Insm1promotes the transition of olfactory progenitors from apical and proliferative to basal, terminally dividing and neuronogenic
© Rosenbaum et al; licensee BioMed Central Ltd. 2011
Received: 1 November 2010
Accepted: 1 February 2011
Published: 1 February 2011
Insm1 is a zinc-finger transcription factor transiently expressed throughout the developing nervous system in late progenitors and nascent neurons. Insm1 is also highly expressed in medulloblastomas and other neuroendocrine tumors.
We generated mice lacking the Insm1 gene and used them to elucidate its role in neurogenic proliferation of the embryonic olfactory epithelium. We found that deletion of Insm1 results in more apical cells and fewer nascent and mature neurons. In the embryonic olfactory epithelium of Insm1 mutants we detect fewer basal progenitors, which produce neurons, and more apical progenitors, which at this stage produce additional progenitors. Furthermore, in the mutants we detect fewer progenitors expressing NEUROD1, a marker of terminally dividing, neuronogenic (neuron-producing) progenitors (immediate neuronal precursors), and more progenitors expressing ASCL1, a marker of the transit amplifying progenitors that migrate from the apical to the basal edges of the epithelium while dividing to generate the terminal, neuronogenic progenitors. Finally, with timed administration of nucleoside analogs we demonstrate that the Insm1 mutants contain fewer terminally dividing progenitors at embryonic day 12.5.
Altogether, these results suggest a role for Insm1 in promoting the transition of progenitors from apical and proliferative to basal, terminal and neuronogenic. This role appears partially conserved with that of its nematode ortholog, egl-46. The similar effects of Insm1 deletion on progenitors of embryonic olfactory epithelium and cortex point to striking parallels in the development of these neuroepithelia, and particularly between the basal progenitors of olfactory epithelium and the subventricular zone progenitors of cortex.
During nervous system development, progenitors divide to produce more progenitors, glia and/or neurons. Decisions to stop proliferating and produce neurons or glia determine shape, size and cellular composition of every part of the nervous system. These decisions can involve transitions between apical and basal sides of a proliferating neuroepithelium. In embryonic olfactory epithelium (OE), apical progenitors generate sustentacular glia and more progenitors, some of which transition basally, divide terminally and produce neurons . Despite its importance, we have limited knowledge of which genes control progenitor transitions from proliferative to neuronogenic.
In Caeneorhabditis elegans, the zinc-finger protein EGL-46 is transiently expressed in certain progenitors about to divide terminally (neuronogenic, N/N divisions) and in their two nascent neurons, but not in the mature neurons or in earlier progenitors that divide to produce one or two progenitors (proliferative, P/P or P/N divisions) (see discussion below). In egl-46 mutants, some of these presumptive N/N progenitors fail to terminally divide and generate additional progenitors [2–4] (see discussion below). Hence, EGL-46 regulates terminal neuronogenic divisions. We hypothesize that a mammalian homolog of EGL-46 also regulates transitions of progenitors from proliferative to terminally dividing and neuronogenic.
In mammals, egl-46 has two orthologs, Insm1 and Insm2 [3, 5]. Insm1 was originally identified as highly expressed in neuroendocrine tumors [6–13], but during normal development it is transiently expressed throughout the embryonic and adult developing nervous system [14–18]. In particular, Insm1 mRNA is expressed in late (not early) progenitors and nascent (not mature) neurons . Thus, the pattern of expression of Insm1 in mice is reminiscent of that of egl-46 in nematode neuronal lineages, suggesting that both genes share a conserved function in regulating neuronal progenitor proliferation. Insm1 has been implicated in the development of the pancreas [19–22], sympatho-adrenal lineages , cortex , and hindbrain . We tested the function of this gene in the embryonic OE.
The OE provides a simple neurodevelopmental model. This placode-derived neuroepithelium contains only one type of neuron, which originates from progenitors located within the epithelium. Embryonic OE is spatially segregated: early progenitors and sustentacular cells localize apically, late progenitors basally, and neurons in between [1, 25]. In embryonic OE, Insm1 mRNA is expressed in intermediate cells (both progenitors and nascent neurons) and basal (but not apical) progenitors . We generated mice entirely lacking Insm1 and used them to conclude that this gene regulates progenitor transitions from apical and proliferative to basal and neuronogenic.
To avoid potentially confounding effects of other genetic variables in our phenotypic assessment, we placed the Insm1 tm1.1Jga allele (from now on referred to as Insm1-) into an isogenic background by performing ten backcrosses into either the inbred C57BL/6 line or the outbred CD1 line.
Deletion of Insm1causes embryonic lethality
Mating between heterozygotes did not result in any Insm1 -/- pups, which indicates that this genotype causes embryonic lethality. This lethality does not seem to result in or from developmental delay, as KO, wild type and heterozygous embryos of the same age did not differ in weight (Additional file 1) or in anatomically defined developmental hallmarks (Additional file 2). In order to determine the embryonic stage of lethality, we generated timed pregnancies and collected embryos at set days post-coitus (from here onwards denoted as E for embryonic day, with the first day after a night of mating considered E0.5). At E11.5, Insm1 -/- embryos appeared at the expected Mendelian ratio (1/4). At E12.5 to E15.5, Insm1 -/- embryos appeared at somewhat lower ratios than expected and by E16.5 there was a severe drop in viability (Additional file 3). Hence, Insm1 -/- embryos suffer lethality around E16. In an attempt to obtain older embryos, we administered L-DOPA (1 mg/ml) to pregnant dams starting no later than 7.5 days post-coitus (dpc), as it has been reported that this treatment increases viability of a similar KO [23, 26]. Although in our hands this treatment did not improve the viability at earlier (E11.5 to E14.5) stages, we were able to obtain a few embryos as old as E20.5 (Additional file 3). We used these embryos to examine the effects of Insm1 deletion on neural development but we did not explore the reasons for this lethality, which others attribute to a catecholamine deficiency resulting from developmental defects of the sympathoadrenal lineage .
