Abstract

Sall1 is a zinc finger containing transcription factor that is highly expressed during mammalian embryogenesis. In humans, the developmental disorder Townes Brocks Syndrome is associated with mutations in the SALL1 gene. Sall1-deficient animals die at birth due to kidney deficits; however, its function in the nervous system has not been characterized. We examined the role of Sall1 in the developing olfactory system. We demonstrate that Sall1 is expressed by cells in the olfactory epithelium and olfactory bulb (OB). Sall1-deficient OBs are reduced in size and exhibit alterations in neurogenesis and mitral cell production. In addition, the olfactory nerve failed to extend past the ventral–medial region of the OB in Sall1-deficient animals. We observed intrinsic patterns of neurogenesis during olfactory development in control animals. In Sall1-mutant animals, these patterns of neurogenesis were disrupted. These findings suggest a role for Sall1 in regulating neuronal differentiation and maturation in developing neural structures.

Introduction

The olfactory system is required for the detection, transmission, and interpretation of scent. Olfactory stimuli are detected by olfactory receptor neurons that are topographically organized within the nasal epithelium (Buck and Axel 1991; Ressler et al. 1993, 1994; Vassar et al. 1993; Sullivan et al. 1995). These neurons project to spatially conserved glomeruli in the olfactory bulb (OB), where they synapse with the dendrites of mitral and tufted cells (Ressler et al. 1994; Mombaerts et al. 1996). Information is further transmitted to the olfactory cortex via the lateral olfactory tract, where it elicits a variety of emotional and behavioral responses (reviewed in Stockhorst and Pietrowsky 2004).

The development, projection pattern, and assembly of the olfactory system have been an area of active research over the last 10 years. In mice, OB morphogenesis initiates on embryonic day 11.5 (E11.5), when the rostral neural epithelium evaginates. Subsequently, olfactory neurogenesis generates the primary projection neurons, the mitral cells, from E11.5–E13.5 (Hinds 1968; Blanchart et al. 2006). Generation of OB interneurons, glomerular, and granule cells begins during late embryogenesis (E14.5), and these populations are continuously replenished throughout life from progenitor cells in the subventricular zone of the dorsal–lateral ganglionic eminence (Lois and Alvarez-Buylla 1994; Doetsch and Alvarez-Buylla 1996; Wichterle et al. 1999, 2001; Stenman et al. 2003). Previous studies have suggested that the olfactory epithelium and OB appear to be independent developmental domains (reviewed in Lopez-Mascaraque and de Castro 2002). However, studies in Xenopus suggest that the olfactory placode is required for OB development (Graziadei and Monti-Graziadei 1992; Byrd and Burd 1993). In rats, innervation of the OB by the olfactory nerve has been shown to influence the cell cycle kinetics of olfactory progenitors (Gong and Shipley 1995), leading to the hypothesis that the olfactory nerve extrinsically influences OB morphogenesis.

Insight into the interactions between the OB and olfactory epithelium/olfactory nerve has been gained from murine knockout analyses. Fibroblast growth factor receptor 1 (FGFR1) is expressed by cells in the olfactory epithelium and OB (Hsu et al. 2001; Hebert et al. 2003). In FGFR1-deficient animals the olfactory epithelium is normal, and olfactory nerve projections are observed (Hebert et al. 2003). However, in these mutant animals the OB fails to evaginate, yet differentiated OB cells accumulate at the anterior tip of the developing telencephalon. These findings suggest that OB evagination is not required for olfactory nerve innervation. Dlx5 is a transcription factor that is also expressed by cells in the olfactory epithelium and OB (Simeone et al. 1994; Stuhmer et al. 2002). In the absence of this transcription factor, the olfactory nerve fails to reach the OB, yet OB evagination and olfactory neurogenesis occur (Long et al. 2003). However, mitral cells were disorganized, and a decrease in interneuron number was observed in these mutant animals (Long et al. 2003). These findings suggest that olfactory nerve innervation is not essential for OB neurogenesis or evagination but may be required for cellular lamination. Because FGFR1 and Dlx5 are expressed by cells in both the olfactory epithelium and OB, it is difficult to dissect the precise role of these factors within the developing olfactory system. Recently, 2 genes have been identified that are only expressed by cells in either the olfactory epithelium (Fez) or OB (Arx) (Yoshihara et al. 2005; Hirata et al. 2006). Mice deficient for either gene have smaller OBs and similar cellular phenotypes. Most cell types are produced, although interneurons are generated in reduced numbers, and laminar organization is disrupted. In addition, the olfactory nerve fails to appropriately contact the OB, remaining outside the structure in a fibrocellular mass. Interestingly, early deficits in progenitor cell proliferation are observed in Arx-deficient animals, which suggests that olfactory innervation may be influenced by the number of differentiated cells. These findings support a role for cross-talk between olfactory epithelium and OB to regulate assembly of the olfactory system during development.

Is appropriate cellular specification required for olfactory nerve innervation? Excitatory and inhibitory neurons in the OB have distinct origins. Excitatory OB neurons have a pallial origin and migrate radially from OB progenitors to form the mitral cell layer (MCL) (Puelles et al. 2000; Moreno et al. 2003). The transcription factor Tbr1 has been implicated in the generation of this population (Bulfone et al. 1998). Tbr1 −/− animals completely lack mitral cells; however, topographic targeting of the olfactory nerve is unaltered. OB interneurons arise from an ER81-positive population in the subpallial dorsal–lateral ganglionic eminence and migrate to the OB via the rostral migratory stream (Lois and Alvarez-Buylla 1994; Doetsch and Alvarez-Buylla 1996; Wichterle et al. 1999, 2001; Stenman et al. 2003). A number of factors that regulate the generation of this diverse population of OB interneurons have been identified including Sp8, GSH1/2, Vax1, and Pax6 (Yun et al. 2003; Soria et al. 2004; Hack et al. 2005; Kohwi et al. 2005; Waclaw et al. 2006). However, deficits in the olfactory nerve have not been reported in these mutant animals. In addition, study of Dlx1/2-deficient animals indicates that, even in the absence of γ-aminobutyric acid-ergic (GABAergic) interneurons, layer organization is maintained and nerve innervation is unaltered (Bulfone et al. 1998). Thus, neither excitatory nor inhibitory neurons alone are required for olfactory nerve targeting.

We are studying the role of the Sall1 gene in the development of the olfactory system. The Sall (Spalt) genes encode a family of zinc finger containing transcription factors originally identified in Drosophila (Jurgens 1988; Kuhnlein et al. 1994). Members of the Sall gene family have been shown to interact at the protein level (Kiefer et al. 2003; Sweetman et al. 2003; Sakaki-Yumoto et al. 2006). Sall1 has been shown to localize to heterochromatized regions (Kiefer et al. 2002; Sato et al. 2004; Lauberth and Rauchman 2006; Netzer et al. 2006; Yamashita et al. 2007), and to interact with histone deacetylases to mediate transcriptional repression (Kiefer et al. 2002; Lauberth and Rauchman 2006). Four mammalian genes have been identified, and are widely expressed throughout development (Kohlhase et al. 1996, 1999, 2000; Ott et al. 1996, 2001; Buck et al. 2000, 2001; Al-Baradie et al. 2002; Kohlhase, Heinrich, Liebers, et al. 2002; Kohlhase, Heinrich, Schubert, et al. 2002). Mutation or deletion of these genes in humans is associated with distinct disorders with abnormalities in the limbs, ear, anus, kidney, and heart (Kohlhase et al. 1996, 1999; Al-Baradie et al. 2002; Kohlhase, Heinrich, Schubert, et al. 2002). In Drosophila and other species Sall proteins have been implicated in processes regulating development, including cell fate specification, neuronal differentiation, migration, and cell adhesion (Jurgens 1988; Kuhnlein and Schuh 1996; de Celis et al. 1999; Cantera et al. 2002; Franch-Marro and Casanova 2002; Toker et al. 2003; Barembaum and Bronner-Fraser 2004). These genes have also been implicated in Wnt and Sonic Hedgehog signaling (Koster et al. 1997; Sturtevant et al. 1997; Carl and Wittbrodt 1999; Farrell and Munsterberg 2000; Onai et al. 2004; Sato et al. 2004; Bohm et al. 2006). These data suggest that members of the Sall gene family are important developmental regulators.

Sall1 (previously named msal3) is expressed by cells in both the OB and olfactory epithelium (Ott et al. 2001), although a detailed pattern of its expression has not been described previously. Here, we characterize expression of Sall1 in the developing olfactory system and show that it is expressed in OB progenitor cells and a subset of differentiated neurons. In order to understand the role of this gene during development, a targeted disruption of Sall1 was generated (Nishinakamura et al. 2001). Deletion of Sall1 results in perinatal lethality due to kidney deficits (Nishinakamura et al. 2001). In addition, a disorganization of OB laminar structure in Sall1-mutant animals was noted (Nishinakamura et al. 2001). Our studies suggest that Sall1 is required to regionally regulate cell differentiation in the developing OB and, consequently, olfactory axon extension and mitral cell organization.

