The association of microcephaly protein WDR62 with CPAP

1

The association of microcephaly protein WDR62 with CPAP/IFT88 is required for cilia

formation and neocortical development.

Belal Shohayeb1, Uda Ho

1, Yvonne Y. Yeap

1,#, Robert G. Parton

2,3, Sean S. Millard

1,

Zhiheng Xu4, Michael Piper

1 and Dominic C. H. Ng

1,*

1School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, St

Lucia, Australia.

2Institute for Molecular Bioscience, The University of Queensland, St Lucia, Australia

3Centre for Microscopy and Microanalysis, The University of Queensland, St Lucia,

Australia.

4State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and

Developmental Biology, Chinese Academy of Sciences, Beijing, China

#Current affiliation: Singapore Immunology Network, Agency for Science Technology and

Research, Singapore.

*Send correspondence to: Dominic C.H Ng; Tel. +61-7-3365 3077; Email: [email protected].

Highlights

Patient-derived missense mutations on WDR62 cause neurodevelopmental defects

related to abnormalities of the primary cilium in mice.

WDR62 mutations cause differentiation and loss of radial glia in ventricular zone.

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WDR62 function in cilia assembly is conserved and required for neocortical

expansion.

WDR62 recruitment of CPAP/IFT88 to the basal body is required for cilia formation.

Abstract

WDR62 mutations that result in protein loss, truncation or single amino-acid substitutions are

causative for human microcephaly, indicating critical roles in cell expansion required for

brain development. WDR62 missense mutations that retain protein expression represent

partial loss-of-function mutants that may therefore provide specific insights into radial glial

cell processes critical for brain growth. Here we utilized CRISPR/Cas9 approaches to

generate three strains of WDR62 mutant mice; WDR62V66M/V66M

and WDR62R439H/R439H

mice

recapitulate conserved missense mutations found in humans with microcephaly, with the third

strain being a null allele (WDR62stop/stop

). Each of these mutations resulted in embryonic

lethality to varying degrees and gross morphological defects consistent with ciliopathies

(dwarfism, anopthalamia and microcephaly). We find that WDR62 mutant proteins (V66M

and R439H) localize to the basal body but fail to recruit CPAP. As a consequence, we

observe deficient recruitment of IFT88, a protein that is required for cilia formation. This

underpins the maintenance of radial glia as WDR62 mutations caused premature

differentiation of radial glia resulting in reduced generation of neurons and cortical thinning.

These findings highlight the important role of the primary cilium in neocortical expansion

and implicates ciliary dysfunction as underlying the pathology of MCPH2 patients.

Introduction

Primary microcephaly is a developmental condition characterized by substantially reduced

head and brain size with associated mild to moderate cognitive impairment [1]. The

identification of microcephaly genes (MCPH) has provided valuable insights into disease

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aetiology and highlighted critical contributions of centrosomal/spindle pole proteins in the

finely-tuned regulation of neural stem and progenitor cell proliferation, survival and

differentiation that underpin brain growth [2]. However, centrosomes integrate complex

cytoskeletal and signal transduction events to influence multiple cellular processes that

contribute to tissue morphogenesis/homeostasis [3]. In addition, the most frequently mutated

MCPH proteins are localized to extracentrosomal compartments with pleiotropic functions

[4]. Thus, whilst defects in cell cycle progression, centriole biogenesis, checkpoint activation,

spindle alignment, asymmetric partitioning of fate determinants and primary cilium formation

have been variously reported as associated with impaired brain growth [5], the molecular

origins of primary microcephaly have remained enigmatic. Here, we characterized mouse

models recapitulating MCPH2 (WDR62) missense mutations found in humans that represent

partial loss-of-function mutants to provide new insights into the molecular and cellular

defects that cause microcephaly.

WDR62 is a large (170 kD) microtubule- and centrosome-associated protein that is highly

expressed in apical and basal neuroprogenitors within the ventricular and subventricular

zones, corresponding with peak neurogenesis in the developing mouse forebrain [6, 7]. The

depletion of WDR62 homologs in mice recapitulate reduced brain size/weight and cortical

thickness associated with precocious differentiation, reduced proliferation and/or excessive

apoptosis of neuroprogenitors, highlighting conserved functions of WDR62 that are required

for brain development [8-10]. However, the mechanisms associated with WDR62 regulation

and function underlying brain growth remain unclear, with WDR62 loss leading to wide

ranging defects in mitogenic kinase signaling, centrosome function, cytoskeletal organization

and cell cycle progression [8-11]. This likely reflects pleiotropic functions of WDR62 which

are collectively disrupted with protein depletion or null mutants [4]. Although the majority of

patient WDR62 mutations are null alleles, genetic studies have additionally revealed

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pathogenic non-synonymous missense mutations that alter evolutionarily conserved residues

that are atypical in MCPH [7, 12]. As the overall tertiary structures of WD40 domain-repeat

proteins are comparatively resistant to single amino acid changes, this subset of missense

mutations may generate mutant protein with partial loss-of-function [13]. An anecdotal report

of a patient with the p.R438H mutation presenting with less severe clinical course compared

to individuals with nonsense mutations lends support to this notion [7]. Importantly, primary

cells isolated from a patient with the p.R438H mutation retained expression of full length

mutant protein, suggesting that it could retain some of the normal WDR62 functions [14]. In

this study, we characterized WDR62 mutant mice with targeted knock-in of c.196G>A in

exon 2 or c.1316G>A in exon 10, resulting in p.V66M and p.R439H single amino-acid

alterations respectively, to recapitulate two frequently reported patient-identified WDR62

missense mutations. In addition, we also generated WDR62 depleted mice through CRIPSR

disruption of exon 2 for comparison.

Here we report overall growth reduction, cortical thinning and anopthalamia as consistent

defects observed across our WDR62 mutant mice. An analysis of the neocortex revealed

precocious differentiation and depletion of radial glia and this was accompanied by ciliary

defects during early neurogenic stages. Given the requirements for intact cilia function for

normal brain development [15], we interrogated ciliary mechanisms perturbed by WDR62

point mutations and demonstrated disrupted recruitment of CPAP (also known as CENPJ) to

the basal body and axonemal loss of intraflagellar transport protein (IFT) 88 required for

ciliary protein trafficking [16]. Our studies reinforces the critical requirement for the cilia of

neuroprogenitors in expansion of the mammalian neocortex and reveal specific functions of

WDR62 protein interactions disrupted by inherited point mutations.

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Results

WDR62 missense mutant mice exhibit embryonic and neocortical growth deficits.

