PDGF-C Signaling Is Required for Normal Cerebellar Development Sara Gillnäs

PDGF-C signaling is required for normal cerebellar development

An analysis of cerebellar malformations in PDGF-C impaired mice

Sara Gillnäs

Degree project in biology, Master of science (2 years), 2021 Examensarbete i biologi 60 hp till masterexamen, 2021 Biology Education Centre and Department of Immunology, Genetics and Pathology, Dag Hammarskjölds väg 20, 751 85 Uppsala, Sweden, Uppsala University Supervisor: Johanna Andrae External opponent: Linda Fredriksson Table of contents Abstract ……………………………………………………………………………………... 2 List of abbreviations ………………………………………………………………………… 3 1. Introduction …………………………………………………………………………. 4 1.1 Platelet derived growth factors and their receptors ……………………………... 4 1.2 The Pdgfc-/-; PdgfraGFP/+ phenotype …………………………………………….. 4 1.3 Cerebellar development and morphology ………………………………………. 5 1.4 Ependymal development and function ………………………………………….. 6 1.5 Cerebellar germinal zones and patterning ………………………………………. 7 1.6 Expression pattern of PDGF-C and PDGFRɑ …………………………………... 8 1.7 Aim of the study ………………………………………………………………… 8 2. Materials and Methods ……………………………………………………………… 8 2.1 Animals …………………………………………………………………………. 8 2.2 Tissue preparation ………………………………………………………………. 9 2.3 Histology and Immunofluorescence staining ………………………………….... 9 2.4 Quantification and statistical analysis of cerebellar vasculature ………………. 11 2.5 Microscopy and imaging ………………………………………………………. 11 3. Results ……………………………………………………………………………... 11 3.1 Pdgfc-/-; PdgfraGFP/+ mice display abnormal cerebellar development …………. 11 3.2 Ependymal disruption in the ventricular zone of the fourth ventricle …………. 13 3.3 Ectopic expression of rhombic lip derived cells ………………………………. 17 3.4 Cerebellar vascularization in Pdgfc-/-; PdgfraGFP/+ mice …………………….... 18 4. Discussion …………………………………………………………………………. 19 4.1 Limitations of this study ………………………………………………………. 19 4.2 PDGF-C impaired mice resembles human Dandy-Walker malformation …….. 20 4.3 Stretching of the ventricular lining due to abnormal development ……………. 20 4.4 Astrocyte accumulation indicate possible repair mechanism ………………….. 23 4.5 PDGF-C signaling important for migration of rhombic lip late derivatives …... 23 4.6 Vascular bed appears normal in the neonatal Pdgfc-/-; PdgfraGFP/+ cerebellum ..25 Acknowledgements ………………………………………………………………………... 25 References …………………………………………………………………………………. 25 Appendix …………………………………………………………………………………... 29

Uppsala University Sara Gillnäs

Abstract Platelet-derived growth factor-C (PDGF-C) and its tyrosine kinase receptor PDGFRɑ have been shown to contribute to several key processes during central nervous system (CNS) development, including normal vascularization and formation of cerebral ventricles and basal membrane of the meninges. Due to redundancy between PDGF-C and PDGF-A, PDGF-C specific roles are sometimes masked and difficult to determine. Using the double mutant Pdgfc-/-;PdgfraGFP/+ mouse (Mus musculus) strain we were able to detect and examine a new, undescribed phenotype of PDGF-C impaired mice, namely cerebellar malformations. These mutant mice displayed an upwards rotation of the cerebellar vermis with a severe posterior vermis hypoplasia and an enlarged fourth ventricle, suggesting PDGF-C/PDGFRɑ signaling as a novel candidate to induce Dandy-Walker malformation (DWM). Due to suspected cerebellar vascular malformation a quantification of diameter, density and number of vessels were performed. A significant increase (P < 0.05) of the number and density of vascular bed in the cerebellar nuclei was detected, however the vessel diameter was not significantly different (P > 0.05) in Pdgfc-/-;PdgfraGFP/+ mice in comparison with the control. Through immunofluorescence staining we detected discontinuation of the ependyma in the acute angle of the ventricular zone adjacent to the rhombic lip, interfacing the fourth ventricle and cerebellar anlagen. We further noted ectopic expression of rhombic lip derived cells in the ventricular zone, suggesting a misguided migration due to ablation of PDGF-C. We conclude that PDGF- C is an essential player in normal cerebellar development.

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Abbreviation

AS - astrocytes CN - Cerebellar nuclei CSF -Cerebrospinal fluid DWM - Dandy-Walker malformation E - Embryonic day EC - Ependymal cells EGZ - External granular zone/layer 4V - Fourth ventricle GCP - Granule cell progenitors IGL - Granular layer IF - Immunofluorescent staining ML - Molecular layer NSC - Neural stem cells P - Postnatal day PCL - Purkinje cell layer PCP - Purkinje cell precursors PDGF-C - Platelet derived growth factor C PDGFRɑ - Platelet derived growth factor receptor alpha r1 - Rhombomere 1 RL - Rhombic lip SVZ - Subventricular zone VZ - Ventricular zone

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1. Introduction

1.1 Platelet derived growth factors and their receptors The platelet-derived growth factors (PDGFs) are widely, but specifically, expressed throughout the body. PDGF signaling has a vast importance during several developmental processes including organogenesis, angiogenesis and hematopoiesis (reviewed by Andrae et al. 2008). There are four PDGF ligands (PDGFA, -B, -C and -D) currently known, composed of polypeptide chains. These polypeptide chains form dimers, most commonly homodimers (reviewed by Andrae et al. 2008). However, heterodimerization of PDGF-AB has been seen in in vitro experiments (Ekman et al. 1999). When the ligand binds to the receptor, a tyrosine residue of the intracellular domain of the receptor is autophosphorylated. This activation leads to a downstream cascade in the cell through a multitude of signaling pathways, such as Ras- MAPK, PI3K and PLCγ (reviewed by Heldin & Westermark 1999). In mammalian in vivo studies, a limited number of PDGF-PDGFR interactions has been confirmed, including restricted interaction of PDGF-AA and PDGF-CC to PDGF-receptor-alpha (PDGFRɑ) (reviewed by Andrae et al. 2008).

1.2 The Pdgfc-/-;PdgfraGFP/+ phenotype PDGF-A and PDGF-C both signal though the PDGFRɑ and they have partially redundant functions. To phenocopy (or mimic) the severe phenotype seen in Pdgfrɑ-/- mice, both PDGF- A and PDGF-C need to be knocked out (Pdgfc-/-; Pdgfa-/-) (Ding et al. 2004). To surpass the redundancy of PDGF-A and PDGF-C, we use the double transgenic mouse strain Pdgfc-/-; PdgfraGFP/+ with only one functional locus of the receptor gene; preventing PDGF-A from rescuing the phenotype. We argue Pdgfc-/-; PdgfraGFP/+ mice to be a sufficient model system to facilitate the study the role of PDGF-C as Pdgfc-/- mice doesn’t exhibit several of the pathological phenotypes, including the cerebellar malformation. Furthermore, as Pdgfa-/-; PdgfraGFP/+ mice doesn’t display the cerebellar phenotype we can be sure that the phenotype is due to insufficient PDGF-C/PDGFRɑ signaling.

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Figure 1. Schematic illustration of cerebellar germinal zones, migration paths and adult morphology in mid-sagittal sections. (A) The main germinal zones during cerebellar development are the rhombic lip (RL) (red area) and the ventricular zone (VZ) (yellow area). From the RL the granule neuron precursors (blue cells) migrate tangentially in a subpial stream (blue arrow), giving rise to the external granular zone/layer (EGZ), while the glutamatergic neurons (red cells), including the cerebellar nuclei neurons, avert radially (red arrow) to reach the cerebellar nuclei (CN) anlagen. The VZ, adjacent to the fourth ventricular (4V), is the source of all GABAergic neurons (yellow cells), such as Purkinje cells. These cells migrate radially (yellow arrow) across the cerebellar anlage, towards the cerebellar cortex. (B) The adult cerebellar grey matter consists of the CN and cortex. The cortex is subdivided into three layers; the molecular layer (ML), Purkinje cell layer (PCL) and internal granular layer (IGL). The anterior vermis, posterior superior vermis and posterior inferior vermis constitute the three cerebellar lobes, which are further subdivided into lobules. A: Anterior, D: Dorsal, V: Ventral, P: Posterior.