Materials and Methods

Animals

Embryos were obtained from matings of Sall1 heterozygote animals and genotyped as previously described (Nishinakamura et al. 2001). No alterations in olfactory development were observed in Sall1 heterozygous (+/−) animals in our study (data not shown), and therefore these embryos were also used as controls. For Sall1 expression studies, embryos were obtained from timed pregnant CD1 mice from Charles Rivers Laboratories (Wilmington, MA). Embryos were collected via cesarean section at embryonic ages from E13.5 to E18.5. The day of vaginal plug was designated as E0.5. Embryos were fixed in either 4% paraformaldehyde (pH 7.4) and processed through increasing sucrose gradients for cryosectioning or Carnoys solution (1:3:6 acetic acid: chloroform: 100% alcohol) and then processed through a butanol series for paraffin sectioning. Cryopreserved embryos were embedded in Tissue-Tek O.C.T. compound (EMS, Hatfield, PA) and sectioned at 20 μm. Paraffin embedded embryos were sectioned at 10 μm. Pregnant dams were injected with 5-bromo-2-deoxyuridine (BrdU) (50 μg/g of body weight), dissolved in sterile 0.9% NaCl and 0.007 M NaOH, at the indicated times before embryo harvest. Short-term BrdU injections were administered 60 min prior to embryo harvest. Animal protocols and procedures were approved by Institutional Animal Care and Use Committee at the University of Pittsburgh and adhered to the National Institutes of Health guidelines.

Measurement of OB Size

The total length of the OB was determined by counting the number of serial sections obtained through the entire OB at E14.5 and E18.5. This was subsequently converted to microns. At E14.5 the length of the OB was 254.4 ± 6.7 μm in control animals compared with 246.7 ± 13.3 μm in Sall1-mutant animals (n = 3, P = 0.7). At E18.5, the length of the OB was 626.7 ± 13.3 μm in control animals, and 493.3 ± 13.3 μm in Sall1-mutant animals (n = 3, P = 0.002). Statistical analysis was performed using an unpaired t-test with InStat 3 software (GraphPad Software, San Diego, CA).

Immunohistochemisty

Paraffin processed sections were deparaffinized in xylene, rehydrated through an ethanol series, and washed in 0.1% triton in phosphate-buffered saline (PBS) pH 7.4. For diaminobenzidine detection, sections were incubated with 3% hydrogen peroxide in methanol for 10 min. Slides were subsequently microwaved in 0.1 M sodium citrate solution pH 6.0, rinsed in PBS, and blocked in 10% heat inactivated goat serum (Jackson ImmunoResearch, West Grove, PA). Cryopreserved tissue was rinsed in PBS and blocked in 10% heat inactivated goat serum. Primary antibodies were incubated overnight at 4 °C. Antibodies used were mouse anti-BrdU (1:25, Amersham Biosciences, Piscataway, NJ); rat anti-BrdU (1:1000, Abcam, Cambridge, MA); rabbit anti-Calretinin (1:1000, Chemicon, Temecula, CA); rabbit anti-glutamic acid decarboxylase 65/67 (Gad65/67) (1:10,000, Sigma, St Louis, MO); mouse anti-GAP43 (1:100, Sigma); rabbit anti-GABA (1:1000, Sigma); rabbit anti-Laminin (1:500, Halfter 1993); rat anti-neural cell adhesion molecule (NCAM) (12F11) (undiluted, Chung et al. 1991); rabbit anti-Neuropilin 1 (1:100, Calbiochem, San Diego, CA); rabbit anti-S100β (1:200, Abcam); mouse anti-Reelin (1:500, Abcam); mouse anti-Sall1 (1:500, PPMX Perseus Proteomics, Tokyo, Japan); rabbit anti-Tbr1 (1:1000, Englund et al. 2005); rabbit anti-Tuj1 (1:1000, Sigma); mouse anti-Tyrosine Hydroxylase (1:5000, Sigma). The tissue was subsequently washed with PBS, incubated with the appropriate biotinylated secondary antibody (Vector Laboratories, Burlingame, CA), and then incubated with Vectastain Elite ABC kit (Vector Laboratories), according to the manufacturer's instructions. Staining was visualized using nickel enhanced diaminobenzidine (Sigma) reaction. Sections were counterstained with hematoxylin and eosin (Fisher Scientific, Pittsburgh, PA) or nuclear fast red (Vector Laboratories). Slides were then dehydrated through ethanol, washed in xylene, and mounted in DPX (Fluka, Sigma). For fluorescent detection of signal, the tissue was washed with PBS and incubated with the appropriate Cy3 (Jackson ImmunoResearch) and/or Alex Fluor 488 (Invitrogen, Carlsbad, CA) secondary antibody, and counterstained with 1,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma), before mounting in fluormount G (Southern Biotechnology Research, Birmingham, AL). For Nissl staining, sections were incubated in 0.5% cresyl violet (Sigma), dehydrated through alcohols, washed in xylene, and mounted in DPX. Sections were visualized on a Nikon (Melville, NY) fluorescent microscope, photographed with a Photometrics (North Reading, MA) Cool Snap digital camera, and IP Lab software (Biovision Technologies, Exton, PA). For confocal analysis, single optical sections were visualized on a Nikon DF0200 confocal microscope at 60× magnification. Composite images were prepared using Photoshop 7.0 (Adobe Systems, San Jose, CA). Contrast, color, and brightness were adjusted in Photoshop 7.0.

Histological Identification of OB Regions

To assign cellular subtypes and boundaries to OB regions, sections were examined at 40× magnification. The progenitor population was identified as the cellular region adjacent to the ventricle containing organized parallel arrays of cells with elongated nuclei arranged perpendicular to the ventricular surface. The differentiating population was identified as a region of heavily stained small cells with round nuclei arranged in a disorganized manner, adjacent to the progenitor population. The MCL was evident from E16.5 and contained cells with large oval-shaped nuclei, superficial to the differentiating field, and arranged perpendicular to the ventricular surface. At E18.5, the glomerular layer (GL) and granule cell layer (GCL) were identifiable. The GL was defined as the region containing round cells between the MCL and the olfactory nerve layer. The olfactory nerve layer was identified by the presence of elongated cells, orientated parallel to the ventricular surface, and perpendicular to the MCL. The GL, as defined in this study, encompasses the GL and the external plexiform layer. The GCL was located on the ventricular side of the MCL and contained small heavily stained round cells.

Cell Counts and Statistical Analysis

For proliferative studies, BrdU and DAPI stained sections were photographed at 40× magnification as described above. The analysis was performed by an observer blind to the genotype. The outermost boundary of the progenitor population was distinguished at 200×, as described above. To examine regional progenitor cell proliferation, the OB was divided into 6 regions, 3 dorsal and 3 ventral (n = 4 per region, Fig. S1A). These regions were defined as 50-μm-wide populations at the medial, central, or lateral extent of the olfactory ventricle, and the number of BrdU-positive and total number of cells within each region was quantified and analyzed, as previously described (Roy et al. 2004). The labeling index was determined, per region, as the number of BrdU-positive cells divided by total cell number. Three sections per animal were counted, and the mean of the 3 sections per animal was compared between genotypes (n = 4 per genotype). The data were subsequently decoded, and statistical analyses of the results were performed using a paired t-test for within genotype comparisons, and an unpaired t-test for between genotype comparisons, with InStat3 software. Based on biological considerations, 1-sided testing was used to examine regional differences of within genotype comparisons. For birth-dating studies (E18.5 embryos injected with BrdU on E11.5 or E14.5), BrdU stained sections were imaged at 20× magnification as described above (n = 3). A 150-μm region in the dorsal–ventral orientation through the center of the OB was examined (Fig. S1B), and heavily labeled BrdU-positive cells were quantified, as single positive or double positive for Tbr1 (E11.5 injected) or GABA (E14.5 injected). Three sections per animal were counted, and the mean of the 3 sections per animal was compared between genotypes (n = 3 per genotype). The data were subsequently decoded and statistical analyses of the results were performed, as described above.

Results

Sall1 is Expressed by Progenitor Cells and Subpopulations of Neurons in the Developing and Adult OB

Neurogenesis and maturation of OB cell types occurs in a precisely timed manner (Altman and Das 1966; Hinds 1968; Altman 1969; Blanchart et al. 2006). In order to more accurately understand the role of Sall1 in the generation of the OB, we first characterized expression of this gene in the developing (n = 3 per age) and adult (n = 2) OB. Development of the OB is initiated when incoming projections from the olfactory placode interact with progenitor cells in the neural epithelium of the developing telencephalon at ∼E11.5 (E13 rat, Gong and Shipley 1995). These projections are believed to stimulate the differentiation of progenitor cells in the telencephalic neural epithelium to form the OB (Gong and Shipley 1995). From E9.5, cells in the neural epithelium of the rostral most tip of the developing telencephalon express Sall1 (data not shown). Sall1 is expressed by cells in the olfactory placode from E9.5 (Buck et al. 2001). The olfactory placode gives rise to the vomeronasal organ and olfactory epithelium. By E13.5, Sall1 is strongly expressed by supporting cells in the olfactory epithelium and vomeronasal organ, and weakly expressed by basal cells in the olfactory epithelium (Fig. 1A,B). At E13.5, Sall1 is expressed by OB progenitors (Fig. 1C), and expression of this gene in OB progenitors continues throughout development (Fig. 1D).

Figure 1.