Deletion of Insm1results in more apical and fewer neuro-basal cells in the embryonic OE
Deletion of Insm1results in fewer nascent and mature neurons in the embryonic OE
Deletion of Insm1only causes a minimal increase in apoptosis at late embryonic stages
Deletion of Insm1does not alter cell cycle length, but results in an increase in apical progenitors and a concomitant decrease in basal ones in the embryonic OE
Because the length of the S-phase is largely invariant and changes in cell cycle length reflect changes in G1, we could estimate changes in overall cell cycle length by calculating the fraction of all dividing cells (Ki67+) that are in S-phase (BrdU+) . A lengthening of the cell cycle, which would result in a delay in the production of neurons, would be reflected in a lower BrdU+/Ki67+ ratio. However, we detected no difference in this ratio when comparing OEs of Insm1 -/- and of Insm1 +/+ littermate embryos (Figure 5). Hence, we conclude that the deletion of Insm1 does not cause an overall lengthening of the cell cycle among OE progenitors.
The above measurements did not distinguish between apical and basal progenitors, and would not have detected a cell cycle lengthening in basal progenitors (which could explain the reduction in neurons) if this was compensated by a cell cycle shortening in apical progenitors. However, we also detected no difference in BrdU+/Ki67+ ratios when considering only basal progenitors (Figure 5). We therefore conclude that the reduction of olfactory receptor neurons in Insm1 -/- embryos is not caused by a lengthening of the cell cycle in basal OE progenitors.
The thickening of the apical layer and thinning of the neuro-basal layer roughly compensate for each other because, as indicated above, the overall thickness of the OE does not change in Insm1 -/- embryos. We also measured the entire surface areas of the OE sections used for estimating number of neurons (Figure 3) and confirmed no difference in the OE size of Insm1 -/- compared to that of Insm1 +/+ littermates (Additional file 5).
Deletion of Insm1results in more ASCL1-expressing and fewer NEUROD1-expressing progenitors in the embryonic OE
Deletion of Insm1results in fewer terminal divisions in the embryonic (E12.5) OE
Transient expression of Insm1from apical cells migrating basally to postmitotic daughters of basal progenitors
Insm1 encodes a zinc-finger protein that is expressed in late progenitors and nascent neurons throughout the developing nervous system, including the embryonic OE [15, 16]. Here we show several lines of evidence indicating that, in embryonic OE, deletion of Insm1 causes a decrease in terminal, neuronogenic progenitors and an increase in earlier, proliferative progenitors. First, at certain embryonic stages the OEs of Insm1 -/- mice (compared with OEs of Insm1 +/+ mice) contain more apical mitoses (E12.5) and fewer basal ones (E12.5 and E14.5). At these stages, progenitors undergoing mitosis apically generate more progenitors, whereas some progenitors undergoing mitosis at the basal lamina generate neurons. Second, at E12.5 the OEs of Insm1 -/- mice display more cells expressing ASCL1 (a marker of TAPs, which migrate from apical to basal locations but still divide to produce progenitors) and fewer cells expressing NEUROD1 (a marker of INPs, which divide terminally next to the basal lamina to produce neurons). Third, by sequential incorporation of nucleoside analogs we demonstrate that, at E12.5, the OEs of Insm1 -/- mice contain fewer progenitors that are terminally dividing. These results indicate that Insm1 promotes the transition of olfactory progenitors from apical, proliferative and uncommitted to basal, terminally dividing and neuron-producing.
Potential molecular mechanisms of Insm1 action
This study does not address the molecular mechanism by which Insm1 regulates the progression from early to late neuronal progenitors. In this regard, studies on EGL-46 offer no clues as its mechanism of action also has not been explored. Some insight, however, might be drawn from previous work revealing that Insm1 has at least two molecular mechanisms of action: it can bind promoter elements to repress transcription of certain genes and it can also promote cell cycle arrest and exit . The basic helix-loop-helix transcription factor ASCL1 binds to an element in the promoter of Insm1, and Ascl1 is required for Insm1 transcriptional activation in the sympathoadrenal lineage , ganglionic eminences  and OE . Furthermore, in the sympathoadrenal lineage, ganglionic eminences and cortex, ablation of Insm1 led to an up-regulation of Ascl1, and these results were interpreted as evidence that Insm1 represses Ascl1 [16, 23]. Similarly, the increase in ASCL1-expressing cells in the OE of Insm1 -/- embryos here reported could result from direct transcriptional repression. However, this increase in ASCL1-expressing progenitors does not explain the decrease in basal progenitors and neurons, both of which require ASCL1.
Insm1 also binds an element in the NeuroD1 promoter and represses its transcription in pancreatic islet cell development [5, 46]. However, this regulatory interaction fails to explain the decrease of NEUROD1-expressing cells in the absence of Insm1. Alternatively, the retention of OE progenitors in a proliferative state in the absence of Insm1 might be more in keeping with the proposed role of this zinc-finger protein in cell cycle exit [5, 49].
Anatomical consequence of Insm1ablation: increase in apical and decrease in basal cells and neurons
Our results do explain the anatomical changes observed in Insm1-/- OEs. The increase in apical progenitors observed at E12.5 may account for the increase in cells observed in the apical layer at subsequent stages (E14.5 and E18.5). Many of these are likely sustentacular cells or their precursors because by E14.5 there is no longer an increase in apical progenitors. Likewise, the decrease of progenitors that are basal, NEUROD1-positive and terminally dividing at E12.5 may explain the decrease in nascent and mature neurons observed at this and later stages. Although apoptosis may also contribute to the decrease in neurons, its later onset (E14.5) and infrequent occurrence (100 times fewer cells appear undergoing programmed cell death than neurons are missing) indicates that it cannot be the sole or principal cause for neuronal decrease. The net increase in apical progenitors and cells and net decrease in basal progenitors and neurons seem to compensate for each other during the stages examined here because we never detected an overall difference in the size (measured in thickness, cell density or surface area in Figure 2 and Additional files 4 and 5) between Insm1 -/- and Insm1 +/+ OEs.