Pdgfc-/-; PdgfraGFP/+ mice exhibit several severe phenotypes; spina bifida occulta, elongated skull, brain and spine hemorrhaging and lung emphysema (Andrae et al. 2016). Detected malformations in the brain consists of vascular malformations, neuronal over-migration in the cerebral cortex due to deficient formation of the meningeal basal membrane and ventricular malformations including hydrocephalus in rare cases (Andrae et al. 2016; Fredriksson et al. 2012). The severity is variable, but the progression of the disease is rapid and very few mice surpass postnatal day 15 (P15), given a stable genetic background (Andrae et al. 2016). Most mice die a few days after birth. Initial observations of cerebellum indicated an irregular foliation and lobules formation as well as hypoplasia of the posterior vermis (Andrae et al. unpublished). These phenotypes have all been described in the pathological umbrella disorder Dandy-Walker malformation (DWM) (Sasaki-Adams et al. 2008; Parisi & Dobyns 2003). Abnormal cerebellar vascular bed was also suspected (Andrae et al. unpublished).

1.3 Cerebellar development and morphology The cerebellum is divided into two hemispheres with the vermis in between, constituting the midline. During development the mammalian cerebellum first divides into three lobes which

5 Uppsala University Sara Gillnäs are subdivided into ten lobules through further foliation, the lobes are continuous between the vermis and cerebellar hemispheres (Leto et al. 2016; Sudarov & Joyner 2007). The adult cerebellar grey matter consists of the cerebellar nuclei (CN) and the cerebellar cortex which in the adult is subdivided into three layers: the molecular layer (ML), the Purkinje cell layer (PCL) and the internal granular layer (IGL) (fig. 1B) (Leto et al. 2016; Marzban et al. 2015). The cerebellum is derived from rhombomere 1 (r1) initiated by the signaling from the isthmic organizer, eg. fgf8, during neural tube closure. The most dorsal part of the neuroepithelium of the r1 becomes the roof plate, while the ventral part gives rise to the ventricular zone (VZ) and the intermediate neuroepithelium becomes the rhombic lip (RL) at the dorsal edge of the fourth ventricle (4V) (fig. 1A) (Leto et al. 2016; Marzban et al. 2015).

1.4 Ependymal development and function………………………………………………… The 4V is lined by a ciliated layer of cells derived from the neuroepithelium known as the ependyma, interfacing the brain parenchyma and cerebrospinal fluid (CSF) in the ventricle system. In the adult mammal the ependyma consists (mostly) of an uninterrupted layer of multiciliated cuboidal ependymal cells (ECs). Through beating with the cilia, the ECs contribute to the flow of the CSF (Bruni 1998, Ringers et al. 2019). The ECs develop during late embryonic development and continue to early postnatal development. The ependymal maturation process entails several degrees of development before the mature ependyma is in place. This process remains largely unknown, however, classifications of the different developmental states have been somewhat elucidated through distribution of ependymal cell types (McAllister et al. 2017; Sarnat 1992; Sarnat 1998; Redmond et al. 2019). Young ependyma can be recognized through a high quantity of mono-ciliated neural stem cells (NSC) with long basal processes expressing glial fibrillary acidic protein (GFAP). The elevated number of cells with short or absent basal process co-expressed with multiple cilia indicate the maturation of the ECs. The mature ependyma is recognized through the high quantity of multiciliated ECs interspersed with only few NSC. Worth mentioning is the tanycytes, which are specialized ependymal cells with long radial processes extended into the neuropil, thought to relay information from CSF to underlying neurons and capillary network (Ringers et al. 2019; Langlet et al. 2013). In differences to the multiciliated ECs, the tanycytes are mono- or bi-ciliated and can only be found within circumscribed areas of the ventricular system (Ringers et al. 2019; Langlet et al. 2013). Discontinuation of the ependyma (denudation) has been described as a deficit maintenance of junction proteins between the ECs and accumulation of astrocytes (AS) (McAllister et al. 2017; Sarnat 1995). Ependymal denudation has been linked

6 Uppsala University Sara Gillnäs to both spina bifida (SB) and hydrocephalus in several studies (Jiménez et al. 2014, McAllister et al. 2017, Sival et al. 2011). It is believed that accumulation of CSF increases the intracerebroventricular pressure causing the ependyma to tear and stretch (McAllister et al. 2017; Sarnat 1995). Another hypothesis is that the denudation causes irregular beating with the cilia which causes the accumulation of CSF (Jiménez et al. 2014, Sival et al. 2011). In other words, seemingly denudation can occur as a cause of mechanical forces posed by the CSF, as well as accumulation of CSF can occur as a consequence of faulty ependymal development.

1.5 Cerebellar germinal zones and patterning Pdgfc-/-; PdgfraGFP/+ mice display a faulty cerebellar patterning, foliation and vermis hypoplasia, hence, the cerebellar precursor migration is of interest. The VZ and RL conduct the two main germinal zones of the cerebellum (fig. 1A), giving rise to the various cell populations during neurogenesis (Leto et al. 2016; Marzban et al. 2015). The VZ derived GABAergic neurons, such as Purkinje cells, migrate radially from the VZ toward the developing cerebellar cortex (yellow cells, fig. 1A). The VZ can be identified through its expression of Ptf1a, which is essential for proliferation of GABAergic neurons (Hoshino et al. 2005; Marzban et al. 2015). The RL is a highly proliferating zone and gives rise to cerebellar granule cell progenitors (GCPs) and glutamatergic neurons, such as projection neurons and unipolar-brush cells. The RL derived precursors migrate tangentially in a subpial stream across the cerebellum; the GCP forms the external germinal layer/zone (EGZ) while the glutamatergic neurons continue their migration further to the CN (blue and red cells, fig. 1A) (Leto et al. 2016; Marzban et al. 2015). The GCP in the EGZ later proliferate and migrate inward to form the internal granular layer and can be recognized through their expression of reelin (Leto et al. 2016; Marzban et al. 2015). Glutamatergic neurons can be recognized through their expression of LIM Homeobox Transcription Factor 1 Alpha (Lmx1a). Cerebellar patterning and migration is tightly controlled by both external and internal signaling. Sonic hedgehog (SHH) expressed into the CSF from choroid plexus is essential for precursor proliferation and cell fate decision of RL derived cells (Leto et al. 2016; Marzban et al. 2015). Signaling from the dorsal mesenchyme in the posterior fossa (the cranial cavity surrounding the developing cerebellum) is essential for both VZ and RL progenitors proliferation and migration, such as forkhead box C1 (Foxc1) signaling (Doherty et al. 2013; Haldipur et al. 2014; Haldipur et al. 2016). Disruption of this signaling leads to impaired cerebellar foliation and lamination. Previous studies have shown that Foxc1 expressed by posterior fossa mesenchyme is essential for proper cerebellar development (Aldinger et al. 2009; Leto et al. 2016; Haldipur et al. 2014; Haldipur et al. 2016). Foxc1 is

7 Uppsala University Sara Gillnäs directly linked to maintenance of mesenchymal SDF1a expression, a chemoattractant important for tangential migration of RL derived cells, starting at embryonic day 12.5 (E12.5). When Foxc1 expression is lost, the expression of SDF1a is downregulated causing ectopic migration of RL precursors into the VZ (Haldipur et al. 2014; Haldipur et al. 2016). Mice with impaired Foxc1 expression display phenotypic similarities to the Pdgfc-/-; PdgfraGFP/+ cerebellum; posterior vermis hypoplasia and deficient foliation.

1.6 Expression pattern of PDGF-C and PDGFRɑ PDGFRɑ is expressed in the meninges and posterior fossa mesenchyme already during embryonic development (Andrae et al. unpublished). During neonatal development the PDGFRɑ expression persists in the meninges and additional expression can be seen throughout the cerebellar anlagen (Andrae et al. 2016; Sievers et al. 1994; Andrae et al. unpublished). Cerebellar expression of PDGF-C during embryonic development is substantially elevated beginning at E12.5 in the EGZ. In Pdgfc+/- control mice lacZ (reflecting Pdgfc) is continuously expressed in EGZ throughout prenatal stages, as well as vaguely expressed in VZ during E14.5 - E15.5 with some dispersion toward the cerebellar nuclei. Meanwhile, Pdgfc-/-; PdgfraGFP/+ mice display lacZ expression throughout most of the cerebellar anlagen beginning at E12.5, with a prominent expression in the VZ during E14.5 - E15.5. Yet, the expression in EGZ is similar to that of the control (Andrae et al. unpublished).

1.7 Aim of the study This study aimed to further elucidate the impact of PDGF-C signaling on the cerebellar phenotype and development. We hypothesized that impaired PDGF-C signaling causes ectopic migration of RL derived precursor cells, and that this is one of the main causes of the posterior vermis hypoplasia and upward vermis rotation. Furthermore, the irregular ventricular lining of the dorsal VZ enticed us to examine whether ependymal denudation was in progress.