WDR62 missense mutations are causative of microcephaly and cortical malformations in

humans [7, 12] but these have not been modelled in vivo. Here, we utilized CRISPR/Cas9

gene-editing to generate WDR62 mutant mice that recapitulate frequently reported mutations

(V65M and R438H in humans) resulting in single amino acid substitutions of conserved

residues (WDR62V66M/V66M


). In addition, we generated mutant mice

with a deletion in exon 2 that results in premature termination and severe truncation of

WDR62 (herein referred to as WDR62stop

) for comparison with the point mutants

(Supplementary Material, Fig. S1A+B). Quantitative PCR analysis confirmed mRNA levels

were maintained in WDR62V66M/V66M


mice but substantially reduced

in WDR62stop

animals (Supplementary Material, Fig. S1C). To evaluate protein expression,

we generated a custom antibody to mouse WDR62 and validated it by immunoblot and

immunofluorescence in MEFs depleted with WDR62 siRNA (Supplementary Material, Fig.

S1D+E). An immunoblot analysis of primary isolated MEFs and embryonic brain tissue from

WDR62 mutant mice indicated WDR62 levels was maintained in WDR62R439H/R439H

mice,

reduced in WDR62V66M/V66M

and significantly depleted in WDR62stop/stop

animals

(Supplementary Material, Fig. S1F+G). Given that mRNA levels are unaltered in

WDR62V66M/V66M

mice, these findings suggests that protein stability and turnover are likely

disrupted by V66M substitution leading to partially reduced protein levels. Our observations

in WDR62R439H/R439H

mice are also consistent with a clinical report of mutant protein

expression in a MCPH patient with the R438H mutation [14] and supports the notion that this

substitution disrupts an important facet of WDR62 function in neurodevelopment. The levels

of WDR62 mRNA and protein in WDR62stop/stop

mice suggests they are likely to be null

animals. We also noted that the expression levels of a WDR62 ortholog, MAPKBP1, was

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increased in brain lysates from WDR62stop/stop

mice but not with missense mutations

(Supplementary Material, Fig. S1G). This may be a compensatory effect of WDR62 loss.

Interestingly, homozygous WDR62R439H/R439H

post-natal animals were rarely observed

whereas homozygous WDR62stop/stop

and WDR62V66M/V66M

mutant mice were born at

marginally lower than expected Mendelian ratios (Fig. 1A). Heterozygous WDR62R439H/+

animals were recovered at expected ratios and were phenotypically normal (Fig. 1A). At

E12.5-17.5, WDR62R439H/R439H

embryos were found at ratios comparable to WDR62stop/stop

and WDR62V66M/V66M

genotypes (Fig. 1A) which suggests homozygous WDR62R439H/R439H

embryos perish during late gestation or at birth from undetermined causes. Embryos from all

three mutant strains were reduced in overall size (Fig. 1B) and showed incomplete penetrance

of a maldevelopment of ocular tissue with striking absence of an eye (anopthalamia) in

homozygous embryos at E15.5-E17.5 (Fig. 1B+F). WDR62 mutant embryos had reduced

brain size with enlarged ventricles and reductions in cortical thickness at E15.5, a phenotype

evident as early as E12.5 for all three genotypes when compared to wild-type littermates (Fig.

1C-E). Our findings are consistent with a requirement for WDR62 in sustaining cortical

expansion and overall embryo growth during development. In addition, the

WDR62R439H/R439H

mutation recapitulated reduced brain growth despite mutant protein

expression (Supplementary Material, Fig. S1F+G) highlighting loss of specific WDR62

functions required for neurodevelopment.

WDR62 missense mutations trigger loss and premature differentiation of radial glia.

The reduction in cortical thickness in WDR62 mutant mice led us to hypothesise that

abnormalities in neural stem and progenitor cell populations during formation of the

neocortex may culminate in the phenotypic abnormalities we had observed late in gestation.

To assess this we analysed the expression of PAX6 and the absence of TBR2 (PAX6+ve

TBR2-ve

), as indicators of apical progenitors (radial glia) that divide along the ventricular

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surface, and TBR2+ve

cells as more committed intermediate progenitors that have delaminated

from ventricular surface. We stained embryonic brains for PAX6 and TBR2, and an analysis

of the medial region of the neocortex revealed that there were significantly fewer radial glia

in the ventricular zone (VZ) and the subventricular zone (SVZ) of WDR62 mutant brain at

E12.5 (Fig. 2A+B) and this persisted throughout later embryonic stages (Fig. 2B). We note

that WDR62 mutations resulted in reduced numbers of radial glia without complete

exhaustion of this stem cell pool at later developmental stages (Fig. 2B).

Previously, conflicting reports of precocious differentiation or cell cycle arrest culminating in

cell death were attributed to the reduction in radial glia numbers following WDR62 depletion

in embryonic brains [8-10, 17]. In this study, we found that the mitotic index of radial glia

(PAX6+ve

TBR2–ve

pHH3+ve

) was elevated at E12.5 and E15.5 in WDR62stop/stop

,

WDR62V66M/V66M


embryos when compared to WDR62+/+

(Fig. 2C)

indicative of an extended duration in mitosis. The reduced numbers and prolonged mitosis of

radial glia was accompanied by a significant increase in TBR2+ve

intermediate (basal)

progenitors in the VZ/SVZ of WDR62 mutant mice at E12.5 (Fig. 2A+E). At E15.5, there

was a trend for increased TBR2+ve

progenitors in mutant embryos compared to wild-type

littermates at E15.5 that was statistically significant for WDR62stop/stop

brains (Supplementary

Material Fig. S2A, Figure 2E). Interestingly, at E17.5 we found an overall decrease in

TBR2+ve

cells in cortical regions of WDR62stop/stop

, WDR62V66M/V66M


embryos (Fig. 2D+E). This was accompanied by significantly reduced numbers of post-

mitotic neurons (TBR1+ve

) in the cortical plate (CP) at E17.5 in WDR62 mutant embryos

(Fig. 2D+F). These findings indicate a wave of precocious differentiation of apical

progenitors that occurs during early neurogenesis (E12.5) that ultimately resulted in the

reduction of intermediate progenitors and reduced generation of new neurons during later

stages of neurodevelopment (E17.5).

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The division orientation of radial glia, relative to the ventricular surface, may predict

proliferative versus self-renewing divisions, with the later generating more committed

progenitors that culminate in neurogenesis [18]. Our analysis of radial glia division

orientation in WDR62 mutant mice indicated a higher propensity for oblique (30-60o) and

horizontal (0-30o) divisions (Figure 3A+B) which are associated with asymmetric divisions

that trigger fate commitment and generation of intermediate progenitors [19]. Conversely,

symmetrical vertical divisions (60-90o) of proliferating radial glia were reduced in WDR62

mutant mice (Fig. 3A+B). An assessment of the proliferative capacity of the progenitor

compartment with single dose BrdU labeling for 24 h revealed a significant increase in cell

cycle exit (defined as the fraction of BrdU+ve

cells which did not immunostain with Ki67

versus all BrdU+ve

cells, Fig. 3C+D). These observations are consistent with the precocious

differentiation and loss of radial glia triggered by WDR62 depletion and point mutations.

