Shep1 is a multidomain signaling protein that contains an SH2 domain followed by a guanine nucleotide exchange factor-like domain (Dodelet et al., 1999
). Shep1 also binds Cas, a key player in integrin signaling. To examine the role of Shep1 in neural development, we generated Shep1 knockout mice (Roselli et al., 2010
). We found that in these mice the olfactory bulbs are reduced in size and have severe defects afferent innervation. Although OSN axons appear to grow normally within the mesenchyme between the olfactory epithelium and the olfactory bulb up to the boundary with the telencephalon, they do not appear to cross the pial basement membrane. Defects are already apparent as early as at E13, soon after wild-type OSN axons have begun to penetrate into the olfactory bulb. We indeed detected high levels of Shep1 expression in wild-type OSN axons at E13 to E14, which is subsequently downregulated by E16. Shep1 immunoreactivity overlaps with that of neuronal markers (such as GAP43, olfactory marker protein and βIII-tubulin), but not glial markers (such as p75NTR and S100β). Furthermore, both Shep1 and Cas proteins are concentrated in the OSN axons compared to the cell bodies.
Interestingly, Cas substrate domain tyrosine phosphorylation is greatly reduced in developing OSN axons of Shep1 knockout mice at E12.5 and E14.5. Both Shep1 and the related family member BCAR3/AND-34 have been shown to promote Src-dependent phosphorylation of the scaffolding protein Cas, which allows Cas to recruit binding partners that promote actin polymerization, cell adhesion and cell migration/invasion (Sakakibara et al., 2002
; Riggins et al., 2003
; Dail et al., 2004
; Geiger, 2006
; Regelmann et al., 2006
; Schrecengost et al., 2007
; Parekh and Weaver, 2009
; Schuh et al., 2009
; Roselli et al., 2010
; Tikhmyanova et al., 2010
). Several studies have also implicated Cas function in neurite outgrowth and axon guidance (Yang et al., 2002
; Huang et al., 2006
; Liu et al., 2007
). Consistent with a role for Shep1 and Cas in OSN axons, we found that in explants cultured from olfactory epithelium Shep1 is needed for efficient axon penetration into a three-dimensional extracellular matrix environment. Forced expression of Shep1 also enhanced HEK293 cell invasion through a collagen gel. These findings suggest that Shep1 in concert with Cas promotes OSN axon invasiveness across the pial basement membrane.
Despite the substantial evidence supporting a role of Shep1 in OSN axons, some Shep1 expression is also detectable in the primordial olfactory bulb during the early establishment of OSN connections. Therefore, we cannot exclude that Shep1 function in the olfactory bulb, or even in the basal lamina, might play a role in OSN connectivity. It is also conceivable that a brief initial innervation of the olfactory bulb may occur before E13 but fail to be stabilized. Further investigations at earlier developmental stages and conditional Shep1 inactivation in developing OSN neurons versus the primordial olfactory bulb will be necessary to conclusively establish the importance of Shep1 in OSN axons.
The molecules that function upstream of Shep1 to regulate OSN connectivity remain unknown. Shep1 could act downstream of cell surface receptors whose decreased function has been shown to also impair the afferent innervation of the olfactory bulb. For example, in FGF8 hypomorphic mice OSN axons grow in the nasal region but fail to extend into the forebrain, presumably because of impaired FGF receptor activation (Chung et al., 2008
). The prokineticin receptor-2 knockout mice also have defects in the olfactory system that are similar to those in Shep1 knockout mice, including OSN axons that appear to grow normally within the nasal mesenchyme but remain outside the forebrain and hypoplastic olfactory bulbs with defective lamination (Matsumoto et al., 2006
). We also found that Shep1 can associate with the activated IGF-1 receptor and promote invasiveness in response to IGF-1. Although IGF-1 receptor knockout mice do not lack olfactory bulb afferent innervation, IGF-1 receptor activation mediates chemoattraction of OSN growth cones and is required for OSN axon targeting to the lateral olfactory bulb (Scolnick et al., 2008
). It will therefore be interesting to investigate whether Shep1 is also involved in other aspects of olfactory system development, such as axon guidance and fasciculation.
