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Regulation of cadherin expression in nervous system development

This review addresses our current understanding of the regulatory mechanisms for classical cadherin expression during development of the vertebrate nervous system. The complexity of the spatial and temporal expression patterns is linked to morphogenic and functional roles in the developing nervous system. While the regulatory networks controlling cadherin expression are not well understood, it is likely that the multiple signaling pathways active in the development of particular domains also regulate the specific cadherins expressed at that time and location. With the growing understanding of the broader roles of cadherins in cell–cell adhesion and non-adhesion processes, it is important to understand both the upstream regulation of cadherin expression and the downstream effects of specific cadherins within their cellular context.


The Cadherin Superfamily of Calcium-Dependent Transmembrane Adhesion Receptors
Cadherin proteins (> 100 in mammals) constitute a large superfamily of transmembrane glycoproteins that includes the classical cadherin, desmosomal cadherin, protocadherin, and cadherin-related protein families, among others. 1,2 Cadherins were first described on the basis of their ability to mediate calcium-dependent intercellular adhesion in tissues (for historical perspective see ref. 3) and subsequently have been shown to play important roles in a variety of other cellular events. For instance, there is evidence that expression of clustered protocadherins in neurons plays a role in self-avoidance during dendritic arborization 4 and that cadherin-6B is involved in non-canonical BMP signaling. 5 The structural characteristic shared by cadherin superfamily members is the presence of a varying number of tandem repeats ~110 amino acids in length referred to as extracellular cadherin (EC) domains. There are five EC domains in the classical cadherins to over 30 EC domains present for the Fat cadherins. 6,7 The spatial and temporal dynamics of cadherin expression patterns in the developing nervous system is complex [8][9][10][11][12][13] and there is much interest in both upstream regulation of cadherin function and downstream effects of specific cadherins. There are several excellent reviews detailing expression patterns and roles of cadherin-based adhesion in the morphogenesis and establishment of functional connections in the nervous system. [14][15][16][17][18] In this review, we will concentrate on what is known about transcriptional regulation of classical cadherin family members in the vertebrate nervous system.

