In this report, we studied the expression and function of Cdh22, a predicted homophilic cell adhesion molecule. Expression of Cdh22 was activated early during establishment of the midbrain–hindbrain boundary. Later, expression of Cdh22 and related cadherin family members was detected in specific yet overlapping regions in the embryonic midbrain and diencephalon. At birth, Cdh22 and Cdh11 were detected in distinct nuclei in forebrain, midbrain, and anterior hindbrain. Expression of Cdh22 and related cadherins in distinct brain areas and nuclei suggests a role in their specification or separation. Postnatal viability of Cdh22 null mutants was decreased, but surviving Cdh22del mutant mice were phenotypically normal and fertile. Brain development appeared normal in Cdh22del mutants suggesting functional redundancy among type II cadherins.
Cdh22 at the Midbrain–Hindbrain Boundary
Isthmic organizer orchestrates patterning of the midbrain and hindbrain. Coherence of this localized signalling center needs to be maintained by cell adhesion molecules. Cdh22 is expressed at the midbrain–hindbrain boundary (Kitajima et al.,1999), which represents a true compartment border in the brain (Zervas et al.,2004; Sunmonu et al.,2011). In addition, expression of Cdh22 was down-regulated at the midbrain–hindbrain border in Fgfr1cko mutants, which have lost FGF signalling and show cell mixing at the midbrain–hindbrain boundary (Trokovic et al.,2003). Our previous (Kala et al.,2008) and current studies show that Cdh22 marks a specific boundary cell population in the most posterior midbrain and most anterior hindbrain. These cells proliferate less, express Fgfr1, and might control brain compartmentalization (Trokovic et al.,2005). If the changes in cell adhesion properties in Fgfr1 mutants are caused by loss of Cdh22 expression, similar compartmentalization defects should have been visible in Cdh22del mutants. However, we could not detect any midbrain–hindbrain boundary defects in Cdh22del mutants. Thus, Cdh22 alone is not required for segregation of neuroepithelial cells between the midbrain and hindbrain.
In addition to Cdh22, Fgf signalling might also regulate expression of other cadherins at the midbrain–hindbrain boundary. At least Cdh6 (Inoue et al.,1997), Cdh8 (Korematsu and Redies,1997b), and Cdh11 (Kimura et al.,1995,1996; Redies and Takeichi,1996) are expressed at the midbrain–hindbrain region during embryogenesis. Indeed, we detected Cdh6 and Cdh11 expression at the midbrain–hindbrain boundary similar to Cdh22 (Fig. 2). Moreover, Cdh11 is also down-regulated in Fgfr1cko similarly to Cdh22 (Jukkola and Partanen, unpublished data). In addition to cadherins, the expression of additional adhesion molecules might be regulated by FGFs. For example, the expression of a cell adhesion molecule Cepu1 has been demonstrated at the midbrain–hindbrain boundary in chicken embryos in a pattern similar to Wnt1 (Jungbluth et al.,2001).
In addition to transcriptional regulation, Fgfrs may also interact with cadherins more directly by modulating their adhesion properties. In the case of widespread cadherin expression, Fgfrs might stabilize cadherin-based adhesion and promote binding specificity and affinity in specific areas. Cadherins can also stimulate neurite outgrowth by binding to Fgfrs. Fgfrs interact with cadherins, at least with N-cadherin (Williams et al.,2001; Sanchez-Heras et al.,2006) and Cdh11 (Boscher and Mege,2008), through their extracellular domain to induce neurite outgrowth and elongation. Moreover, inhibition of FGF signalling via N-cadherin (Lom et al.,1998) disrupts pathfinding of trochlear motoneurons and projection in the isthmus region (Irving et al.,2002). Interestingly, loss of zebrafish N-cadherin does not affect antero-posterior or dorso-ventral patterning during early embryogenesis but leads to abnormal positioning of neurons and axon guidance defects in the midbrain–hindbrain region (Lele et al.,2002). The critical role of N-cadherin might indicate that the other cadherins could play just a modulatory role in this process.