Furthermore, we measured radial glial cell death and revealed a modest but statistically

significant increase in number of apoptotic cells (TUNEL+ve

) in the cortex of WDR62stop/stop

mice whilst the extent of cell death was unchanged with WDR62 point mutations (Fig.

3E+F). Thus, the compromised survival of radial glia, perhaps related to mitotic arrest,

appears to be a feature of severe WDR62 depletion but not following missense mutations.

WDR62 is also required for neuronal migration and normal lamination of the neocortex [10].

Whilst total numbers of TBR1+ve

neurons in the cortical region were reduced at E17.5 (Fig.

2F), we also observed an increase in TBR1+ve

neurons specifically in the intermediate zone

(IZ) in WDR62stop/stop

and WDR62V66M/V66M

mice but not WDR62R439H/R439H

brains

(Supplementary Material, Fig. S2B, DAPI stain demarcating IZ from CP layers and TBR1+ve

fluorescence channel from Fig. 2D shown in Supplementary Material Fig. 2C for clarity)

which suggests a defect in neuronal migration to the CP. Immunostaining for an additional

marker (Ctip2) for newly post-mitotic cortical neurons revealed similar findings of CTIP2+ve

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neurons in the IZ of WDR62stop/stop

and WDR62V66M/V66M

but not WDR62R439H/R439H

mice

(Supplementary Material, Fig. S2D+E). Given the reduced protein levels in WDR62stop/stop

and WDR62V66M/V66M

embryos, our results indicate that WDR62 may also be required for the

migration of newly post-mitotic neurons to the CP. In contrast, cell movements appear

unperturbed by R439H mutation (Supplementary Material, Fig. S2B+E). Our findings reveal

migration of immature neurons that are differentially impacted by WDR62 point mutations

and gene deletion.

WDR62 regulates cilia in the developing cortex

The anopthalamic phenotype observed with WDR62 mutations was reminiscent of mice with

defects in the non-motile primary cilium [20]. Primary cilia are cellular sensory projections

that have defined roles in neurogenesis, neural patterning and cortical development [21].

While ciliary defects have been previously reported in WDR62 knock-out mice [9], how

WDR62 mechanisms were involved in cilia regulation remain unresolved. Thus, we next

investigated the organization of primary cilia in WDR62 missense mutant mice.

We stained for Arl13b, which is cilia membrane marker, at E12.5 and E15.5 to investigate

ciliary defects in neuroprogenitors within the developing cortex. We observed a decrease in

the number of ciliated cells in the SVZ of WDR62 mutant mice compared to wild-type brains

(Fig. 4A+B). Quantification of the percentage of ciliated cells revealed significant reductions

in WDR62 mutant mice compared to wild-type littermates (Fig. 4B). In addition, ciliary

lengths in cells that retained Arl13b staining were significantly reduced in WDR62 mutant

mice at both E12.5 and E15.5 (Fig. 4C+D). Similar to a previous report in WDR62 knock-out

mice [9], we observed an increased incidence of ciliary remnants (Fig. 4E, white arrowheads)

in WDR62 mutant mice at E12.5. As ciliary membrane dissociation from centrosome is a

feature of differentiated cells in late neurogenesis [22], our observation is consistent with the

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premature differentiation of apical progenitors with WDR62 mutation. In evaluating the

broader requirement for WDR62 in proper cilia formation, we performed neural-specific

depletion (RNAi) of the WDR62 ortholog in Drosophila melanogaster and utilized Elav-

Gal4>GFP to visualize ciliated sensory neurons in the legs and in the antennae [23]. The loss

of dWDR62 severely disrupted cilia formation and decreased axonemal length in femoral

chordotonal sensory neurons in the leg and within chemosensory neurons that innervate the

hairs in the antennae (Supplementary Material, Fig. S3). Taken together, these results suggest

that WDR62 has a conserved role in cilia regulation that is disrupted in mice by microcephaly

mutations.

To more closely interrogate ciliary defects arising from WDR62 mutations, we isolated

mouse embryonic fibroblasts (MEFs) at E14.5 and synchronized cells through serum

deprivation to promote ciliogenesis. We found that WDR62 localized to the basal body of

ciliated wild-type MEFs but were absent in MEFs isolated from WDR62stop/stop

embryos (Fig.

5A). Interestingly, WDR62 R439H and, despite its reduced cellular levels, V66M mutant

proteins were detected on basal bodies at the base of the primary cilium (Fig. 5A) which

indicates that ciliary defects associated with WDR62 point mutations are not due to overt

mislocalization of the protein. The basal body is derived from the mother centriole and

WDR62 localization to centrosomes was previously reported to be involved in centriole

biogenesis [9]. However, we did not find significant changes in centriole numbers following

WDR62 depletion or mutation in MEFs (Supplementary Material, Fig. S4A+B).

Furthermore, the ultrastructure of centrioles in the VZ of WDR62stop/stop

brains appeared

normal (Supplementary Material, Fig. S4C). This indicates that WDR62 mutations in our

mouse models was not sufficient to trigger abnormal centriole duplication and excludes this

process as the potential cause of the neurological and ciliary defects.

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Similar to the ciliary deficiencies observed in vivo, we observed a reduction in the percentage

of ciliated cells (Fig. 5B), shortening in cilia length (Fig. 5C) and increased ciliary membrane

remnants (Supplementary Material, Fig. S4D+E) in WDR62 deleted and point mutant MEFs.

Reductions in cilia length were rescued with the return of WDR62-GFP to WDR62stop/stop

MEFs (Fig. 5D+E) which confirmed that WDR62 was specifically required for normal cilia

growth. We also observed a significant proportion (~10%) of WDR62 mutant MEFs were

biciliated with two closely associated Arl13b positively-stained perinuclear ciliary structures

clearly evident (Fig. 5F+G). On closer inspection bicilated cells were also present in vivo in

the SVZ of WDR62stop/stop

, WDR62V66M/V66M


brains (Fig. 4A, white

arrowheads). Immunostaining with acetylated tubulin confirmed double microtubule

axonemal structures in biciliated MEFs (Fig. 5F). Duplicated axonemes were associated with

independent basal bodies that were positively stained with CEP170, a distal appendage and

mother centriole marker (Fig. 5H). Quantification of CEP170-stained cells indicated that

~15% of WDR62stop/stop

, WDR62V66M/V66M


MEFs possessed two

mature centrioles in close proximity (Fig. 5I) which indicates centriole maturation is

perturbed by WDR62 loss or microcephaly-linked mutations.

