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. Author manuscript; available in PMC: 2025 Apr 26.
Published in final edited form as: Cells Dev. 2024 May 9;179:203926. doi: 10.1016/j.cdev.2024.203926

Evaluating neural crest cell migration in a Col4a1 mutant mouse model of ocular anterior segment dysgenesis

Corinna Cozzitorto a,1, Zoe Peltz a, Lourdes M Flores a, Luca Della Santina a,2, Mao Mao a, Douglas B Gould a,b,*
PMCID: PMC12032921  NIHMSID: NIHMS2069909  PMID: 38729574

Abstract

The periocular mesenchyme (POM) is a transient migratory embryonic tissue derived from neural crest cells (NCCs) and paraxial mesoderm that gives rise to most of the structures in front of the eye. Morphogenetic defects of these structures can impair aqueous humor outflow, leading to elevated intraocular pressure and glaucoma. Mutations in collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) cause Gould syndrome – a multisystem disorder often characterized by variable cerebrovascular, ocular, renal, and neuromuscular manifestations. Approximately one-third of individuals with COL4A1 and COL4A2 mutations have ocular anterior segment dysgenesis (ASD), including congenital glaucoma resulting from abnormalities of POM-derived structures. POM differentiation has been a major focus of ASD research, but the underlying cellular mechanisms are still unclear. Moreover, earlier events including NCC migration and survival defects have been implicated in ASD; however, their roles are not as well understood. Vascular defects are among the most common consequences of COL4A1 and COL4A2 mutations and can influence NCC survival and migration. We therefore hypothesized that NCC migration might be impaired by COL4A1 and COL4A2 mutations. In this study, we used 3D confocal microscopy, gross morphology, and quantitative analyses to test NCC migration in Col4a1 mutant mice. We show that homozygous Col4a1 mutant embryos have severe embryonic growth retardation and lethality, and we identified a potential maternal effect on embryo development. Cerebrovascular defects in heterozygous Col4a1 mutant embryos were present as early as E9.0, showing abnormal cerebral vasculature plexus remodeling compared to controls. We detected abnormal NCC migration within the diencephalic stream and the POM in heterozygous Col4a1 mutants whereby mutant NCCs formed smaller diencephalic migratory streams and POMs. In these settings, migratory NCCs within the diencephalic stream and POM localize farther away from the developing vasculature. Our results show for the first time that Col4a1 mutations lead to cranial NCCs migratory defects in the context of early onset defective angiogenesis without affecting cell numbers, possibly impacting the relation between NCCs and the blood vessels during ASD development.

Keywords: Neural crest cells, Cell migration, Type IV collagen, Col4a1, Ocular anterior segment dysgenesis

1. Introduction

Ocular anterior segment dysgenesis (ASD) is a clinically and genetically heterogeneous group of developmental disorders affecting the structures in the front of the eye: cornea, iris, lens, ciliary body, and ocular drainage tissues (trabecular meshwork and Schlemm’s canal) (Sowden, 2007; Gould et al., 2004; Reis et al., 2012). Alterations of these structures can cause significant visual impairment and obstruct aqueous humor outflow leading to elevated intraocular pressure (IOP), which is a major risk factor for glaucoma (Gould et al., 2004; Gould and John, 2002; Ito and Walter, 2014). The most common genetic causes of ASD are mutations in genes encoding the winged helix/forkhead transcription factor Forkhead Box C1 (FOXC1) and the bicoid-like homeodomain protein Pituitary Homeobox 2 (PITX2) (Mears et al., 1998; Mirzayans et al., 2000; Nishimura et al., 2001; Nishimura et al., 1998). The prevalence of FOXC1 or PITX2 mutations ranges from 40 % to 70 % of ASD cases, suggesting the existence of additional genes involved in ASD pathogenesis (Strungaru et al., 2007; Lines et al., 2002; Weisschuh et al., 2011; Weisschuh et al., 2006). Notably, mutations in genes encoding the extracellular matrix (ECM) proteins collagen type IV alpha 1 and alpha 2 (COL4A1 and COL4A2) have been identified in individuals with ASD and developmental glaucoma (Gould and John, 2002; Jeanne and Gould, 2017; Kuo et al., 2012; Coupry et al., 2010).