Cadherins Specifying Distinct Brain Nuclei
Cdh22 and related cadherin family members were expressed throughout the ventral midbrain including the dopaminergic, gabaergic, and glutamatergic nuclei, both at early and late stages of embryonic development. Interestingly, Cdh22 and Cdh11 showed largely complementary expression patterns in several regions of the forebrain, midbrain, and hindbrain. This suggests a role in specification and separation of distinct neuronal populations and layers. Also in the developing spinal cord, different type II cadherins are expressed in separate motoneuron populations and regulate segregation of these motoneuron pools (Price et al.,2002). Similar to Cdh22del mutants, Cdh6 mutants are viable and fertile and show no alterations in brain morphology. However, at the cortico-striatal boundary region in endogenous cadherin-expressing background, Cdh6 and R-Cdh overexpressing cells are sorted to lateral ganglionic eminence or cortex, respectively, according to the expression boundaries of these genes (Inoue et al.,2001). This sorting of over-expressing cells does not occur in Cdh6 mutants. Thus, expression of different cadherins can regulate cell sorting at the boundary region and separation of brain nuclei. Similar mechanisms could apply to other cadherins, such as Cdh22, in other neuronal populations in the central nervous system. However, we were unable to detect any major changes in neural populations in Cdh22del mutant brains.
In embryonic diencephalon, expression of type II cadherins may indicate specification of diencephalic nuclei. For example, Cdh22, Cdh11, and Cdh8 were expressed in postmitotic cells at anterior P2, which later gives rise to ventrolateral geniculate nucleus (Puelles and Rubenstein,2003; Vue et al.,2007; Kataoka and Shimogori,2008), where these cadherins were also expressed. Similarly, at E18.5 we observed Cdh22, Cdh11, and Cdh8 expression in the pretectum, anterior pretectal nucleus, which could be the most anterior postmitotic population in P1 at E12.5. Postmitotic P3 population, where Cdh8 was expressed, could be developing subthalamic nucleus, where Cdh8 is also expressed later (Korematsu and Redies,1997a; Suzuki et al.,1997; Korematsu et al.,1998).
Some type II cadherins, such as Cdh11 (Manabe et al.,2000) and Cdh8 (Korematsu and Redies,1997a), participate in neuronal specification and contact formation in the limbic system. Expression of Cdh22 in medial habenular nucleus, amygdala, ventral thalamus, hippocampus, hypothalamus, and interpeduncular nucleus suggests a role in the limbic system. As Cdh22 was expressed in several developing nuclei in the same neuronal circuitry, Cdh22 might also participate in axon guidance and synaptogenesis as suggested for other type II cadherins (Suzuki et al.,1997; Inoue et al.,1998; Korematsu et al.,1998; Redies,2000).
The cadherins are a large group of adhesion molecules, and many of them are expressed in similar areas in the brain. In many cases, abolishing a function of a single cadherin is not enough to result in a phenotypic change. Despite specific expression patterns, there also was significant overlap in the expression of Cdh22, Cdh11, Cdh8, and Cdh6. In regions like indesium griseum, bed nucleus of stria terminalis, pyramidal cell layer in hippocampus (CA3–CA1), ventrolateral geniculate nucleus, and anterior pretectal nucleus, Cdh22 and Cdh11 were co-expressed and might function together. Similarly, expression of other cadherin genes shows overlap and the Cdh22-positive regions always seemed to co-express at least one other type II cadherin, Cdh11, Cdh8, or Cdh6 (Korematsu and Redies,1997b; Suzuki et al.,1997). Also an evolutionarily closely related cadherin-12 appears to be expressed in some of these populations (Mayer et al.,2010). There are several additional type II cadherins, Cdh7, Cdh9, Cdh10, and Cdh20 (Fushimi et al.,1997; Kools et al.,1999; Bekirov et al.,2002; Takahashi and Osumi,2008), expressed in forebrain and midbrain during embryogenesis, but their expression still remains to be characterized in more detail. It is also possible that Cdh22 inactivation leads to compensatory up-regulation of one or several other family members by an unknown mechanism. Redundancy with related type II cadherins could explain why no phenotypic alterations were seen in Cdh22del mutants.