Vertebrate classical cadherins
There are 18 classical cadherins in humans, many of which are expressed in the embryonic and adult nervous system. The classical cadherins as a family are defined by five EC domains (EC1-5) in the extracellular portion, followed by a single-pass transmembrane domain and two well-conserved catenin-binding domains in the cytoplasmic portion. In general, cadherins are thought to participate in homophilic interactions with the EC1 domain contributing to the specificity of this interaction. 19,20 In this scenario, cells expressing the same cadherin aggregate and sort from cells expressing other cadherins. In addition, as cells expressing the same cadherin in different quantities segregate, 21 it appears that segregation of cells can be a consequence of qualitative (cadherin type) and/or quantitative (level of cadherin protein) differences. 22 Based on sequence comparison, the classical cadherins are divided into two subfamilies: type I and type II. 1,23 The type I classical cadherins (cdh1-4 and 15) participate in strong cell-cell adhesion and include the founding member of the classical cadherin family, E-cadherin (cdh1), in addition to N-cadherin, P-cadherin, R-cadherin, and M-cadherin (cdh2-4 and 15, respectively). 2 In 1991, Suzuki and colleagues reported partial cDNA sequences for eight cadherin family members (cdh4-11) from neural tissues. 24 Sequence comparison suggested that most of these identified (with the exception of cdh4) formed a subfamily to the classical cadherin family, now referred to as type II cadherins. To date, the members of the type II cadherins This review addresses our current understanding of the regulatory mechanisms for classical cadherin expression during development of the vertebrate nervous system. The complexity of the spatial and temporal expression patterns is linked to morphogenic and functional roles in the developing nervous system. while the regulatory networks controlling cadherin expression are not well understood, it is likely that the multiple signaling pathways active in the development of particular domains also regulate the specific cadherins expressed at that time and location. with the growing understanding of the broader roles of cadherins in cell-cell adhesion and non-adhesion processes, it is important to understand both the upstream regulation of cadherin expression and the downstream effects of specific cadherins within their cellular context. include the following: cadherins 5-12, 18-20, 22, and 24. 2 Table 1 lists the classical cadherins in the type I and type II subfamilies and indicates some of the known heterotypic interactions between different cadherin proteins or other transmembrane receptors.
Type II cadherins are associated with less robust cell-cell adhesion 25,26 and appear to be more likely to participate in heterophilic binding than type I cadherins. 27,28 However, weak heterophilic interactions have also been reported between cells expressing type I N-cadherin and R-cadherin 29,30 and cells expressing N-cadherin and E-cadherin. 31 The trans heterophilic interaction between different cadherins may be significant for cells that are migrating through tissues or interacting with other cell types not expressing the same cadherin. Cadherin proteins are also reported to interact with other transmembrane proteins. One example is that both N-cadherin and cadherin-11 have been shown to interact with and activate signaling downstream of the FGF receptor (FGFR). This engagement and activation of FGFR appears to play a positive role in neurite outgrowth. 32,33 Alternative isoforms and cleavage Cadherin isoforms Variant forms of the full-length cadherin protein are produced by transcription from multiple promoters, alternative splicing of the transcript, or cleavage of translated protein to form biologically active fragments. We will provide a brief description of some of these variants. Thus far, isoforms for numerous type II cadherins have been reported, including cadherin-6, -7, -8, -11, -20, -22, and -24. [34][35][36][37][38][39][40][41] The significance for many of these different isoforms remains to be characterized. In addition, alternative splicing has been observed for the type I E-cadherin in cancer cells, resulting in premature termination, degradation, and subsequent downregulation of expression. 42,43 Another recent study reported alternative promoter usage for E-cadherin, producing a protein differing in the N terminus. 44 While three isoforms are observed for Drosophila N-cadherin, [45][46][47] a type III cadherin similar in structure to chicken cHz-cadherin, there are no reports of alternative splicing for the vertebrate N-cadherin.
Several of the cadherin isoforms identified are expressed in patterns that allow for a potential role in the development of the nervous system, although no isoform-specific functional data defines such a role at this time. The soluble isoform of cadherin-7 produced via alternative splicing inhibits full-length cadherin-7-mediated cell adhesion. 38 Its expression in the dermomyotome may serve as an inhibitory cue for cadherin-7-expressing migrating neural crest cells, which migrate dorsoventrally between the dermomyotome and the neural tube or dorsolaterally between the dermomyotome and the ectoderm. 27 Two soluble cadherin-8 isoforms, truncated in the EC5 domain, have been identified. 41 The full-length and short isoforms are spatially and temporally regulated during brain development, suggesting they may play distinct roles in development. There is a short cadherin-11 variant expressed in brain and other tissues for which the inclusion of an alternative exon produces a truncated cytoplasmic domain. 37 While it does not support adhesive activity alone, the short variant cadherin-11 enhances the calcium-dependent aggregation kinetics of mouse fibroblastic L cells also transfected with the full-length cadherin-11. In the avian developing spinal cord, a short variant for cadherin-20 or short motor neuron-cadherin (sMN-cad) has been identified that, likely due to alternative promoter usage, lacks the first two EC domains on the N terminus and does not support calcium-dependent adhesive properties. 40 The expression level of the short form of cadherin-20 is less than that for the long form and peaks at embryonic day 5 (E5) compared with peak expression of the long form at E6. It would be interesting to correlate the changes in isoform expression spatially and temporally with motor neuron development at these developmental stages. Finally, a short isoform for cadherin-22 capable of mediating calcium-dependent cell adhesion and lacking the β-catenin binding domain was identified by Sugimoto et al. 35 in rat brain. Both the short form and long form of cadherin-22 are  expressed in fetal and adult rat brain, with the long form mRNA expressed more predominantly.