Sall1 expression in the developing and adult olfactory system. Immunohistochemistry of Sall1 expression in coronal (AF, H, I) and sagittal (G) sections in the developing (AG) and adult (H, I) olfactory system. Sall1 is expressed by cells in the olfactory epithelium (OE) (A, B) and vomeronasal organ (VNO) (A) at E13.5. Sall1 is strongly expressed by supporting cells (SC) and weakly expressed by basal cells (BC) in the OE (B). (B) is higher magnification of boxed region in (A). At E13.5 (C) and E17.5 (D), Sall1 is expressed by progenitor cells and differentiating cells. In addition, at E17.5 Sall1 is also expressed by cells in the MCL and GCL, and a subpopulation of cells in the GL (D, E). At E17.5, punctuate staining of Sall1 was observed in the MCL (arrowheads, F), whereas expression in other cellular populations was diffuse (arrows, F). Expression of Sall1 was also observed in the RMS (G). Sall1 expression is maintained in the adult (H, I) in the GL, MCL, and GCL. ORN: olfactory receptor neuron; P: progenitor cell; D: differentiating field; RMS: rostral migratory stream. Scale bar (in I) represents: 40 μm in (B); 70 μm in (F); 150 μm in (E); 275 μm in (C, I); 400 μm in (A); 425 μm in (D); 550 μm in (G); 1000 μm in (H).

Figure 1.

Sall1 expression in the developing and adult olfactory system. Immunohistochemistry of Sall1 expression in coronal (AF, H, I) and sagittal (G) sections in the developing (AG) and adult (H, I) olfactory system. Sall1 is expressed by cells in the olfactory epithelium (OE) (A, B) and vomeronasal organ (VNO) (A) at E13.5. Sall1 is strongly expressed by supporting cells (SC) and weakly expressed by basal cells (BC) in the OE (B). (B) is higher magnification of boxed region in (A). At E13.5 (C) and E17.5 (D), Sall1 is expressed by progenitor cells and differentiating cells. In addition, at E17.5 Sall1 is also expressed by cells in the MCL and GCL, and a subpopulation of cells in the GL (D, E). At E17.5, punctuate staining of Sall1 was observed in the MCL (arrowheads, F), whereas expression in other cellular populations was diffuse (arrows, F). Expression of Sall1 was also observed in the RMS (G). Sall1 expression is maintained in the adult (H, I) in the GL, MCL, and GCL. ORN: olfactory receptor neuron; P: progenitor cell; D: differentiating field; RMS: rostral migratory stream. Scale bar (in I) represents: 40 μm in (B); 70 μm in (F); 150 μm in (E); 275 μm in (C, I); 400 μm in (A); 425 μm in (D); 550 μm in (G); 1000 μm in (H).

Two major neuronal populations are present in the OB, excitatory projection neurons that comprise the MCL, and inhibitory interneurons that populate the GCL and GL. These populations are sequentially produced, with excitatory mitral cells born prior to interneuron populations (Altman and Das 1966; Hinds 1968; Altman 1969; Blanchart et al. 2006). Cells destined for the MCL are born from E11.5 to E13.5 (Hinds 1968; Blanchart et al. 2006). At E13.5, Sall1 is expressed by a large number of differentiating cells that are primarily destined for the MCL (D in Fig. 1C). By E17.5, Sall1 is robustly expressed by cells in the MCL (Fig. 1DF). Interestingly, at E17.5, we observed punctuate staining of Sall1 in mitral cells (arrowheads, Fig. 1F), whereas expression in other cell types was diffuse (arrows, Fig. 1F). Sall1 has been shown to localize to heterochromatized regions within the nucleus and to interact with histone deacetylases to mediate transcriptional repression (Kiefer et al. 2002; Sato et al. 2004; Lauberth and Rauchman 2006; Netzer et al. 2006; Yamashita et al. 2007). This difference in cellular localization of Sall1 could suggest that it has a distinct functional role in mitral cells at this age.

Interneuron populations arise from the ventral telencephalon and migrate to the OB via the rostral migratory stream (Lois and Alvarez-Buylla 1994; Doetsch and Alvarez-Buylla 1996; Wichterle et al. 1999, 2001; Stenman et al. 2003). Sall1 is expressed by cells within the rostral migratory stream at E15.5 (Fig. 1G). At E17.5, interneurons destined for the GL and GCL are differentiating, and a subpopulation has already reached their final laminar position. Sall1 is expressed by a subset of cells in the GL and GCL at this age (Fig. 1D,E). Expression of Sall1 is maintained in the MCL, GCL, and GL of the adult OB (Fig. 1H,I). In summary, Sall1 is expressed by OB progenitors from the onset of olfactory development, and expression continues in a subpopulation of differentiated cells. These data suggest a role for Sall1 in the development and function of olfactory structures.

Cellular Disorganization in the OB of Sall1-Mutant Animals

To determine whether this gene is required for distinct phases of OB development, mice with a targeted disruption of the Sall1 gene were examined. Sall1-deficient (Sall1−/−) animals do not thrive after birth and die shortly thereafter due to kidney deficits (Nishinakamura et al. 2001); thus Sall1-mutant embryos were examined up to E18.5. An analysis of gross OB morphology at E14.5 and E18.5 was conducted to determine the effect of loss of this gene on OB size. No difference in OB length was observed at E14.5 (P = 0.7, n = 3), however, by E18.5 a 21.3% decrease in length of OB (P = 0.002, n = 3) was observed in Sall1-mutant animals compared with controls.

To characterize the contribution of Sall1 to distinct cell types, coronal sections of control and Sall1−/− animals were examined from E14.5 to E18.5 (n = 3 per age). At E14.5, 2 distinct histological regions are visible in control OBs, the progenitor population and the differentiating field (Fig. 2A,A′). Both cell types were present in Sall1-mutant animals (Fig. 2B,B′). Interestingly, in Sall1−/− animals the OB ventricle appeared narrowed, elongated, and extended ventral–medially (Fig. 2B), which suggests an abnormal pattern of proliferation or differentiation. This slanting of the ventricle continued to E18.5 (Fig. 2D,F). By E16.5, a layer of differentiated neurons is observed outside the differentiating field. Mitral cells represent the major cellular population that has differentiated at this time and can be distinguished by their large oval-shaped cell body and light staining with Nissl (arrowheads, Fig. 2C′), as compared with small intensely stained cell bodies in the differentiating field. We therefore assigned this population as the MCL (Fig. 2C,C′). Cells within this population are tightly laminated and arranged perpendicular to the ventricular surface. In Sall1-mutant animals, the progenitor population and a population of differentiating cells were evident at this age (Fig. 2D). High-power examination of the OB revealed the presence of a cellular population with large oval-shaped cell bodies, lightly stained with Nissl, outside the differentiating field (arrowheads, Fig. 2D′). However, this population was not tightly laminated, as in controls, and thus was termed MCL-like (MCLl, Fig. 2D′). These data suggest an alteration in mitral cell number, specification, or organization in Sall1-mutant animals. By E18.5, a subset of interneurons have migrated past the MCL to reach their laminar position in the GL, superficial to the MCL (Fig. 2E,E′). In Sall1-mutant animals at E18.5 it was difficult to histologically distinguish laminar cell types (Fig. 2F), although similar to E16.5 a population of disorganized cells with oval-shaped cell bodies, lightly stained with Nissl, was present outside the differentiating field at this age (MCLl, Fig. 2F′). These observations suggest a role for Sall1 in cell type specification or laminar organization in the OB.

Figure 2.

Histological examination of the developing OB in control and Sall1-mutant animals. Nissl staining of Sall1 control (A, A′, C, C′, E, E′) and Sall1-mutant (B, B′, D, D′, F, F′) animals at E14.5 (AB′), E16.5 (CD′), E18.5 (EF′). Differentiated cells are present in wild type (A, A′) and Sall1-mutant (B, B′) animals from E14.5. From E16.5, a tightly laminated MCL is present consisting of large oval-shaped, lightly stained cells (arrowheads, C′) in control animals (C, C′, E, E′). In Sall1-mutant animals large oval cells (arrowheads, D′) are present from E16.5, but these cells are not tightly laminated (D, D′, F, F′). In the absence of Sall1, the ventricle appears slanted (B, D, F). (A′) Higher magnification of (A), etc. Dorsal is top and medial right, as indicated. P: progenitor cell; D: differentiating field; DL: dorsal; M: medial. Scale bar (in F′) represents: 100 μm in (A′, B′, C′, D′, E′, F′); 200 μm in (A, B); 300 μm in (C, D, E, F).

Figure 2.

Histological examination of the developing OB in control and Sall1-mutant animals. Nissl staining of Sall1 control (A, A′, C, C′, E, E′) and Sall1-mutant (B, B′, D, D′, F, F′) animals at E14.5 (AB′), E16.5 (CD′), E18.5 (EF′). Differentiated cells are present in wild type (A, A′) and Sall1-mutant (B, B′) animals from E14.5. From E16.5, a tightly laminated MCL is present consisting of large oval-shaped, lightly stained cells (arrowheads, C′) in control animals (C, C′, E, E′). In Sall1-mutant animals large oval cells (arrowheads, D′) are present from E16.5, but these cells are not tightly laminated (D, D′, F, F′). In the absence of Sall1, the ventricle appears slanted (B, D, F). (A′) Higher magnification of (A), etc. Dorsal is top and medial right, as indicated. P: progenitor cell; D: differentiating field; DL: dorsal; M: medial. Scale bar (in F′) represents: 100 μm in (A′, B′, C′, D′, E′, F′); 200 μm in (A, B); 300 μm in (C, D, E, F).