WDR62 is required to recruit CPAP and IFT88 to the basal body

The similarities in ciliary deficiencies between isolated MEFs and neuroprogenitors in the

neocortex suggest that WDR62 mutant MEFs may provide valuable insights into WDR62

mechanisms underlying cilia regulation in vivo. We first considered WDR62 interactions with

CEP170 [12], a subdistal appendage protein that is required for centrosome-microtubule

anchoring [24] and thus tethering of the mother centriole to the plasma membrane for

ciliogenesis. However, the extent of CEP170 localization on mother centrioles was

unchanged in the three different WDR62 mutant MEF lines (Supplementary Material, Fig.

S5A+B). In addition, the density of microtubules in the immediate vicinity of basal body was

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not markedly altered in WDR62 mutant MEFs which suggests normal microtubule anchoring

at centrosomes (Supplementary Material, Fig. S5C). EM visualization of the basal body distal

appendages of radial glia in WDR62stop/stop

brain sections appeared normal (Supplementary

Material, Fig. S4C). Moreover, depletion of CEP170 did not phenocopy decreased cilia

growth observed with WDR62 loss (Supplementary Material, Fig. S5D). This indicated

disruption of cilia in WDR62 mutant cells was not due to lost CEP170 or abnormal

microtubule attachments at the sub-distal appendages of mother centrioles.

We next considered that the ciliary defects observed with WDR62 loss or point mutations,

such as biciliation from duplicated basal bodies, were reminiscent of perturbed CPAP

localization/interaction with axonemal microtubules [25]. Given CPAP was a previously

described downstream target [26] and interacting partner of WDR62 (Supplementary

Material, Fig. S6A), we investigated whether WDR62 interactions with CPAP were involved

in cilia regulation. CPAP localization to the basal body in the progenitor compartment, as

defined by staining intensity and co-localization with -tubulin, was significantly reduced in

WDR62stop/stop

, WDR62V66M/V66M


brains compared to wild-type

(Fig.Figure 6A-C). Similarly, the localization of CPAP to centrosomes was reduced in

WDR62 mutant MEFs (Fig. 6D+E). CPAP localization at duplicated basal bodies in

biciliated cells was also reduced (Supplementary Material, Fig. S6B). The co-

immunoprecipitation of CPAP with WDR62, indicative of partner protein interaction, was

substantially reduced in MEFs with WDR62 loss (WDR62stop/stop

and WDR62V66M/V66M

) and

in WDR62R439H/R439H

MEFs despite mutant protein expression (Fig. 6F). This indicates

WDR62 loss or mutation disrupts CPAP recruitment to centrosomes during cilia formation

and elongation. CPAP localization to centrosomes in WDR62stop/stop

MEFs was rescued with

expression of full-length WDR62-GFP (Fig. 6G+H) or a truncated WDR62 mutant lacking

C-terminal region (WDR62 N-GFP aa 1-841, Fig. 6I+J). In contrast, the centrosomal

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localization of CPAP was not rescued with a truncated WDR62 mutant lacking the N-

terminal region (WDR62 C-GFP aa 842-1523, Fig. 6I+J). This corresponded with a capacity

for WDR62-N, but not WDR62-C, to interact with CPAP (Supplementary Material, Fig.

S6D). This indicates that the WD40-repeat region of WDR62 is specifically required for

interaction and recruitment of CPAP to centrosomes and consistent with CPAP defects

observed with R439H mutation.

Previous studies have demonstrated that CPAP interactions with tubulin, and potentially

intraflagellar transport (IFT) proteins, regulate polymerization and elongation of axonemal

microtubules for cilia growth [25]. We found that IFT88 co-precipitated with WDR62 and

CPAP indicating interactions with these partner proteins (Supplementary Material, Fig. S6A).

Therefore, we next investigated whether IFT88, a core component of IFT-B anterograde

transport complex, was perturbed by WDR62 missense mutations. We found that IFT-88 was

present on basal bodies and ciliary axonemes of progenitor cells in VZ/SVZ cortical layers in

wild-type mice (Fig. 7A). The localization of IFT88 to the primary cilium, indicated by

intensity of IFT88 staining and co-localization with -tubulin, was disrupted by WDR62 loss

or missense mutation (Fig. 7A-C). Similarly, IFT88 was strongly localized to the basal body

and to the axoneme in wild-type MEFs (Fig. 7D). The loss of WDR62 resulted in decreased

IFT88 levels at the basal body supporting perturbed tubulin transport required for elongation

(Fig. 7D+E). In addition, IFT88 levels at the basal body were similarly reduced in the

presence of WDR62V66M/V66M


mutants (Fig. 7D+E). The decrease in

IFT88 localization at the basal body was reversed with WDR62-GFP expression confirming

that WDR62 was specifically required for the ciliary localization of IFT88 (Fig. 7F+G). This

highlights WDR62-CPAP interactions as required for anterograde tubulin transport and

normal cilia growth. Taken together, our studies have revealed that patient-identified

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missense mutations in WDR62 disrupt its interaction with CPAP which leads to cilia defects

that may contribute to the development of microcephaly.

Discussion

WDR62 was first identified, nearly a decade ago, as a factor required for normal human

embryonic brain growth [6]. Whilst genetic models resulting in protein loss have revealed

several cellular processes requiring WDR62 expression [9, 10], the specific functions of

WDR62 impaired by pathogenic missense mutations and required for neocortical

development remain unclear. Here we report on the first mouse models recapitulating

WDR62 missense mutations that cause microcephaly in humans. Our studies confirm that the

conserved V66 and R439 residues on WDR62 are required for overall embryonic growth,

neocortical expansion and normal ocular development; the later not having been previously

reported in studies with WDR62 gene-trap animals [8, 9, 11]. Mutant protein levels, but not

mRNA levels, were reduced in WDR62V66M/V66M

mice consistent with computational

predictions that this mutation may disrupt protein stability. Thus, WDR62V66M/V66M

mice are

hypomorphic mutants with neurological defects likely arising from insufficient WDR62

protein expression. In contrast, WDR62R439H/R439H

animals retained substantial levels of

mutant protein and this is consistent with clinical studies of MCPH2 patients harbouring the

equivalent mutation (p.R438H). Moreover, we show that CRISPR disruption of WDR62

expression (WDR62stop/stop

) resulted in largely comparable abnormalities which reinforces the

observed phenotypes are due to WDR62 loss-of-function. These mouse lines represent

valuable in vivo models of human MCPH2 mutations to study WDR62 mechanisms in order

to provide insights into protein function.