Cadherin proteolysis
Following translation and delivery to the cell surface, the functional properties of cadherin proteins in the developing nervous system can be further regulated by additional modifications, including proteolytic cleavage. Using N-cadherin as an example, during translation the pre-sequence of pre-pro-Ncadherin is cleaved in the rough endoplasmic reticulum. The resulting pro-N-cadherin lacks adhesive properties as a result of steric hindrance 48 and is thought to be cleaved to remove the pro-domain prior to localization at the cell surface. Recently, using cell surface biotinylation, it has been demonstrated that pro-domain cleavage of N-cadherin can occur after translocation to the cell surface. 49,50 Potentially, this pro-domain cleavage may serve as a mechanism for regulating the timing and rate of synaptogenesis as N-cadherin pro-domain cleavage occurs coincidentally with the onset of synaptogenesis. 50 In support, Rohon-Beard synapse formation is delayed in zebrafish embryos expressing an uncleavable pro-N-cadherin. 51 Likewise, in rat hippocampal neurons, the ratio of ProN to N-cadherin is developmentally regulated and expression of an uncleavable pro-N-cadherin resulted in fewer synapses formed. 51 Furthermore, a higher ratio of proNcadherin to total N-cadherin at the cell surface is observed in tissue samples taken from high grade human brain tumor, melanoma, and carcinoma, and in highly invasive human melanoma and brain tumor cell lines, decreasing cell adhesion and increasing cell motility. 49 Soluble ectodomain and cytoplasmic fragments of cadherins produced by proteolytic processing have biological activity, including signaling and transcriptional roles in development and disease. The full-length mature N-cadherin protein can be regulated by ADAM10, a disintegrin and metalloproteinase, that cleaves N-cadherin to release a soluble ectodomain as well as generate a 40 kDa C-terminal fragment (CTF1), which is further processed by γ-secretase into a 35 kDa intracellular CTF2. 52 The CTF2 fragment is involved in regulating gene expression by redistributing β-catenin from the cytoplasmic domain of N-cadherin to the cytoplasm, which in turn, alters cell adhesion through β-catenin/LEF-1 (lymphoid enhancer-binding factor)-regulated gene expression. [52][53][54] An example of this in development is found at the step of neural crest cell delamination from the neural tube. 55 Prior to neural crest cell delamination, BMP-4 promotes ADAM10 cleavage of N-cadherin into CTF1, which is further processed to CTF2. CTF2 acts to promote the transcription of cyclinD1, stimulating passage through the G1/S cell cycle transition, and therefore, the delamination of neural crest cells. In disease, a recent study has also shown that accumulation of the CTF1 fragment of N-cadherin may play a role in Alzheimer's disease by inducing the amyloid-β-triggered synapse damage. 56 Lastly, Xenopus cadherin-11 is cleaved by ADAM13 between the EC3 and EC4 extracellular domains during cranial neural crest migration. 57 The biologically active 40 kDa fragment released promotes cranial neural crest migration. In addition, the membrane-bound cytoplasmic domain of Xcadherin-11 also plays a role in promoting cranial neural crest cell migration through activation of Rho GTPases. 58 This activation is accomplished by its interaction with Trio, a guanine exchange factor known to be important in axon guidance, as also reported for mouse cadherin-11. 58-60

Regulation of Cadherin Expression
What regulates expression of cadherins during nervous system development?
Cadherin function may be regulated at many points, including transcription, membrane transport, proteolysis and phosphorylation, interaction with specific binding partners, endocytosis, and degradation (for review, see refs. 61 and 62). One approach used to understand the mechanisms regulating cadherins at the transcriptional level during development of the nervous system has been to look at signaling systems that drive and coordinate these developmental processes. This has led to the identification of transcription factors and signaling pathway components responsible for cadherin expression (see Table 2). Likewise, genomic level analyses have been used to identify cis-regulatory elements in cadherin genes that are active in neural development. Thus far, N-cadherin, 63-65 cadherin-6 66 in mouse, cadherin-6B in avians, 67,68 cadherin-7, 69 and cadherin-11 70 have been analyzed for cis-regulatory elements specifically related to expression in the nervous system.