A number of transcriptional regulators have been shown to regulate olfactory bulb innervation by OSN axons and development, including the homeodomain transcription factors Dlx5 (Levi et al., 2003
; Merlo et al., 2007
) and Emx2 (Yoshida et al., 1997
), the zinc finger transcription factor Klf7 (Laub et al., 2005
), and the zinc-finger transcriptional repressor Fezf1 (Hirata et al., 2006
; Watanabe et al., 2009
). Similar to Shep1 knockout mice, the basement membrane surrounding the olfactory bulb lacks fenestrations in the Dlx5 knockout mice (Merlo et al., 2007
). In addition, Fezf1-deficient OSN axons fail to penetrate the basal lamina surrounding the olfactory bulb and loss of Fezf1 also impairs axonal growth from olfactory epithelium explants embedded in Matrigel (Watanabe et al., 2009
). Thus, Shep1 might be one of the functional targets of these transcriptional regulators.
The Shep1 knockout mice express, in at least some tissues, a truncated form of Shep1 that lacks the N-terminal portion, including the SH2 domain (Roselli et al., 2010
). This truncated form of Shep1, if it is expressed in the developing olfactory system, does not appear to interfere with the function of wild-type Shep1 in a dominant negative manner because heterozygous mice do not display obvious defects in OSN axon connectivity. Furthermore, a Shep1 fragment similar to that expressed in the knockout mice does not promote cell invasiveness when ectopically expressed in HEK 293 cells, suggesting that the truncated Shep1 expressed in the mutant mice is functionally deficient. Constistent with this, a truncated form of BCAR3/AND-34 without the N-terminus and the SH2 domain has also been recently shown to have impaired function (Makkinje et al., 2009
Although the Shep1 knockout mice die perinatally, we have not detected major anatomical defects in newborn pups except for small olfactory bulbs. Insufficient feeding due to a defective sense of smell might explain the lethality observed in the C57BL/6 background (Contos et al., 2000
; Matsumoto et al., 2006
). Indeed, we have observed that many of the Shep1 knockout mice have little or no milk in their stomachs (Roselli et al., 2010
). Furthermore, most of the surviving mice exhibit asymmetric abnormalities with one less affected olfactory bulb and may thus have retained some olfactory ability.
Olfactory bulb defects have not been reported for BCAR3/AND34 knockout mice (Near et al., 2009
) and we did not detect BCAR3/AND34 mRNA in E15 olfactory neurons (data not shown). Consistent with a distinctive role of Shep1 in developing OSNs, we observed a dramatic reduction in Cas tyrosine phosphorylation in OSN axons of Shep1 knockout mice. However, Shep1 and BCAR3/AND34 might have redundant functions in cells where they are both expressed (Sakakibara and Hattori, 2000
; Vervoort et al., 2007
; Near et al., 2009
). In agreement with this hypothesis, the BCAR3/AND34 knockout mice also do not exhibit major abnormalities, except in the eye (Near et al., 2009
). Since the third member of the family, NSP1, does not appear to be expressed in human neural tissue (Vervoort et al., 2007
), Shep1 may also have a distinctive function in the human olfactory system.
The olfactory bulb hypoplasia, defects in GnRH neuron development and small testes in the Shep1 knockout mice resemble the abnormalities observed in Kallmann syndrome patients. Kallmann syndrome is a developmental disease characterized by an impaired ability to smell often accompanied by hypoplasia of the olfactory bulb, and by hypogonadism due to defective gonadotropin-releasing hormone (GnRH) neuron migration and/or differentiation (Hardelin and Dode, 2008
; Dode and Hardelin, 2009
). Although this syndrome was first described more than 60 years ago, only ~30% of the genes whose mutations are responsible for the hereditary form of the disease have been identified, including mutations in the extracellular protein anosmin-1, FGF receptor 1 and its ligand FGF8, and prokineticin-2 and the prokineticin-2 receptor (Hardelin and Dode, 2008
; Dode and Hardelin, 2009
). Since our data suggest that Shep1 is a key regulator of OSN connectivity, it will be interesting to determine whether mutations in the human SHEP1 gene may be involved in the etiology of Kallmann syndrome.