Formation of the ocular anterior segment results from a coordinated series of events involving migration, inductive interactions, and differentiation of the surface ectoderm, neural ectoderm, and periocular mesenchyme (POM) (Gould et al., 2004). The POM derives from cranial neural crest cells (NCCs) and paraxial mesoderm, and plays a major role in anterior segment formation (Gage et al., 2005). Defects in POM patterning and differentiation contribute to ASD and glaucoma (Gage et al., 2005; Kupfer and Kaiser-Kupfer, 1978; Kupfer and Kaiser-Kupfer, 1979; Noden, 1975; Trainor and Tam, 1995). In the mouse, cranial NCCs originating from the rostral diencephalon and mesencephalon migrate laterally toward the prospective eye region at the optic vesicle stage around embryonic day (E) 9.0–9.5. The lens vesicle detaches from the surface ectoderm at E10.5, when POM cells start invading the space between the surface ectoderm and neural ectoderm (Gage et al., 2005; Cvekl and Tamm, 2004). The POM subsequently migrates across the anterior surface of the lens to establish the presumptive corneal stroma (Hay et al., 1979; Pei and Rhodin, 1970; Haustein, 1983). Between E13.5 and E15.5, the POM cells closest to the lens condense into the corneal endothelium, while others differentiate into ECM-secreting keratocytes, forming the corneal stroma (Cvekl and Tamm, 2004). Other anterior segment structures, including the iris stroma, ciliary body (CB), and trabecular meshwork (TM) also arise from the POM. While altered POM differentiation can lead to ASD (Williams and Bohnsack, 2015; Martino et al., 2016; Walker et al., 2021), the roles of impaired NCC migration, survival and/or proliferation are less well understood (Kupfer and Kaiser-Kupfer, 1978; Kupfer and Kaiser-Kupfer, 1979).

Semi-dominant COL4A1 and COL4A2 mutations cause Gould syndrome, a multi-system disorder that includes cerebrovascular, ocular, neuromuscular, and renal manifestations (Jeanne and Gould, 2017; Kuo et al., 2012; Labelle-Dumais et al., 2011; Labelle-Dumais et al., 2019; Kuo et al., 2014; Gould et al., 2005; Marion et al., 2015). Highly variable cerebral small vessel disease is the most prominent feature of Gould syndrome and ranges from porencephaly and fetal intracerebral hemorrhages to multifocal and recurrent intracerebral hemorrhages that may involve age-related cerebrovascular dysfunction. (Gould et al., 2005; Marion et al., 2015; Yamasaki et al., 2023a; Yamasaki et al., 2023b; Thakore et al., 2023) Approximately one-third of individuals with Gould syndrome have ASD including cataract, microphthalmia, optic nerve hypoplasia, and pediatric or juvenile glaucoma (Jeanne and Gould, 2017; Coupry et al., 2010; Sibon et al., n.d.; Livingston et al., 2011; Yoneda et al., 2013; Xia et al., 2014).

Heterotrimers composed of two COL4A1 and one COL4A2 proteins [α1α1α2(IV)] are integral components of specialized ECM structures called basement membranes (BMs) (Yurchenco et al., 2004). Collagen α1α1α2(IV) assembles within the endoplasmic reticulum before being secreted into the extracellular space (Trüeb et al., 1982) where they polymerize into cross-linked networks that interact with other ECM molecules, growth factors, and cell-surface proteins to influence cellular behaviors. The primary consequence of COL4A1 or COL4A2 mutations is impaired secretion of mutant collagen α1α1α2(IV) into BMs (Jeanne and Gould, 2017; Kuo et al., 2012; Gould et al., 2005; Gould et al., 2007).

