Stage-specific functions of Semaphorin7A during adult hippocampal neurogenesis rely on distinct receptors

The guidance protein Semaphorin7A (Sema7A) is required for the proper development of the immune and nervous systems. Despite strong expression in the mature brain, the role of Sema7A in the adult remains poorly defined. Here we show that Sema7A utilizes different cell surface receptors to control the proliferation and differentiation of neural progenitors in the adult hippocampal dentate gyrus (DG), one of the select regions of the mature brain where neurogenesis occurs. PlexinC1 is selectively expressed in early neural progenitors in the adult mouse DG and mediates the inhibitory effects of Sema7A on progenitor proliferation. Subsequently, during differentiation of adult-born DG granule cells, Sema7A promotes dendrite growth, complexity and spine development through β1-subunit-containing integrin receptors. Our data identify Sema7A as a key regulator of adult hippocampal neurogenesis, providing an example of how differential receptor usage spatiotemporally controls and diversifies the effects of guidance cues in the adult brain

The homeodomain transcription factor Phox2 in the stellate ganglion of the squid Loligo pealei

Homeodomain transcription factors regulate development of embryos and cellular physiology in adult systems. Paired-type homeodomain genes constitute a subclass that has been particularly implicated in establishment of neuronal identity in the mammalian nervous system. We isolated fragments of eight homeodomain genes of this subclass expressed in the stellate ganglion of the North Atlantic long finned squid Loligo pealei (lp) [Note: Loligo pealei has been officially renamed Doryteuthis pealei. For reasons of uniformity and clarity Loligo pealei (lp) is used here]. Of the most abundant ones, we cloned a full length cDNA which encoded the squid ortholog of the paired-type homeodomain proteins Phox2a/b. The homology of lpPhox2 to invertebrate and mammalian Phox2 was limited to the homeodomain. In contrast to mouse Phox2b, lpPhox2 was unable to transactivate the dopamine beta-hydroxylase (DBH) promoter in a heterologous mammalian transfection system. In vivo, lpPhox2 was expressed in the developing stellate ganglion of stage 27 squid embryos and continued to be expressed in the adult stellate neurons where expression was confined to the giant fiber lobe containing the neurons that form the giant axons. The expression of lpPhox was similarly timed and distributed as the Fmrf gene. Furthermore, the Fmrf upstream region contained putative Phox2a/b binding sites. These results suggest a role of lpPhox2 in the developmental specification of neuronal identity and regulation of neurons of the squid giant axon

Spatiotemporal Expression of Repulsive Guidance Molecules (RGMs) and Their Receptor Neogenin in the Mouse Brain

<div><p>Neogenin has been implicated in a variety of developmental processes such as neurogenesis, neuronal differentiation, apoptosis, migration and axon guidance. Binding of repulsive guidance molecules (RGMs) to Neogenin inhibits axon outgrowth of different neuronal populations. This effect requires Neogenin to interact with co-receptors of the uncoordinated locomotion-5 (Unc5) family to activate downstream Rho signaling. Although previous studies have reported RGM, Neogenin, and/or Unc5 expression, a systematic comparison of RGM and Neogenin expression in the developing nervous system is lacking, especially at later developmental stages. Furthermore, information on RGM and Neogenin expression at the protein level is limited. To fill this void and to gain further insight into the role of RGM-Neogenin signaling during mouse neural development, we studied the expression of RGMa, RGMb, Neogenin and Unc5A-D using <em>in situ</em> hybridization, immunohistochemistry and RGMa section binding. Expression patterns in the primary olfactory system, cortex, hippocampus, habenula, and cerebellum were studied in more detail. Characteristic cell layer-specific expression patterns were detected for RGMa, RGMb, Neogenin and Unc5A-D. Furthermore, strong expression of RGMa, RGMb and Neogenin protein was found on several major axon tracts such as the primary olfactory projections, anterior commissure and fasciculus retroflexus. These data not only hint at a role for RGM-Neogenin signaling during the development of different neuronal systems, but also suggest that Neogenin partners with different Unc5 family members in different systems. Overall, the results presented here will serve as a framework for further dissection of the role of RGM-Neogenin signaling during neural development.</p> </div

Differential expression of the FMRF gene in adult and hatchling stellate ganglia of the squid Loligo pealei

