development. In the OE, loss of Rb leads to ectopic proliferation of late-born progenitors (Tuj-1+), abnormal radial migration and terminal maturation of olfactory sensory neurons (OSNs). In the OB, deletion of Rb causes severe lamination defects with loss of clear boundaries between distinct layers. Importantly, starting around E15.5 when OB glomerulogenesis is initiated, many OSNs axons that project along the olfactory nerve layer (ONL) fail to properly innervate the nascent bulb, thus resulting in partial loss of connectivity between OE-OB and gradual neuronal degeneration in both tissues peaking at birth. This deficiency correlates with deregulated expressions of two key chemo-repellant molecules, Robo2/Slit1 and Nrp2/Sema3F that control the formation of dorsal-ventral topographic map of OSNs connections with OB glomeruli. This study highlights a critical requirement for Rb during neurogenesis and the establishment of proper synaptic connections inside the OS during development.

No MeSH data available.


Related in: MedlinePlus