Col4a1 and Col4a2 mutant mice recapitulate the pathophysiological hallmarks of Gould syndrome, including cerebrovascular and ocular pathologies, and represent useful tools to study the pathogenic processes contributing to disease (Kuo et al., 2012). We previously described a Col4a1 splice site mutation that results in skipping of exon 41 (Col4a1+/Δex41) and leads to severe pathology (Kuo et al., 2012; Kuo et al., 2014; Gould et al., 2005). Homozygous Col4a1Δex41 mutant mice die embryonically, while heterozygotes littermates have highly penetrant cerebrovascular disease (Labelle-Dumais et al., 2019; Gould et al., 2005; Marion et al., 2015; Gould et al., 2007; Mao et al., 2022; Jeanne et al., 2012), severe ASD, elevated IOP, and progressive loss of retinal ganglion cells that models glaucoma (Gould et al., 2007; Van Agtmael et al., 2005; Mao et al., 2015; Mao et al., 2021). Moreover, conditional expression of the Col4a1Δex41 mutation showed that the primary pathogenic events contributing to ASD occur during early embryonic development (Marion et al., 2015; Alavi et al., 2016; Mao et al., 2017). In this study, we report severe growth retardation and lethality near mid-gestation in Col4a1-Δex41/Δex41 embryos. In addition, using a combination of three-dimensional (3D) confocal microscopy imaging and morphological and quantitative analyses, we show that Col4a1+/Δex41 mutant embryos have impaired cerebrovascular plexus remodeling, together with altered NCC migratory behavior to and within the POM. We propose abnormal vascular development as a potential primary defect altering NCC biology underlying ASD in Col4a1 mutant mice. Moreover, our findings suggest that a Col4a1 mutant uterine environment might have detrimental effect on embryonic development which could have potential clinical implications.

2. Material and methods

2.1. Animals

All experiments were conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the Institutional Animal Care and Use Committee at the University of California, San Francisco (Protocols AN159737 and AN182181). The Col4a1Δex41 mouse strain has been described previously (Kuo et al., 2014; Gould et al., 2005; Mao et al., 2022). All adult Col4a1 mutant mice used in this study were heterozygous for the Col4a1Δex41 mutation and backcrossed to C57BL/6J (B6) mice for at least 37 generations. All animals were maintained in full-barrier facilities free of pathogens on a 12 h light/dark cycle with ad libitum access to food and water. Both male and female mice were used for all experiments and no samples were excluded from the study. For timed mating, sires and dams were placed into a breeding cage overnight and plug check was performed daily. The presence of a vaginal plug in the next morning was noted as E0.5. For NCC analyses, embryos were staged by somites counting using Theiler staging and emouse atlas (http://www.emouseatlas.org/emap/ema/home.html). E9.0: 13–20 somites; E9.5: 21–29 somites.

2.2. Immunohistochemistry

Mouse embryos were dissected and fixed in 4 % paraformaldehyde (PFA) in phosphate buffered saline (PBS) for 2 h at room temperature or overnight at 4 °C. For whole-mount immunofluorescence labeling, fixed embryos were bleached in Dent’s bleach for 2 h and blocked for additional 2 h in blocking buffer (5 % Donkey serum, 0.1 % TritonX-100 in PBS). Samples were incubated overnight at 4 °C with primary antibodies diluted in staining buffer (1:5 blocking buffer in PBS) followed by overnight incubation at 4 °C with secondary antibodies. Whole-mount immunolabeled embryos were dehydrated in a methanol dilution series and clarified before imaging with methyl salicylate. Clarified embryos were imaged on a paraffin-sealed glass depression slide with Zeiss LSM 700 or LSM 900 Airyscan confocal microscopes using a 10× water immersion objective. Antibodies: rat anti-CD31 (BD Pharmigen 553370, 1:150), rabbit anti-cleaved caspase 3 (Cell Signaling 9664S, 1:600), anti-phosphorylated histone H3 (Ser10) (Millipore 06–570, 1:1000), goat anti-Sox10 (SantaCruz sc-17342, 1:40; R&D AF2864, 1:100).