Alterations in Cellular Lamination are Observed in the Absence of Sall1

Histological analyses of the developing OB identified alterations in the laminar structure or cell type specification of the OB in the absence of Sall1. To exclude the possibility that these alterations were due to cell death, TdT-mediated dUTP nick end labeling (n = 3; E15.5, E18.5) and activated caspase 3 (n = 3; E18.5) staining were examined in developing embryos. No gross differences were observed between control and Sall1-mutant animals (data not shown), which suggests that Sall1 is not required for OB cell survival. In Sall1-mutant animals, a disorganized population of cells was present outside the progenitor population from E16.5 (Fig. 2D,D′,F,F′). To verify these cells were neurons, expression of the pan neuronal marker β-tubulinIII (Tuj1) was examined at E18.5. In control animals, Tuj1-positive cells were present throughout the OB and exhibited a characteristic striated staining pattern in the MCL (Fig. 3A). In Sall1-mutant animals, Tuj1-positive cells were present, but the striated staining pattern in the MCL was not observed (n = 4) (Fig. 3B), which suggests that neurons are differentiating, but that development of the MCL may be compromised.

Figure 3.

Examination of cell type and layer specification in control and Sall1-mutant animals. Tuj1 (A, B), Reelin (CF), and TH (G, H) expression (brown) in Sall1 control (A, C, E, G) and Sall1-mutant (B, D, F, H) animals at E16.5 (E, F) and E18.5 (AD, G, H). Sections were counterstained with hematoxylin and eosin. Cells in the MCL appear scattered and disorganized in the absence of Sall1 (B, MCLl: D, F). The GL is present in superficial regions in Sall1-mutant animals (H). Scale bar (in H) represents 70 μm in (DF); 100 μm in (A, B, G, H).

Figure 3.

Examination of cell type and layer specification in control and Sall1-mutant animals. Tuj1 (A, B), Reelin (CF), and TH (G, H) expression (brown) in Sall1 control (A, C, E, G) and Sall1-mutant (B, D, F, H) animals at E16.5 (E, F) and E18.5 (AD, G, H). Sections were counterstained with hematoxylin and eosin. Cells in the MCL appear scattered and disorganized in the absence of Sall1 (B, MCLl: D, F). The GL is present in superficial regions in Sall1-mutant animals (H). Scale bar (in H) represents 70 μm in (DF); 100 μm in (A, B, G, H).

To identify distinct laminae of the OB and to distinguish between early versus late generated populations, cell type specific markers were examined. Expression of Reelin (Fig. 3C-F), a marker of the early born MCL, and Tyrosine Hydroxylase (TH) (Fig. 3G,H), a marker of the later born GL, was examined. In Sall1−/− animals, Reelin-positive cells were present in superficial regions of the OB at E18.5 (n = 4) (MCLl, Fig. 3D), although the staining pattern was not tightly laminated, but appeared disorganized, in contrast to controls. Reelin expression is first detected in the MCL at E16.5. To determine whether the onset of Reelin expression was normal in Sall1-mutant animals, E16.5 embryos were examined (n = 2). Disorganized Reelin-positive staining was observed in Sall1−/− embryos at E16.5 (MCLl, Fig. 3F). These data suggest that in the absence of Sall1, mitral cells are generated but that these cells fail to appropriately organize. In wild type animals, TH is expressed by a subpopulation of cells in the GL, adjacent to the olfactory pial surface (Fig. 3G). In Sall1-mutant animals numerous TH-positive cells were detected adjacent to the pial surface at E18.5 (n = 3), which suggests appropriate generation and positioning of cells in the GL in the absence of Sall1 (Fig. 3F). These findings suggest that Sall1 is required for laminar organization of the MCL.

In the absence of Sall1, the OB is reduced in size, the olfactory ventricle is elongated and extends ventral–medially, and mitral cells appear disorganized. To further investigate the effect of loss of Sall1 on the MCL, we examined the orientation and organization of this cellular population using expression of Tbr1 to label cell bodies and Reelin to label apical dendrites (n = 4) (Fig. 4A,B). In control animals, Tbr1-positive cell bodies in the MCL were arranged in parallel arrays and Reelin-positive dendrites extended toward the pial surface (Fig. 4A). In the absence of Sall1, some Reelin-positive mitral cell dendrites were orientated toward the pial surface, although these dendrites appeared thicker and shorter than in control animals (arrows, Fig. 4B). Furthermore, many Reelin-positive mitral cell dendrites were found to be misorientated toward the basal, medial, and lateral surfaces (arrowheads, Fig. 4B). These data indicate that, in the absence of Sall1, mitral cells are misorientated and disorganized. Projections from mitral cells to the olfactory cortex via the lateral olfactory tract can be visualized with Calretinin (Fig. 4C). Despite the disorganization and misorientation of mitral cells observed in Sall1−/− animals, mitral cell projections via the lateral olfactory tract were present, although somewhat reduced (n = 3) (Fig. 4D).

Figure 4.

Cellular organization in control and Sall1-mutant animals. Confocal image of single optical slice of Tbr1 (green) and Reelin (red) expression at E18.5 (A, B). In control animals Reelin-positive dendrites extend toward the pial surface (A). In Sall1-mutant animals, Reelin-positive dendrites orientated toward the pial surface were thicker and shorter than in controls (arrows, B). In addition, many dendrites were misorientated toward the medial, lateral, and basal surfaces in Sall1−/− animals (arrowheads, B). Calretinin immunohistochemistry (black, C, D) indicated that the LOT projected to the olfactory cortex in Sall1-deficient animals at E18.5 (D). GAD65/67 (red, E, F) and Calretinin (red, G, H) immunohistochemistry at E18.5 revealed a disorganization of interneuron populations in Sall1−/− animals (F, H) compared with controls (E, G). Pial (P) surface is up, ventricular (V) surface is down in (A, B, EH), as indicated in (A). Sections were counterstained with nuclear fast red (C, D) or DAPI (EH). P: pial surface; V: ventricular surface; LOT: lateral olfactory tract; ON: olfactory nerve, CR: calretinin. Scale bar (in H) represents 30 μm in (A, B); 75 μm in (EH); 200 μm in (C, D).

Figure 4.

Cellular organization in control and Sall1-mutant animals. Confocal image of single optical slice of Tbr1 (green) and Reelin (red) expression at E18.5 (A, B). In control animals Reelin-positive dendrites extend toward the pial surface (A). In Sall1-mutant animals, Reelin-positive dendrites orientated toward the pial surface were thicker and shorter than in controls (arrows, B). In addition, many dendrites were misorientated toward the medial, lateral, and basal surfaces in Sall1−/− animals (arrowheads, B). Calretinin immunohistochemistry (black, C, D) indicated that the LOT projected to the olfactory cortex in Sall1-deficient animals at E18.5 (D). GAD65/67 (red, E, F) and Calretinin (red, G, H) immunohistochemistry at E18.5 revealed a disorganization of interneuron populations in Sall1−/− animals (F, H) compared with controls (E, G). Pial (P) surface is up, ventricular (V) surface is down in (A, B, EH), as indicated in (A). Sections were counterstained with nuclear fast red (C, D) or DAPI (EH). P: pial surface; V: ventricular surface; LOT: lateral olfactory tract; ON: olfactory nerve, CR: calretinin. Scale bar (in H) represents 30 μm in (A, B); 75 μm in (EH); 200 μm in (C, D).

Our results suggest a role for Sall1 in the lamination and orientation of mitral cells. In the absence of Sall1, TH-positive interneurons destined for the GL can migrate past the MCLl to their appropriate laminar position (Fig. 3F). The OB contains independent interneuron populations, which can be distinguished by expression of molecular markers (Kosaka et al. 1995; Toida et al. 2000; Parrish-Aungst et al. 2007). To determine whether other olfactory interneuron populations were altered in the absence of Sall1, we examined expression of 2 independent olfactory interneuron populations, GAD65/67 (n = 3) and Calretinin (n = 3), at E18.5. GAD65/67 is expressed by GABAergic cells in the GL and GCL, as well as the projections of these cells through the MCL (Fig. 4E). In Sall1−/− animals, GAD65/67 expression was present, although disorganized, throughout this region (Fig. 4F). Calretinin is expressed by a nondopaminergic olfactory interneuron population (Kosaka et al. 1995), and at E18.5 is expressed by olfactory nerve axons and cells within the GL, MCL, and GCL (Fig. 4G). In Sall1-mutant animals, similar to the GAD65/67 staining, Calretinin-positive cells were scattered throughout the entire region (Fig. 4H). Taken together, these results indicate that interneuron populations are appropriately generated in the absence of Sall1. These data suggest that Sall1 is not required for interneuron specification or migration to the OB via the rostral migratory stream; however, Sall1 expression may be required for laminar organization of subsets of interneurons.