Potential roles of Insm1in cell survival and neuronal differentiation
The slight increase in cell death detected in Insm1 -/- OEs might also be suggestive of a role of Insm1 in cell survival. This role would be modest, since it affects a very small fraction of Insm1 -/- OE cells during the period examined (from E10.5 to E18.5). However, the increase in cell death is observed at a stage (E14.5) when olfactory receptor neurons innervate their targets in the olfactory bulb. In the wild type, the peak of cell death at this moment is thought to reflect the elimination of neurons that failed to establish proper synaptic contacts . Hence, given that the Insm1 -/- embryos may also experience a reduction in olfactory bulb neurons (as they do in cortical neurons ), the slight increase in OE cell death may be an indirect consequence and not reflect a cell autonomous role of Insm1 in promoting neuronal survival.
The decrease in OMP-expressing, mature olfactory receptor neurons is more pronounced than the decrease in TuJ1-expressing, immature neurons (approximately 40% versus approximately 92%, respectively; Figure 3). The increase in cell death mentioned above could contribute to this difference if the cells dying are neurons prior to maturation. We were not able to elucidate which cells undergo programmed cell death in the OE, but previous studies have shown these to be TuJ1-expressing, young neurons . Although the increase of dying (that is, ACC3-expressing) cells (0.3 cells per 0.1 mm2 at E14.5) is ten-fold lower than the decrease in OMP-expressing neurons (3.1 cells per 0.1 mm2 at E14.5; after correcting for the 39% decrease in immature, TuJ1-expressing neurons), the transient nature of apoptotic cells underestimates their frequency. For example, if an apoptotic cell expresses ACC3 during a 3-hour period (a guess, since this has not been measured), then the density of missing cells (dying at a density of 0.3 cells per 0.1 mm2 mentioned above) would reach 3 per 0.1 mm2 in 30 hours, a value consistent with the observed reduction in OMP-expressing neurons.
The disproportionately larger reduction in OMP-expressing mature neurons than in TuJ1-expressing young neurons might also suggest a role for Insm1 in neuronal differentiation. In fact, Insm1 has been implicated in the differentiation of pancreatic and intestinal endocrine cells , adrenal chromaffin cells  and hindbrain monoaminergic neurons . However, the disproportionate reduction in OMP-expressing cells may simply reflect a more steep change in the onset of OMP expression than in the onset of TuJ1 expression during the observed stages (E14.5 and E18.5). Due to the lethality of Insm1 -/- embryos, we cannot determine whether all TuJ1-expressing nascent neurons will mature to express OMP, or whether many of them will fail to differentiate (thus revealing a role for Insm1 in olfactory neuron differentiation).
Phenotypic similarities between Insm1 and egl-46mutants suggest functional conservation in neurogenic proliferation from nematodes to mammals
Mammalian Insm1 and nematode egl-46 have a similar pattern of expression in neuronal lineages: both are transiently expressed in neuronogenic progenitors and nascent neurons and not in many of the earlier, proliferative progenitors (Figure 10A, C). In particular, nematode egl-46 is expressed in progenitors of the motorneuron and Q-neuroblast lineages undergoing N/N but not P/P or P/N divisions, whereas mouse Insm1 is expressed in OE progenitors undergoing mitosis basally (many of which are neuronogenic) but not apically (many of which are proliferative). Although our data cannot determine whether Insm1 is exclusively expressed in terminally dividing N/N progenitors of OE, they indicate that Insm1 is expressed in late neuronogenic progenitors and not in many of the earlier, proliferative progenitors. In nematodes, mutation of egl-46 results in a transient decrease in neuronogenic (N/N) divisions and a concomitant increase in proliferative (P/N or P/P) divisions (Figure 10B). This occurs because progenitors that by lineage are expected to terminally divide fail to do so, and instead some of their progeny divide one or more times. Similarly we find that, in the mammalian OE, deletion of Insm1 results in fewer terminally dividing neuronogenic progenitors and in a concomitant increase in proliferative (apical and ASCL1-expressing) progenitors (Figure 10D). Hence, we propose that Insm1 and egl-46 play evolutionarily conserved roles in promoting terminal, neuronogenic divisions.
It should be noted that, in both organisms, the mutant effects are modest. In egl-46 mutants, only some terminal progenitors are delayed, and only by one or two cell divisions [2–4]. In Insm1 mutants, the increase in apical progenitors is transient (detected at E12.5 but not at E10.5 or E14.5) and small (42% increase; Figure 6), as is the decrease in basal progenitors (45% at E12.5 and 35% at E14.5; Figure 6). The subtlety of both phenotypes demonstrates that these two genes are not essential for the transition from proliferative to neuronogenic progenitors, and instead suggests that their function is to fine tune their occurrence.
A difference between the egl-46(loss-of-function) and Insm1 -/- phenotypes is that, in mutant nematodes, an increase in proliferative divisions eventually results in additional neurons, whereas mouse Insm1 -/- OEs still contain fewer neurons than the wild type at the latest stages examined (E18.5). The embryonic lethality of Insm1 -/- prevents determining whether more neurons would be eventually produced. However, in these mutant OEs we also detected an increase in apical cells, which would eventually become sustentacular glia. Because apical progenitors stop transitioning basally by E16.5 [1, 25], the increase in apical progenitors observed at E12.5 may eventually result in more sustentacular cells and not in more neurons.