2.3. Image analysis

NCCs cell counts were obtained by using the automatic spot detection function of the Imaris v9.7 and 9.8 software (Oxford Instruments) on 3D reconstruction of entire whole-mount images followed by manual removal of spots in case sporadic errors from the detection algorithm were identified by visual inspection. Diencephalic stream and POM volumes were analyzed using the manual surface creation function of the Imaris v9.7 and 9.8 software (Oxford Instruments). Cells at the edges of the streams or POM were used as “boundaries” to manually draw a 3D region of interest around them. POM was identified as cells in direct contact or distant up to two cells farther away from the optic vesicle. The vascular density of diencephalic streams and POMs were calculated as the ratio of the volume occupied by the blood vessels within the diencephalic streams or POMs volume on the entire diencephalic streams or POMs volume, respectively. Distance between NCCs and blood vessels was analyzed with the Distance Transformation tool within the Imaris software.

2.4. Statistics

Statistical analyses were performed using Graph-Pad Prism v8 and 9 (GraphPad, La Jolla, CA). Statistical differences between two groups were determined using two-tailed unpaired Student’s t-test or Mann-Whitney test and adjusted for the high number of samples, when necessary, using the Bonferroni method. Multiple-group comparisons were performed using ordinary one-way ANOVA and Tukey’s multiple comparison test. Data were presented as mean ± SE and n refers to biological replicates. p values <0.05 were considered statistically significant. The sample size was selected on the basis of previous experience in the laboratory and the literature. No statistical methods were used to predetermine sample size. No method of randomization was followed, and no animals were excluded from this study. The investigators were not blinded to sample allocation during the experiments and assessment of results.

3. Results

3.1. Col4a1Δex41 homozygosity causes severe growth retardation and lethality between E9.5 and E10.5

To investigate NCC behavior during early ocular development, we first intercrossed Col4a1+/Δex41 sires and dams and analyzed E9.5 and E10.5 embryos. We found that Col4a1Δex41/Δex41 embryos had severe growth retardation, resulting in embryonic lethality around E10.5 (Fig. 1). Gross morphological analysis of embryos collected at E9.5 and E10.5 highlights that Col4a1Δex41/Δex41 embryos showed severe developmental delay compared to Col4a1+/Δex41 and Col4a1+/+ embryos (Fig. 1A–F). Col4a1Δex41/Δex41 embryos were remarkably smaller than Col4a1+/+ embryos at both stages. At E9.5 Col4a1Δex41/Δex41 embryos failed to undergo embryonic turning, and resembled E8.5 embryos (Fig. 1A–C). Col4a1Δex41/Δex41 embryos eventually turned by E10.5, but still resembled embryos that were 24 h younger (Fig. 1D–F). Analysis of genotype frequency showed that Col4a1Δex41/Δex41 embryos were recovered at the expected frequency at E9.5 but were reduced at E10.5 (Fig. 1G) indicating that embryonic lethality occurs around this stage for Col4a1Δex41/Δex41 embryos. Since embryos of the same gestational age can differ in their stage of development, we next performed staging of the embryos using somites formation as a proxy of embryonic development (Chan et al., 2004). Somites are transient mesodermal structures formed between E8.0 and E11.5 (ref. (Saga and Takeda, 2001; Tam, 1981)). In the mouse, a 2 h time period corresponds to the formation of a somite, allowing for a more precise calculation of the embryonic stage at the time of embryo collection (Chan et al., 2004; Saga and Takeda, 2001; Tam, 1981; Roellig et al., 2011). Consistent with the gross morphological analysis, the number of somites in E9.5 Col4a1Δex41/Δex41 embryos was significantly reduced compared to that observed in Col4a1+/Δex41 and Col4a1+/+ littermates (Fig. 1A–C, 1H). By E10.5, we recovered only one severely developmentally delayed Col4a1Δex41/Δex41 embryo (17 somites compared to the mean of 35 at E10.5) (Fig. 1I). Together with the small number of embryonic resorptions at both E9.5 and E10.5 (Fig. 1G), this data sets the time of embryonic lethality in Col4a1Δex41/Δex41 embryos between E9.5 and E10.5. Analysis of crosses between Col4a1+/Δex41 sires with Col4a1+/+ dams and vice versa revealed no morphological differences between Col4a1+/Δex41 and Col4a1+/+ embryos at both E9.5 and E10.5 (Fig. 1A–B and D-E), and the distribution of genotypes did not deviate from the expected in both cases (Fig. 1J, M). Surprisingly, somite staging comparison between E9.5 embryos derived from the reciprocal mating schemes revealed a small but significant developmental delay for Col4a1+/Δex41 offspring of Col4a1 mutant dams only (Fig. 1N), suggesting a potential effect of the maternal environment. Due to this, together with the severity of growth retardation (Fig. 1C, F) and the impossibility to compare stage matched Col4a1+/+ and Col4a1Δex41/Δex41 embryos, the remaining experiments were performed exclusively on Col4a1+/+ and Col4a1+/Δex41 embryos derived from matings between Col4a1+/Δex41 sires and Col4a1+/+ dams.