Regulation of type I cadherins in neural development
In regard to regulation of cadherin expression during establishment of the central and peripheral nervous system, we will first address what is known about the type I N-cadherin and R-cadherin. While E-cadherin is also expressed in the developing nervous system, 71   the neuroepithelium. 76 One of the early proneural transcription factors, Sox2 (sry-box containing gene 2), is expressed prior to N-cadherin in the neural plate and placodes 63,77 and has been shown to activate N-cadherin expression in these regions. 63,78 To address the mechanism of N-cadherin regulation by SoxB1 group members, Matsumata and colleagues 63 identified three enhancers for the chicken N-cadherin gene that are regulated by Sox2 during early neural and placode formation. There are also reports of the activation of N-cadherin expression by Paired Box transcription factor Pax2 in the otic placode, 64 Pax6/Sox2 in the lens placode, 78 and Pax6 during retina development. 79 Pax6 is also implicated in activation of the expression for another type I cadherin, R-cadherin, in the embryonic zebrafish brain, 80 and during axonal guidance in the mouse forebrain, 81,82 while Shh signaling was shown to inhibit R-cadherin expression in zebrafish brain. 80 The appropriate spatiotemporal expression of N-cadherin during neural development is important during a number of processes. For example, N-cadherin is required for structural integrity of the neural tube and in cortical structures. [83][84][85][86] N-cadherin also contributes to the increasing complexity of the central nervous system as it develops by having roles in cell migration direction and velocity, circuit formation and synapse formation, and maintenance of progenitor pools. [87][88][89][90][91][92][93][94][95] In regard to this last function, there is evidence that N-cadherin may contribute to maintaining a pool of neural progenitors in neurogenic niches of the developing and adult brain by controlling delamination and subsequent differentiation. 65,[96][97][98] The expression pattern of N-cadherin is consistent with this idea as proliferating cells in the subgranular and subventricular zone neural progenitor pools express N-cadherin, while postmitotic cells that detach from these populations downregulate N-cadherin. 96,97,99 Moreover, N-cadherin loss-of-function leads to premature departure and differentiation of neural progenitors 98,99 while overexpression can block differentiation, 65 implicating N-cadherin adhesion in regulation of the neurogenic process. One recent study, using chicken spinal cord and mouse cerebral cortex, provides support for the role of forkhead box protein transcription factor Foxp2/4 in direct as well as indirect (via repression of Sox2) repression of N-cadherin expression in this context. 65 The authors proposed that Foxp4 and Sox2 directly regulate N-cadherin levels to control the rate of neurogenesis and that Foxp2/4 repression of N-cadherin allows for the emigration and subsequent differentiation of the newly born neurons.

Regulation of type II cadherins in neural development
In this section, we will review what is known about the transcriptional regulation of type II cadherins -6/6B, -7, and -11 in the developing nervous system, focusing on the neural tube and hindbrain and the neural crest for which there is more information regarding transcriptional regulators. For detailed review of expression patterns in the neural crest, see references 100 and 101; for a discussion of cadherins in motor neuron pools in the spinal cord, see reference 102; for brain, see references 14, 17, and 18.