Phenotypic and other similarities suggest a common mechanism of neurogenic proliferation between OE and cortex
In embryonic cortex, progenitors undergoing mitosis at the apical side of the ventricular zone generate more progenitors, some of which migrate in the basal direction. These basally located progenitors divide one or more times to produce neurons, which migrate to the cortical plate and differentiate [50–55]. The effects of Insm1 deletion in OE reported here share similarities with those reported in dorsal telencephalon . In both embryonic OE and cortex, Insm1 deletion results in an enlargement of the area with apical progenitors (the ventricular zone in cortex and the apical layer in OE), a decrease in basal, neuronogenic progenitors (the intermediate progenitor cells of the cortical subventricular zone and the INPs of OE), and a decrease in neurons. Furthermore, both neuroepithelia share striking similarities that extend beyond the Insm1 -/- phenotype. At embryonic stages, both have progenitors dividing apically (near the airways in OE and near the ventricle in cortex), some of which display radial glia characteristics . These apical progenitors undergo interkinetic nuclear migration and generate glial-like epithelial cells (ependymal in cortex and sustentacular in OE) as well as additional progenitors that migrate basally (to the basal lamina in OE and to the subventricular zone in cortex). In both embryonic neuroepithelia, many of these basal progenitors divide terminally to produce neurons. After embryogenesis, proliferation subsides in apical areas but is maintained in basal areas (subventricular zone and base of OE), where adult neural stem cells reside and neurogenesis occurs. All these similarities suggest that both neuroepithelia share a common mechanism of progenitor migration, proliferation and differentiation orchestrating neurogenesis and gliogenesis. We propose that common molecular pathways involving Insm1 and other factors may direct the development of OE and cortex and, perhaps, other neural tube and placodal neuroepithelia.
Our results indicate that, in the mouse embryonic OE, Insm1 promotes the transition of progenitors from apical, proliferative and uncommitted to basal, terminally dividing and neuron-producing. This role is similar to that of Insm1's C. elgans ortholog, egl-46, in certain neuronal lineages, and therefore appears to be evolutionarily conserved (Figure 10). Furthermore, the role of Insm1 in embryonic OE is similar to its role in embryonic cortex, in both cases facilitating the progenitors' transition from apical, proliferative and uncommitted to basal, terminally dividing and neuron-producing (Figure 10). Based on this and other similarities, we propose that both the neural-tube derived cortex and the placode-derived OE contain homologous neuroepithelial organization and types of progenitors whose progression is governed by common factors such as Insm1.
Materials and methods
Generation of Insm1knockout mouse
Construction of Insm1 targeting vector. A mouse 129/SvEv genomic BAC library, RPCI-22, was screened using a mouse Insm1 3' UTR probe isolated from pCRII7  by digesting with EcoRI. Three BAC clones were isolated and mapped using restriction enzymes. Two subclones from BAC clone 439G2 were used to generate the Insm1 targeting vector (Figure 1A). A 15.2 kb BglII genomic fragment containing the Insm1 coding sequence, 6.9 kb of 5' sequence and 6.7 kb of 3' sequence was cloned into the BglII site of pCMV-Myc (Clontech, Mountain View, California, USA) to generate pCMV-Myc G2Bgl. A 6-kb EcoRI fragment from the G2 BAC was cloned into the EcoRI site of pBluescript-SK- (Stratagene, Lost Pines, Texas, USA) to generate pSK-G2Eco24. A 1.05-kb XbaI-EcoRI genomic fragment from pSK-G2Eco24, the 3' homology arm, was cloned into pSK-, which had been cut with SmaI and EcoRI, to generate pSK-3'arm. The thymidine kinase cassette of pSK-PGKTK (a gift of Dr L Jameson) was subcloned as an EcoRI-XhoI fragment into pSK-3'arm to generate pSK-3'armTK. The floxed neomycin (Neo) cassette was released from pSK-FloxPGKNeo (a gift of Dr L Jameson) by cutting with XbaI; after blunting, the floxed Neo cassette was subcloned in an inverted orientation into pSK3'armTK that had been cut with BamHI and blunted to create pSKneo3'armTK. A 4-kb 5' fragment, the 5' homology arm, was cut from pCMV-Myc G2Bgl and subcloned into pSKneo3'armTK, which had been cut with XbaI and blunted, to generate the targeting vector, pSK insm1KO#2.
Generation of mouse ES cells and chimeric mice containing a targeted deletion of Insm1 The targeting vector pSKinsm1KO#2 was linearized with NotI prior to electroporation. Electroporation, ES cell culture and blastocyst injections were performed by the Northwestern University Transgenic and Targeted Mutagenesis Laboratory. Homologous recombination in positive ES cell clones was confirmed by PCR with external primer insm1 A7(CGAACATCCATACATTGCCCTGTAGCTCAG) and primer neoA1(CTGCTAAAGCGCATGCTCCAGACTGCCTTG), which yield a 1.3-kb band from the recombined allele, and by Southern hybridization of BglII digested genomic DNA with a 168-bp 5' probe, which detects a 15-kb wild-type band and a 6.35-kb recombined band, and a 3' probe that detects a 15-kb wild-type band and a 7.5-kb recombined band.
Chimeric males were mated to C57Bl/6 (Taconic) females, and germline transmission was indicated by agouti offspring. PCR with primers insm1 A7 and neoA1 demonstrated the presence of the targeted allele. The resulting mixed background 129P/Ola+C57Bl/6 Insm1- Neo + line (Insm1 tm1Jga ) was crossed against a C57Bl/6 E2a-Cre trasnsgenic line in order to excise the Neo cassette. The resultant Insm1- transline (Insm1 tm1.1Jga ) was then backcrossed into C57Bl/6 mice (strain code 027, Charles River, Wilmington, MA, USA) for ten generations to achieve effective line purity. Additionally, mice from the mixed-background Insm1- line were out-crossed into the CD-1 strain (strain code 002, Charles River) and back-crossed for ten generations.
Mouse genotypes were ascertained by tail samples purified by DNEasy DNA purification kit (69506, Qiagen, Gaithersburg, MD, USA), followed by PCR-based genotyping for the KO allele. PCR for the KO allele used the forward primer 5'-TTTGGAAGCCAGCCAGCA-3' and the reverse primer 5'-CCCGATTCTTGTGAGAGAGGAGGA-3'. PCR for the wild-type allele used the same forward primer, but used reverse primer 5'-GGGAACGCGCAACTCAACTC-3'. Primers were synthesized by Integrated DNA Technologies (Coralville, IA, USA). Due to the GC-rich nucleoside content of the Insm1 sequence, 1M betaine (B2629, Sigma-Aldrich, St Louis, MO, USA) was added to the reaction mixture to promote DNA melting.