Fig. 1. Col4a1Δex41 homozygosity causes severe growth retardation and lethality between E9.5 and E10.5.

Fig. 1.

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(A-F) Representative images of control (+/+), heterozygous (+/Δex41), and homozygous (Δex41/Δex41) mutant embryos collected at E9.5 and E10.5. (G-I) Analysis of embryos derived from matings between Col4a1+/Δex41 sires and dams at E9.5 and E10.5. Frequency distribution of genotypes and embryonic resorptions (G), and somites quantification (H-I). (J-L) Analysis of embryos derived from matings between Col4a1+/Δex41 sires and Col4a1+/+ dams at E9.5 and E10.5. Frequency distribution of genotypes and embryonic resorptions (J), and somite quantification (K-L). (M-N) Analysis of embryos derived from matings between Col4a1+/+ sires and Col4a1+/Δex41 dams at E9.5 and E10.5. Frequency distribution of genotypes and embryonic resorptions (M), and somites quantification (N). Scale bar: 1 mm. Sample sizes as stated. Data are presented as mean ± SE. *p < 0.05; **p < 0.01, Brown-Forsythe and Welch ANOVA test for H, Student’s t-test for all others.

3.2. Col4a1Δex41 mutation causes impaired cerebrovascular plexus remodeling.

Similar to individuals with Gould syndrome, Col4a1+/Δex41 mice exhibit highly penetrant cerebrovascular disease and abnormal vascular development (Gould et al., 2005; Marion et al., 2015; Yamasaki et al., 2023a; Jeanne et al., 2012; Gould et al., 2009). Here we used whole-mount CD31 immunolabeling and confocal microscopy to visualize vascular endothelial cells. Because of the rapid changes taking place during early embryogenesis, and to avoid confounding effects that could be attributed to developmental stage variation within the same litter, here and in all subsequent experiments, we used somite staging to distinguish E9.0, E9.5 and E10.5 embryos for more refined analyses. Gross morphological analysis revealed delayed intersomitic vessel remodeling in E9.0 Col4a1+/Δex41 embryos compared to wild type littermates that resolves by E9.5 (Fig. 2A–B, G, H–I, N). While intersomitic vessels were correctly arranged in segments located between somite boundaries, on the dorsal side, the bilateral process of arborization of the capillary network has not yet started in E9.0 Col4a1+/Δex41 embryos as shown by the reduced capillary network density in the trunk region (arrowheads in Fig. 2A). No obvious vascular abnormality was observed in the developing endocardium (Fig. 2C–D, G, J–K, N). Importantly, we show that cerebrovascular abnormalities in Col4a1+/Δex41 mice were detected as early as E9.0 with abnormal cerebrovascular remodeling observed in more than half of Col4a1+/Δex41 embryos compared to their Col4a1+/+ littermates at E9.0 and in all but one Col4a1+/Δex41 embryo at E9.5, revealing an earlier onset of cerebral angiogenesis defects than previously described (Marion et al., 2015). These defects were characterized by unresolved plexus with large-caliber unrefined blood vessels (Fig. 2E–G, L–N). By E10.5, Col4a1+/Δex41 embryos showed altered arborization of the cerebral vasculature and reduced number of connections between the major vessels (Fig. 2O–S). These vascular abnormalities are consistent with impaired or delayed cerebral plexus remodeling in which the diameter of the vessels does not evolve over time and hierarchy of the arborization is not achieved (Adams et al., 1999; Udan et al., 2013).These data are consistent with previous reports (Jeanne et al., 2012; Favor et al., 2007) and demonstrate that abnormal cerebrovascular development occurs as early as E9.0 in Col4a1 mutant mice and persists throughout the developmental window in which NCCs migrate to the prospective eye field.