2. Materials and Methods

2.1 Animals All experiments on mice used in this study were in accordance to Swedish animal welfare legislation ethical permits were approved by the Uppsala animal ethics committee (C225-12, C115/15, 5.8.18-03029-2020). Mice strains used: PdgfraGFP/+ (Pdgfratm11(EGFP)Sor Hamilton et.

8 Uppsala University Sara Gillnäs al 2003); Pdgfc knockout (Pdgfctm1Nagy Ding et. al 2004). These two lines were then crossed to generate double transgenic mice Pdgfc-/-; PdgfraGFP/+. Mice used in this study was of the ages E14.5, E15.5, E17.5, P0, P1 and P2. Samples collected 2018 and earlier have a pure genetic background of C57BL/6J, corresponding to the paraffin and OCT embedded samples used in this study. Due to genetic drift the mutant strain became unviable and to obtain new samples the C57BL/6J mice were crossed with 129S1 mice, producing a viable, however mixed genetic background. The C57BL/6J;129S1 mice correspond to the vibratome sectioned tissues in this study. The mice with Pdgfc-/-; PdgfraGFP/+ genotype are easily recognized at birth due to the hemorrhagic stripe over the lower spine, with the exception of one mouse of the mixed background, which was dismissed from this study. All mice were genotyped for Pdgfc (wildtype and/or knockout alleles) through PCR.

2.2 Tissue preparation All mice in this study were sacrificed by decapitation. Embryonic mice were dissected out at days 14.5, 15.5 and 17.5 after plugging. Heads were deskinned and emerged into 4% paraformaldehyde (PFA) in 1x phosphate-buffered saline (PBS) for fixation overnight at +4℃. The heads were washed in 1x PBS overnight at +4℃. Fixed heads were embedded in 4% low- melting agarose gel and sectioned in a vibratome; sagittal with a thickness of 70µm. For paraffin embedding and cryosections the brains were dissected out of the cranium prior to fixation, and dehydrated and embedded in paraffin and sectioned sagittal with a thickness of 5µm in a microtome or soaked in 30% sucrose and embedded in OCT for sagittal cryosectioning to a thickness of 14µm.

2.3 Histology and Immunofluorescence (IF) staining Paraffin embedded sections were heated at 60℃ for 15min followed by deparaffinized in Xylene and rehydrated in a EtOH series (99.5%, 96%, 70%, dH20). Antigen retrieval in either Dako TRS Citrate buffer pH 6 (S2369, Dako, Glostrup, Denmark) or Dako Target Retrieval Solution pH 9 (S2367, Dako, Glostrup, Denmark), see table for specific primary-antibody and antigen retrieval treatment (sup. table 1). Sections were washed in 1x PBS (3x2min). Sections were blocked in PS (1% bovine serum albumin (BSA), 0.5% Triton X-100 (T8787, Sigma) in 1x PBS) with 2.5% normal donkey serum (017-000-121, Jackson ImmunoResearch) for a minimum of 30 min at room temperature (RT). Sections were incubated overnight in primary antibodies diluted in PS/2 (0.5% BSA, 0.25% Triton X-100 (T8787, Sigma) in 1x PBS) at +4℃. After washing in 1x PBS (3x5min) secondary antibodies diluted in PS/2 were applied and

9 Uppsala University Sara Gillnäs incubated for a minimum of 1h at RT. The sections were then washed in 1x PBS (3x5min) before adding Hoechst (Invitrogen) diluted to 1:10 000 in 1x PBS to incubate for 10min at RT. Wash in 1x PBS (2 min) and mounted with ProLongGold antifade reagent (Invitrogen).

Cryosections were blocked for 1h in PS with 2.5% donkey serum (017-000-121, Jackson ImmunoResearch) at RT and incubated 1h in RT and overnight at +4℃ with primary antibodies diluted in PS/2. The sections were then washed in 1x PBS for 3x15min prior and following incubation in secondary antibodies diluted in PS/2 for 2h at RT, and mounted in ProLongGold with DAPI (Invitrogen).

Free-floating vibratome sections were blocked overnight in PS with 2.5% donkey serum (017- 000-121, Jackson ImmunoResearch) at +4℃. Incubated with primary antibodies for 48h at +4℃. Washed in PBS-T (1x PBS with 0.1% Tween 20 (P1379, Sigma)) for 3x1h and incubated with secondary antibody overnight at +4℃. Sections were then washed in PBS-T (3x1h), incubated in 1:10 000 Hoechst (Invitrogen) in 1xPBS for 15 min at RT, washed in PBS-T (3x30min) and mounted in ProLongGold antifade reagent (Invitrogen)

All staining procedures were compared to negative controls using only secondary antibodies, to eliminate any possible false positive immunoreactivity.

The primary antibodies (sup.table 1) used were anti-Podocalyxin (1:200, goat polyclonal, AF1556, R&D Systems), anti-Glial fibrillary acidic protein (GFAP) (1:100, rat monoclonal, 13-0300, Invitrogen), anti-S100B (1:100, rabbit polyclonal, ZO311, Dako), anti-βIV-tubulin (1:500, rabbit monoclonal, ab179509, Abcam), anti-N-cadherin (1:50, rat monoclonal, MNCD2-C, Developmental Studies Hybridoma Bank), anti-Connexin 43 (1:50, mouse monoclonal, sc-271837, Santa Cruz Biotechnology), anti-Reelin (1:100, goat polyclonal, af- 3820, Novus biologicals), anti-Lmx1a (1:200, rabbit polyclonal, ab10533, Sigma-Aldrich). Alexa-Flour conjugated secondary antibodies (Cy3, A647, A488) produced in donkey were used (Invitrogen).

Hematoxylin and Eosin staining were done using paraffin embedded sections. The sections were heated at 60℃ for 15min, deparaffinized in Xylene and rehydrated in an EtOH series (99.9%, 96%, 70%, dH20). The sections were then immersed in Mayer’s hematoxylin (HTX) (01820, HistoLab, Västra Frölunda, Sweden) for 2min and rinsed in dH2O. Running tap water

10 Uppsala University Sara Gillnäs for 5 min was used as a bluing reagent. Sections were then rinsed in dH2O. Sections were then immersed in Eosin (01650, HistoLab, Västra Frölunda, Sweden) for 30 seconds before dehydrating the sections (95% EtOH, 99.5% EtOH, Xylene) and mounted in PERTEX (00811, HistoLab, Västra Frölunda, Sweden).

2.4 Quantification and statistical analysis of cerebellar vasculature All vessel measurements were done in Fiji and statistically analyzed in R-studio. In total 6 samples were included in the quantification, three Pdgfc-/-; PdgfraGFP/+ mice and three controls. The mice were at stage P0 and embedded in paraffin and IF stained with anti-Podocalyxin and appropriate secondary antibody. Vessel diameter, vessel density and the amount of vessels in the lobes of the anterior vermis and the CN were analyzed. To standardize the measurement, only cross-sections of the vessels were included. The diameter of slightly elliptical vessels was quantified for the shortest diameter. For each parameter and location, the mean of each sample was calculated before the two groups were compared using an independent two-sample t-test, statistical significance was defined as p-value < 0.05.

2.5 Microscopy and imaging Images were captured using Leica DMi8 (for bright-field and overview images) and Leica TCS SP8 X confocal (for z-stacks and high-resolution images) microscope. No image alteration was done, with the exception of minor brightness and contrast adjustments.

3. Results

3.1 Pdgfc-/-; PdgfraGFP/+ mice display abnormal cerebellar development This study was conducted on Pdgfc-/-; PdgfraGFP/+ mice. These mice developed spinal hemorrhage, recognizable via a red stripe on their lower spine, and genotyped through PCR. Only mice clearly exhibiting this red stripe were chosen for this study. Controls for this study were heterozygote or homozygote for the Pdgfc allele (Pdgfc+/+; PdgfraGFP/+ or Pdgfc+/-; PdgfraGFP/+). In total three mutants and three controls were examined for each treatment (with the exception for Lmx1a, only two mutants and two controls were examined).