The Olfactory Nerve Fails to Extend to the Dorsal–Lateral OB Surface in Sall1−/− Animals

Defects in olfactory nerve innervation have previously been shown to be associated with MCL deficits and OB disorganization (Long et al. 2003; Yoshihara et al. 2005; Hirata et al. 2006; Laub et al. 2006). We therefore examined innervation of the OB by the olfactory nerve using GAP43 expression (Fig. 5AD). At ∼E11.5 (E13 rat) the olfactory nerve innervates the neural epithelium of the developing telencephalon, and this is believed to stimulate the evagination of the bulb from this region (Gong and Shipley 1995). By E15.5, the nerve has contacted the ventral surface of the OB and has begun to extend laterally and medially in control animals (arrows, Fig. 5A), with a few fibers extending dorsally (*, Fig. 5A). In the absence of Sall1 the nerve innervated the ventral surface but did not extend as far medially and laterally as in controls (n = 4) (arrows, Fig. 5B), although similar to controls a few fibers extended toward the dorsal surface (*, Fig. 5B). In wild type animals at E17.5, the nerve has extended laterally, medially, and dorsally (Fig. 5C). In Sall1-mutant animals, the nerve remains in the ventral–medial position, failing to extend laterally (n = 3), with very few fibers extending dorsally (*, Fig. 5D). Furthermore, GAP43 staining demonstrated the presence of glomeruli-like structures in Sall1-mutant animals in the ventral OB at E17.5 (n = 3) (arrows, Fig. 5E,F), which suggests that where the olfactory nerve is present, appropriate innervation is established.

Figure 5.

Examination of olfactory nerve extension in control and Sall1-mutant animals. GAP43 expression at E15.5 (A, B) and E17.5 (C, D) identified a failure of olfactory nerve extension in Sall1-mutant animals (B, D). The arrows demarcate the medial and lateral most extension of the olfactory nerve (A, B, D). At E15.5, a few GAP43-positive fibers extended dorsally in both control and Sall1−/− animals (*, A, B). At E17.5, the olfactory nerve was confined to the ventral–medial OB (arrows, D), with only a few GAP43-positive fibers observed in dorsal regions (*, D). Glomeruli-like structures were observed with GAP43 staining in control and Sall1-deficient animals (arrows, E, F). S100β (G, H) and laminin (I, J) expression identified that olfactory ensheathing cells extended to the dorsal surface in both controls (G, I) and Sall1−/− (H, J) animals in sagittal sections at E18.5 (arrows). Neuropilin1 (Npn1)–positive axons were appropriately segregated in control (K) and Sall1-mutant animals (L), avoiding the region corresponding to Semaphorin3a expression (*, K, L). Dorsal is up and medial is right (AD, K, L), as indicated in (A). Dorsal is up and rostral is right (GJ), as indicated in (G). DL: dorsal; M: medial; R: rostral. Scale bar (in L): 75 μm in (E, F); 300 μm in (AD); 550 μm in (K, L); 600 μm in (GJ).

Figure 5.

Examination of olfactory nerve extension in control and Sall1-mutant animals. GAP43 expression at E15.5 (A, B) and E17.5 (C, D) identified a failure of olfactory nerve extension in Sall1-mutant animals (B, D). The arrows demarcate the medial and lateral most extension of the olfactory nerve (A, B, D). At E15.5, a few GAP43-positive fibers extended dorsally in both control and Sall1−/− animals (*, A, B). At E17.5, the olfactory nerve was confined to the ventral–medial OB (arrows, D), with only a few GAP43-positive fibers observed in dorsal regions (*, D). Glomeruli-like structures were observed with GAP43 staining in control and Sall1-deficient animals (arrows, E, F). S100β (G, H) and laminin (I, J) expression identified that olfactory ensheathing cells extended to the dorsal surface in both controls (G, I) and Sall1−/− (H, J) animals in sagittal sections at E18.5 (arrows). Neuropilin1 (Npn1)–positive axons were appropriately segregated in control (K) and Sall1-mutant animals (L), avoiding the region corresponding to Semaphorin3a expression (*, K, L). Dorsal is up and medial is right (AD, K, L), as indicated in (A). Dorsal is up and rostral is right (GJ), as indicated in (G). DL: dorsal; M: medial; R: rostral. Scale bar (in L): 75 μm in (E, F); 300 μm in (AD); 550 μm in (K, L); 600 μm in (GJ).

The olfactory nerve is segregated into inner and outer regions (Au et al. 2002), which can be distinguished by expression of NCAM and S100β. NCAM is expressed by the entire olfactory nerve (Chung et al. 1991), whereas S100β is strongly expressed by olfactory ensheathing cells in the outer portion of the olfactory nerve (Astic et al. 1998) (Fig. S2A). To determine whether Sall1 was required for segregation or layering of the olfactory nerve, we examined expression of these markers in sagittal sections at E18.5 (n = 3). In control animals, coexpression of NCAM and S100β was observed in the outer region of the olfactory nerve (**, Fig. S2A), whereas NCAM alone was expressed within the inner olfactory nerve (*, Fig. S2A). In the absence of Sall1, the olfactory nerve was thicker in ventral regions; however, segregation of NCAM and S100β expression was observed within this region (Fig. S2B). These findings indicate that Sall1 is not required for segregation of the olfactory nerve into outer and inner nerve layers.

Olfactory ensheathing cells are hypothesized to produce guidance factors required for olfactory nerve innervation (Treloar et al. 1996; Kafitz and Greer 1999; Tisay and Key 1999). To determine whether an absence of olfactory ensheathing cells in the dorsal–lateral OB contributed to the observed phenotype, we examined expression of S100β and laminin at E18.5 in sagittal sections (Fig. 5EH). S100β is strongly expressed by olfactory ensheathing cells (Astic et al. 1998), and at E18.5, S100β-positive cells were observed surrounding the OB, extending from the ventral surface up to the dorsal surface (arrows, Fig. 5G). In the absence of Sall1, a layer of S100β-positive olfactory ensheathing cells was observed extending to the dorsal surface (n = 3) (arrows, Fig. 5H). We confirmed that olfactory ensheathing cells extended the entire surface of the OB in Sall1-mutant animals at E18.5 using the olfactory ensheathing cell marker laminin (Doucette 1990) (n = 2) (arrows, Fig. 5I,J). These findings indicate that olfactory ensheathing cells are appropriately specified in the absence of Sall1. Taken together, these data suggest that extension of the olfactory nerve to lateral and dorsal surfaces is dependent on Sall1.

Axon guidance molecules expressed by cells within the OB have been shown to guide and sort incoming olfactory nerve fibers (reviewed in Lin and Ngai 1999; St John, Clarris, et al. 2002; Nedelec et al. 2005). In the absence of Sall1, the olfactory nerve contacts the OB but remains in the ventral–medial region, failing to extend laterally or dorsally (Fig. 5B,D). We hypothesized that alterations in signaling molecules in this region could account for this phenotype. We identified 2 key molecules that are expressed in complimentary patterns in the nerve layer of the developing OB, Semaphorin3a, and Neuropilin1. Semaphorin3a is expressed by cells in the olfactory nerve layer, and is hypothesized to act as a repulsive signal to direct and segregate Neuropilin1-expressing axons to lateral and medial regions of the central OB (Schwarting et al. 2000, 2004). In Semaphorin3a-mutant mice, Neuropilin1 axons do not segregate and fail to extend to lateral regions in the central and caudal OB (Schwarting et al. 2000), similar to the Sall1−/− phenotype. At E18.5, Neuropilin1 axons segregated into lateral and medial populations in both control and Sall1-deficient animals, avoiding the region corresponding to Semaphorin3a expression (*, Fig. 5K,L), although these axons did not extend as far laterally in Sall1-mutant animals as in controls (n = 2) (Fig. 5K,L). These data indicate that Semaphorin3a/Neuropilin1-mediated axonal sorting is relatively normal in Sall1-mutant animals.

The olfactory nerve arises from 4 distinct zones in the olfactory epithelium, and these zones project to topographically distinct glomeruli in the OB (Ressler et al. 1993, 1994; Vassar et al. 1993; Mombaerts et al. 1996). To determine whether the failure of the olfactory nerve to extend dorsally and laterally in Sall1−/− animals was a consequence of olfactory epithelium deficiencies, we examined development of this structure and zonal organization of the olfactory epithelium. The olfactory epithelium appears normal in Sall1-mutant animals at E15.5 and E17.5, as visualized by Nissl (Fig. S3AD) and Tuj1 staining (Fig. S3EL). Nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity is located in zone 1 of the olfactory epithelium (Alenius and Bohm 2003), which projects to the rostral–dorsal region of the OB (Ressler et al. 1994; Alenius and Bohm 2003), the region that is deficient in Sall1-mutant mice. No difference in NADPH diaphorase histochemistry was observed at E18.5 (data not shown, n = 4), which indicates that altered zonal organization is not responsible for deficiencies observed in Sall1 −/− animals. These data suggest that alterations in olfactory nerve innervation are not due to an intrinsic lack of Sall1 in the olfactory epithelium. Sall1 is not expressed by olfactory receptor neurons, which suggests that the deficits in olfactory nerve extension are due to a noncell autonomous effect.