Fig. 2. Col4a1Δex41 mutation causes impaired cerebrovascular plexus remodeling.

Fig. 2.

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(A-F) Representative images of maximum intensity projections of Z-stack confocal acquisitions of E9.0 whole-mount controls (+/+) and mutant (+/Δex41) embryos immunolabeled for the vascular marker CD31. Intersomitic vessels (A-B), heart (C-D) and cerebral vasculature (E-F). Arrowheads in 2A indicate areas of reduced capillary network density in the trunk region. Boxed magnifications in E and F indicate a region where mutant embryos show less remodeled cerebral vasculature. Scale bars: 100 μm. (G) Table summarizing morphological analyses of the vasculature in E9.0 embryos immunolabeled for CD31. Data are presented as the number of embryos showing proper developmental features and total number of embryos analyzed. (H-N) Representative maximum intensity projections of whole-mount E9.5 embryos immunolabeled for CD31 (H-M) and summary of corresponding morphological analyses (N). Scale bars: 100 μm. (O-S) Representative maximum intensity projections of whole-mount E10.5 embryos immunolabeled for CD31 (O, P) and corresponding quantitative morphological analyses (Q-S). Scale bar: 150 μm. Sample sizes are indicated below the graphs. Data are presented as mean ± SE. Student’s t-test.

3.3. Altered NCC migration in the diencephalic stream and POM in Col4a1 mutant embryos.

Vascular defects are common consequences of COL4A1 and COL4A2 mutations and can influence NCCs survival and migration (Lewis et al., 2015; Milgrom-Hoffman et al., 2014). To test whether impaired NCC migration, proliferation, or survival could contribute to ASD in Col4a1 mutant mice, we started evaluating the relationship between NCCs and the cerebral vasculature in Col4a1+/Δex41 embryos. We performed immunolabeling of whole mount embryos at E9.0 and E9.5, when cranial NCCs derived from the rostral diencephalon and mesencephalon migrate laterally toward the prospective eye region (Gage et al., 2005; Cvekl and Tamm, 2004), to evaluate their number and distribution. Using whole-mount confocal imaging and 3D reconstruction of embryos labeled to observe migratory NCCs (SOX10), we measured the distance between SOX10-labeled NCCs and CD31-labeled blood vessels (Fig. 3). Although the vascular densities were similar between Col4a1+/+ and Col4a1+/Δex41 embryos (Fig. 3A–C, F–H; Suppl. Fig. 1A–C, F–H), diencephalic and POM NCCs localized farther away from the vasculature (Fig. 3D–E, I–J; Suppl. Fig. 1D–E, I–J) in E9.0 Col4a1+/Δex41 embryos which may perturb a potential crosstalk between the NCCs and blood vessels.

Fig. 3. Altered proximity of NCCs to cerebral vasculature in E9.0 Col4a1 mutant embryos.

Fig. 3.