Expression pattern overview
In the neural tube, type II cadherins are expressed in distinct domains. In the dorsal region of the neural tube, cadherin-6/6B mRNA expression is high while in comparison, N-cadherin protein levels are low. 27,[103][104][105][106] For N-cadherin, this appears to be regulated at the protein level in avians since N-cadherin mRNA levels are not downregulated. 55 In mouse embryos, cadherin-6 expression is maintained by migrating neural crest cells, 66,103 and for avian embryos, cadherin-6B is downregulated 27,104 either prior to delamination in the cranial neural crest cells 107 or following delamination in the trunk neural crest cells. 108 The migrating neural crest cells express other cadherins, including cadherin-7 (chicken 27,104 ), cadherin-11 (Xenopus 109,110 ; chicken 111 ), and cadherin-19 (rat, 112 chicken 113 ).
Cadherin-6/6B is expressed in the roof plate as described above, the floorplate, motor neuron pools, 104,114-116 the branchiomotor neurons (BMN), 114,117 and brain. 118 In the neural tube, Cadherin-7 is expressed in a lateral domain correlating to the dorsal boundary of the basal plate in the spinal cord and hindbrain, the floorplate, and early in the motor neuron (MN) pool. 104,114,119 Cadherin-11 is also expressed in a lateral domain of the spinal cord and motor neurons, 111,116,[120][121][122][123] with cadherin-20 expression found ventral to cadherin-7 in spinal cord, lateral motor column (LMC), and brain. 115,124 This diverse expression pattern of different cadherins in the developing CNS is accompanied by their dynamic regulation as discussed below.
Cadherin-6/6B BMP and Wnt signaling pathways are active in the dorsal neural tube and studies in neural crest and neural tube development have pointed to the regulation of cadherin-6B by both of these signaling pathways. [105][106][107][108]111 Wnt signaling induces several dorsal neural tube/pre-migratory neural crest genes in intermediate neural plate explant cultures and in trunk neural crest cell cultures, including that of cadherin-6B. 107,111 In addition, there are several reports of BMP signaling inducing cadherin-6B expression. 105,106,108 In the cranial neural crest, the repression of cadherin-6B expression is reported to be mediated by Slug/Snail2 together with PHD12 and Sin3A, followed by deacetylation at the cadherin-6B promoter. 67,68 Interestingly, a recent paper places FoxD3 repression of tetraspanin18 upstream of cadherin-6B as another mechanism of cadherin-6B downregulation on the posttranslational level during this transition. 125 In the mouse embryo, multiple cis-acting elements regulating cadherin-6 expression in the developing nervous system have been characterized. One element regulating cadherin-6 expression in neural crest derivatives was described to include putative Sox5 (D group), Sox9 (E group), RP58 and TAL1-related, Pax6, and Meis1A/B binding sites, 66 thus, suggesting a role of multiple transcription factors in regulating cadherin-6 expression.

Cadherin-7
In the avian embryo, cadherin-7 expression is induced in the migrating neural crest cell population and, as with cadherin-6B, factors known to be important in neural crest cell development have an impact on cadherin-7 expression. 126 Wnt3a signaling increases the expression of cadherin-7 in trunk neural crest cell cultures. 111 In addition to regulation by Wnt/β-catenin signaling, there is evidence by gain-of-function studies that Sox10 and FoxD3 (Forkhead box D3) also play a role in regulating cadherin-7 expression. [127][128][129] In support, genomic analysis of a neural minimal enhancer identified for cadherin-7 revealed clustered binding sites for SoxE factors Sox9 and Sox10, FoxD3, and β-catenin/TCF/ LEF 69 (T-cell factor/lymphoid enhancer-binding factor) that were bound by these factors in the neural explant cultures during the neural crest cell migratory stages (unpublished data, Prasad and Paulson). This suggests a possible combinatorial role of these factors in regulating cadherin-7 expression at this time.
In addition to its expression in avian neural crest cells during migration, 27,104 cadherin-7 is similarly expressed in association with the migration/outgrowth phases of cranial branchiomotor neurons 114,117 and early spinal motor neurons, 119 and during reorganization of the song nuclei in finches during the learning stages of vocal system development. 130,131 The transient expression of cadherin-7 during progenitor/migratory stages switches to expression of other cadherins for differentiation such as cadherin-6B in the BMN and song nuclei or cadherin-20 in the LMC. It will be useful to define regulatory mechanisms of cadherin-7 expression during these dynamic processes involving cadherin switching.
In the neural tube, a combination of enhancer and silencer elements restricts cadherin-7 expression to the lateral or intermediate domain of the neural tube where the radial glia are located. 69 Signaling pathways and transcription factors directing dorsalventral patterning of the neural tube are well-documented 132 and there is evidence that some of these contribute to the distinct spatiotemporal expression pattern of cadherin-7 in this region. In the dorsal neural tube, Pax7 is implicated in repression of cadherin-7 while intermediate to low levels of Shh signaling activate its expression in the lateral/intermediate domain. 133,134 This regulation may be specific to the lateral domain as the expression of cadherin-7 in early LMC motor neuron pools or floorplate does not appear to be regulated by Pax7 or by Shh signaling. 133 Reflecting differences in the dorsal-ventral pattern of expression in the neural tube/hindbrain, 135 cadherin-7 and cadherin-20 are regulated differently by Shh signaling, with cadherin-7 induced at low levels of Shh signal and cadherin-20 at high levels of Shh signal in the motor column and hindbrain. 124,133 This differential sensitivity to Shh may be one factor in the switch from cadherin-7 to cadherin-20 in the LMC MN pools. 115,119 Similar to cadherin-6, these data again point toward a role of multiple transcription factors in regulating cadherin-7 expression.