Interestingly, we showed that different WDR62 mutations caused variable lethality despite

defined genetic backgrounds. A percentage of homozygous mutants were lost in mid-late

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gestation and this was presumably associated with severe dwarfism and anopthalamia as

these defects were not observed in post-natal animals. Embryonic survival was compromised

by WDR62R439H/R439H

mutation, in particular, with only rare instances of homozygous

animals born compared to WDR62V66M/V66M

and WDR62stop/stop

mice which were reduced in

frequency but generally survived to post-natal stages. Our findings suggest that expression of

WDR62 R439H mutant protein may be less well tolerated compared to protein depletion in

WDR62V66M/V66M

and WDR62stop/stop

animals. This could indicate that R439H is a novel gain

of function mutation that is detrimental during late gestation. However, neomorphic

mutations are typically dominant/semi-dominant [27] and we did not observe detrimental

effects in heterozygous animals (WDR62R439H/+

) which we recovered at expected numbers

and were phenotypically normal. In contrast, recent studies suggest that the variable lethality

may be the result of cell-autonomous compensatory expression of paralogs with redundant

functions in knock-out mice [28] and triggered through a nonsense mediated decay pathway

[29]. In support of this notion, we observed reduced mRNA levels in WDR62stop/stop

mice and

this coincided with increased expression of MAPKBP1, an ortholog reported to share

functions with WDR62, for example in the regulation of stress responses [30] and spindle

microtubules [31]. Importantly, MAPKBP1 was reported to be dispensable for cilia formation

[31] and thus would not compensate for WDR62 loss in ciliogenesis.

The possibility that the increased lethality in WDR62R439H/R439H

animals may be the result of

an off-target mutation is highly unlikely. The incidence of off-target gene editing in in vivo

experiments is extremely low [32] and our mice are extensively back-crossed to segregate

off-target mutations. An exception may be gene mutations that are in close chromosomal

vicinity to R439H (genetic linkage) and thus resistant to homologous recombination.

However, this has not impaired studies in early embryonic development and our

identification of morphological and neurological abnormalities in all three mutant lines

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implicates WDR62 specifically. In addition, our investigation of neuropathological

mechanisms in multiple independent mutant lines provide insights into specific WDR62

functions required for neurodevelopment. Notably, the primordial dwarfism, cortical

thinning, ocular abnormalities and infertility phenotypes in WDR62 mutant mice are

consistent with embryonic defects associated with ciliary deficiencies [33-35].

Primary cilia are important regulators of neurodevelopment and this is exemplified by

manifestations of microcephaly and cortical malformations as pathophysiological

characteristics of ciliopathies [36]. Ciliary structures in radial glia serve critical functions in

defining apical-basal polarity, morphogen sensing and hedgehog signaling control of

neuroprogenitor expansion, differentiation and migration within proliferative zones of the

neocortex [15]. Moreover, the inheritance of ciliary remnants may specify self-renewal by

enhancing ciliogenesis in daughter cells following mitosis [22]. Here we showed that

pathological WDR62 missense mutations caused reductions in the pool of proliferating radial

glia during early-mid gestation (E12.5), premature differentiation into intermediate

progenitors and immature neurons, resulting in microcephaly without overt increases in cell

death which is consistent with our previous in utero knockdown studies and following

conditional deletion of WDR62 in the brain [10, 37]. Neural defects in WDR62 deleted or

mutant mice were associated with clear ciliary abnormalities in the VZ/SVZ including

increased dissociation of ciliary membranes from centrosomes which is characteristic of

precocious differentiation of radial glia [9, 22]. Impairments in cilia assembly or timely

reabsorption in radial glia result in cell cycle exit and the onset of neuronal differentiation

[38, 39]. Therefore, in response to WDR62 mutation, the reduction of ciliated cells at the

ventricular membrane is likely the trigger for radial glia loss and their premature

differentiation to intermediate progenitors.

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It was recently reported that global deletion of WDR62 using CMV-Cre excision of a floxed

allele caused a delay in cilia resorption without apparent defects in ciliogenesis [40]. The

reasons underlying the phenotypic differences between this recent report and our study are

undetermined but may be related to the specific genetic background of WDR62 knock-out

mice. The floxed WDR62 allele was generated by mating C57BL/6 chimeras with

129S1/SvImJ resulting in a mosaic strain that was then subsequently crossed with CMV-Cre

mice [40]. In contrast, our WDR62 mutant mice, generated using CRISPR/Cas9 editing and

extensively backcrossed, are isogenic C57BL/6 strains. It is possible that strain-specific

expression of genetic modifiers may account for differences in severity of impaired

ciliogenesis observed between our mouse models. Importantly, our findings do not discount a

role for WDR62 in the disassembly of cilia. Moreover, we reported deficiencies in

ciliogenesis that were consistent between three independent WDR62 mouse strains

(WDR62stop/stop

, WDR62V66M/V66M


). This indicates WDR62 expression

and the R439 residue are involved in cilia formation and required to maintain a pool of

proliferative neuroprogenitors during expansion of the mammalian neocortex.

Cilia-regulated polarization of radial glial processes are also required for oriented migration

and proper placement of cells in the neuroepithelium [41]. We observed reduced migration of

TBR1+ve

neurons to the CP in WDR62V66M/V66M

and WDR62stop/stop

, but not

WDR62R439H/R439H

brains. As WDR62 levels are reduced in WDR62V66M/V66M

and

WDR62stop/stop

brains, this suggests neuronal migration requires normal WDR62 expression.

This is aligned with previous findings of supressed migration of immature neurons and lost

apical-basal polarity of radial glia following in utero depletion of WDR62 [10]. However,

cilia contributions in this context are unclear as the R439H mutation results in comparable

severity and range of ciliary defects but neuronal migration appears unperturbed. It remains

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possible that WDR62 mutations may delineate between multiple cilia functions however this

requires clarification in future work.

WDR62 loss perturbs cilia formation in Drosophila sensory neurons which suggests WDR62

regulation of cilia is evolutionarily conserved. We previously characterized WDR62 as a

spindle microtubule-associated protein but, in ciliated cells, WDR62 was localized

specifically to the basal body and not to the axoneme which suggests WDR62 may not

directly regulate axonemal microtubules. However, WDR62 interacts with CPAP [9] and the

later determines axoneme length through tubulin interactions and control of microtubule

dynamics in cilia [25]. Notably, WDR62 mutations phenocopy primordial dwarfism and

microcephaly observed in CPAP mutant mice [42] which suggests shared functions in

neurodevelopment. We find that WDR62 R439 is involved in recruiting CPAP to the basal

body and the proper function of anterograde (IFT-B) tubulin transport complexes required for

cilia elongation. While further investigation of patient-derived cells/tissue is required, our

results suggest that CPAP dysfunction and subsequent cilia malformations may underlie the

development of microcephaly and range of cortical defects in human patients with WDR62

mutations.