Sall1 Regulates the Rate of Proliferation in the Developing OB

One striking phenotype observed in Sall1-mutant animals was the altered shape of the olfactory ventricle, which was elongated and extended ventral–medially from E14.5 (Fig. 2B,D,F). In Sall1-deficient animals, very few differentiated cells were observed in the ventral–medial region at this age (Fig. 2B). At this time, the nerve is localized to ventral–medial regions in control and mutant animals, which suggests that the initial targeting of the nerve is not affected by loss of Sall1. By E15.5, the olfactory nerve extends dorsally and laterally; however, in Sall1-mutant animals, the nerve remains predominately in the ventral–medial region, with only few fibers extending dorsally. In light of the absence of differentiated cells and failure of nerve extension past ventral–medial regions, we postulated that regional alterations in progenitor cell proliferation or differentiation could account for these deficiencies in Sall1-mutant animals. To investigate this possibility, we first quantified the number of progenitor cells in the OB at E14.5 and observed no difference in total progenitor cell number between wild type and mutant animals (713.8 ± 43.5 progenitor cells in controls versus 764.7 ± 25.4 progenitor cells in Sall1-mutant animals, n = 4, P = 0.4). We next examined the rate of proliferation of OB progenitors using short-term BrdU labeling. To determine whether regional differences in OB progenitor proliferation contributed to the observed phenotype, we divided the OB into 6 regions, medial, central, and lateral, in both the dorsal and ventral OB (Fig. S1A). In each region, we calculated the labeling index and compared wild type with mutant animals. In wild type animals, an intrinsic regional difference in the labeling index was observed. The labeling index was higher in dorsal compared with ventral progenitor populations (31.96% [dorsal–central] versus 19.01% [ventral–central], P = 0.02, n = 4, Table 1) and lateral compared with medial progenitor cells (38.98% [dorsal–lateral] versus 30.84% [dorsal–medial], P = 0.05, n = 4; 31.12% [ventral–lateral] versus 23.82% [ventral–medial], P = 0.04, n = 4; Table 1). These regional differences in the labeling index were preserved in Sall1-mutant animals (33.61% [dorsal–central] versus 29.61% [ventral–central], P = 0.007, n = 4; 45.41% [dorsal–lateral] versus 32.48% [dorsal–medial], P = 0.03, n = 4, Table 1; 40.49% [ventral–lateral] versus 23.39% [ventral–medial], P = 0.001; n = 4; Table 1), although the dorsal–ventral gradient was not as robust in Sall1-mutant animals as in controls (12.95% difference dorsal–central versus ventral–central in controls versus 3.9% difference dorsal–central versus ventral–central in Sall1-mutant animals, P = 0.04, n = 4). Interestingly, in ventral–central and ventral–lateral regions, where the olfactory nerve failed to extend at E15.5, we observed an increase in the labeling index at E14.5 in Sall1−/− animals compared with wild type littermates (Table 1). These findings suggest that, in the absence of cell death, Sall1 regionally regulates either the rate of proliferation or the rate of cell cycle exit in olfactory progenitors.

Table 1

Labeling index of Sall1+/ and Sall1−/− animals at E14.5

Region of study Labeling Index ± SEM % of WT P value 
 Sall1+/ Sall1−/−   
Ventral–medial 23.82 ± 2.41% 23.39 ± 1.68% 98.19% 0.8870 
Ventral–central 19.01 ± 1.13% 29.61 ± 0.84% 155.76% 0.0003 
Ventral–lateral 31.12 ± 0.96% 40.49 ± 2.22% 130.10% 0.0088 
Dorsal–medial 30.84 ± 2.65% 32.48 ± 1.75% 105.31% 0.6269 
Dorsal–central 31.96 ± 2.69% 33.61 ± 1.19% 105.16% 0.5923 
Dorsal–lateral 38.98 ± 1.50% 45.41 ± 3.06% 116.49% 0.1090 
Region of study Labeling Index ± SEM % of WT P value 
 Sall1+/ Sall1−/−   
Ventral–medial 23.82 ± 2.41% 23.39 ± 1.68% 98.19% 0.8870 
Ventral–central 19.01 ± 1.13% 29.61 ± 0.84% 155.76% 0.0003 
Ventral–lateral 31.12 ± 0.96% 40.49 ± 2.22% 130.10% 0.0088 
Dorsal–medial 30.84 ± 2.65% 32.48 ± 1.75% 105.31% 0.6269 
Dorsal–central 31.96 ± 2.69% 33.61 ± 1.19% 105.16% 0.5923 
Dorsal–lateral 38.98 ± 1.50% 45.41 ± 3.06% 116.49% 0.1090 

Sall1 Regionally Regulates Differentiation in the Developing OB

To determine the consequences of these changes in the labeling index in control versus Sall1-mutant animals, we labeled a population of proliferating cells on E14.5 using BrdU incorporation and examined the position of these cells at E18.5. Heavily labeled BrdU-positive cells represent the population of cells that differentiated on E14.5; cells that contain more lightly labeled BrdU represent cells that underwent further rounds of cell division, thus diluting the BrdU label. In control animals, heavily labeled BrdU-positive cells are predominately detected in the superficial GCL, as expected (Fig. 6A). Consistent with the observed regional differences in the rate of proliferation, more heavily labeled BrdU-positive cells were located in the ventral OB (65.4% [37.3 cells]) than in dorsal regions (34.6% [19.7 cells]) (n = 3, P = 0.02, Table 2, Fig. 6A) in control animals. These findings suggest that the regional differences in the labeling index correlate to regional changes in neurogenesis. In Sall1-mutant animals, the number of cells born on E14.5 and their laminar position in the GCL at E18.5 was similar to controls (n = 3, P = 0.4, Table 2, Fig. 6B), suggesting that loss of Sall1 does not alter initial interneuron production or their migration in the rostral migratory stream. However, the ventral–dorsal pattern of neurogenesis was not maintained in Sall1-mutant animals (48.3% [29.2 cells] ventral versus 51.7% [31.2 cells] dorsal, n = 3, P = 0.1, Fig. 6B, Table 2). Furthermore, a distinct change in the medial to lateral distribution of labeled cells was observed in Sall1-mutant animals, with very few heavily labeled BrdU-positive cells present in lateral regions (*, Fig. 6B). This area contained only lightly labeled BrdU cells, which suggests that they did not differentiate on E14.5 and underwent further rounds of cell division. These findings suggest that the increased labeling index at E14.5 in ventral and lateral regions is associated with decreased neuronal differentiation in Sall1-mutant animals. Furthermore, these data indicate that at E14.5 intrinsic gradients of proliferation and neurogenesis are observed in the OB in controls animals and that in the absence of Sall1 these gradients are disrupted.

Table 2

Cellular birth-dating in Sall1+/ and Sall−/− animals at E18.5

 Sall1+/a Sall1−/−a % of WT P Value 
Total: BrdU E14.5 57.0 ± 1.3 60.4 ± 3.4 105.9% 0.3759 
Ventral: BrdU E14.5 37.3 ± 1.6 29.2 ± 1.6 78.3% 0.0231 
Dorsal: BrdU E14.5 19.7 ± 1.7 31.2 ± 2.0 158.4% 0.0126 
Total: BrdU E11.5 5.8 ± 0.8 14.2 ± 1.6 244.8% 0.0102 
Ventral: BrdU E11.5 1.7 ± 0.3 4.3 ± 0.3 268.7% 0.0064 
Dorsal: BrdU E11.5 4.1 ± 0.6 9.9 ± 1.5 239.0% 0.0266 
 Sall1+/a Sall1−/−a % of WT P Value 
Total: BrdU E14.5 57.0 ± 1.3 60.4 ± 3.4 105.9% 0.3759 
Ventral: BrdU E14.5 37.3 ± 1.6 29.2 ± 1.6 78.3% 0.0231 
Dorsal: BrdU E14.5 19.7 ± 1.7 31.2 ± 2.0 158.4% 0.0126 
Total: BrdU E11.5 5.8 ± 0.8 14.2 ± 1.6 244.8% 0.0102 
Ventral: BrdU E11.5 1.7 ± 0.3 4.3 ± 0.3 268.7% 0.0064 
Dorsal: BrdU E11.5 4.1 ± 0.6 9.9 ± 1.5 239.0% 0.0266 
a

Values indicate the number of BrdU-positive cells ± SEM.

Figure 6.

Examination of position and number of early (E11.5) and later (E14.5) born cells in the developing OB at E18.5. BrdU staining (red) of E18.5 embryos labeled with BrdU on E14.5 (A, B). In control animals, heavily labeled BrdU-positive cells are found throughout the circumference of the OB, with highest concentration in the ventral OB (A). In Sall1−/− animals, heavily labeled cells are located in ventral and dorsal populations but are absent from lateral regions (*, B). BrdU staining (red) of E18.5 embryos labeled with BrdU on E11.5 (C, D). In control animals, heavily labeled cells are located in the MCL, although more cells are present dorsally than ventrally (C). In Sall1−/− animals, many more heavily labeled cells were observed in the MCLl in ventral, dorsal, and lateral regions than in control animals; however, very few heavily labeled cells were present in the medial OB (*, D). Staining with the mitral cell marker Tbr1 (red) confirmed that in Sall1-mutant animals (F), more mitral cells were present in the OB at E18.5 than in controls (E), and that the medial OB contained very few mitral cells. Sections were counterstained with DAPI. Dorsal is up and medial is right, as indicated in (E). DL: dorsal; M: medial. Scale bar = 300 μm.

Figure 6.

