Alopecia in Harlequin mutant mice is associated with reduced AIF protein levels and expression of retroviral elements.

We investigated the contribution of apoptosis-inducing factor (AIF), a key regulator of mitochondrial biogenesis, in supporting hair growth. We report that pelage abnormalities developed during hair follicle (HF) morphogenesis in Harlequin (Hq) mutant mice. Fragility of the hair cortex was associated with decreased expression of genes encoding structural hair proteins, though key transcriptional regulators of HF development were expressed at normal levels. Notably, Aifm1 (R200 del) knockin males and Aifm1(R200 del)/Hq females showed minor hair defects, despite substantially reduced AIF levels. Furthermore, we cloned the integrated ecotropic provirus of the Aifm1Hq allele. We found that its overexpression in wild-type keratinocyte cell lines led to down-regulation of HF-specific Krt84 and Krtap3-3 genes without altering Aifm1 or epidermal Krt5 expression. Together, our findings imply that pelage paucity in Hq mutant mice is mechanistically linked to severe AIF deficiency and is associated with the expression of retroviral elements that might potentially influence the transcriptional regulation of structural hair proteins.

Alopecia in a viable phospholipase C delta 1 and phospholipase C delta 3 double mutant.

BACKGROUND: Inositol 1,4,5trisphosphate (IP(3)) and diacylglycerol (DAG) are important intracellular signalling molecules in various tissues. They are generated by the phospholipase C family of enzymes, of which phospholipase C delta (PLCD) forms one class. Studies with functional inactivation of Plcd isozyme encoding genes in mice have revealed that loss of both Plcd1 and Plcd3 causes early embryonic death. Inactivation of Plcd1 alone causes loss of hair (alopecia), whereas inactivation of Plcd3 alone has no apparent phenotypic effect. To investigate a possible synergy of Plcd1 and Plcd3 in postnatal mice, novel mutations of these genes compatible with life after birth need to be found. METHODOLOGY/PRINCIPAL FINDINGS: We characterise a novel mouse mutant with a spontaneously arisen mutation in Plcd3 (Plcd3(mNab)) that resulted from the insertion of an intracisternal A particle (IAP) into intron 2 of the Plcd3 gene. This mutation leads to the predominant expression of a truncated PLCD3 protein lacking the N-terminal PH domain. C3H mice that carry one or two mutant Plcd3(mNab) alleles are phenotypically normal. However, the presence of one Plcd3(mNab) allele exacerbates the alopecia caused by the loss of functional Plcd1 in Del(9)olt1Pas mutant mice with respect to the number of hair follicles affected and the body region involved. Mice double homozygous for both the Del(9)olt1Pas and the Plcd3(mNab) mutations survive for several weeks and exhibit total alopecia associated with fragile hair shafts showing altered expression of some structural genes and shortened phases of proliferation in hair follicle matrix cells. CONCLUSIONS/SIGNIFICANCE: The Plcd3(mNab) mutation is a novel hypomorphic mutation of Plcd3. Our investigations suggest that Plcd1 and Plcd3 have synergistic effects on the murine hair follicle in specific regions of the body surface.

Specific expression of annexin A8 in adult murine stratified epithelia.

Annexin A8 is a member of the annexin family of calcium-regulated membrane-binding proteins. In this report, we investigated the expression of annexin A8 in adult mouse organs. Northern blot analysis of adult mouse organs showed that a single annexin A8 transcript of 1.9 kb is expressed most strongly in skin, eye and tongue. In situ hybridisations using annexin A8-specific probes revealed that in the stratified epithelia of the tongue and the early postnatal epidermis, annexin A8 transcription could be detected in basal and suprabasal layers of these stratified epithelia. Western blot analyses using a murine ANXA8-specific antiserum showed, that the 36 kD ANXA8 protein was most abundant in the skin and tongue. The abundance of ANXA8 protein in the skin increased during postnatal days 1-18 and was immunohistochemically localised in suprabasal layers of the epidermis. In the tongue epithelium as well, ANXA8 protein was found in suprabasal layers. ANXA8 immunoreactivity was also found in suprabasal layers of the stratified epithelia of the oesophagus and the forestomach, while it was detected in all layers of the cornea epithelium and in the cornea endothelium of the eye. We also investigated the expression of retinoic acid receptor alpha protein (RARA) and ANXA8 in the epidermis immunohistochemically. While RARA immunoreactivity was exclusively detected in the basal layer, ANXA8 immunoreactivity was restricted to suprabasal layers of the epidermis. Thus, ANXA8 protein is most abundant in stratified epithelia of the postnatal mouse. Its location in the suprabasal layers suggests that ANXA8 may be associated with the terminal differentiation of epithelial cells in these tissues.

Fxyd3 and Lgi4 expression in the adult mouse: a case of endogenous antisense expression.

We have investigated the expression of Fxyd3 and Lgi4 in the adult mouse by Northern blot analyses and in situ hybridization. Murine Fxyd3 and Lgi4 have been mapped to the same locus on mouse Chromosome (Chr) 7, where the last exon of Fxyd3 completely overlaps with the 3'UTR in the last exon of Lgi4, which is transcribed in the opposite orientation. The Fxyd3 gene (formerly called Mat-8) encodes an 8-kDa transmembrane protein that is upregulated in mammary tumors and can induce a chloride conductance upon RNA injection into Xenopus oocytes. Fxyd3 is a member of the Fxyd family of which several members are tissue-specific regulators of ion channels. Murine Lgi4 is a recently described member of the leucine-rich-repeat gene family Lgi. Northern blot analyses demonstrated a 0.6-kb Fxyd3 transcript with abundant expression in the murine skin, colon, and mammary gland, but low level expression in the brain. In contrast, a 3.2-kb Lgi4 transcript was abundant in brain, with lower level expression in colon. Lgi4 transcription in the skin was detectable only by RT-PCR. A Fxyd3-specific sense cRNA probe hybridized to a transcript in Northern blots of brain and colon RNA that co-migrated with the Lgi4 mRNA. In situ hybridization experiments revealed that both Fxyd3 and Lgi4 were expressed in the same tissue compartments in skin, uterus, intestine, mammary gland, and brain. These results demonstrate that Fxyd3 and Lgi4 transcripts potentially form double-stranded RNA molecules in many cell types in vivo, which may impact on their respective expression.

Murine calmodulin binding protein 1 (Calmbp1): tissue-specific expression during development and in adult tissues.

Expression of the 1.9 kb cDNA of murine Calmbp1 has been shown to interfere with the mitotic S-M checkpoint in yeast (J. Cell Sci. 111 (1998) 3609). The physiological function and expression pattern of Calmbp1 in mice, however, are unknown. We have investigated the expression of Calmbp1 in mid-gestation and late-gestation fetuses and in adult organs of the mouse. In Northern blot analyses, using a Calmbp1-specific probe, a single mRNA of more than 7.4 kb was found that showed a progressive decline in total RNA preparations of fetal heads during the period from day E12 to E16. In the adult, this Calmbp1 transcript was detectable by Northern blot analysis exclusively in testis, ovary and spleen of all organs examined. In situ hybridizations revealed that Calmbp1 is expressed (a) in the differentiating central and peripheral nervous system, (b) in the epithelial cells lining the crypts of the small intestine in late gestation and adult mice, (c) in the fetal, but not the adult liver, (d) in both the fetal and adult spleen, where the signal colocalized with hematopoetic cells in the red pulp, (e) in late gestation embryos in the thymus, S-shaped tubules in the kidney, epidermis, and (f) in leptotene, zygotene and pachytene spermatocytes of the adult testis and the follicle epithelium of the activated follicles in the adult ovary.