Molecular and functional analysis of Popeye genes: A novel family of transmembrane proteins preferentially expressed in heart and skeletal muscle

Abstract

Popeye (Pop) genes encode novel transmembrane proteins, of which three family members are present in vertebrates, while in Drosophila a single gene is found. By northern blot analysis a restricted expression pattern is observed; Pop genes are predominantly expressed in the heart, skeletal and smooth muscle. Using homologous recombination, a null mutation was generated in the case of Pop1. The homozygous mutants are viable and do not display any obvious phenotype. They display an impaired ability to regenerate skeletal muscle while the hypertropic response of the heart after isoproterenol infusion revealed no difference between genotypes. Recently a function for Pop1 as a prototype of a novel class of cell adhesion molecules was proposed. Further work is required to substantiate these findings and to extend it to other members of the family.

Keywords: Cell adhesion; Membrane proteins; Null mutation; Popeye genes.

Figure 1)

Northern blot analysis of Popeye (Pop)1 gene expression in an embryonic day 10 (dE 10) chick embryo. Poly (A)⁺ RNA was isolated from various organs and probed with a Pop1-specific probe. Two hybridizing mRNA species of approximately 2 and 1.6 kb were exclusively present in heart (H). B Brain; G Gut; K Kidney; Li Liver; Lu Lung; M Muscle; St Stomach; W Wing tissue

Figure 2)

Dendogram of the Popeye (pop) gene family. The protein sequences derived from the cDNA sequence of Pop genes of human (Hs), mouse (Mm), chicken (Gg), Xenopus (Xl), zebrafish (Dr), Branchiostoma (Bf) and Drosophila (Dm) origin were aligned by the clustal method present in the Lasergene program. In most vertebrates three Pop proteins, Pop1, Pop2 and Pop3, with high interspecific sequence conservation are found. Pop2 and Pop3 are more similar to each other than to Pop1

Figure 3)

Popeye (Pop)3 gene expression in a HH stage 24 chicken embryo. (A) Right side, (B) a frontal view and (C) left side of the embryo. Arrow and arrowhead in (B) demarcate the borders between the strong expression domain of Pop3 in atrium (AT) and left ventricle (LV), intermediate expression level in right ventricle (RV) and no expression in outflow tract (OT) myocardium, respectively. In panel (D) a section through the trunk region of the embryo depicted in (A-C) is shown. Strong expression is seen in the myotome. (E) Section through the heart showing strong expression in the LV and in AT. Note differential expression in the ventricle, strong expression in the compact layer (CL) and absence of Pop3 expression in the trabecular layer (TL). Pop3 is only expressed in muscle cells. The atrioventricular cushion (AVC) and epicardium (arrows) are devoid of Pop3 expression

Figure 4)

Working model of the structure of Pop1 protein. Glycosylation sites are indicated in red at the N-terminus (N). Three transmembrane proteins (green) are predicted to be present in the Pop1 protein. The large cytoplasmic domain contains a putative cyclic nucleotide-binding domain, which is not depicted here

Figure 5)

Pop1-LacZ expression in a sagittal section of a 14.5 dpc homozygous Pop1-LacZ mouse embryo. LacZ expression was detected in the heart (Ht), tongue (To), diaphragm (Di), intercostal musculature (Ic), tail musculature (Tm), and smooth muscle cells of the bladder (Bl), intestine (In), oesophagus (Oe) and lung (Lu). In addition, the pancreas (Pa), the pituitary gland (Hy), the choroid plexus (Cp) and neurons in the spinal chord (Sc) displayed LacZ activity