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Previous experiments have shown that the foliation of the vermis in the P0-P2 Pdgfc-/-; PdgfraGFP/+ mice was clearly disorganized and with a notably underdeveloped posterior vermis. This malformation was described more in detail. In control mice, five primary fissures were seen at P0, dividing the vermis into five cardinal lobes, and secondary fissures started to appear already at P1 to become even more apparent at P2 as the folia lengthened (fig. 2D-F). Foliation of the anterior vermis in Pdgfc-/-; PdgfraGFP/+ mice was observed from P0. However, primary fissures of the posterior vermis were not developing, resulting in fewer cardinal lobes of the vermis; giving the posterior inferior vermis a flat appearance (fig. 2J-L). Secondary fissures at P2 could not be observed (fig. 2L). It is difficult to determine whether the onset of

Figure 2. Vermis rotation and posterior vermis hypoplasia in Pdgfc-/-; PdgfraGFP/+ mice Hematoxylin/Eosin staining of mid-sagittal paraffin section of Pdgfc+/+; PdgfraGFP/+ control mice (A-F, M) and Pdgfc-/-; PdgfraGFP/+ mutant mice (G-H). The Pdgfc-/-; PdgfraGFP/+ cerebellar development appears normal during E14.5 (A, G) and E15.5 (B, H). Beginning at E17.5 and onward (I-L), PDGF-C impaired mice display posterior vermis hypoplasia indicated by lack of fissure (white asterisks) and lobe development in the posterior inferior vermis (J-L). Four cardinal fissures (white asterisks) divide the vermis into five cardinal lobes in the control mice (D-F). The relative angle off the second cardinal lobe in the anterior vermis, ɑ° < β° (F, L), and the relative position of the rhombic lip (red arrow) indicates an upward rotation of the vermis and similarities with the human Dandy-Walker “tail”. Cb: Cerebellum. Scale bars = 200 µm (A-L) and 1 mm (M).

secondary fissures were delayed in Pdgfc-/-; PdgfraGFP/+ mice or if they did not develop at all, as most mutant mice died a few days after birth. Furthermore, an upwards rotation of the vermis could be seen when comparing the relative position of the RL and the angle of which the lobes

12 Uppsala University Sara Gillnäs develop in Pdgfc-/-; PdgfraGFP/+ mice (fig. 2F, L). The first sign of hypoplasia and upward rotation of the vermis was indicated embryonically, prior foliation, as the anlagen for the posterior inferior vermis is underdeveloped (fig. 2C, I). Moreover, the size of the 4V in the midline was seemingly larger in the Pdgfc-/-; PdgfraGFP/+ mice in comparison with the control (fig. 2). This has however not been quantified.

Taken together these abnormalities of the mutant cerebellum, including the elongated skull and brain (Andrae et al. 2016) with an suggested enlargement of the posterior fossa: the Pdgfc-/-; PdgfraGFP/+ mice cerebellar phenotype is consistent with the key features of the human syndrome Dandy-Walker malformation (DWM). However, more analyses are needed to clearly associate defect PDGF-C signaling with DWM. For example, quantification of the posterior fossa and 4V is needed. Nevertheless, this led us to further investigate ectopic migration from the RL in Pdgfc-/-; PdgfraGFP/+ mice.

3.2 Ependymal disruption in the ventricular zone of the fourth ventricle Ependymal alterations in the Pdgfc-/-; PdgfraGFP/+ mice were initially detected through partial loss of immunoreactivity with podocalyxin (Podx1), normally labelling the apical side of the ependymal cells. The alteration could be seen beginning at E17.5, while younger specimens displayed normal epithelial structure, similar to the control (fig. 4). In all Pdgfc-/-; PdgfraGFP/+ samples examined, the alteration of the ependyma was always observed in the acute angle of the VZ adjacent to the RL, corresponding to the posterior inferior vermis anlagen (fig. 3A-G). In contrast with the control mice, the ECs were no longer stacked in an epithelial manner in the interface of the 4V and the underlying cerebellar brain tissue (fig. 3E-G). We also observed an increase of the area affected by the alteration, expanding ventrally over the VZ of the 4V as development progressed (fig. 4).

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Figure 3. Ependymal alteration in ventricular zone of the fourth ventricle Immunofluorescent (IF) staining with podocalyxin (red) counterstained with nuclear Hoechst staining (blue) of P1 mid-sagittal paraffin section of Pdgfc+/+; PdgfraGFP/+ control mice (A-C) and Pdgfc-/-; PdgfraGFP/+ mice (D-H, H’) revealed alteration of the ventricular lining of the fourth ventricle. The normal ependyma have an epithelial structure and express podocalyxin throughout the VZ (yellow dotted line) (A-C). Pdgfc- /-; PdgfraGFP/+ mice lack epithelial structure and expression of Podx1 in the dorsal VZ, adjacent to the RL. White arrowhead indicates the border between normal and altered ependyma (D, G). Irregular clusters of cells could be observed in the subventricular zone in Pdgfc-/-; PdgfraGFP/+ mice (H). Through IF staining the ependymal junction proteins n-cadherin (green) and connexin 43 (white) were detected in these cell clusters (H’). CP: Choroid Plexus, 4V: Fourth ventricle, RL: Rhombic lip, VZ: Ventricular zone.

Furthermore, we could observe a loss of junction proteins in the altered area. N-cadherin (Cdh2) is normally expressed in neuroepithelium/ependyma, on the lateral-apical pole of the cells of the intact ependyma consistent with the apical expression of Podx1. In the altered ependyma of Pdgfc-/-; PdgfraGFP/+ mice, the n-cadherin was generally not present in the lining of the ventricle (fig. 4F-H). The gap-junction protein connexin 43 (Cx43) is considered to be an ependymal specific marker, as it in difference from n-cadherin is not expressed in other neuroepithelial cells. Connexin 43 is expressed laterally between the normal ependymal cells, and similar to n- cadherin, connexin 43 is not present in the disrupted ependyma (fig. 4F-H). Notably, the expression of connexin 43 is fully consistent with podocalyxin and only present between cells in epithelium structure, n-cadherin expression persists a few cells into the disrupted VZ. Due to the loss of junction proteins we determined the affected area in the Pdgfc-/-; PdgfraGFP/+ mice as disrupted, undergoing denudation. Indeed, in the disrupted area, the epithelial properties

14 Uppsala University Sara Gillnäs were lost. Furthermore, just beneath the ventricular lining in the subventricular zone (SVZ), clusters of cells positive for both n-cadherin and connexin 43 could be observed (fig. 3H). These clusters were only seen in a few samples examined, imposing a random and more rare pathological feature due to loss of PDGF-C.

Figure 4. Loss of ependymal junction proteins in ventricular lining Immunofluorescent staining of ependymal specific gap-junction protein connexin 43 (white) and neuroepithelial junction protein n-cadherin (green) together with podocalyxin (red) on mid-sagittal paraffin sections of Pdgfc+/+; PdgfraGFP/+ control mice (A-D) and Pdgfc-/-; PdgfraGFP/+ mice (E-H). E17.5-P1 Pdgfc-/-; PdgfraGFP/+ mice display a loss of junction proteins in the dorsal VZ, consistent with the loss of podocalyxin expression, implying a loss of cell-to-cell communication. This infer a loss of ependymal properties and classified as an ependymal disruption (F-G). The ependymal disruption began in the acute angle between the dorsal VZ, adjacent to the RL, and expanded ventrally as development progressed (F-G). Ependymal disruption could not be detected at E15.5 (E). CP: Choroid Plexus, 4V: Fourth ventricle, RL: Rhombic lip, VZ: Ventricular zone.

Next, we wanted to see if the cells of the disrupted ependyma retained EC specific features, through IF staining with ependymal specific markers βIV-tubulin (βIV-tub) and S100B. Due to the mixed ependymal cell population, 70 µm thick vibratome sections were made to be able to

15 Uppsala University Sara Gillnäs sufficiently detect the distribution of the different ependymal cell types. βIV-tubulin is a microtubule component and strongly expressed in the cilia of ECs. In Pdgfc-/-; PdgfraGFP/+ mice βIV-tubulin was only expressed in cells in intact ependyma with a gradual decline in the number of multiciliated cells going from ventral to dorsal of the VZ, to completely disappear before the denuded area (fig. 5A-B, E-F). Indeed, cells displaying epithelial structure connected via junction proteins adjacent to the disrupted ependyma lacked cilia. In other words, the multiciliated feature of the ECs appeared greatly affected in the Pdgfc-/-; PdgfraGFP/+ mice. Meanwhile, control mice exhibited a continuous βIV-tubulin expression throughout the VZ, with perhaps a slight decline most dorsally, close to the ventricular angle adjacent to the RL.

Throughout the normal ependyma of the VZ, S100B was expressed in a typical cuboidal manner in the cytoplasm of the ECs. The Pdgfc-/-; PdgfraGFP/+ mice displayed a weak and irregular expression of S100B in the dorsal VZ and completely lost the cuboidal expression pattern in the dorsal edge of the VZ (fig. 5C, G). No ectopic expression of βIV-tubulin was detected in neighboring tissue (fig. 5E), indicating that ependymal rosette formation doesn’t occur in Pdgfc- /-; PdgfraGFP/+ mice. It is however possible that it is too early in pathological development to detect ependymal rosettes, or possibly a random feature, too rare for detection with the small sample count used in this study. The cell clusters positive for junction proteins could possibly indicate early rosette formation, although this study could not elucidate whether these clusters were also βIV-tubulin positive.