Generation of homozygous knockout embryos
Heterozygous mice were cross-bred in timed pregnancies in order to produce embryos of specific, known ages. Males and females were placed together for a single night and the following morning were checked for plugs and separated whether a plug was observed or not. Midnight of the night of mating was considered the beginning of E0. In order to rescue prenatal lethality of homozygous KO embryos collected at E12.5 and later, pregnant dams were supplied daily with L-DOPA (1 mg/mL, 0.25% ascorbic acid) starting no later than 7 dpc (TCD0600, VWR Scientific, West Chester, PA, USA; D9628, Sigma-Aldrich) [23, 26]. To obtain embryos at E11.5 and earlier, females in which plugs had been observed were collected for harvest. In order to collect embryos of developmental stages E12.5 and later, females were weighed at least once prior to 10.5 dpc, then every day at 10.5 dpc and later in order to track weight gain. Females in which consistent weight gain of at least 5 to 10% per day was observed were considered pregnant and were collected for harvest.
Dams were sacrificed by isoflurane overdose, followed by cervical dislocation. Embryos were harvested, and their tails were removed, washed in PBS, and processed for genotyping by PCR. KO embryos and either wild type (WT) or heterozygous (HET) littermates were assayed for developmental stage , decapitated, and placed immediately into 4% paraformaldehyde fixative (1008A, Tousimis, Rockville, MD, USA). Following dehydration in sucrose gradient, embryos were weighed, and the heads were embedded in OCT compound (25608-930, VWR Scientific) and cryosectioned.
In situhybridizatioin, immunohistochemistry, nucleoside analog labeling, and EdU detection chemistry
In situ hybridization and IHC were performed on 10-μm or 12-μm sections as previously described . For IHC, the following primary antibodies were used: mouse anti-neuronal class III β-tubulin (1:500; clone Tuj1, Covance, Princeton, NJ, USA), goat anti-OMP (1:1,000; Wako Chemicals, Richmond, VA, USA), rabbit anti-pH3 (1:100; Millipore, Billerica, MA, USA), rabbit anti-ACC3 (1:1,000; Cell Signaling, Beverly, MA, USA), rat anti-BrdU (1:400; AbD Serotec, Raleigh, NC, USA), mouse anti-BrdU clone MoBu (1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), mouse anti-Ki67 (1:200; BD Pharmingen, San Diego, CA, USA), mouse anti-Mash1 (1:100; BD Pharmingen), goat anti-NEUROD1 (1:50; Santa Cruz Biotechnology, Inc.).
Anti-Tuj1, anti-Ki67, and anti-ASCL1 staining required antigen retrieval with 10 mM sodium citrate in 0.25% Triton X-100 at 96°C, pH6.0 for 20 minutes, with a 30-minute cool-down, prior to blocking. Anti-BrdU staining required antigen exposure in 5 M HCl in 1% Triton X-100 for 15 minutes, prior to blocking. DAPI counterstain was applied to all IHC experiments, with the exception of those requiring the detection of BrdU because the acid antigen exposure ablates DAPI staining. Staining for ACC3 was performed using a horseradish peroxidase conjugated secondary antibody and a colorimetric 3,3'-diaminobenzadine (DAB) reaction kit (SK-4100, Vector Labs, Burlingame, CA, USA) to visualize positively stained cells. Detection followed the supplied protocol.
For BrdU/Ki67 double labeling, and for analysis of S-phase alone (BrdU only) 10 mM BrdU (B23151, Invitrogen, Carlsbad, CA, USA) was injected into pregnant dams (50 mg/kg) 30 minutes prior to sacrifice. For EdU/BrdU timed pulse labeling, 10 mM EdU (A10044, Invitrogen) was injected at 12.5 dpc (25 mg/kg), followed 12 hours later (13 dpc) by 10 mM BrdU (30 mg/kg). Embryos were allowed to develop until E14.5, at which point they were harvested and cryosectioned as described above. The mouse primary antibody to BrdU (clone MoBu) was used for IHC detection of BrdU because of demonstrated lack of cross-reactivity with EdU. Following IHC detection of BrdU, EdU was detected according to a modified protocol supplied by Invitrogen. Briefly, following BrdU IHC, slides were washed in 1× PBS, incubated in the EdU reaction cocktail (for 1.5 ml of reaction cocktail: 7.5 μl component B (AlexaFluor 488 azide), 30 μl component H (copper sulfate), 1,313 μl component G (reaction buffer), 150 μl component I (buffer additive), washed in 1% bovine serum albumin in 1× PBS, then cover-slipped.
Quantification and statistics
Littermates were paired, and stained sections were visualized and photographed on a Nikon Eclipse E600 microscope with an RT Slider SPOT camera (version 2.3.1; Diagnostic Instruments, Sterling Heights, MI, USA). Coronal sections were aligned anatomically to assure that similar regions of OE were being compared between KO embryos and their littermates. Images were counted blindly using Image J software (available at ) with the Cell Counter plugin (available at ). BrdU/EdU co-labeling and BrdU/Ki67 co-labeling were evaluated using the Colocalization plugin (available at ). Images were optimized for brightness and contrast using Image J and CorelDraw software. Data were cataloged and analyzed using Microsoft Excel 2007. For measurements of the thickness of the OE, anatomically aligned section pairs were measured multiple times (seven for E12.5, eight for E14.5, and nine for E18.5) along the straight portion of the septal OE. These measurements were summed to generate an average OE thickness. These paired averages were then compared to generate a percent difference in thickness between the KO and wild-type littermates. All other measurements were obtained from all areas of OE. A one-sample Student's t-test was used to evaluate the percent difference between the measurements of experimental (KO) and control (wild-type or HET) groups for both cell counts and for thickness measurements. Due to comparatively infrequent expression of ACC3, sections were not anatomically aligned, and all of the sections from one or more whole slides were quantified for positive staining of ACC3. Because the sections were not aligned anatomically for ACC3 expression, an unpaired Student's t-test was used for statistical analysis. To compare the mean weights of KO embryos to wild-type+HET embryos, an unpaired t-test was used. For all graphs, error bars reflect standard error of the mean (SEM) and *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
activated cleaved caspase 3
bacterial artificial chromosome
immediate neuronal precursor
olfactory marker protein
transit amplifying progenitor
We are especially grateful to Jennifer Ray for extensive mouse husbandry, genotyping and cryosectioning. Alfred Rademaker and Nanjiang Hou, from the Biostatistics core at Northwestern University, advised on statistical analysis. Supported by grants from NIH-NINDS (R01 NS44363) and from the Brain Research Foundation (BRFSG 2009-6) to JG-A.