Summary The giant fiber system of the squid Loligo pealei mediates the escape response and is an important neurobiological model. Here, we identified an abundant transcript in the stellate ganglion (SG) that encodes a FMRFamide precursor, and characterized FMRFamide and FI/LRF-amide peptides. To determine whether FMRFamide plays a role in the adult and hatchling giant fiber system, we studied the expression of the Fmrf gene and FMRFamide peptides. In stage 29 embryos and stage 30 hatchlings, Ffmr transcripts and FMRFamide peptide were low to undetectable in the SG, in contrast to groups of neurons intensely expressing the Fmrf gene in several brain lobes, including those that innervate the SG. In the adult SG the Fmrf gene was highly expressed, but the FMRFamide peptide was in low abundance. Intense staining for FMRFamide in the adult SG was confined to microneurons and fibers in the neuropil and to small fibers surrounding giant axons in stellar nerves. This shows that the Fmrf gene in the SG is strongly regulated post-hatching, and suggests that the FMRFamide precursor is incompletely processed in the adult SG. The data suggest that the SG only employs the Fmrf gene post-hatching and restricts the biosynthesis of FMRFamide, demonstrating that this peptide is not a major transmitter of the giant fiber system. This contrasts with brain lobes that engage FMRFamide embryonically as a regulatory peptide in multiple neuronal systems, including the afferent fibers that innervate the SG. The biological significance of these mechanisms may be to generate diversity within Fmrf-expressing systems in cephalopods

RGM and Neogenin expression in the mouse olfactory system.

<p><i>In situ</i> hybridization on coronal mouse brain sections at E16.5 (A–C′) and P5 (L–N′). Panels A′–C′ and L′–N′ show higher magnifications of boxed areas in A–C and L–N, respectively. Immunohistochemistry (D–F, H–J) and RGMa-AP section binding (G, K) on E16.5 coronal mouse brain sections. Sections in D–F and H–J are counterstained in blue with fluorescent Nissl. (A–C′) <i>In situ</i> hybridization shows differential expression patterns of <i>RGMa</i>, <i>RGMb</i> and <i>Neogenin</i> in the olfactory bulb and olfactory epithelium (OE). In line with this, immunohistochemistry reveals that axons of olfactory sensory neurons in the OE stain strongly for RGMb and weakly for RGMa and Neogenin. Furthermore, RGMa, RGMb and Neogenin are expressed on olfactory bulb axon projections such as the lateral olfactory tract (LOT). a, apical; ACa, anterior commissure pars anterior; AOB, accessory olfactory bulb; b, basal; CP, cortical plate; CRP, cribriform plate; EPL, external plexiform layer; GL, glomerular layer; GR, granule cell layer; IPL, internal plexiform layer; IZ, intermediate zone; LV, lateral ventricle; MCL, mitral cell layer; ONL, olfactory nerve layer; OVZ, olfactory ventricular zone; S, septum; STR, striatum; VN, vomeronasal nerve. Scale bar A–C 200 µm, A′–C′ 100 µm, D–F 300 µm, G 500 µm, H–J 400 µm, K 500 µm, L–N 400 µm and L′–N′ 200 µm.</p

Postnatal expression of RGMa, RGMb and Neogenin in the cortex and corticospinal tract.

<p><i>In situ</i> hybridization (A–C′) and immunohistochemistry (D–I) on coronal mouse brain sections at P5. Panels A′–C′ show higher magnifications of boxed areas in A–C. Sections in D-I are counterstained in blue with fluorescent Nissl. (A–C′) <i>In situ</i> hybridization detects strong expression of <i>RGMa</i> in cortical layers 1–3, 5 and 6. <i>RGMb</i> is mainly expressed in layer 5 in a medial to lateral gradient and <i>Neogenin</i> is expressed in layers 1–5. (D–F) RGMa protein is expressed in layers 1–3 and 5. Very weak staining is detected for RGMb and Neogenin is strongly expressed in cortical layers 1–3. (G–I) High levels of RGMa and Neogenin are detected in the corticospinal tract. AC, anterior commissure; CC, corpus callosum; LV, lateral ventricle; PD, pyramidal decussation; S, septum; STR, striatum. Scale bar A–C 600 µm, A′–C′ 200 µm, D–F 200 µm and G–I 250 µm.</p

Unc5 expression in the cortex.