Discontinuation (or denudation) of ependyma has been associated with an accumulation of astroglia processes. Through IF staining with GFAP we detected an excessive expression of AS processes in the denuded areas of P0 Pdgfc-/-; PdgfraGFP/+ mice (fig. 5D, H). GFAP expression is expected in the ependyma at P0, as the ependyma is not yet fully mature and still houses a significant number of NSC interspersed between the ECs. In the normal ependyma the GFAP expression was visible as short projections, as seen in the control mice (fig. 5D). The GFAP processes seen in the Pdgfc-/-; PdgfraGFP/+ mice were irregular and lacked the typical radial direction expected of NSCs (fig. 5H). Due to the lack of co-expression with other ependymal markers, the GFAP positive glial accumulation in the denuded VZ is likely to be of AS descent. The disrupted ependyma was seemingly replaced with a patch of AS to hinder the neural progenitors in the parenchyma of coming in contact with the CSF.

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Figure 5. Loss of ependymal cell properties and astrocytic reaction in disrupted ependyma Immunofluorescence (IF) staining of ependymal specific markers βIV-tubulin (A-B, E-F) and S100B (C, G) and glial projection marker GFAP (D, H) in P0 mice. Normal ependymal cells co-express βIV-tubulin (A, B) and S100B (C) throughout the ventricular zone, with a slight decline most dorsally. Pdgfc-/-; PdgfraGFP/+ mice display a gradual decline in the VZ of cell expressing βIV-tubulin (high ventral expression, low dorsal expression) to be completely lost in the disrupted ependyma (E, F). No ectopic βIV-tubulin expression in the SVZ was detected (E). The expression of S100B is irregular and vague in the dorsal VZ of Pdgfc-/-; PdgfraGFP/+ mice (G), in comparison with the cubic expression in Pdgfc+/+; PdgfraGFP/+ control mice (C). Note that S100B is additionally expressed by glial cells in the parenchyma (C, G). GFAP expression visualized an accumulation of AS in the disrupted ependyma (H), not displaying radial projections expected of NSC in intact ependyma (D, H (co-expressed with Podx1)). Podocalyxin (red) staining visualizes intact ependyma (A-H), white arrow marks border of disrupted ependyma (E-H). CP: Choroid Plexus, 4V: Fourth ventricle, RL: Rhombic lip, VZ: Ventricular zone. Vibratome sections 70µm.

3.3 Ectopic expression of RL derived cells Reelin (Reln) is an extracellular glycoprotein critical for cerebellar corticogenesis (cortex development). Reln is expressed by RL derived GCPs in the EGZ to attract Purkinje cell precursors (PCP) to form the PCL during postnatal development. In P0 control mice a dispersion of reelin expression could be seen throughout cerebellum, with a prominent expression in the EGZ (fig. 6A). The PCP clusters in the subpia could be distinguished by their large size and lack of reelin expression. Pdgfc-/-; PdgfraGFP/+ mice showed a similar expression of reelin in the EGZ in the anterior vermis. However, the expression in the posterior vermis was both irregular and dispersed, and the normally distinct layers of EGZ and PCP-cells were sometimes hard to distinguish (fig. 6B). Furthermore, the Pdgfc-/-; PdgfraGFP/+ mice had a highly elevated expression of reelin in the VZ compared to the control (fig. 6B). This ectopic

17 Uppsala University Sara Gillnäs expression of reelin indicates a deviation in the subpial migration of GCP from the RL. All Pdgfc-/-; PdgfraGFP/+ mice examined displayed a clear elevation of reelin expression in the VZ in comparison with the control, however, some variation in the distribution of the ectopic expression between the PDGF-C mutant mice was observed.

To further examine ectopic expression of RL derived cell populations, we investigated glutamatergic neuron precursors through immunoreactivity with Lmx1a. No clear deviation in the migratory path of Lmx1a-positive cells could be detected in Pdgfc-/-; PdgfraGFP/+ mice at P0 (fig. 6C-D). Accumulation of Lmx1a-positive cells was observed in the CN anlagen, consistent with the control, without ectopic expression in the most dorsal VZ. These results indicate normal migration of RL derived glutamatergic neurons.

Figure 6. Ectopic expression of rhombic lip derived precursors Immunofluorescent (IF) staining of GCP expressed Reelin in P0 Pdgfc-/-; PdgfraGFP/+ mice visualize an irregular and dispersed expression in the posterior vermis, and a ectopic expression in the VZ, indicating ectopic migration of late derivatives from the RL (A), in comparison with Pdgfc+/+; PdgfraGFP/+ control mice (B). IF staining of Lmx1a visualized an accumulation of glutamatergic neurons in the CN in both P0 Pdgfc-/-; PdgfraGFP/+ and control mice (C,D). No ectopic migration through the VZ in PDGF-C impaired mice is indicated (D). CN: Cerebellar nuclei, CP: Choroid Plexus, 4V: Fourth ventricle, RL: Rhombic lip, VZ: Ventricular zone. Vibratome sections 70 µm (A, B), cryosections 14 µm (C, D).

3.4 Slightly increased cerebellar vascularization in Pdgfc-/-; PdgfraGFP/+ mice Vascular alterations and bleedings have previously been described in the cerebrum of Pdgfc-/-; PdgfraGFP/+ mice (Andrae et al. 2016). Here, potential alterations of the cerebellar vessel

18 Uppsala University Sara Gillnäs development due to impaired PDGF-C signaling was investigated through quantifications of several parameters including: diameter, density and number of vessels. These measurements were done in the lobes of the anterior vermis and the nuclei center in P0 mice. In the cerebellar nuclei, both the total number of vessels (p-value = 0.0038) and vessel density (p-value = 0.0319) was increased in Pdgfc-/-; PdgfraGFP/+ mice in comparison with the control mice (sup. fig. 2). No difference was detected in the anterior vermis. No significant difference in vessel diameter (p-value > 0.05) can be reported.

4. Discussion The current study demonstrates that impaired PDGF-C signaling during development causes vast cerebellar malformations including (1) hypoplasia and upwards rotation of the posterior vermis, (2) discontinuation of the VZ ependyma and (3) ectopic expression of RL derived neural progenitors. These features were all present in Pdgfc-/-; PdgfraGFP/+ mice displaying spinal hemorrhaging and spina bifida occulta. This study presents novel characterization of the PDGF-C role during cerebellar development as well as further insight into the pathological features associated with the Pdgfc-/-; PdgfraGFP/+ strain of mice. Moreover, the cerebellar phenotype of Pdgfc-/-; PdgfraGFP/+ mice propose that lack of PDGF-C contribute to Dandy- Walker malformations.

4.1 Limitations of this study Regeneration of the Pdgfc-/-; PdgfraGFP/+ mice with a pure genetic background C57BL/6J has been maintained for approximately 15 years at our facility. Recently, genetic drift in the population caused a severe increase in lethality. This lethality limited the number of samples used in this study with a pure genetic background to samples collected 2018 and earlier. To regain a stable Pdgfc-/-; PdgfraGFP/+ strain we crossed C57BL/6J mice with 129S1 mice; half of the Pdgfc-/-; PdgfraGFP/+ samples used in this study have a heterogeneous genetic background. In total three controls and three Pdgfc-/-; PdgfraGFP/+ mice were used for each staining treatment. Due to the redundancy between PDGF-C and other PDGFs via PDGFRɑ signaling, the strain used in this study aims to unmask Pdgfc specific phenotypes by replacing one of the Pdgfra wildtype alleles with a PdgfraGFP knock-in allele (Andrae et al. 2016). However, as there is yet much to learn about PDGF signaling, there might be additional features not uncovered by this strain.

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4.2 PDGF-C impaired mice resembles human Dandy-Walker malformation Dandy-Walker malformations is an umbrella diagnosis of cerebellar abnormalities including the key features: enlarged posterior fossa, vermis hypoplasia with an upward rotation and an enlarged 4V (Sasaki-Adams et al. 2008; Parisi & Dobyns 2003). This study report Pdgfc-/-; PdgfraGFP/+ mice to display both hypoplasia of the posterior inferior vermis as well as an upward rotation of the vermis and an enlarged 4V in the cerebral mid-line. Quantification of the size of the entire 4V is needed to fully assess the extent of the enlargement, hence, in this study we cannot conclude whether the enlargement is due to accumulation of CSF or simply an effect of the overall cerebellar malformation. Nevertheless, the observed enlargement of the 4V in the mid-line is consistent with previous studies on DWM (Parisi & Dobyns 2003; Sasaki- Adams et al. 2008; Aldinge et al. 2009; Haldipur et al. 2014; Haldipur et al. 2017).