Further to cilia regulation, CPAP has defined roles in procentriole elongation and centriole

biogenesis [43] and centriole loss can cause microcephaly [44]. However, we did not observe

significant changes in the ultrastructure or numbers of centriole following WDR62 loss or

mutation with the exception of duplicated mature centrioles associated with biciliated cells in

a percentage of primary MEFs and neuroprogenitors in vivo. Therefore, our findings suggest

that WDR62 recruitment of CPAP to centrosomes is required for ciliogenesis but may not be

critical for centriole duplication. Biochemical alterations in CPAP-tubulin interaction has

been shown to cause ectopic formation of cilia on daughter centrioles leading to biciliation

[25] which underscores complex mechanisms underlying CPAP control of tubulin delivery

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for cilia organization. It is interesting to note that a recent study that deleted CPAP in murine

radial glia resulted in microcephaly associated with long cilia [45]. It is tempting to speculate

that WDR62-CPAP complex formation may contribute to CPAP function in tubulin transport

at the basal body although this requires further investigation. Taken together, our studies

demonstrate WDR62-CPAP complexes are disrupted by microcephaly mutations and this

leads to dysfunction of the primary cilium and maintenance of proliferating progenitors

Materials and Methods

WDR62 mutant mice

WDR62stop/stop

, WDR62V66M/V66M


mice (C57BL/6) were generated by

the Australian Phenomics Network through CRISPR/Cas9 editing of the Wdr62 gene.

Homology directed repair donor oligonucleotides 5’-

gagctgctggagcaaatatttttctgacagcccttttctttgcaggtgacacttgagaagAtgcttggcatcacagcccagaacagcag

cgggctaacctgtgaccctggcacaggccatg – 3’ and 5’ -

accagagagcttgcctgccgtccgggacttttctgacttgttcctcagacaataccatccActtctggaatttggatagcgcctctgacac

tcgatggcaaaagaacatcttcagcgatgt were utilized to generate the c.196G>A (V66M) and

c.1316G>A (R439H) mutations respectively, recapitulating pathogenic mutations identified

in MCPH patients (Suppl. Fig. 1A). Guide RNAs were microinjected into C57BL/6

blastocysts and transferred into pseudopregnant female C57BL/6 mice. In screening for

founder animals carrying point mutations, we also identified a mouse (WDR62stop/stop

) with a

1 base pair deletion in exon 2 (190delG) resulting in pre-termination codon

(p.Glu64Argfs*77). Founder animals were bred to generate N1 progeny and mutant alleles

determined by sequence analysis of amplified PCR products from genomic DNA isolated

from tail tips. We utilized 5’ CTGAAGTGACTTTCTGACCCTCC 3’ and 5’

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CGCCACAGAGCATGGTGAGG 3’ primer pairs in genotyping 1316G>A and 5’

TGCAGCAGACAGGGGAATACT 3’ and 5’ TCCCCTCCATAGCCCTCACA 3’ primers

for both 196G>A and 190delG. Heterozygous animals were backcrossed for 10 generations

prior to experimentation and maintained in accordance with National Health and Medical

Research Council of Australia’s code of practice for the care and use of animals for scientific

research and approved by the University of Queensland Anatomical Biosciences Animal

Ethics committee (Ethics ID #452/18, #445/18). For the cell cycle exit experiments, pregnant

dams were intraperitoneal injected with 100uL/10g ready-to-use BrdU labelling reagent

(Thermo Fisher Scientific) 24 h prior to embryo collection.

Brain fixation and sectioning.

Embryonic (E15.5-17.5) brains were dissected and fixed in 4% (w/v) paraformaldehyde

(PFA) for a minimum of 48 hrs, washed in phosphate buffered saline (PBS, 4 hrs) and

processed with a Leica ASP300S enclosed tissue processor instrument. Embryonic brains

were embedded in paraffin wax and sectioned at the thickness of 6 μm for

immunohistochemical or histological staining. Embryos collected at E12.5 were fixed in 4%

(w/v) PFA for 48 hrs, washed in PBS and immersed in 15% sucrose and Tissue Tek O.C.T

compound for cryo-sectioning using the Thermo cyrostar NX70. E12.5 brains were kept at -

20 C° and sectioned at the thickness of 10 μm. Embryos from cell cycle exit study were

collected at E15.5 and 24 h following BrdU administration, fixed in 4% (w/v) PFA (72 h),

washed in PBS and embedded into 3% Noble agar for sectioning.

Hematoxylin and eosin staining

Tissue sections embedded in paraffin wax were dewaxed and stained with hematoxylin for 2

minutes, washed with running water and 70% ethanol. Sections were then counter-stained

with eosin for 25 seconds and washed in 90% ethanol. Brain sections were then immersed in

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xylene for fixation and mounted on to glass slides using DePex mounting media. Images

were collected using Leica Aperio digital slide scanners.

Murine embryonic fibroblasts

Mouse embryonic fibroblasts (MEF) were isolated from freshly dissected E14.5 embryos by

trypsin (0.25%) digest for 30 mins. Embryonic tissues were then disrupted by passing

through a G18 needle (10-12 times) and trypsin neutralized with culture media (DMEM)

supplemented with 10% (v/v) FCS, 1% (v/v) penicillin/streptomycin, 10 mM L-glutamine, 1

x non-essential amino acids and 0.01% (v/v) -mercaptoethanol. Cells were maintained at

37°C and 5% CO2 in a humidified incubator. To induce cilia formation, MEFs were plated

(4x105 cells/well) for 24 hrs in serum-containing media, washed with PBS and then incubated

in serum-free media for 72 hours to synchronize cells at G0. In rescue experiments previously

generated pEGFP-WDR62, pEGFP-WDR62 N (aa 1-841) and pEGFP-WDR62 C (aa 842-

1523) [17] were transiently expressed in WDR62stop/stop

MEFs using Lipofectamine 2000. At

48 h post-transfection, primary cilia were induced with serum-free media and cells fixed and

analysed by immunofluorescence.