- Murdoch B, Roskams AJ: Olfactory epithelium progenitors: insights from transgenic mice and in vitro biology. J Mol Histol. 2007, 38: 581-599. 10.1007/s10735-007-9141-2.View ArticlePubMed
- Desai C, Horvitz HR: Caenorhabditis elegans mutants defective in the functioning of the motor neurons responsible for egg laying. Genetics. 1989, 121: 703-721.PubMed CentralPubMed
- Duggan A: Studies of neural development in Caenorhabditis elegans. PhD thesis. 1998, New York: Columbia University, 145-
- Wu J, Duggan A, Chalfie M: Inhibition of touch cell fate by egl-44 and egl-46 in C. elegans. Genes Dev. 2001, 15: 789-802. 10.1101/gad.857401.PubMed CentralView ArticlePubMed
- Lan MS, Breslin MB: Structure, expression, and biological function of INSM1 transcription factor in neuroendocrine differentiation. FASEB J. 2009, 23: 2024-2033. 10.1096/fj.08-125971.PubMed CentralView ArticlePubMed
- Breslin MB, Zhu M, Notkins AL, Lan MS: Neuroendocrine differentiation factor, IA-1, is a transcriptional repressor and contains a specific DNA-binding domain: identification of consensus IA-1 binding sequence. Nucleic Acids Res. 2002, 30: 1038-1045. 10.1093/nar/30.4.1038.PubMed CentralView ArticlePubMed
- De Smaele E, Fragomeli C, Ferretti E, Pelloni M, Po A, Canettieri G, Coni S, Di Marcotullio L, Greco A, Moretti M, Di Rocco C, Pazzaglia S, Maroder M, Screpanti I, Giannini G, Gulino A: An integrated approach identifies Nhlh1 and Insm1 as Sonic Hedgehog-regulated genes in developing cerebellum and medulloblastoma. Neoplasia. 2008, 10: 89-98. 10.1593/neo.07891.PubMed CentralView ArticlePubMed
- Goto Y, De Silva MG, Toscani A, Prabhakar BS, Notkins AL, Lan MS: A novel human insulinoma-associated cDNA, IA-1, encodes a protein with "zinc-finger" DNA-binding motifs. J Biol Chem. 1992, 267: 15252-15257.PubMed
- Lan MS, Russell EK, Lu J, Johnson BE, Notkins AL: IA-1, a new marker for neuroendocrine differentiation in human lung cancer cell lines. Cancer Res. 1993, 53: 4169-4171.PubMed
- Parent AS, Matagne V, Westphal M, Heger S, Ojeda S, Jung H: Gene expression profiling of hypothalamic hamartomas: a search for genes associated with central precocious puberty. Horm Res. 2008, 69: 114-123. 10.1159/000111815.View ArticlePubMed
- Pomeroy SL, Tamayo P, Gaasenbeek M, Sturla LM, Angelo M, McLaughlin ME, Kim JY, Goumnerova LC, Black PM, Lau C, Allen JC, Zagzag D, Olson JM, Curran T, Wetmore C, Biegel JA, Poggio T, Mukherjee S, Rifkin R, Califano A, Stolovitzky G, Louis DN, Mesirov JP, Lander ES, Golub TR: Prediction of central nervous system embryonal tumour outcome based on gene expression. Nature. 2002, 415: 436-442. 10.1038/415436a.View ArticlePubMed
- Taniwaki M, Daigo Y, Ishikawa N, Takano A, Tsunoda T, Yasui W, Inai K, Kohno N, Nakamura Y: Gene expression profiles of small-cell lung cancers: molecular signatures of lung cancer. Int J Oncol. 2006, 29: 567-575.PubMed
- Zhu M, Breslin MB, Lan MS: Expression of a novel zinc-finger cDNA, IA-1, is associated with rat AR42J cells differentiation into insulin-positive cells. Pancreas. 2002, 24: 139-145. 10.1097/00006676-200203000-00004.View ArticlePubMed
- Lukowski CM, Ritzel RG, Waskiewicz AJ: Expression of two insm1-like genes in the developing zebrafish nervous system. Gene Expr Patterns. 2006, 6: 711-718. 10.1016/j.modgep.2005.12.008.View ArticlePubMed
- Duggan A, Madathany T, de Castro SC, Gerrelli D, Guddati K, Garcia-Anoveros J: Transient expression of the conserved zinc finger gene INSM1 in progenitors and nascent neurons throughout embryonic and adult neurogenesis. J Comp Neurol. 2008, 507: 1497-1520. 10.1002/cne.21629.View ArticlePubMed
- Farkas LM, Haffner C, Giger T, Khaitovich P, Nowick K, Birchmeier C, Paabo S, Huttner WB: Insulinoma-associated 1 has a panneurogenic role and promotes the generation and expansion of basal progenitors in the developing mouse neocortex. Neuron. 2008, 60: 40-55. 10.1016/j.neuron.2008.09.020.View ArticlePubMed
- Xie J, Cai T, Zhang H, Lan MS, Notkins AL: The zinc-finger transcription factor INSM1 is expressed during embryo development and interacts with the Cbl-associated protein. Genomics. 2002, 80: 54-61. 10.1006/geno.2002.6800.PubMed CentralView ArticlePubMed
- Breslin MB, Zhu M, Lan MS: NeuroD1/E47 regulates the E-box element of a novel zinc finger transcription factor, IA-1, in developing nervous system. J Biol Chem. 2003, 278: 38991-38997. 10.1074/jbc.M306795200.PubMed CentralView ArticlePubMed
- Gierl MS, Karoulias N, Wende H, Strehle M, Birchmeier C: The zinc-finger factor Insm1 (IA-1) is essential for the development of pancreatic beta cells and intestinal endocrine cells. Genes Dev. 2006, 20: 2465-2478. 10.1101/gad.381806.PubMed CentralView ArticlePubMed