Cadherin-11
As with cadherin-6B and cadherin-7, cadherin-11 expression is upregulated in neural crest cultures in response to Wnt3a signaling. 111 While cadherin-11 is known to be expressed in the brain and spinal cord, 111,116,[121][122][123]136 little is known about its transcriptional regulation in the developing nervous system. A recent study demonstrated repression of cadherin-11 by the Bhlh5/Prdm8 neuronal repressor complex as necessary for proper migration/targeting of corticospinal motor neurons. 70 The authors identified gene targets using ChIP-seq and verified the binding of Bhlh5/Prdm8 to an intronic region of the cadherin-11 gene locus. In absence of the Bhlh5/Prdm8 repressor complex, the axons terminate prematurely in cadherin-11 positive regions without reaching the spinal cord, illustrating the role cadherins play in establishing the architecture of the central nervous system. As Twist1 is reported to regulate cadherin-11 expression in the endocardial cushion, it would be useful to know if this regulatory role of Twist is conserved in the cranial neural crest where cadherin-11 is expressed. 137

Conclusion
Understanding the transcriptional regulation of cadherins is an important aspect of understanding how the complexity of the nervous system is generated. Despite the fact that not much is known about the regulatory mechanisms driving cadherin expression in the central and peripheral nervous system, there is much data about the signaling pathways and transcription factors that are involved in neural development. Based on the current evidence we have so far about cadherin gene regulation, it appears that these genes are regulated in a distinct temporal and spatial manner by the signaling pathways involved in those regions. This regulatory mechanism is supported by studies with N-cadherin, cadherin-6, and cadherin-7 that show multiple transcription factors regulate their expression in specific domains of developing CNS. For instance, factors known to be involved in dorsal-ventral patterning of the neural tube, such as Pax7, SoxE factors, and Shh, control the regional expression of cadherin-7 in the developing spinal cord. In addition, data available from other developmental and cancer related studies can be used to implicate the role of specific transcription factors in regulating cadherin gene expression in the nervous system. For example, reports that cadherin-11 expression is regulated by Twist1 in endocardial cushions and N-cadherin is upregulated by Dlx2 in the branchial arch ectomesenchyme may be relevant to regulation of these cadherins in neural tissues. 137, 138 The cadherins have a diverse and dynamic expression pattern that contributes to the morphological and functional architecture of the nervous system. However, further work is required to understand how the signaling pathways that orchestrate the development of the central and peripheral nervous system control the expression of effector molecules such as cadherins. Furthermore, progress needs to be made in understanding how these dynamic cadherin expression patterns modulate cell behavior during different stages of neural development. In development and in disease, cadherins are being discovered to play not only adhesive roles, but also roles in signal transduction and transcription. Thus, it becomes important to understand the regulatory mechanisms controlling their expression during development of the CNS.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.

Acknowledgments
We gratefully acknowledge funding support by NIH Center of Biomedical Research Excellence (COBRE) Grant P20 RR015567.