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(A) Schematic of the murine embryonic head at E9.0–9.5 showing the anatomical topology of the analyzed structures. ov: optic vesicle; ba: branchial arch. (B-E, G-J) Three dimensional reconstructions and quantification of the vascular density and the distance of migratory NCCs relative to the developing vasculature in E9.0 whole mount embryos immunolabeled for the endothelial cell marker CD31 (red) and migratory NCCs marker SOX10 (pink and yellow) within the diencephalic stream (B-E) and POM (G-J). In B and C, yellow dots indicate NCCs that are within 5 μm from the vasculature, while pink dots are located >5 μm away. In G and H, the threshold for colour change was set at 4 μm. (F, K) Frequency distribution of NCCs proximity to the vasculature of the diencephalic stream (F) and POM (K). Scale bars: 20 μm. Data are presented as mean ± SE (D, I). Sample sizes are indicated below the graphs. ****: p < 0.0001. For all analyses Student’s t-tests were performed, Bonferroni correction was also applied for analyses in E and J.

We next focused our analysis on properties of the diencephalic stream of NCCs migrating toward the eye region. At E9.0, we detected the same number of migrating NCCs in Col4a1+/Δex41 and Col4a1+/+ littermates, however there was a trend for higher cell density and reduced stream volume in Col4a1+/Δex41 embryos (Fig. 4A–E). The more cohesive NCC distribution observed at E9.0 was resolved by E9.5 although there was a trend toward reduced numbers of NCCs (Suppl. Fig. 2A–E). Next, we focused on NCCs localized around the optic vesicle forming the POM. At E9.0 the POM was smaller but constituted by an equal number of Col4a1+/Δex41 NCCs resulting in a slightly denser POM compared to Col4a1+/+ littermates (Fig. 4F–J). In E9.5 Col4a1+/Δex41 embryos, NCCs number, POM volume, and density were restored to control levels (Suppl. Fig. 2F–J). This data suggests a delay in the development of the Col4a1+/Δex41 eye.

Fig. 4. Altered NCC migration in the diencephalic stream and POM in E9.0 Col4a1 mutant embryos.

Fig. 4.

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(A-B, F-G) Representative images of maximum intensity projections of Z-stacks confocal acquisitions of whole-mount immunofluorescence of controls (+/+) and mutant (+/Δex41) embryos immunolabeled for the migratory NCC marker SOX10. Yellow dashed lines outline the diencephalic migratory streams of NCCs (A-B) and orange dashed lines outline the optic vesicles (F-G). Scale bars: 100 μm. (C-E, H-J) Quantification of diencephalic stream (C-E) and POM (H-J) parameters. Sample sizes are indicated below the graphs. Data are presented as mean ± SE. *: p < 0.05, Student’s t-test. ov: optic vesicle.

Because vascular defects can influence NCC migration and survival (Lewis et al., 2015; Milgrom-Hoffman et al., 2014), we evaluated proliferation and survival of SOX10+ NCCs by co-labeling with phosphorylated histone H3 (pHH3) and cleaved Caspase 3 (clCASP3), respectively (Suppl. Fig. 3–5). We identified a small but significant increase in the number of proliferating NCCs within the diencephalic stream of Col4a1+/Δex41 embryos compared to control littermates at E9.0 but not E9.5 (Suppl. Fig. 3). On the other hand, proliferation assessments found no difference between Col4a1+/+ and Col4a1+/Δex41 NCCs within the POM (Suppl. Fig. 4). We also did not identify SOX10/clCASP3 co-labeling in the diencephalic streams and periocular region of Col4a1+/+ or Col4a1+/Δex41 embryos at either stage (Suppl. Fig. 5 A–B), despite observing evidence of expected cell death in the trigeminal ganglia (another NCCs-derived tissue) of the same embryos (Suppl. Fig. 5C–D). Collectively, these results suggest that Col4a1 mutation influences migration of both diencephalic and POM NCCs (Fig. 3), while potentially impacting NCC proliferation of diencephalic NCC at early embryonic stages (Suppl. Fig. 3).

