A model of how cell differentiation of non-neuronal cells is affected by the neurogenic failure in <i>Ascl1</i> knockout mice.

<p>The model depicts proposed cell lineage relationships during <b>normal development</b> in the embryo (<b>left side</b>) as compared to <i>Ascl1</i> knockout (<b>Ascl1 KO</b>) animals (<b>right side</b>). Italics designate genes expressed by the various cell types. Arrow thickness on the <i>left</i> and <i>right</i> sides of the model represent differences in differentiation pathways utilized in normal development as compared to the <i>Ascl1</i> knockout – <b>thicker arrows</b> indicate a pathway that is used more, while <b>thinner arrows</b> indicate less. <b>Faded cell types</b> on the knockout panel (<b>right</b>) symbolize the loss of those cell stages because of <i>Ascl1</i> knockout (Cau et al., 1997). <i>Ascl1</i> knockout pushes the epithelium toward the expansion of upstream OPP/GBCs and away from HBCs (which are normally generated by OPP/GBCs; Packard et al., 2011) in the face of aborted production of neurons. However, to a limited degree, neurons are made by, or in apparent association with, cKIT-expressing progenitors. Abbreviations: OPP/GBC = olfactory placodal progenitor/globose basal cell, GBC_TA-OSN = transit amplifying globose basal cell producing olfactory sensory neurons, GBC_INP = immediate neuronal precursor, HBC = horizontal basal cell.</p

βIV TUBULIN strongly labels respiratory epithelium.

<p>Sections of nasal epithelium were harvested from <b>E14.5 HET</b> and <b><i>Ascl1</i></b><b> KO</b> animals and stained for <b>PGP9.5</b>, a marker of neuronal differentiation, and <b>βIV TUBULIN</b>, which strongly labels respiratory epithelium. Dotted lines identify the olfactory/respiratory border. Boxed region in <b>A</b> is shown at higher magnification in <b>B</b>. Note that the <b>βIV TUBULIN</b> labeling is strictly limited to ventral epithelium and has minimal overlap with the <b>PGP9.5</b>(+) olfactory epithelium, substantiating its usefulness as a respiratory epithelial marker. <b>C.</b> An image of the olfactory/respiratory border in age-matched <i>Ascl1</i> KO littermates, showing the expected lack of neurons (lack of <b>PGP9.5</b> labeling) in the olfactory area and a region of <b>βIV TUBULIN(+)</b> epithelium, which marks the respiratory epithelium in these animals as well. Scale bar in <b>A</b> is 100 µm. Scale bar in <b>C</b> panel is 25 µm and applies to <b>B</b> and <b>C</b>.</p

SOX2 is expressed by the overwhelming majority of the cells in the <i>Ascl1</i> KO epithelium.

<p>OE sections harvested from <b>E14.5</b> animals were stained for <b>SOX2</b>, <b>K18</b> and <b>Hoechst 33258</b>. (<b>A</b>), (<b>D</b>) Single channel green images of SOX2 staining. (<b>B</b>)<b>,</b> (<b>E</b>) Merged images of <b>SOX2</b> (green), <b>K18</b> (red), and <b>Hoechst 33258</b> (blue, to label nuclei). (<b>C</b>)<b>,</b> (<b>F</b>) Blue-only images showing labeled nuclei. (<b>A–C</b>) In <b>HET</b> epithelium anti-<b>SOX2</b> labels two major populations, apical Sus cells (identified by co-labeling with K18), and GBCs. (<b>D–F</b>) In <b><i>Ascl1</i></b><b> KO</b> epithelium anti-<b>SOX2</b> stains cell nuclei throughout the entire apical to basal extent of the epithelium with only a very few exceptions (e.g., <b>white arrow</b>). <b>Arrowheads</b> indicate basal lamina. <b>Scale bar</b> in <b>H</b> is 25 µm and applies to all panels.</p

NOTCH1-labeled cells are absent from the <i>Ascl1</i> KO epithelium.

<p>OE harvested at various embryonic ages were stained for NOTCH1. Boxed regions are shown at higher magnification in the insets. Arrowheads in insets indicate basal lamina. (<b>A–D</b>) <b>NOTCH1</b> is expressed by basal cells throughout the developing OE of heterozygous mice; (<b>E–H</b>) Notch1 staining is largely absent from the epithelium of <b><i>Ascl1</i></b><b> KO</b> mice. Higher magnification examination of the tissue on both sides of the basal lamina (insets <b>B–D</b>, <b>F–H</b>) demonstrate that <b>NOTCH1</b> staining in the <b><i>Ascl1</i></b><b> KO</b> is found within the lamina propria, and not the epithelium, indicating that the absence of stained cell in the OE of knockout mice is not an artifact. Scale bar in lower magnification image of <b>H</b> (10 µm) applies to all panels. Scale bar for the high magnification inset in <b>H</b> is 25 µm and applies to all insets.</p

Bowman’s duct and gland development appear normal in the <i>Ascl1</i> KO OE.