The foliation of the posterior inferior vermis is severely underdeveloped, lacking developing fissures and features reminiscent of the typical “tail” of the posterior vermis defined as pathognomonic features in human DWM (Bernardo et al. 2015; Haldipur et al. 2017). Previous quantification of the Pdgfc-/-; PdgfraGFP/+ mice describes an elongated skull and brain (Andrae et al. 2016), which is in line with an enlarged posterior fossa. The cause of human DWM is largely unknown, seemingly a defect implicated by a number of different genes where only a few have been determined to contribute to the diagnosis. One of those genes is Foxc1 (Aldinger et al. 2009; Doherty et al. 2013; Haldipur et al. 2014; Haldipur et al. 2017). Mice with deficient Foxc1 signaling display several cerebellar phenotypic similarities with the Pdgfc-/-; PdgfraGFP/+ mice, including the key DWM features and irregular and ectopic migration of cerebellar cell populations.

4.3 Stretching of the ventricular lining due to abnormal development causes disruption This study demonstrates a discontinuation of the ependyma of the cerebellar VZ in Pdgfc-/-; PdgfraGFP/+ mice, interfacing the 4V. This discontinuation is visible mid-sagittally in the dorsal VS adjacent to the RL, beginning at E17.5 (sup fig. 1) and becomes increasingly larger during prenatal and natal development. EC features are lost in the discontinued site, evident by the lack of βIV-tubulin and S100B. The loss of cilia (indicated by the lost βIV-tubulin expression) in the ventricular lining infer the loss of one of the most characteristic functions of the ependyma, namely the propulsion of the CSF. The disrupted ependyma in Pdgfc-/-; PdgfraGFP/+ mice also lack an epithelial structure, and due to the lack of immunoreactivity to connexin 43

20 Uppsala University Sara Gillnäs and n-cadherin, a loss of junctions is evident in the disrupted site. This deficiency in maintaining junction proteins is consistent with previous studies on ependymal denudation, implying both the loss of ependymal structure and function in the disrupted area (Xu et al. 2001; Ma et al. 2007; Sival et al. 2011; McAllister et al. 2017). This cell-coupling is not just important for maintaining a structure, but cell-to-cell communication through gap-junctions (connexin 43) might organize the synchronized ciliary beating. It has been suggested that even small disruptions can have implications on CSF flow through (Sival et al. 2011). This is probable to have implications on the flow through the ventricular system of Pdgfc-/-; PdgfraGFP/+ mice, with the possibility to influence the pressure and accumulation of fluids; which alludes to the hydrocephalus seen in some of the PDGF-C impaired postnatal mice (earliest seen in P7), displaying vast accumulation of CSF in the lateral ventricles (Fredriksson et al. 2012; Andrae et al. unpublished). Previous studies have shown that there is an association between hydrocephalus and ependymal denudation (Sival et al. 2011; Jiménez et al. 2014).

The relationship between ependymal discontinuation and hydrocephalus is complex. It is difficult to establish whether the increased pressure conducted by accumulation of CSF causes the ependyma to stretch and disrupt, or if the accumulation of CSF is a consequence of denudation of ependyma (Sarnat 1995; Sival et al. 2011; Jiménez et al. 2014; McAllister et al. 2017). We reason that in neonatal Pdgfc-/-; PdgfraGFP/+ mice it is more likely that the disruption is due to stretching and tearing conducted by the upwards rotation of the vermis, rather than increased pressure imposed by the CSF in the 4V. As described by Sarnat, the site of disruption in hydrocephalus is unpredictable and unlikely to only occur in one position, and the acute angle between the VZ and RL is less susceptible to disruption compared to smooth ventricular surface (Sarnat 1995). Based on this reasoning, it’s improbable that the disruption in the 4V VZ in embryonic and neonatal Pdgfc-/-; PdgfraGFP/+ mice would be a consequence of accumulation of CSF; as the site of the disruption is consistently found in the acute angle between the VZ and RL in all Pdgfc-/-; PdgfraGFP/+ mice examined, being both predictable and unlikely in site.

Interestingly, in human patients spina bifida has been identified as a cause of hydrocephalus by disturbing the CSF outflow from the 4V (Bigio 2010). In the current study we could see an enlargement of the 4V in the midline, perhaps alluding to an altered flow through of the CSF due to inadequate and/or unorganized beating of EC cilia caused by the denudation. Needless to say, there is evidently an association between spina bifida, hydrocephalus and ependymal

21 Uppsala University Sara Gillnäs denudation, however, the underlying mechanisms and triggering effects cannot be elucidated by this study.

A previous study of adult Pdgfc-/- mice have described that malformed and denuded ependyma in the lateral ventricles with loss of cuboidal appearance of the ECs, as well as partial deflation of the lateral ventricles (Fredriksson et al. 2012). Notably, the denudation seen in adult PDGF- C impaired mice is evenly distributed in the ventricular lining of the lateral ventricular while in embryonic and neonatal mice, examined in this study, the site of the disruption was predictable. In postnatal and adult mice PDGFRɑ-positive cells have been observed in the ventricular lining of the fourth (Andrae et al. unpublished) and lateral ventricle (Jackson et al. 2006). The presence of PDGFRɑ-positive cells in the VZ or SVZ of the 4V in neonatal mice could not be seen (data not shown). This implies that PDGF-C/PDGFRɑ signaling is important during late development and maintenance of the ependyma. Furthermore, the Pdgfc-/- mice don’t exhibit the pathological cerebellar phenotype seen in Pdgfc-/-; PdgfraGFP/+ mice (Fredriksson et al. 2012). We reason that the malformation seen in the adult lateral ventricular and neonatal fourth ventricular are separate events, although more studies are needed to infer this statement.

Ependymal rosettes positive for βIV-tubulin were not detected in the SVZ in the Pdgfc-/-; PdgfraGFP/+ mice, contradictory to other studies on ependymal denudation (Sarnat 1995; Sival et al. 2011; Jiménez et al. 2014; McAllister et al. 2017). Clusters of cells positive for n-cadherin and connexin 43 in the SVZ of the 4V were detected in some of the mice examined, however, due to low sample number and the rare appearances of these clusters we cannot conclude if these are rosettes or not.

The constricted single area of the ependyma together with the apparent lack of rosette formation in the SVZ are important differences to the more classical description of discontinuation of ependyma. As an alternative hypothesis, we propose that the abnormal cerebellar development itself causes the disruption, through upwards rotation of the vermis, stretching and tearing the ependyma in the acute angle between the VZ and RL. Ectopic migration of neural progenitors from the RL to the VZ might play a part in this, in accordance with studies on DWM (Haldipur et al. 2014; Haldipur et al. 2017).

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4.4 Astrocyte accumulation in disrupted ependyma indicate possible repair mechanism Neonatal Pdgfc-/-; PdgfraGFP/+ mice were consistently displaying immunoreactivity to GFAP in the disrupted ventricular lining. Ependymal injuries, regardless of cause, display an accumulation of glial processes, immunoreactive with GFAP antibodies (Sarnat 1995; Sival et al. 2011; McAllister et al. 2017), in accordance with results presented in this study. The projections of the GFAP-positive cells lack direction, in contrast to the radial direction normally expressed by NSC during the maturation of the ependyma. The process in disrupted ependyma is more likely an astrocyte response to the injury, creating a patch/scar to hinder neural progenitors from coming in contact with the CSF and falling into the ventricle. Even though many features of the ependyma are lost in this scar formation, it keeps the ventricle lining intact; keeping the function of the ependyma in some regards viable (Sarnat 1995; McAllister et al. 2017). It is unknown to what extent astroglia can retain the properties of the ependymal cells of the VZ (Sarnat 1995). This astrocyte response to the injury could be seen as a repair mechanism, necessary to restructure the VZ lining as there is no evidence for regeneration of ependyma after injury (Sarnat 1995; McAllister et al. 2017).