Examination of position and number of early (E11.5) and later (E14.5) born cells in the developing OB at E18.5. BrdU staining (red) of E18.5 embryos labeled with BrdU on E14.5 (A, B). In control animals, heavily labeled BrdU-positive cells are found throughout the circumference of the OB, with highest concentration in the ventral OB (A). In Sall1−/− animals, heavily labeled cells are located in ventral and dorsal populations but are absent from lateral regions (*, B). BrdU staining (red) of E18.5 embryos labeled with BrdU on E11.5 (C, D). In control animals, heavily labeled cells are located in the MCL, although more cells are present dorsally than ventrally (C). In Sall1−/− animals, many more heavily labeled cells were observed in the MCLl in ventral, dorsal, and lateral regions than in control animals; however, very few heavily labeled cells were present in the medial OB (*, D). Staining with the mitral cell marker Tbr1 (red) confirmed that in Sall1-mutant animals (F), more mitral cells were present in the OB at E18.5 than in controls (E), and that the medial OB contained very few mitral cells. Sections were counterstained with DAPI. Dorsal is up and medial is right, as indicated in (E). DL: dorsal; M: medial. Scale bar = 300 μm.

Olfactory interneuron populations are born from E14.5 (Hinds 1968), and the OB contains distinct interneuron populations (Kosaka et al. 1995; Parrish-Aungst et al. 2007). GABAergic cellular populations represent the largest interneuron population in the OB (Parrish-Aungst et al. 2007). To verify that cells born on E14.5 differentiated into interneurons, we examined the proportion of cells born on E14.5 that were GABAergic at E18.5 (BrdU+/GABA+). In control animals, 72.4 ± 1.8% of cells born on E14.5 expressed GABA at E18.5. In Sall1-mutant animals, 84.4 ± 0.7% of cells born on E14.5 expressed GABA at E18.5. These findings indicate that GABAergic cells are the major population born on E14.5 in control and Sall1-mutant animals. Interestingly, a 16.6% increase in the proportion of GABAergic cells born on E14.5 was observed in the absence of Sall1 (P = 0.003, n = 3). These data suggest that Sall1 may influence the relative proportion of differentiating interneuron subtypes.

In the absence of Sall1, alterations in OB development are already apparent from E14.5, which suggests a role for Sall1 in MCL differentiation. To examine this possibility, we performed birth-dating studies on E11.5 animals, when MCL cells are born, and examined the position of heavily labeled cells at E18.5. In control animals, heavily labeled cells are located in the MCL, as expected, and encompass the entire OB. In Sall1-deficient animals, labeled cells were located outside the differentiating field, in the MCLl (Fig. 6D). More heavily labeled BrdU-positive cells were observed in dorsal regions (70.7% [4.1 cells]) than ventral (29.3% [1.7 cells]) in control animals (n = 3, P = 0.03, Table 2, Fig. 6C). Similar to control animals, more labeled cells were found dorsally in Sall1-mutant animals (69.7% [4.3 cells] dorsal versus 30.3% [9.9 cells] ventral, n = 3, P = 0.04, Table 2, Fig. 6D). Interestingly, labeled cells were noticeably absent from the ventral–medial region (*, Fig. 6D). In addition, a 144.8% increase in the number of cells born on E11.5 was observed in Sall1-deficient animals compared with control littermates (n = 3, P = 0.01, Table 2, Fig. 6D). This increase in the number of cells born on E11.5 in Sall1-deficient animals was observed in both dorsal and ventral populations (Table 2).

To verify that the cells born on E11.5 differentiated into mitral cells in the absence of Sall1, we examined coexpression of Tbr1, a MCL marker (Fig. 6E), and BrdU. The percent of cells that differentiate on E11.5 that express Tbr1 at E18.5 was similar in Sall1−/− (95.4 ± 1.6%) and control animals (94.5 ± 1.0%) (n = 3, P > 0.6). In addition, we quantified the total number of Tbr1-positive cells in dorsal–ventral regions at E18.5 and observed a 27.5% increase in the total number of Tbr1-positive cells in Sall1-mutant animals compared with controls (494.2 ± 6.5 in controls versus 630.2 ± 12.3 in Sall1-mutant animals, n = 3, P = 0.001, Fig. 6E,F). This is accompanied by a distinct reduction in the number of Tbr1-positive cells in the ventral–medial region (n = 4) (*, Fig. 6F). These findings indicate that Sall1 does not regulate mitral cell fate specification, but regionally regulates mitral cell neurogenesis in the developing OB. We therefore hypothesize that Sall1 is temporally and spatially required to regulate cellular differentiation in the developing OB.

Discussion

The role of the Sall1 gene was examined in the development of the olfactory system. We demonstrated that Sall1 is expressed by progenitor cells and subpopulations of differentiated neurons in the olfactory epithelium and OB. Sall1-mutant animals die at birth and have smaller OBs. We identified regional alterations in the pattern of proliferation and neurogenesis in the developing OB in the absence of Sall1. Our findings are consistent with a role for Sall1 in the temporal and spatial regulation of cellular differentiation during olfactory development. An increase in the number of early born neurons, mitral cells, was observed in the absence of Sall1. In addition, this cellular population was disorganized and misorientated. Despite the alterations in the mitral cell population, projections of these neurons to the olfactory cortex, via the lateral olfactory tract were present, although reduced in size. We postulate that the decreased size of the OB in Sall1-mutant animals contributes to the reduction in size of the lateral olfactory tract, although, it is possible that not all of the misorientated mitral cells project to the olfactory cortex.

Interneuron populations were specified in Sall1-deficient animals, and TH-positive cells migrate past the MCLl to their appropriate laminar position. However, alterations in the laminar position of GABAergic and Calretinin populations were observed in Sall1−/− animals. These alterations in laminar position may be a consequence of the disorganized MCL or may reflect a role for Sall1 in the lamination of distinct subsets of interneuron populations. We did not observe an absence of any distinct interneuron population in the absence of Sall1. However, more cells born at E14.5 differentiated into GABAergic subtypes, which suggests that a shift in interneuron subtype specification may exist in Sall1-deficient animals.

In Sall1-mutant animals, the olfactory nerve contacted the OB but failed to extend to dorsal and lateral OB regions. We hypothesize that the altered olfactory nerve extension is a consequence of Sall1’s role in OB neurogenesis, and that the disorganization of mitral cells is due to altered innervation of the OB by the olfactory nerve.

Sall1 Regulates OB Neurogenesis

Sall1 is required from as early as E11.5 to regulate olfactory neurogenesis. One of the first deficits observed in the absence of Sall1 is an increase in the number of neurons produced at E11.5 relative to control animals. Mitral cells are born from E11.5 to E13.5 in mice (Hinds 1968; Blanchart et al. 2006), and birth-dating studies indicate that these cells are overproduced in mutant animals. At E18.5, we observed spatial differences in the position of mitral layer cells born on E11.5 in mutant animals. In control animals, BrdU birth-dating studies indicated mitral cells born on E11.5 were localized in dorsal, ventral, medial, and lateral positions at E18.5. However, significantly more cells were localized in dorsal regions than ventral. In Sall1-mutant animals, more mitral cells were born on E11.5; nonetheless, the dorsal–ventral pattern of neurogenesis was maintained. However, very few mitral cells were observed in the ventral–medial OB at E18.5. This decrease in cell number in ventral–medial OB is obvious from E14.5, when very few neurons were observed in ventral–medial regions and the ventricle was elongated and extended ventral–medially.

From E14.5, cells destined for the GCL and GL are born (Hinds 1968). Our birth-dating studies indicate that cells born on E14.5 in control animals are predominantly localized in ventral, medial, and lateral regions on E18.5, with fewer cells born dorsally. Consistent with our findings, a recent study identified a number of factors, including Dlx1, GAD67, ER81, and Sp8, that are expressed in a “ventrolateral crescent” of the OB ventricular/subventricular zone at E15.5 (Long et al. 2007). These studies suggest that interneuron populations predominately differentiate in ventral regions at E15.5. In Sall1-mutant animals, cells born on E14.5 were localized in dorsal, medial, and ventral regions but were noticeably absent from lateral regions at E18.5. We hypothesized that regional alterations in cellular proliferation or differentiation could account for the observed differences in Sall1-deficient animals. We found that there is an intrinsic difference in the labeling index in dorsal/ventral and medial/lateral progenitor cells in control animals at E14.5. The labeling index is higher in dorsal and lateral than ventral and medial regions, in control animals. Because there is no significant difference in the number of progenitor cells in dorsal versus ventral, or lateral versus medial regions (data not shown), these findings suggest that there are fewer cells in S-phase of the cell cycle in ventral and medial regions, and that therefore, the cell cycle is longer. Previous studies in the cerebral cortex have shown that the cell cycle lengthens as progenitor cells mature and increased neuronal output is associated with lengthening of the cell cycle (Takahashi et al. 1999; Caviness et al. 2003). Consistent with these findings, increased neuronal differentiation (E14.5 to E18.5) is associated with a decrease in the labeling index at E14.5 in ventral regions in control animals. In Sall1-mutant animals, decreased neuronal differentiation (E14.5 to E18.5) in the ventral OB is associated with an increased labeling index at E14.5, compared with controls, in the ventral–central and ventral–lateral OB. Taken together, these observations suggest that progenitor cells in ventral–central and ventral–lateral regions continue to proliferate as opposed to differentiate in the absence of Sall1. We therefore hypothesize that Sall1 is required to temporally and spatially regulate olfactory neurogenesis.