Immunofluorescence

Paraffin embedded sections were rehydrated or cryo-preserved sections washed briefly in

PBS prior to antigen retrieval (10 mM sodium tri-citrate pH 6.0, 95°C, 51 minutes). Sections

were then washed in PBS twice, blocked in a solution containing 20% (v/v) FCS, 2% (w/v)

BSA and 0.2% (w/v) Triton X-100 in PBS for 1 hour at room temperature before overnight

incubation with primary antibodies diluted in blocking buffer. Stained sections were then

washed three times in cold PBS, incubated with appropriate Alexa Fluor conjugated

secondary antibodies followed by DAPI staining and mounting in 80% glycerol for

fluorescence imaging. Images were collected using Leica TCS SP8 or Diskovery Spinning

Disk confocal microscopes. Primary antibodies included mouse anti-γtubulin (1:400, T5326

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Sigma), rabbit anti-γtubulin (1:400, T5192 Sigma), rabbit anti-Arl13b (1:300, 17711-1-AP

Proteintech), mouse anti-Arl13b (1:300, 75287, Antibodies Inc.), mouse anti-αtubulin (1:300,

T5168 Sigma), rabbit anti-CPAP (1:200, 11517-1-Ap Proteintech), rabbit anti-IFT88 (1:200,

13967-1-AP Proteintech), rabbit anti-Tbr1 (1:200, ab31940 Abcam), rat anti-Tbr2 488

(1:200, 53-4875-80 eBioscience), rabbit anti-phospho-histoneH3 (1:300, ab47297 Abcam),

rat anti-Ctip2 (1:300, ab18465), rabbit anti-Satb2 (1:300, ab51502), rat anti-ki67-FITC

(1:200, eBioscience), mouse anti-BrdU (1:200, DSBH) and mouse anti-Pax6 (1:200, DSBH).

Polyclonal anti-WDR62 was generated by immunizing rabbits with a synthetic peptide

corresponding to amino acids 1150-1166 of murine WDR62 (VGQGGNQPKAGPLRAGT)

conjugated to Keyhole Limpet Hemocyanin. Custom antibody production including affinity

purification was performed by the Monash Antibody Technologies Facility (Monash

University, VIC, Australia).

Immunofluorescence analyses of MEFs were performed similarly. Following fixation in 4%

(w/v) paraformaldehyde or cold (-20°C) methanol as appropriate, MEFs were permeabilized

(0.2% [v/v] Triton X-100 in PBS, 20 min) and blocked (20% [v/v] FCS, 1% [w/v] BSA in

PBS, 60 min) prior to incubation with primary and Alexa Fluor conjugated secondary

antibodies diluted in blocking buffer. MEFs were counterstained with DAPI, mounted on

glass slides in ProLong Gold mounting media and imaged using confocal microscopes.

Protein lysates, immunoblots and immunoprecipitation

Protein lysates were prepared by homogenization and lysis of whole brain tissue or isolated

MEFs in RIPA buffer [150 mM NaCl, 100 mM Na3VO4, 50 mM Tris-HCl pH 7.3, 0.1 mM

EDTA, 1% v/v Triton X-100, 1% w/v sodium deoxycholate and 0.2% w/v NaF]

supplemented with protease inhibitors. Protein lysates were cleared by centrifugation (16,000

g, 10 min) and protein concentrations determined using Bradford assays. Proteins were

separated by SDS-PAGE, transferred onto a polyvinylidene fluoride (PVDF) membrane and

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analysed by immunoblotting. For immunoprecipitation experiments, cleared cell lysates were

incubated with rabbit polyclonal primary antibodies to WDR62 (custom antibody) or CPAP

(Proteintech), followed by affinity isolation with Protein-A agarose beads for 16 h at 4°C on

an end-to-end rotator. After repeated washing with lysis buffer, bound proteins were eluted

with Laemelli buffer and separated by SDS-PAGE for immunoblotting.

Acknowledgments

DN acknowledges funding support from the National Health and Medical Research Council

(GNT1046032, GNT1162652), Australian Research Council (FT120100193) and Cancer

Council (GNT1101931). BS is a recipient of a UQ International Scholarship from the

University of Queensland. We thank Prof Carol Wicking (Institute of Molecular Biosciences,

University off Queensland) for providing anti-Arl13b and anti-IFT88. We extend our

gratitude to Dr Richard Webb (Centre for Microscopy and Microanalysis) for assistance with

electron microscopy and Dr Shaun Walters (School of Biomedical Science Imaging Facility)

for support with confocal microscopes.

Conflicts of Interest

The authors declare no competing interests.

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Y.D. and Nigg, E.A. (2009) Control of centriole length by CPAP and CP110. Curr. Biol.,

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44. Nano, M. and Basto, R. (2017) Consequences of centrosome dysfunction during brain

development. Adv. Exp. Med. Biol., 1002, 19-45.

45. Ding, W., Wu, Q., Sun, L., Pan, N.C. and Wang, X. (2019) Cenpj regulates cilia

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2010.

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Figure legends

Figure 1. WDR62 deletion and missense mutations cause neural growth defects in mice.

A) Measured ratios of wild-type, heterozygous and homozygous WDR62 mutant mice at P0

and at E15.5. B) Reduced length of WDR62 mutant embryos at E15.5 compared to wild-type

littermates. C) Cortical thickness was measured in WDR62 mutant embryos and compared to

wild-type littermates at E15.5 and D) E12.5. Scale bars - 500 m. E) Ventricular area as a

ratio of brain size was measured in WDR62 mutant embryos and compared to wild-type

littermates at E15.5. F) Number of WDR62 mutant mice with anopthalmic defects observed

at E15.5 and 17.5. Mean values ± SEM are depicted. *p<0.05, **p<0.01, ***p<0.001 and

****p<0.0001 (t-test)

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Figure 2. WDR62 missense mutations trigger premature differentiation and loss of self-

renewed radial glia. A) Coronal brain sections from WDR62+/+

, WDR62stop/stop

,

WDR62V66M/V66M


E12.5 embryos were stained with PAX6 (red),

TBR2 (green) and phospho-Histone H3 (pHH3, blue). B) Quantification of PAX6+ve

, TBR2-ve

radial glia (red bars) in the VZ/SVZ of wild-type and WDR62 mutant embryos at E12.5-17.5

and E15.5. C) Measured mitotic index of radial glia (PAX6+ve

, TBR2-ve

, pHH3+ve

) in wild-

type and WDR62 mutant embryos at E12.5 and E15.5. D) Coronal sections of wild-type and

WDR62 mutant mice at E17.5 stained with PAX6 (red), TBR2 (green) and TBR1 (Magenta)

for radial glia, intermediate progenitors and immature neurons respectively. E) Quantification

of TBR2+ve

cells in the VZ/SVZ, intermediate zone (IZ) and the cortical plate (CP) at E12.5-

17.5. F) Quantification of immature neurons (TBR1+ve

) in the CP of wild-type and WDR62

mutant brains at E17.5. All scale bars - 20 m. Mean values ± SEM are depicted. *p<0.05,

**p<0.01, ***p<0.001 and ****p<0.0001 (t-test).

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Figure 3. WDR62 missense mutations perturb division orientation of apical progenitors

and increase cell cycle exit. A) Mitotic radial glia (PAX6+ve

, pHH3+ve

) undergoing vertical

(60°-90°) and horizontal (0°-30°) divisions typical of symmetric and asymmetric divisions

respectively. Scale bars - 5 m. Division angles of mitotic radial glia in the VZ of WDR62

mutant embryos (E15.5) were quantified and expressed within 0°-30°, 30°-60° and 60°-90°

ranges or B) expressed in a dot plot with mean angles indicated (red line). C) Cell cycle exit

analysis of 24 h BrdU labelled coronal brain sections from wild-type and WDR62 mutant

mice (E15.5) and stained for BrdU (green) labeling and Ki67 (red) as a proliferation marker.