<p>OE sections harvested at various ages were stained for <b>K18</b> to mark sustentacular cells and developing Bowman’s duct and gland units. In sections harvested from <b>E16.5</b> and later, examples of developing duct/gland units and gland acini are indicated by <b>arrows</b> and shown at higher magnification in insets. At <b>E12.5</b> and <b>E14.5</b>, ducts are not yet distinct. However, occasional intensely stained cells that are indicated by <b>arrowheads</b> extend toward the basal lamina, and are presumably developing ducts (<b>B)</b>. By <b>E16.5</b>, epithelial spanning ducts in the lamina propria are seen in both <b>HET</b> and <b>Ascl1 KO</b> (arrows in <b>C</b>, <b>D</b>, <b>H, I, J</b>). Insets show the duct/gland structures identified by <b>arrows</b>. Scale bar in <b>J</b> (50 µm) applies to <b>A–J</b>. Scale bar in the inset of panel <b>J</b> (25 µm) applies to all insets.</p

HBC Emergence is aberrant but not aborted completely as a consequence of <i>Ascl1</i> KO.

<p>Matched sections from E19.5 <b>HET</b> and <b>Ascl1 KO</b> nasal epithelium were stained for <b>K14</b> to mark HBCs and <b>βIV TUBULIN</b> to mark respiratory epithelium. <b>K14</b> staining was highlighted and cartooned in black and <b>βIV TUBULIN</b> was highlighted and cartooned in red. Representative sections were arranged from <b>Rostral</b> to <b>Caudal</b> levels (<i>top</i> to <i>bottom</i>, matched levels numbered <b>1</b> to <b>5</b>). Coincident black and red indicate regions of respiratory epithelium that contain a dense population of K14 (+) basal cells, while areas of black only represent the emergence of <b>K14</b>(+) HBCs in olfactory epithelium. In the <b>HET</b> (<i>left side</i>) HBCs are relatively abundant in the caudal olfactory areas, while few if any are seen in comparable regions of the OE in the <b><i>Ascl1</i></b><b> KO</b> (<i>right side</i>).</p

The labeling intensity of Hes1 is reduced in Ascl1 knockout compared to heterozygote.

<p>Multiple sections from various ages were stained for Hes1 and imaged. The intensity of nuclear labeling was sampled from 50 consecutive nuclei and pixel intensity for 50 single-pixel intensity readings were measured for Hes1(+) consecutive nuclei in the olfactory domain and the respiratory domain using ImageJ (in other words, unstained cells were not measured). A ratio of intensity values was calculated for olfactory epithelium to respiratory epithelium for each genotype at each embryonic time point described. At all time points examined the calculated OE/RE intensity ratio is less for <b>Ascl1 knockout</b><b>mice</b> than <b>heterozygotes</b>.</p

Sus cells differentiate normally in developing OE of Ascl1 KO mice.

<p>Sustentacular (Sus) cells are marked in adult wild-type mice (<b>A–C</b>) and in wild-type embryos (<b>WT; </b><b>D–F</b>) and knockout (<b><i>Ascl1</i></b><b>KO; </b><b>G–I</b>) littermates at E19.5 by staining with <b>SUS4</b> (<b>A, D, G</b>), anti-<b>ezrin</b> (<b>B, E, H</b>) and anti-<b>REEP6</b> (<b>C, F, I</b>), as shown here on three consecutive sections from each animal. Prominent cytoplasmic labeling of both <b>SUS4</b> and anti-<b>REEP6</b> at the apical processes of Sus cells (<i>large white arrows</i>) is comparable between wild-type and knockout mice. Anti-<b>ezrin</b> heavily labels the microvilli at the apical surface of the Sus cells (<i>black triangles</i>) as well as Bowman’s duct/gland cells in the adult (<b>B</b>) and the staining pattern is consistent in wild-type (<b>E</b>) and knockout (<b>H</b>) mouse embryos. <i>White arrowheads</i> indicate the basal lamina. Scale bar in <b>I</b> is 50 µm, and also applies to <b>A–H</b>.</p

A subset of TuJ1(+) cells are proliferating.

<p>Sections of OE harvested from E16.5 wild type animals were stained with <b>TuJ1</b> and co-stained with phospho-histone 3 (<b>pH3</b>, <b>A–C</b>) or proliferating cell nuclear antigen (<b>PCNA</b>, <b>D–F</b>) and Hoechst 33258 to label nuclei. (<b>A, D</b>) Red channel only. (<b>B, E</b>) Merged images including Hoechst nuclear stain (blue). (<b>C, F</b>) Green channel only. <b>Arrows</b> indicate double positive cells. Scale bar in <b>F</b> is 25 µm and applies to all panels.</p

NOTCH1-labeled cells are proliferating and upstream of differentiated neurons.

<p>OE from <b>E16.5</b> wild type embryos was stained for <b>NOTCH1</b> and co-stained with <b>Ki67</b>, a marker of proliferating cells, and <b>NCAM</b>, a marker of neuronal differentiation. (<b>A</b>) Many <b>NOTCH1</b>(+) cells are <b>Ki67</b>(+) (vertical <b>arrows</b>). (<b>B</b>) The vast majority of <b>NOTCH1</b>(+) cells are <b>NCAM</b> (–). Horizontal <b>arrows</b> in <b>B</b> illustrate the border between <b>NOTCH1</b>(+) basal cells and the <b>NCAM</b> (+) neuronal layer. Arrowheads indicate basal lamina. Scale bar in <b>B</b> (20 µm) applies to both panels.</p