In this study we characterized woe2, a novel mouse mutation which arose spontaneously. Genetic analysis of the woe2 locus identified a 1308 bp deletion in the Ppp1r13l gene encompassing exons 9 through 11 resulting in an aberrant Ppp1r13l
transcript. The putative woe2 PPP1R13L protein encoded by the Ppp1r13l
transcript lacks 223 amino acids from the C-terminal end. PPP1R13L, at the C-terminal end, is characterized with ankyrin (AKN) repeats, and a SRC homology (SH3) domain . The biochemical analyses of PPP1R13L have established that the ANK repeats and the SH3 domain are essential for the PPP1R13L binding with its partners: p53 , p65RelA , and p63 . The 223 amino acids missing from the mutant PPP1R13L from woe2 encompass both the ANK repeats and the SH3 domain. Although we did not functionally evaluate the truncated woe2 PPP1R13L protein, we expect that a loss of 223 amino acids may compromise the stability of the mutant woe2 PPP1R13L protein. Even if the mutant woe2 PPP1R13L protein remains s, the loss of the ANK repeats and the SH3 domain most likely would result in a complete loss of PPP1R13L function. This is consistent with the findings previously reported for wa3 mice that carry a 14-bp deletion in Ppp1r13l resulting in a loss of the SH3-domain from the C-terminal end . The mutant PPP1R13L protein from wa3 mice exhibited a loss of binding to the p65RelA subunit of NF-кB , a previously established PPP1R13L binding partner . As such, wa3 was established as a Ppp1r13l loss-of-function mutation . These findings collectively suggest that woe2 is a novel Ppp1r13l loss-of-function mutation.
The wavy fur and cardiac phenotypes identified in woe2 resemble the cardiac and wavy fur phenotypes previously characterized in the wa3 mice . Abnormalities in the fur and heart were also reported for cattle affected with cardiomyopathy and wooly hair coats (CWH) syndrome that carry a frame-shift mutation in the bovine ortholog of the Ppp1r13l gene . Although the EOB phenotype was also reported for wa3 mice, it was not further investigated . As a part of this study we established that in woe2 the EOB phenotype is due to a defect in embryonic eyelid closure. Even though the morphological analysis of woe2 embryonic eyelids did not identify any obvious abnormalities in the formation of the primitive eyelid, the formation of the leading edge or accumulation of peridermal cells at the leading edge, the woe2 embryonic eyelids failed to close. Immunohistochemical analysis showed that PPP1R13L was expressed in the eyelid epithelial sheet including epidermis, palpebral and bulbar conjunctiva, as well as in the cells of the leading edge. It has been shown before that the embryonic eyelid closure process depends on the movements of the epithelial sheet of the developing eyelids in a morphogenic process similar to dorsal closure in Drosophila . In the developing epidermis of the skin, via its interaction with p63, PPP1R13L regulates the expression of genes that play an essential role in cell-matrix adhesion, as well as in epithelial cell junctions . In addition, PPP1R13L was identified to regulate cell senescence and is required for epithelial stratification . Given that the role of PPP1R13L during embryonic eyelid closure has never been investigated, our current hypothesis is that PPP1R13L interacts with p63 in a similar way as it does in the developing epidermis of the skin and regulates the expression of the adhesion genes and/or genes involved in the differentiation of the cells of the leading edge. Our hypothesis is further supported by the observation that p63
mice also exhibit the EOB phenotype even though the development of the eyelids proceeds normally prior to the embryonic eyelid closure . Although our current hypothesis implicates a role of PPP1R13L via interactions with p63, we cannot exclude the possibility that PPP1R13L may also be involved in molecular processes via interactions with the two other partners p65RelA and/or p53. Therefore, the role of PPP1R13L, as well as which molecular pathways are affected during embryonic eyelid closure requires further investigation.
Results from this study also identified the absence of meibomian glands in the woe2 eyelids. Meibomian glands along with hair, teeth, nails and exocrine glands are epidermal appendages formed from an ectodermal placode . Ectodermal dysplasia syndromes comprise a group of greater than 175 highly diverse disorders characterized by abnormalities of at least two ectodermal tissues where at least one involves hair, teeth, nails or sweat glands. In ectodermal dysplasia patients, other structures exhibiting abnormalities may include the mammary glands, thyroid gland, thymus, anterior pituitary, adrenal medulla, central nervous system, external ear, melanocytes, cornea, conjunctiva, lacrimal gland and lacrimal duct and meibomian glands . Interestingly, alterations of or the absence of meibomian glands was identified as one of the most reliable clinical ocular indications in ectodermal dysplasia patients . In addition to the absence of meibomian glands, evaluation of woe2 eyes revealed abnormalities of the cornea and the anterior structures. The corneal epithelium is a tissue also originating from the surface ectoderm ; proper maturation of the corneal epithelium plays an essential role in ocular development, especially for the development of the anterior segment . Taken together, these findings, along with the structural hair abnormalities previously identified as responsible for the wavy fur in wa3 mice , prompted us to morphologically evaluate other epidermal appendages in woe2 mice. However, our analysis did not identify any obvious defects in teeth, nails, lacrimal, salivary, mammary, sebaceous and sweat glands in woe2 mice. Our results suggest that PPP1R13L may have unique roles in the development of meibomian glands, cornea and hair follicles although PPP1R13L may have overlapping roles in the development of other ectodermal appendages.
It should be noted that the abnormalities in the cornea and anterior segment structures identified in woe2 mice may be solely related to the premature exposure to the environment consequent to the failure of the embryonic eyelid closure. Furthermore, the absence of meibomian glands may be further contributing to the abnormalities of the cornea--especially the corneal epithelium. Meibomian glands produce a lipid-rich secretion called meibum that is released from the orifices of the glands; the meibum is spread across the ocular surface and mix with tears produced by lacrimal glands to produce tear film that covers the entire ocular surface and has a protective, lubricatory, nutritional, and antimicrobial roles . A dysfunction of meibomian glands results in abnormalities of the tear film and consequently ocular irritation, inflammation and ocular surface disease . At this point, the molecular etiology of the anterior segment defects in woe2 remains unclear. It should be also noted that woe2 EOB and ocular phenotypes reported in this study are very similar to phenotypes reported for mice with defects in EGFR signaling pathway [5–14]. While no functional relationship between PPP1R13L and EGFR signaling has been reported, the similarities in the eyelid and ocular phenotypes as well as hair phenotypes identified in woe2 and mice with EGFR signaling defects suggest a common molecular pathway. Taken together, woe2 mice provide an excellent resource for determining the role of PPP1R13L especially during development as well as for elucidating the molecular mechanisms associated with PPP1R13L function.