4.5 PDGF-C signaling important for migration of rhombic lip late derivatives Dandy-Walker malformation is associated with ectopic migration of RL derived cells, suggesting misguided migrations to be one of the main causes of the posterior vermis hypoplasia in Foxc1 impaired mice (Haldipur et al. 2017). To analyze if the Pdgfc-/-; PdgfraGFP/+ mice displayed a similar ectopic migration we investigated the immunoreactivity of reelin and Lmx1a in neonatal mice (P0). During this stage of development, a pronounced reelin expression is expected in GCP in the EGZ and no or little expression in the VZ, which can be seen in the control mice. The Pdgfc-/-; PdgfraGFP/+ mice exhibit an elevated expression of reelin in the VZ, suggesting an ectopic migration of GCP into the VZ. Moreover, in the posterior vermis the reelin expression was not restricted to the EGZ, but rather irregularly distributed throughout the lobes. This can possibly explain the irregular layer formation and clusters of Purkinje cells in the posterior vermis (Andrae et al. unpublished), as reelin signaling is critical for formation of the monolayer of Purkinje cells in the developing subpia (Marzban et al. 2015). We suggest that misguided migration of reelin-positive RL derived cells contribute to the posterior vermis hypoplasia seen in Pdgfc-/-; PdgfraGFP/+ mice as well. However, further investigations in earlier stages are needed to be able to fully conclude the path and timing of the ectopic migration, and to exclude any fate switch resulting in reelin expression of VZ derived cells. Notably, Lmx1a-positive glutamatergic neuron precursors did not exhibit any

23 Uppsala University Sara Gillnäs obvious ectopic expression in neonatal Pdgfc-/-; PdgfraGFP/+ mice. Accumulation of Lmx1a- positive cells could be seen in CN, similarly to the control, without any indication of ectopic migration via the dorsal VZ. However, as almost all Lmx1a positive cells had reach CN in neonatal mice, it was difficult to determine any migratory path (normal or faulty); seemingly P0 mice have developed too far to be able to detect ectopic migration of glutamatergic neurons due to impaired PDGF-signaling. Further investigation during embryonic development is needed to conclude the migration route of Lmx1a-positive cells in Pdgfc-/-; PdgfraGFP/+ mice.

We hypothesize that only late derivatives from the RL are dependent on PDGF-C/PDGFRɑ signaling from the posterior fossa mesenchyme and EGZ; explain the posterior vermis hypoplasia. Pdgfc+/LacZ mice have revealed that PDGF-C becomes increasingly and exclusively expressed in the EGZ in the healthy cerebellar anlagen between E12.5-E17.5, with no expression in the posterior vermis anlagen (Andrae et al. unpublished), adjacent to the PDGFRɑ-positive cells in the meninges and posterior fossa mesenchyme covering cerebellum during this stage (data not shown). In Pdgfc-/-; PdgfraGFP/+ mice, cells in the VZ display additional and intense expression of lacZ (reflecting Pdgfc) in the VZ during E14.5-E15.5 (Andrae et al. unpublished). This ectopic expression further imply the irregular patterning of the cerebellar anlagen due to the lack PDGF-C signaling. Anterior vermis fated GCP migrate from the RL around E12.5, leaving only posterior vermis fated GCP remaining after around E14-E15, which elucidates to a time point crucial for posterior vermis development (Machold & Fishell 2005; Leto et al. 2016; haldipur et al. 2017). The spatial-temporal expression of PDGF-C together with the timing of late RL derivative migration rationalizes the posterior vermis phenotype of Pdgfc-/-; PdgfraGFP/+ mice, leaving the anterior vermis relatively unaffected; only the late derivatives in the RL appears dependent on PDGF-signaling. The vast phenotypic similarities, including the ectopic expression of RL derived cells, seen in both Pdgfc-/-; PdgfraGFP/+ mice and Foxc1 impaired mice (Haldipur et al. 2017) might be due to a common mechanism. A previous study on zebrafish cerebral hemorrhaging (French et al. 2014), has linked Foxc1 with PDGFRɑ. They saw a downregulation of both Pdgfrɑ and Pdgfrβ in Foxc1 morpholino inhibited zebrafish, as well as similar pathogenic phenotype when investigating the PDGFRɑ morphant, as the Foxc1 morphant. This suggests a genetic interaction between the two genes, positioning Pdgfrɑ downstream of Foxc1. Further examination of the genetic and transcriptional relationship between Foxc1 and the PDGF signaling pathways is needed to fully elucidate this hypothesis.

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4.6 Vascular morphology appears normal in the neonatal Pdgfc-/-; PdgfraGFP/+ cerebellum An increased number of vessels and vessel density was detected in the CN in neonatal mice. No difference could be observed in the lobes of the anterior vermis, while areas in the posterior vermis was not measured due to the vast malformations in the Pdgfc-/-; PdgfraGFP/+ strain, hindering relevant comparison to the control mice. The significant difference in the CN could possibly indicate an abnormal cerebellar vessel development, however, it cannot be excluded that the increased amount and density is due to the general malformations and reduced size of Pdgfc-/-; PdgfraGFP/+ cerebellum, in comparison with the control. Furthermore, several measuring points were excluded prior quantification (only cross-sectioned vessels were included, dismissing longitudinally sectioned vessels), implying a risk of false positives due to the limited number of samples.

Defects in cerebral vessel development has previously been described in adult Pdgfc-/- mice (Fredriksson et al. 2012), thus, the overall normal vascular morphology seen in neonatal Pdgfc- /-; PdgfraGFP/+ mice used in this study might be due to a late onset of PDGF-C signaling for vessel development. The vessel quantification was originally intended to be performed in the mutant strain Pdgfc-/-; Pdgfa+/- ; Cldn5-GFP, were the Claudin-5 (Cldn5) reporter visualizes the vessels, but the regeneration was not successful within the given time plan for this project.

Acknowledgements I am grateful to the members of Christer Betsholtz’s group for their support and helpful suggestions, especially Jana Chmielniakova and Pia Petersson who have provided technical support. A special thanks goes to Johanna Andrae for supervising the project, contributing with interesting discussion, suggestions and constructive feedback during the writing process.

References Andrae J, Gallini R, Betsholtz C. 2008. Role of platelet-derived growth factors in physiology and medicine. Genes & Development 22: 1276–1312. Andrae J, Gouveia L, Gallini R, He L, Fredriksson L, Nilsson I, Johansson BR, Eriksson U, Betsholtz C. 2016. A role for PDGF-C/PDGFRα signaling in the formation of the meningeal basement membranes surrounding the cerebral cortex. Biology Open 5: 461– 474.

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Aldinger KA, Lehmann OJ, Hudgins L, Chizhikov VV, Bassuk AG, Ades LC, Krantz ID, Dobyns WB, Millen KJ. 2009. FOXC1 is required for normal cerebellar development and is a major contributor to chromosome 6p25.3 Dandy-Walker malformation. Nature Genetics 41: 1037–1042. Bernardo S, Vinci V, Saldari M, Servadei F, Silvestri E, Giancotti A, Aliberti C, Porpora MG, Triulzi F, Rizzo G, Catalano C, Manganaro L. 2015. Dandy–Walker Malformation: is the ‘tail sign’ the key sign? Prenatal Diagnosis 35: 1358–1364. Bigio MRD. 2010. Neuropathology and structural changes in hydrocephalus. Developmental Disabilities Research Reviews 16: 16–22. Bruni JE. 1998. Ependymal development, proliferation, and functions: A review. Microscopy Research and Technique 41: 2–13. Ding H, Wu X, Boström H, Kim I, Wong N, Tsoi B, O’Rourke M, Koh GY, Soriano P, Betsholtz C, Hart TC, Marazita ML, Field LL, Tam PPL, Nagy A. 2004. A specific requirement for PDGF-C in palate formation and PDGFR-α signaling. Nature Genetics 36: 1111–1116. Doherty D, Millen KJ, Barkovich AJ. 2013. Midbrain and hindbrain malformations: advances in clinical diagnosis, imaging, and genetics. The Lancet Neurology 12: 381–393. Ekman S, Thuresson ER, Heldin C-H, Rönnstrand L. 1999. Increased mitogenicity of an αβ heterodimeric PDGF receptor complex correlates with lack of RasGAP binding. Oncogene 18: 2481–2488. Fredriksson L, Nilsson I, Su EJ, Andrae J, Ding H, Betsholtz C, Eriksson U, Lawrence DA. 2012. Platelet-Derived Growth Factor C Deficiency in C57BL/6 Mice Leads to Abnormal Cerebral Vascularization, Loss of Neuroependymal Integrity, and Ventricular Abnormalities. The American Journal of Pathology 180: 1136–1144. French CR, Seshadri S, Destefano AL, Fornage M, Arnold CR, Gage PJ, Skarie JM, Dobyns WB, Millen KJ, Liu T, Dietz W, Kume T, Hofker M, Emery DJ, Childs SJ, Waskiewicz AJ, Lehmann OJ. 2014. Mutation of FOXC1 and PITX2 induces cerebral small-vessel disease. The Journal of Clinical Investigation 124: 4877–4881. Haldipur P, Gillies GS, Janson OK, Chizhikov VV, Mithal DS, Miller RJ, Millen KJ. 2014. Foxc1 dependent mesenchymal signalling drives embryonic cerebellar growth. eLife, doi 10.7554/eLife.03962. Haldipur P, Dang D, Aldinger KA, Janson OK, Guimiot F, Adle-Biasette H, Dobyns WB, Siebert JR, Russo R, Millen KJ. 2017. Phenotypic outcomes in Mouse and Human Foxc1