<p><i>In situ</i> hybridization on coronal mouse brain sections at E16.5 (A–D) and P5 (E–H). (A–D) <i>Un5A-C</i> are expressed in the cortical plate (CP), and <i>Unc5A</i> and <i>Unc5C</i> in the subplate (SP). All <i>Unc5</i>s are expressed in the subventricular zone (SVZ) and ventricular zone (VZ). (E–H) At P5, <i>Unc5A</i>, <i>Unc5C</i> and <i>Unc5D</i> are expressed in the cortex. CC, corpus callosum; LV, lateral ventricle; S, septum; STR, striatum. Scale bar A–D 300 µm and E-H 600 µm.</p

Subregion-specific expression of RGMs and Neogenin in the hippocampus.

<p><i>In situ</i> hybridization (A–C, J–L) and immunohistochemistry (D–I, M–O) on coronal mouse brain sections at E16.5 (A–I) and P5 (J–O). Sections in D–I and M–O are counterstained in blue with fluorescent Nissl. (A–F) RGMa mRNA and protein are expressed in the ventricular zone (VZ), dentate gyrus (DG) and cornu ammonis (CA) region. Strong expression of RGMb mRNA and protein is detected in the pial surface lining the hippocampal fissure (HF). Neogenin transcripts and protein are widely expressed in the developing hippocampus (Hip). (G–I) Immunostaining with isotype matched controls. (J–L) <i>In situ</i> hybridization at P5 shows strong but differential expression patterns of <i>RGMa</i>, <i>RGMb</i> and <i>Neogenin</i> in the CA pyramidal cell layers (Pyr). In addition, strong expression of <i>Neogenin</i> is detected in the granular layer (GC) of the DG. (M–O) Immunohistochemistry reveals expression of RGMa and weak expression of RGMb in the stratum lacunosum moleculare (SLM) and fimbria (FIM). Neogenin strongly labels different hippocampal layers. CX, cortex; Hb, habenula; HC, hippocampal commissure; PO, polymorph layer; SO, stratum oriens; SR, stratum radiatum; Th, thalamus. Scale bar A–C: 400 µm, D–F: 300 µm, G–I: 300 µm, J–L: 500 µm and M–O: 400 µm.</p

Differential expression of RGMs and Neogenin in the cerebellum.

<p><i>In situ</i> hybridization (A–C, G–I, M–O) and immunohistochemistry (D–F, J–L, P–R) on coronal mouse brain sections at E16.5 (A–F), P5 (G–L) and in the adult (M–R). (A–I, M–O) <i>In situ</i> hybridization and immunohistochemistry reveals strong and broad expression of RGMa, RGMb and Neogenin in the cerebellum (CB). (J–L) Immunostaining shows expression of RGMa and Neogenin in all cerebellar layers at P5. RGMb is expressed in the internal granular layer (IGL), Purkinje cell layer (PCL) and external granular layer (EGL). (P–R) In the adult, RGMa, RGMb and Neogenin protein are expressed in Purkinje cells (PCs) and axons in the granular cell layer (GCL), PCL and molecular layer (ML). Neogenin strongly labels PC dendrites in the ML. DCN, deep cerebellar nuclei; WM, white matter; VZ, ventricular zone. Scale bar A–C: 300 µm, D–F: 250 µm, G–I: 150 µm, J–L: 150 µm, M–O: 100 µm and P–R: 50 µm.</p

Unc5 expression in the habenula.

<p><i>In situ</i> hybridization on coronal mouse brain sections at E16.5 (A–D), P5 (E–H) and adult (I–L). (A–D) At E16.5 <i>in</i> situ hybridization shows expression of <i>Unc5A-D</i> in the habenula (Hb). <i>Unc5B</i> expression labels blood vessels but not habenular neurons. (E–H) At P5, <i>Unc5A</i> is weakly expressed in the Hb and <i>Unc5D</i> expression is restricted to the lateral habenula (LHb). (I–L) In the adult Hb, weak <i>Unc5B</i> expression is detected. 3V, third ventricle; CX, cortex; MHb, medial habenula; PVT paraventricular thalamic nucleus. Scale bar A–D: 300 µm and E–L: 200 µm.</p