Congenital microcephaly‐linked CDK5RAP2 affects eye development

Biallelic mutations in the cyclin‐dependent kinase 5 regulatory subunit‐associated protein 2 gene CDK5RAP2 cause autosomal recessive primary microcephaly type 3 (MCPH3). MCPH is characterized by intellectual disability and microcephaly at birth, classically without further organ involvement. Only recently, congenital cataracts were reported in four patients of one pedigree with MCPH3. Given the lack of a further pedigree with this phenotype, it remained unclear whether this was a true causal relationship. Here we support the link between CDK5RAP2 and eye development by showing that most Cdk5rap2 mutant mice (an/an) exhibit eye malformations ranging from reduced size of one or both eyes (microphthalmia) to total absence of both eyes (anophthalmia). We also detected increased apoptosis in the an/an retinal progenitor cells associated with more mitotic cells. This indicates an important role of Cdk5rap2 in physiologic eye development.


INTRODUCTION
Biallelic mutations in the cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene CDK5RAP2 cause autosomal-recessive primary microcephaly type 3 (MCPH3) (reviewed in Kraemer et al., 2011;Zaqout, Morris-Rosendahl, & Kaindl, 2017b). Patients with MCPH3 display nonprogressive microcephaly at birth and nonsyndromic intellectual disability, classically without further organ involvement This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.  (Kaindl et al., 2010). Only recently, congenital cataracts were reported in four patients of one pedigree with MCPH3 (Alfares et al., 2018;Pagnamenta et al., 2012). Given the presence of only one pedigree with this finding, it remained unclear whether this was a true causal relationship.
Cdk5rap2 mutant or Hertwig's anemia mice (an/an) show congenital microcephaly, peripheral blood cytopenia, spontaneous aneuploidy, predisposition to hematopoietic tumors, and lack of germ cells leading to infertility (Lizarraga et al., 2010;Zaqout, Bessa, Kramer, Stoltenburg-Didinger, & Kaindl, 2017a;reviewed in Kraemer et al., 2015). Because Cdk5rap2 is a centrosomal protein ubiquitously expressed in various tissues (Issa et al., 2013;Lizarraga et al., 2010), we hypothesized that other organs are also likely affected. Cdk5rap2 is also affecting cell-cycle progression (Zaqout et al., 2017a), and therefore abnormalities during retinal progenitor cell (RPC) proliferation and differentiation are highly expected. Here we report that an/an mice exhibit eye malformations already at early developmental stages. These include cataract and a spectrum of anomalies ranging from unilateral or bilateral microphthalmia (reduced size of one or both eyes) to anophthalmia (total absence of both eyes). Moreover, we found a potential mitotic delay in the an/an RPCs, which also show more apoptosis as compared to the wild type (+/+).

MATERIALS AND METHODS
Cdk5rap2 mutant mice (Hertwig's anemia mice; an/an) were generated by crossing heterozygous (+/an) mice (C57BL/6 background; Jackson Laboratory, stock no. 002306). The breeding was performed at the animal facility of the Charité -Universitätsmedizin Berlin, Berlin, Germany, and all experiments were carried out in accordance with national ethics principles (registration no. T0309.09).

RESULTS
We observed the eye phenotype in an/an from E12.5 to adult age. Most an/an show either uni-or bilateral eye deformity ranging from microphthalmia (reduced eye size) to anophthalmia (absence of an eye) (Figure 1a-c). In around 20% of adult mice, we detected white lenses as correlates of cataracts (Figure 1a, white arrow). H&E-stained E14.5 transverse head sections revealed signs of developmental eye defects with some animals showing rudimentary eyes openly connected to the diencephalon (Figure 1b, E14.5, III), while others lacked eye tissue (Figure 1b, E14.5, IV). We also found a reduction in the an/an P0 retina thickness (Figure 1d,e).
We then looked for potential defects in the proliferation and/or apoptosis in RPCs that might lead to the resultant eye phenotype in an/an. We found an increase in the number of pH3 + mitotic RPCs (Figure 2a,b) and Caspase3 + apoptotic RPCs (Figure 2a,c) in P0 an/an retinas.

DISCUSSION
MCPH3 is widely known as a model disorder for nonsyndromic congenital microcephaly, an isolated brain phenotype. However, Cdk5rap2 is ubiquitously expressed (Issa et al., 2013), and previous studies on an/an as a mouse model for MCPH3 indicate that Cdk5rap2 functions not only as a regulator of neural progenitor proliferation but also affects the development of other organs in mice (Kraemer et al., 2011;Megraw, Sharkey, & Nowakowski, 2011;Zaqout et al., 2017a). In support of a recent report linking CDK5RAP2 to congenital cataracts in humans (Alfares et al., 2018), here we found cataracts and further eye malformations in an/an from reduced retinal thickness, via microphthalmia to anophthalmia.
The eye development in vertebrate starts with the formation of an optic vesicle, which subsequently contacts the ectoderm and promotes the proliferation of the lens placode. The lens placode separates as a lens vesicle and invaginates within the enfolded optic vesicle to form the mature eye (Wawersik & Maas, 2000). Our findings of a wide variation of eye malformation from rudimentary eyes openly connected to the diencephalon to a lack of eye tissue in an/an indicate that these critical steps are likely affected. One factor that is critical for RPCs to reach normal eye size that could contribute to this phenotype is an abnormal control of proliferation and apoptosis. We report in an/an mice that the number of pH3 + mitotic cells and Caspase3 + apoptotic cells is increased. The increased number of mitotic cells in an/an can be the result of an accumulation and delay in mitotic progression as we recently demonstrated for germ cells in an/an mice (Zaqout et al., 2017a). Similarities between eye and brain development will open the door to study the mechanism behind eye defects in an/an on the one hand and to further understand the role of Cdk5rap2 in central nervous system development on the other hand.