The atypical cadherin Celsr3 and axon guidance

In Drosophila, the gene named starry night (stan, cloned by the Adler lab: Chae et al., 1999) or flamingo (fmi, identified by the Uemura lab: Usui et al., 1999) encodes a seven pass atypical cadherin that plays critical roles in three developmental processes. First, homozygous mutant larvae die with defective longitudinal axonal tracts in the brain. Second, hypomorphic alleles have an anomaly of hair (trichome) polarity on wings, and abnormal rotation of eye onmatidia that are typical of defects in "planar cell polarity" (PCP) (for a review on PCP, see Adler, 2003 or Fanto and McNeill, 2004). Third, viable flies have an anomaly of dendritic development, in that some neurons extend their dendritic arbor beyond the midline, which normal neurons never do.

The mammalian genome contains three Flamingo orthologs named Celsr1, 2 and 3, all of which are expressed in the developing brain. Celsr1 is expressed in ventricular zones of stem cell proliferation. It is a very important protein: two point mutations were shown to lead to an haplo-insufficient phenotype (Curtin et al., 2003). Whereas homozygous mice are embryo lethal due to defective neural tube closure, heterozygous mice have defective organization of the inner ear epithelium. These observations point to a role of Celsr1 in mammalian PCP. Celsr2 is expressed in ventricular zones as well as in postmitotic neurons during development and in the adult brain, and in some organs outside the brain. It plays a role in regulation of dendritic development (Shima et al., 2004). Celsr3 is expressed in several embryonic structures of the early embyo but becomes rapidly restricted to postmitotic neurons, and is down regulated after birth, with residual expression in a few neurons (Tissir et al., 2002).

We inactivated Celsr3 (Tissir et al., 2005) and showed that mutant mice die at birth with a very intriguing anomaly of fiber tracts, with absence of anterior commissure, internal capsule, medial lemniscus and several other longitudinal bundles in all parts of the CNS, including spinal cord. Most interestingly, this phenotype is identical to the one described in mice with inactivation of frizzled3 (Fz3 or Fzd3), made by the Nathans lab (Wang et al., 2002).

These observations point to a genetic pathway that is responsible for the development of the axonal blueprint and that bears analogy to the genetic pathway that controls PCP in flies. A possibility is that a non canonical Wnt pathway (Habas & Igor, 2005) is implicated.

To study the role of Celsr3 in axon guidance, we made mice in which two loxP sites flank the region that was deleted in the null mutans. By crossing "floxed" Celsr3 mice with mice that express the recombinase Cre in sectors of the developing forebrain, we could show that axons are guided to their target by guidepost cells and that the Celsr3/Fzd3 system is critical to interactions between axons and guidepost cells (Zhou et al., 2008).

Role of the atypical cadherin Celsr2

There is indication from work on Fmi/stan in Drosophila and RNA interference in brain slices, that Celsr2 regulates the development of dendrites. To study the role of Celsr2, we have used a genetrap mutant. Homozygous animals are viable and fertile, showing that Celsr2 is less important than Celsr3 for survival. Celsr2 mutants have an interesting brain phenotype, with hydrocephalus and atrophy of some structures such as septum, hippocampus and amygdala. We showed that Celsr2 collaborates with Celsr3 to regulate the docking of ependymal cilia (Tissir et al., 2010). Celsr2 also regulates migration of facial branchiomotor neurons in the brainstem (Qu et al., 2010). Since our genetrap allele seems to have some dominant negative action, we produced null and floxed Celsr2 alleles that we use to understand the action of the protein further.

Role of Celsr1in neural stem cells and neuronal migration

Two Celsr1 mutants were obtained from an ENU screen by the group of Murdoch (Curtin et al, 2003). Both are missense mutations with substitutions of one aminoacid. Intriguingly, both single residue substitutions generate florid phenotypes: heterozygous animals have PCP-like anomalies in the inner ear and homozygotes have defective neural tube closure. This indicates that Celsr1 plays a key role in PCP-related processes, like Fzd3 & 6, and like Vangl2. ENU mutants are likely to have some dominant negative action. To investigate the role of Celsr1 further, we made a floxed Celsr1 mouse and a null mutant. We are now using those alleles to investigate the role of Celsr1 in the planar polarity of the ependymal epithelium and in neural stem cells. In addition to its role in the CNS, Celsr1 also regulates the disposition of body hairs (Ravni et al., 2009), an observation that complements more extensive investigations of the role of Celsr1 is skin development by the Fuchs lab.

Leucine repeat receptor neuronal 1-3 (Lrrn1-3)

Leucine-rich repeats are found is many proteins. We became interested in a family of three genes/proteins named Lrrn1-3 because they are quite specifically expressed in the brain, particularly during development, and because two orthologs in Drosophila, Tartan and Capricious, play important roles in development. Constitutive and conditional mutants have been produced for the three genes and are under analysis.

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