Interactions between the Olfactory Epithelium and OB

Complex regulatory interactions between the olfactory epithelium/olfactory nerve and OB have been described (Mombaerts et al. 1996; Hebert et al. 2003; Long et al. 2003; Yoshihara et al. 2005; Hirata et al. 2006). Furthermore, it has been shown that the type of neuron produced in the OB is not critical for olfactory nerve innervation, because neither projection neurons nor GABAergic neurons are required for topographic targeting (Bulfone et al. 1998). The olfactory nerve appropriately exits the olfactory epithelium and contacts the OB in Sall1-deficient animals suggesting that initial olfactory nerve development is not dependent on Sall1; however, from E15.5 the olfactory nerve fails to target to lateral and dorsal regions. Sall1 is not expressed by olfactory receptor neurons in the olfactory epithelium. In addition, no alterations in olfactory epithelium development were observed in Sall1-mutant animals, which suggests that the deficits observed are due to a requirement for Sall1 expression in the OB. Yoshihara et al. hypothesized that radial glial cells may be instructive in the guidance of the olfactory nerve (Yoshihara et al. 2005). In Sall1-mutant animals, no alterations in radial glial fibers were observed at E14.5/E15.5, when the first differences in olfactory ventricular shape and olfactory nerve innervation were observed (unpublished observations). Furthermore, at all ages examined radial glial fibers extended to the pial surface. Although we cannot exclude a role for radial glial in the initial process of olfactory nerve innervation, the absence of altered radial glial fibers at the onset of the Sall1-mutant phenotype suggests that they are not required for targeting or extension of the olfactory nerve to at least the lateral and dorsal olfactory surface. Previous studies have suggested that olfactory ensheathing cells produce guidance factors that influence olfactory nerve innervation (Treloar et al. 1996; Kafitz and Greer 1999; Tisay and Key 1999). Interestingly, in Sall1-mutant mice, olfactory ensheathing cells were present and extended to the dorsal surface of the OB. Our data suggest that olfactory ensheathing cells are not sufficient to induce olfactory nerve extension.

Studies in Xenopus have shown that the timing of production of ventral and dorsal olfactory neurons differs (Fritz et al. 1996). Cells destined for the ventral OB are born first, and this differentiation appeared to be independent of olfactory innervation. However, cells destined for the dorsal OB are born later, and this differentiation is dependent on olfactory innervation. In rats, rostral to caudal differences in the timing of glomeruli formation have also been identified (Bailey et al. 1999). We observed differences in the pattern of neurogenesis of cells born on E11.5 in control animals. More cells born at this age are destined for the dorsal OB than the ventral OB, which suggests that dorsal populations are born first in early olfactory neurogenesis. This would suggest that early olfactory neurogenesis in mice is opposite to that in Xenopus. However, more cells born at E14.5 are destined for ventral regions. The regional differences in proliferation and differentiation are apparent before the olfactory nerve extends to the dorsal surface at E14.5. It is interesting therefore to speculate that regional differences in the rate of proliferation/neurogenesis in ventral/dorsal regions in control animals influence olfactory nerve extension. In support of this hypothesis, in Sall1-mutant animals, a significant increase in the labeling index in progenitor populations (on E14.5) and a decrease in the number of neurons produced (by E18.5), was observed compared with controls, in ventral–central and ventral–lateral regions. By E15.5, the olfactory nerve had failed to extend past the ventral–central domain toward the ventral–lateral OB in Sall1-mutant animals, which suggests that regional patterns of neurogenesis may influence olfactory nerve extension. Moreover, we propose that like Xenopus, early olfactory neurogenesis is independent of olfactory nerve innervation, whereas an interaction between the olfactory nerve and the OB is required during late neurogenesis (from E14.5). Consistent with this hypothesis, early born neurons (mitral cells) are generated in a variety of mutant mice that lack olfactory nerve innervation, but varying degrees of disturbance in the later born interneuron populations are observed (Long et al. 2003; Yoshihara et al. 2005; Hirata et al. 2006). We postulate that OB neurons produce trophic factors that influence innervation, as has been observed in other systems (Tucker et al. 2001; Markus et al. 2002; Zhou et al. 2004), and Sall1-dependent regulation of cellular differentiation is required to influence olfactory nerve extension. A number of axon guidance molecules, including NCAM, olfactory cell adhesion molecule, Ephrin, and Ephrin receptors, are expressed in the developing OB (Treloar et al. 1997; Kafitz and Greer 1998; St John, Pasquale, et al. 2002; Cutforth et al. 2003; Treloar et al. 2003). However, knockout analysis of mice deficient for these molecules does not result in olfactory nerve axon extension abnormalities, similar to those observed in Sall1-mutant animals (Treloar et al. 1997; Cutforth et al. 2003; Walz et al. 2006). Based upon expression and knockout analyses, the most likely candidates were the Semaphorin3a/Neuropilin1 proteins (Schwarting et al. 2000, 2004). However, our studies indicate that these molecules were unchanged in Sall1-deficient animals. Although previous studies have shown that mitral cells are not required for topographic targeting, we cannot exclude the possibility that an over production of mitral cells in lateral and dorsal regions is inhibitory to olfactory nerve extension in Sall1-mutant animals. Previously, a model has been proposed in which 2 overlapping gradients, located in dorsal and ventral regions of the OB, specify topographic olfactory nerve targeting (Gierer 1998; St John, Clarris, et al. 2002). Our findings indicate that intrinsic patterns of neurogenesis exist in the dorsal/ventral OB, which could establish a molecular gradient of guidance cues. These patterns of neurogenesis are altered in Sall1−/− animals. We therefore hypothesize that regulation of olfactory neurogenesis is required for olfactory nerve extension, and that the abnormalities observed in Sall1-deficient animals are a consequence of the altered pattern of neurogenesis.

Olfactory Nerve Innervation is Required for Cellular Lamination of the OB

A correlation between MCL lamination and altered olfactory nerve innervation has been previously observed (Long et al. 2003; Yoshihara et al. 2005; Hirata et al. 2006; Laub et al. 2006). In Dlx5-, Arx-, and Fez-deficient animals, olfactory nerve innervation was altered and mitral cells were disorganized, with apical dendrites misorientated (Long et al. 2003; Yoshihara et al. 2005; Hirata et al. 2006). Long et al. proposed that olfactory nerve innervation is not required for olfactory cell type generation, although, olfactory nerve innervation may be required for lamination of olfactory cell types. However, it has also been proposed that disorganization of mitral cells may be a consequence of altered interneuron generation (Yoshihara et al. 2005; Hirata et al. 2006). Interneuron subtypes were specified in Sall1-mutant animals, which suggests that interneuron specification may not be required for mitral cell organization. In mice deficient for the transcription factor Klf7, the OB was decreased in size and altered cellular lamination was observed (Laub et al. 2006). Moreover, the olfactory nerve only partially innervated the OB, in the ventral–medial region, and the shape of the olfactory ventricle was altered, similar to Sall1−/− animals. In a subset of Klf7−/− mice that survived postnatally, focal restoration of cellular lamination was observed in regions where the olfactory nerve innervated the OB (Laub et al. 2006). In Sall1-mutant animals, we observed similar alterations in lamination of olfactory cellular population. Although we cannot exclude an intrinsic requirement for Sall1 in mitral cell organization, taken together, these data suggest that olfactory nerve innervation is required for olfactory cellular lamination.

A Conserved Role for Sall during Development

We identified a role for Sall1 in regulating neurogenesis during murine olfactory development. A similar role for Sall has been identified in Drosophila. In the developing thorax expression of Sall must be eliminated in sensory organ precursor cells in order for these cells to differentiate (de Celis et al. 1999), which suggests that Sall inhibits differentiation. Consistent with this role for Sall in regulating differentiation, early in olfactory neurogenesis, we observed an increased production of mitral cells in the absence of Sall. Taken together, these data suggest a conserved role for Sall1 in regulating neural differentiation.

Our data indicate that the olfactory nerve penetrates the OB in Sall1-mutants, and we observed glomeruli-like structures in regions where the nerve contacts the OB. Because Sall1−/− animals die at birth, it is not possible to determine if these are functional connections or if the reduced innervation present in these animals leads to abnormalities in olfactory perception. However, previous studies have indicated that loss of olfactory input in neonatal rats is associated with weight loss and altered social interaction and behavior (Hofer 1976; Stewart et al. 1983). Deficits in the central nervous system have not been described in detail in patients with SALL1 mutations, Townes Brocks Syndrome. Our studies suggest that perturbed SALL1 function in these patients could alter olfactory perception.

Supplementary Material

Supplementary material can be found at: http://www.cercor.oxfordjournals.org/.

Funding

National Institutes of Health grant (NIMH NIH2RO1MH60774); National Institute on Drug Abuse grant (NIDA grant RO1AA13004) (A.P.M.); and Ministry of Education, Culture, Sports, Science, and Technology and the Ministry of Health, Labor, and Welfare of Japan research grants to R.N.

We thank K. Mauro for her technical assistance, and Dr N. Roy for creating the cell counting program. We thank Dr S. Watts for his assistance with the confocal imaging. We thank the following for providing antibodies: Dr C. Lagenaur (NCAM [12F11]) and Dr R. Hevner (Tbr1). We thank E. Drill, and Drs W. Halfter, C. Lance-Jones, L. Lillen, and E. Thiels for helpful discussion. Conflict of Interest: None declared.

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