- Horb LD, Jarkji ZH, Horb ME: Xenopus Insm1 is essential for gastrointestinal and pancreatic endocrine cell development. Dev Dyn. 2009, 238: 2505-2510. 10.1002/dvdy.22071.PubMed CentralView ArticlePubMed
- Zhang T, Wang H, Saunee NA, Breslin MB, Lan MS: Insulinoma-associated antigen-1 zinc-finger transcription factor promotes pancreatic duct cell trans-differentiation. Endocrinology. 2010, 151: 2030-2039. 10.1210/en.2009-1224.PubMed CentralView ArticlePubMed
- Mellitzer G, Bonne S, Luco RF, Van De Casteele M, Lenne-Samuel N, Collombat P, Mansouri A, Lee J, Lan M, Pipeleers D, Nielsen FC, Ferrer J, Gradwohl G, Heimberg H: IA1 is NGN3-dependent and essential for differentiation of the endocrine pancreas. EMBO J. 2006, 25: 1344-1352. 10.1038/sj.emboj.7601011.PubMed CentralView ArticlePubMed
- Wildner H, Gierl MS, Strehle M, Pla P, Birchmeier C: Insm1 (IA-1) is a crucial component of the transcriptional network that controls differentiation of the sympatho-adrenal lineage. Development. 2008, 135: 473-481. 10.1242/dev.011783.View ArticlePubMed
- Jacob J, Storm R, Castro DS, Milton C, Pla P, Guillemot F, Birchmeier C, Briscoe J: Insm1 (IA-1) is an essential component of the regulatory network that specifies monoaminergic neuronal phenotypes in the vertebrate hindbrain. Development. 2009, 136: 2477-2485. 10.1242/dev.034546.PubMed CentralView ArticlePubMed
- Kawauchi S, Beites CL, Crocker CE, Wu HH, Bonnin A, Murray R, Calof AL: Molecular signals regulating proliferation of stem and progenitor cells in mouse olfactory epithelium. Dev Neurosci. 2004, 26: 166-180. 10.1159/000082135.View ArticlePubMed
- Thomas SA, Matsumoto AM, Palmiter RD: Noradrenaline is essential for mouse fetal development. Nature. 1995, 374: 643-646. 10.1038/374643a0.View ArticlePubMed
- Smart IH: Location and orientation of mitotic figures in the developing mouse olfactory epithelium. J Anat. 1971, 109: 243-251.PubMed CentralPubMed
- Graziadei PPC, Monti Graziadei GA: Continuous nerve cell renewal in the olfactory system. Handbook of Sensory Physiology. Edited by: Jacobson M. 1978, Berlin: Springer, IX: 55-82.
- Farbman AI, Margolis FL: Olfactory marker protein during ontogeny: immunohistochemical localization. Dev Biol. 1980, 74: 205-215. 10.1016/0012-1606(80)90062-7.View ArticlePubMed
- Miragall F, Monti Graziadei GA: Experimental studies on the olfactory marker protein. II. Appearance of the olfactory marker protein during differentiation of the olfactory sensory neurons of mouse: an immunohistochemical and autoradiographic study. Brain Res. 1982, 239: 245-250. 10.1016/0006-8993(82)90846-0.View ArticlePubMed
- Baker H, Grillo M, Margolis FL: Biochemical and immunocytochemical characterization of olfactory marker protein in the rodent central nervous system. J Comp Neurol. 1989, 285: 246-261. 10.1002/cne.902850207.View ArticlePubMed
- Voyron S, Giacobini P, Tarozzo G, Cappello P, Perroteau I, Fasolo A: Apoptosis in the development of the mouse olfactory epithelium. Brain Res Dev Brain Res. 1999, 115: 49-55. 10.1016/S0165-3806(99)00055-3.View ArticlePubMed
- Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H: Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol. 1984, 133: 1710-1715.PubMed
- Gerdes J, Li L, Schlueter C, Duchrow M, Wohlenberg C, Gerlach C, Stahmer I, Kloth S, Brandt E, Flad HD: Immunobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67. Am J Pathol. 1991, 138: 867-873.PubMed CentralPubMed
- Gerdes J, Schwab U, Lemke H, Stein H: Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer. 1983, 31: 13-20. 10.1002/ijc.2910310104.View ArticlePubMed
- Cubelos B, Sebastian-Serrano A, Kim S, Moreno-Ortiz C, Redondo JM, Walsh CA, Nieto M: Cux-2 controls the proliferation of neuronal intermediate precursors of the cortical subventricular zone. Cereb Cortex. 2008, 18: 1758-1770. 10.1093/cercor/bhm199.View ArticlePubMed
- Brenner RM, Slayden OD, Rodgers WH, Critchley HO, Carroll R, Nie XJ, Mah K: Immunocytochemical assessment of mitotic activity with an antibody to phosphorylated histone H3 in the macaque and human endometrium. Hum Reprod. 2003, 18: 1185-1193. 10.1093/humrep/deg255.View ArticlePubMed
- Calof AL, Chikaraishi DM: Analysis of neurogenesis in a mammalian neuroepithelium: proliferation and differentiation of an olfactory neuron precursor in vitro. Neuron. 1989, 3: 115-127. 10.1016/0896-6273(89)90120-7.View ArticlePubMed