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dependent Dandy-Walker cerebellar malformation suggest shared mechanisms. eLife, doi 10.7554/eLife.20898. Hamilton TG, et al., Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms. Mol Cell Biol. 2003 Jun;23(11):4013-25 Heldin C-H, Westermark B. 1999. Mechanism of Action and In Vivo Role of Platelet-Derived Growth Factor. Physiological Reviews 79: 1283–1316. Hoshino M, Nakamura S, Mori K, Kawauchi T, Terao M, Nishimura YV, Fukuda A, Fuse T, Matsuo N, Sone M, Watanabe M, Bito H, Terashima T, Wright CVE, Kawaguchi Y, Nakao K, Nabeshima Y. 2005. Ptf1a, a bHLH Transcriptional Gene, Defines GABAergic Neuronal Fates in Cerebellum. Neuron 47: 201–213. Jackson EL, Garcia-Verdugo JM, Gil-Perotin S, Roy M, Quinones-Hinojosa A, VandenBerg S, Alvarez-Buylla A. 2006. PDGFRα-Positive B Cells Are Neural Stem Cells in the Adult SVZ that Form Glioma-like Growths in Response to Increased PDGF Signaling. Neuron 51: 187–199. Jiménez AJ, Domínguez-Pinos M-D, Guerra MM, Fernández-Llebrez P, Pérez-Fígares J-M. 2014. Structure and function of the ependymal barrier and diseases associated with ependyma disruption. Tissue Barriers, doi 10.4161/tisb.28426. Langlet F, Mullier A, Bouret SG, Prevot V, Dehouck B. 2013. Tanycyte-like cells form a blood–cerebrospinal fluid barrier in the circumventricular organs of the mouse brain. Journal of Comparative Neurology 521: 3389–3405. Leto K, Arancillo M, Becker EBE, Buffo A, Chiang C, Ding B, Dobyns WB, Dusart I, Haldipur P, Hatten ME, Hoshino M, Joyner AL, Kano M, Kilpatrick DL, Koibuchi N, Marino S, Martinez S, Millen KJ, Millner TO, Miyata T, Parmigiani E, Schilling K, Sekerková G, Sillitoe RV, Sotelo C, Uesaka N, Wefers A, Wingate RJT, Hawkes R. 2016. Consensus Paper: Cerebellar Development. Cerebellum (London, England) 15: 789–828. Ma X, Bao J, Adelstein RS. 2007. Loss of Cell Adhesion Causes Hydrocephalus in Nonmuscle Myosin II-B–ablated and Mutated Mice. Molecular Biology of the Cell 18: 2305–2312. Machold R, Fishell G. 2005. Math1 Is Expressed in Temporally Discrete Pools of Cerebellar Rhombic-Lip Neural Progenitors. Neuron 48: 17–24. Marzban H, Del Bigio MR, Alizadeh J, Ghavami S, Zachariah RM, Rastegar M. 2015. Cellular commitment in the developing cerebellum. Frontiers in Cellular Neuroscience, doi 10.3389/fncel.2014.00450. McAllister JP, Guerra MM, Ruiz LC, Jimenez AJ, Dominguez-Pinos D, Sival D, den Dunnen W, Morales DM, Schmidt RE, Rodriguez EM, Limbrick DD. 2017. Ventricular Zone

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Disruption in Human Neonates With Intraventricular Hemorrhage. Journal of Neuropathology and Experimental Neurology 76: 358–375. Parisi MA, Dobyns WB. 2003. Human malformations of the midbrain and hindbrain: review and proposed classification scheme. Molecular Genetics and Metabolism 80: 36–53. Redmond SA, Figueres-Oñate M, Obernier K, Nascimento MA, Parraguez JI, López- Mascaraque L, Fuentealba LC, Alvarez-Buylla A. 2019. Development of Ependymal and Postnatal Neural Stem Cells and Their Origin from a Common Embryonic Progenitor. Cell Reports 27: 429-441.e3. Ringers C, Olstad EW, Jurisch-Yaksi N. 2019 The role of motile cilia in the development and physiology of the nervous system. Phil. Trans. R. Soc. B 375: 20190156. http://dx.doi.org/10.1098/rstb.2019.0156 Sarnat HB. 1992. Regional Differentiation of the Human Fetal Ependyma: Immunocytochemical Markers. Journal of Neuropathology 51: 58–75. Sarnat HB. 1995. Ependymal Reactions to Injury. A Review. Journal of Neuropathology 54: 1–15. Sarnat HB. 1998. Histochemistry and immunocytochemistry of the developing ependyma and choroid plexus. Microscopy Research and Technique 41: 14–28. Sasaki-Adams D, Elbabaa SK, Jewells V, Carter L, Campbell JW, Ritter AM. 2008. The Dandy–Walker variant: a case series of 24 pediatric patients and evaluation of associated anomalies, incidence of hydrocephalus, and developmental outcomes. Journal of Neurosurgery: Pediatrics 2: 194–199. Sival DA, Guerra M, Dunnen WFA den, Bátiz LF, Alvial G, Castañeyra‐Perdomo A, Rodríguez EM. 2011. Neuroependymal Denudation is in Progress in Full-term Human Foetal Spina Bifida Aperta. Brain Pathology 21: 163–179. Sievers J, Pehlemann FW, Gude S, Berry M. 1994. Meningeal cells organize the superficial glia limitans of the cerebellum and produce components of both the interstitial matrix and the basement membrane. Journal of Neurocytology 23: 135–149. Sudarov A, Joyner AL. 2007. Cerebellum morphogenesis: the foliation pattern is orchestrated by multi-cellular anchoring centers. Neural Development 2: 26. Xu X, Li WEI, Huang GY, Meyer R, Chen T, Luo Y, Thomas MP, Radice GL, Lo CW. 2001. N-Cadherin and Cx43α1 Gap Junctions Modulates Mouse Neural Crest Cell Motility via Distinct Pathways. Cell Communication & Adhesion 8: 321–324.

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Appendix

Supplementary table 1: Primary antibodies including proficient antigen retrieval treatment Antibody Abbreviation Protein type Target tissue Host Company Ref. nr. Dilution AR (pH) Mouse Podxl Type 1 Endothelium, goat R&D Systems AF1556 1:200 6.0 or podocalyxin transmembrane Hematopoietic 9.0 antibody glycoprotein precursors, Ependyma GFAP monoclonal GFAP Glial fibrillary Astrocytes, rat Invitrogen 13-0300 1:100 - antibody acidic protein, Neural stem class III cells intermediate filament protein family PolyclonalRabbit S100B S100 calcium- Glial cells, rabbit Dako ZO311 1:100 - anti-S100 binding protein ependyma B Anti-beta IV βIV-tub Microtubules, Multiciliated rabbit Abcam ab179509 1:500 - Tubulin antibody cilia and ependymal flagella cells Cadherin, N- Cdh2 Cell adhesion Neural rat Developmental MNCD2-C 1:50 9.0 (neural) antibody protein epithelium Studies Hybridoma Bank Anti-connexin 43 Cx43 Gap junction Ependyma mouse Santa Cruz sc-271837 1:50 9.0 antibody protein Biotechnology Reelin antibody Reln Serine Rhombic lip goat Novus af-3820 1:100 - protease, migrating cells biologicals Extracellular (Cerebellar matrix nuclei cells, glycoprotein external secreted by granular layer) neurons Anti-Lmx-1 Lmx1a Transcription Roof plate, rabbit Sigma-Aldrich ab10533 1:200 - antibody factor Choroid plexus, belonging to Rhombic lip LIM- migrating cells homeodomain (Cerebellar family nuclei cells, external granular layer)

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Supplementary figure 1: Ependymal disruption visible in mid-sagittal sections in E17.5 mice Immunofluorescent staining of podocalyxin (red), n-cadherin (green) and connexin 43 (white) of paraffin section (lateral to mid-sagittal) of E17.5 Pdgfc-/-; PdgfraGFP/+ mice, counterstained with nuclear Hoechst (blue) (A-O). Ependymal disruption (yellow line) is visible only in mid-sagittal section (K-O), white arrowhead marks the border of intact and disrupted ependyma. CP: Choroid Plexus, 4V: Fourth ventricle, RL: Rhombic lip, VZ: Ventricular zone. Sections 5 µm (C, D).

Supplementary figure 2: Quantification of cerebellar vascularization in neonatal mice. Quantification of number of podocalyxin positive vessels (A) and vessel density (B) shows a significant (P < 0.05) increase in the number of vessels and a more compact vascular bed in the cerebellar nuclei of Pdgfc-/-; PdgfraGFP/+ mice (n = 3) compared to Pdgfc+/+; PdgfraGFP/+ control mice (n = 3). The quantification of vessel diameter (C) showed no significant difference (P > 0.05) between Pdgfc-/-; PdgfraGFP/+ mice and control. Error bars represent SEM.