Identification of γA‐like protocadherin expressed during chick development

Abstract

The protocadherins are calcium-dependent cell adhesion molecules of the cadherin superfamily that have been described in numerous species. Although less well characterized than classical cadherins, the protocadherins are also thought to facilitate critical cell-cell interactions during embryonic development. We have cloned a novel protocadherin from the embryonic chick utilizing a monoclonal antibody produced against a peanut agglutinin-binding fraction of cultured chick limb tissue to screen a lambdaZAP cDNA expression library from the stage 25 limb. A 2.8 kb cDNA clone was obtained that encoded multiple cadherin-like ectodomains. Northern blotting revealed a single 4.6 kb RNA transcript that was highly enriched in the stage 43 chick brain. Utilization of 3' Rapid amplification of cDNA ends (RACE) identified the entire 2.4 kb reading frame. The chick protocadherin contained five cadherin-like extracellular repeats and a highly conserved cytoplasmic domain. Amino acid alignment of the extracellular domains revealed marked identity to the human gammaA protocadherin subfamily. In situ hybridization showed low levels of mRNA localization in several developing chick tissues, but stronger expression in the neural tube and dorsal root ganglia at stage 27. In the stage 43 chick brain, protocadherin mRNA was noted in discrete regions, particularly within the developing optic lobe. As for protocadherins described in other species, these results suggest that this novel gammaA-like protocadherin may also play a role in chick neural development.

Gamma Protocadherin Expression in the Embryonic Chick Nervous System

“…Expression was also observed in dorsal root ganglia (DRG). Expression in the dorsal horn and DRG is consistent with that reported for a single member of the pcdh-γA subfamily in chick, γA-like pcdh (pcdh-γA5; 14 ), and suggests a role for one or more members of the pcdh-γ family in interaction between interneurons and the DRG during establishment of peripheral sensory connections with the CNS. In embryonic mouse spinal cord, expression of the pcdh-γ family was widespread in gray matter, but preferentially associated with the dorsal horn in the adult 20 and appropriate spinal cord interneuron survival and synapse formation or maintenance have been demonstrated dependent on the pcdh-γ family 8 , 9 .…”

“…In addition, pcdh-γ protein was demonstrated in early axonal and dendritic growth cones of cultured murine e12.5 spinal cord neurons, later becoming progressively restricted to the somatodendritic compartment indicating that pcdh-γ members may also participate in neuronal maturation and neurite outgrowth 20 . Expression in the chick floorplate as seen with the pcdh-γ constant region in situ hybridization probe used in the present study was not observed earlier for γA-like pcdh 14 , but in mouse, pcdh-γ isoforms and pcdh 15 have been reported in floorplates of brain ventricles 24 and the neural tube 25 . The difference in chick neural tube localization between the common γ family probe used in the present study and isoform specific γA-like pcdh probe reported previously supports the notion that more than one pcdh-γ family member is expressed in the developing chick spinal cord.…”

“…The region of pcdh-γ cytoplasmic domain used as probe template for in situ hybridization represents 100% sequence identity between the two members of this family in chick present in GenBank at the current time (pcdh-γA5, AY325274 bases 3064-3400 and pcdh-γC5, NM_214674/AB004689 bases 2582-2918). While only two members of the pcdh-γ family have thus far been described in chick 14 it is likely that additional pcdh-γ genes will be identified as found in other amniotes 3 - 5 .…”

“…Samples were fixed in 85% ethanol, 10% formaldehyde, 5% glacial acetic acid, embedded in paraffin and sectioned at 8 μm. In situ hybridization was performed according to Breitschopf et al 15 as modified by Capehart and Kern 14 . Briefly, cDNA prepared by RT-PCR from stage 43 16 ) chick brain RNA was utilized to generate a 337 bp cDNA probe template specific for the γ protocadherin constant cytoplasmic domain (GenBank accession # AY325274, bases 3064-3400) using primers 5'-CTTCTCTCAGACCCAGAGAC-3' and 5'-CTTCTTCTCCTTCTTGCCAG-3', which was verified by sequencing and subcloned into pGEMT Easy (Promega).…”

“…Only few studies of protocadherins in the chick developmental model have been reported to date. Expression of the OL-protocadherin family 10 - 12 and members of the protocadherin α family 13 show a dynamic distribution in the developing avian CNS; however, at present only one published study has identified a member of the pcdh-γ family in chick 14 and little is known of pcdh-γ expression at early stages of CNS development in this species. Spatial and temporal localization of pcdh-γ family expression is therefore an important initial step toward identifying potential function in the avian.…”