- Manglapus GL, Youngentob SL, Schwob JE: Expression patterns of basic helix-loop-helix transcription factors define subsets of olfactory progenitor cells. J Comp Neurol. 2004, 479: 216-233. 10.1002/cne.20316.View ArticlePubMed
- Suzuki Y, Mizoguchi I, Nishiyama H, Takeda M, Obara N: Expression of Hes6 and NeuroD in the olfactory epithelium, vomeronasal organ and non-sensory patches. Chem Senses. 2003, 28: 197-205. 10.1093/chemse/28.3.197.View ArticlePubMed
- Calof AL, Bonnin A, Crocker C, Kawauchi S, Murray RC, Shou J, Wu HH: Progenitor cells of the olfactory receptor neuron lineage. Microsc Res Tech. 2002, 58: 176-188. 10.1002/jemt.10147.View ArticlePubMed
- Balmer CW, LaMantia AS: Noses and neurons: induction, morphogenesis, and neuronal differentiation in the peripheral olfactory pathway. Dev Dyn. 2005, 234: 464-481. 10.1002/dvdy.20582.View ArticlePubMed
- Cau E, Casarosa S, Guillemot F: Mash1 and Ngn1 control distinct steps of determination and differentiation in the olfactory sensory neuron lineage. Development. 2002, 129: 1871-1880.PubMed
- Cau E, Gradwohl G, Fode C, Guillemot F: Mash1 activates a cascade of bHLH regulators in olfactory neuron progenitors. Development. 1997, 124: 1611-1621.PubMed
- Tucker ES, Lehtinen MK, Maynard T, Zirlinger M, Dulac C, Rawson N, Pevny L, Lamantia AS: Proliferative and transcriptional identity of distinct classes of neural precursors in the mammalian olfactory epithelium. Development. 2010, 137: 2471-2481. 10.1242/dev.049718.PubMed CentralView ArticlePubMed
- Liu WD, Wang HW, Muguira M, Breslin MB, Lan MS: INSM1 functions as a transcriptional repressor of the neuroD/beta2 gene through the recruitment of cyclin D1 and histone deacetylases. Biochem J. 2006, 397: 169-177. 10.1042/BJ20051669.PubMed CentralView ArticlePubMed
- Wildner H, Gierl MS, Strehle M, Pla P, Birchmeier C: Insm1 (IA-1) is a crucial component of the transcriptional network that controls differentiation of the sympatho-adrenal lineage. Development. 2008, 135: 473-481. 10.1242/dev.011783.View ArticlePubMed
- Castro DS, Skowronska-Krawczyk D, Armant O, Donaldson IJ, Parras C, Hunt C, Critchley JA, Nguyen L, Gossler A, Gottgens B, Matter JM, Guillemot F: Proneural bHLH and Brn proteins coregulate a neurogenic program through cooperative binding to a conserved DNA motif. Dev Cell. 2006, 11: 831-844. 10.1016/j.devcel.2006.10.006.View ArticlePubMed
- Zhang T, Liu WD, Saunee NA, Breslin MB, Lan MS: Zinc finger transcription factor INSM1 interrupts cyclin D1 and CDK4 binding and induces cell cycle arrest. J Biol Chem. 2009, 284: 5574-5581. 10.1074/jbc.M808843200.PubMed CentralView ArticlePubMed
- Haubensak W, Attardo A, Denk W, Huttner WB: Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: a major site of neurogenesis. Proc Natl Acad Sci USA. 2004, 101: 3196-3201. 10.1073/pnas.0308600100.PubMed CentralView ArticlePubMed
- Miyata T, Kawaguchi A, Saito K, Kawano M, Muto T, Ogawa M: Asymmetric production of surface-dividing and non-surface-dividing cortical progenitor cells. Development. 2004, 131: 3133-3145. 10.1242/dev.01173.View ArticlePubMed
- Noctor SC, Martinez-Cerdeno V, Ivic L, Kriegstein AR: Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases. Nat Neurosci. 2004, 7: 136-144. 10.1038/nn1172.View ArticlePubMed
- Wu SX, Goebbels S, Nakamura K, Nakamura K, Kometani K, Minato N, Kaneko T, Nave KA, Tamamaki N: Pyramidal neurons of upper cortical layers generated by NEX-positive progenitor cells in the subventricular zone. Proc Natl Acad Sci USA. 2005, 102: 17172-17177. 10.1073/pnas.0508560102.PubMed CentralView ArticlePubMed
- Englund C, Fink A, Lau C, Pham D, Daza RA, Bulfone A, Kowalczyk T, Hevner RF: Pax6, Tbr2, and Tbr1 are expressed sequentially by radial glia, intermediate progenitor cells, and postmitotic neurons in developing neocortex. J Neurosci. 2005, 25: 247-251. 10.1523/JNEUROSCI.2899-04.2005.View ArticlePubMed
- Pontious A, Kowalczyk T, Englund C, Hevner RF: Role of intermediate progenitor cells in cerebral cortex development. Dev Neurosci. 2008, 30: 24-32. 10.1159/000109848.View ArticlePubMed
- Murdoch B, Roskams AJ: A novel embryonic nestin-expressing radial glia-like progenitor gives rise to zonally restricted olfactory and vomeronasal neurons. J Neurosci. 2008, 28: 4271-4282. 10.1523/JNEUROSCI.5566-07.2008.View ArticlePubMed
- EMAP Embryo Stage Selector. [http://genex.hgu.mrc.ac.uk/Atlas/intro.html]
- ImageJ. [http://rsbweb.nih.gov/ij/]
- Cell Counter. [http://rsbweb.nih.gov/ij/plugins/cell-counter.html]
- Colocalization. [http://rsbweb.nih.gov/ij/plugins/colocalization.html]
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.