Scale bars - 20 m D) Cell cycle exit index in WDR62 mutant and wild-type mice

determined by counting BrdU+ve

/Ki67-ve

cells and expressed as a proportion of total BrdU+ve

cells. E) TUNEL staining of apoptotic cells in coronal brain sections of wild-type and

WDR62 mutant embryos at E15.5 F) Quantification of TUNEL+ve

cells in the neocortex.

Scale bars - 20 m. Mean values ± SEM are depicted. *p<0.05, **p<0.01, ***p<0.001,

****p>0.0001 and ns; not significant (t-test).

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Figure 4. WDR62 mutations disrupt normal cilia regulation in the developing cortex.

A) Cortical sections from WDR62stop/stop

, WDR62V66M/V66M

, WDR62R439H/R439H

E15.5

embryos were stained for primary cilia (Arl13b/γ-tubulin) and representative images from the

VZ and SVZ shown. Scale bars - 20 μm. B) Percentage of ciliated cells within the SVZ at

E15.5. C) Measured length of primary cilia at E15.5 within the VZ/SVZ of developing

cortex. D) Measured length of primary cilia within the ventricular zone of the developing

cortex of WDR62stop/stop

, WDR62V66M/V66M

, WDR62R439H/R439H

embryos at E12.5 compared to

wild-type (+/+). Mean values ± SEM. **p<0.01, ***p<0.001 and ****p<0.0001 (t-test). E)

Radial glia in the VZ at E12.5 were stained with Arl13b (green) and γ-tubulin (red) to

visualize primary cilia. Arrow heads indicate ciliary remnants in WDR62 mutant mice. Scale

bars - 30 μm.

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Figure 5. WDR62 missense mutations cause axonemal and basal body defects A)

Primary MEFs were isolated from WDR62stop/stop

, WDR62V66M/V66M

, WDR62R439H/R439H

or

wild-type (WDR62+/+

) embryos (E14.5) and serum starved to induce cilia formation. WDR62

expression and localization (green) on primary cilia (marked by Arl13b, red) was determined

by immunostaining. B) Percentage of ciliated MEFs derived from WDR62 mutant or wild-

type embryos. C). Measured length of primary cilia in MEFs. D) Quantification of cilia

length following transient expression of WDR62-GFP in WDR62stop/stop

MEFs. E)

Representative images of primary cilia (Arl13b/tubulin) and WDR62-GFP expression in

WDR62stop/stop

MEFs. F) Representative images and G) quantification of primary MEFs

derived from WDR62 mutant mice with multiple axonemes. H) Representative images and I)

quantification of MEFs with multiple basal bodies indicated by the mother centriole marker

CEP170. All scale bars - 5 μm. Insets show individual fluorescence channels or higher

magnification images of cilia. Values are *p<0.05, **p<0.01 ***p<0.001 and ****p<0.0001.

Mean values ± SEM.

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Figure 6. CPAP recruitment to the basal body is perturbed by WDR62 mutations. A)

Centrosomal localization (marked by tubulin, red) of CPAP (green) in VZ/SVZ cortical

layers of WDR62stop/stop

, WDR62V66M/V66M

, WDR62R439H/R439H

or wild-type (WDR62+/+

)

embryonic brains at E12.5. B) Intensity of CPAP staining at the centrosome in WDR62

mutant embryos compared to wild-type. C) Evaluation of CPAP co-localized with tubulin at

centrosomes using Pearson’s correlation coefficient. D) CPAP localization in primary MEFs

isolated from WDR62stop/stop

, WDR62V66M/V66M

, WDR62R439H/R439H


)

embryos (E14.5). Inset indicates reduced CPAP (green) (scale bars - 5 μm). E) Intensity of

CPAP staining at the centrosome in WDR62 mutant MEFs compared to wild-type. F)

Immunoprecipiation of WDR62 from primary MEFs. WDR62 pulldown, co-precipitation of

CPAP and protein expression in total cell lysates (TCL) were determined by immunoblot

analysis. G) Representative images of CPAP (cyan) centrosomal localization and H)

quantification of CPAP intensity at the centrosome following WDR62-GFP expression in

WDR62-depleted MEFs. I) Representative images and J) quantification of CPAP (cyan)

localization at the centrosome following expression of WDR62 N-GFP (aa 1-841) and

WDR62 C-GFP (aa 842-1523) truncation mutants in WDR62stop/stop

MEFs. Insets show

higher magnification or individual fluorescence channels at centrosomes. All scale bars - 5

μm. Mean value ± SEM. ****p<0.0001, (t-test).

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Figure 7. WDR62 mutations disrupt IFT-88 localization to primary cilia. A) IFT88

localization to primary cilia in VZ/SVZ cortical layers of WDR62stop/stop

, WDR62V66M/V66M

,

WDR62R439H/R439H


) embryonic brains at E12.5. B) IFT88 staining

intensity (region measured indicated within insets in A.) in WDR62 mutant and wild-type

brains. C) Evaluation of IFT88 co-localized with tubulin at centrosomes using Pearson’s

correlation coefficient. D) IFT88 co-localization with markers of axonemes (Arl13b) and

basal body (-tubulin) in WDR62 mutant and wild-type MEFs. E) IFT88 fluorescence

intensity at the basal body in WDR62 mutant MEFs was quantified and compared with wild-

type (WDR62+/+

). F) Representative images and G) quantification of IFT88 staining intensity

at the centrosome following WDR62-GFP expression in WDR62-depleted MEFs

(WDR62stop/stop

). Mean values are indicated (red line). ****p<0.0001. Insets show higher

magnification or individual fluorescence channels at centrosomes. All scale bars - 5 μm. H)

Illustrative diagram of WDR62 function in recruiting CPAP/IFT88 for cilia formation in

neuroprogenitors.

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Documents

The association of microcephaly protein WDR62 with CPAP