4. Discussion

Type IV collagens are fundamental BM components and mutations in COL4A1 and COL4A2 cause Gould syndrome (Labelle-Dumais et al., 2011; Weng et al., 2012; Branyan et al., 2022; Boyce et al., 2021), a multisystem disorder that includes cerebrovascular, ocular, muscular, and renal manifestations. Of particular interest, the POM, which give rise to the ocular anterior segment is derived from NCCs and paraxial mesoderm. Studies over the years have shown that differentiation defects in the POM lead to ASD but earlier pathogenic defects affecting NCCs behaviors have never been explored (Kupfer and Kaiser-Kupfer, 1978; Kupfer and Kaiser-Kupfer, 1979). In addition, vascular defects are common in patients with COL4A1 and COL4A2 mutations and can influence NCC survival and migration (Marion et al., 2015; Lewis et al., 2015; Milgrom-Hoffman et al., 2014). For these reasons, we hypothesized that alteration in NCC behaviors could contribute to ASD in Col4a1 mutant mice.

We have previously reported defects during vascular angiogenesis within the central nervous system in Col4a1 mutant mice including increased density and tortuosity of the vascular plexus in the embryonic hindbrain at E10.5 and altered development and patterning of the retinal vascular network starting at postnatal day 7(refs (Marion et al., 2015; Branyan et al., 2022)). In this study, we identified cerebrovascular defects as early as E9.0 in Col4a1+/Δex41 embryos, together with concomitant abnormal NCCs migration and altered proximity of NCCs to the developing cerebral vasculature within the diencephalic stream and the POM. At E9.5 the migratory properties of NCCs of Col4a1+/Δex41 embryos seem to be indistinguishable from their control counterparts. Our results suggest that Col4a1 mutations may lead to cranial NCC migratory defects in the context of early onset defective angiogenesis and altered spatial relation between NCCs and blood vessels in E9.0 embryos. This data suggests that NCC migratory defects could represent an early pathogenic mechanism contributing to ASD and possibly other aspects of Gould syndrome. One possibility is that migrating NCCs are not exposed to necessary cues at the proper time and/or space, leading to subsequent differentiation defects. This hypothesis, as well as if early cerebrovascular defects are the cause of the described NCCs migratory defects, remain to be explored.

Early impairment in vascular development is associated with increased NCCs death and/or decreased proliferation (Lewis et al., 2015; Milgrom-Hoffman et al., 2014). Thus, we analyzed NCCs survival and proliferation in Col4a1+/Δex41 embryos. Here we show that, in contrast to other molecules such as ephrins (ephrin-B2) (Lewis et al., 2015) and vascular endothelial growth factor 2 (Flk1) (Milgrom-Hoffman et al., 2014), Col4a1 mutation does not appear to affect NCC survival or cell numbers. While we saw a small proliferation increase in diencephalic NCCs proliferation at E9.0 in Col4a1+/Δex41 embryos, we did not see any difference in diencephalic NCC numbers or proliferation at E9.5. These two pieces of data could be reconciled taking into consideration that NCCs proliferation within the diencephalic stream remained minimal in both Col4a1+/+ and Col4a1+/Δex41 embryos (with 2 % and 4 % proliferation on an average NCCs population of ~300 cells), suggesting that this increase could reflect a faster cell-cycle and a faster cell-cycle exit for NCC in Col4a1+/Δex41 embryos compared to controls that seem not to have meaningful consequences. Alternatively, the increase in proliferative diencephalic NCCs at E9.0 coupled with the trend of reduced NCCs within the same region at E9.5, could suggest the hypothesis that NCCs might have migrated to other locations. This speculation will need to be experimentally addressed.

Interestingly, Col4a1+/Δex41 diencephalic and POM-localized migratory NCCs localized further away from the vasculature at E9.0 when the Col4a1+/Δex41 cerebral vasculature is still almost completely immature, suggesting a possible perturbation of a potential crosstalk between the NCCs and blood vessels. It is possible that the defective plexus has different soluble and/or insoluble cues compared to controls, and it will be interesting to further test the possibility that inappropriate crosstalk with the vasculature influences NCCs migration and/or differentiation. In addition, our previous work demonstrated that the primary consequence of COL4A1 or COL4A2 mutations is impaired collagen α1α1α2(IV) secretion (Jeanne and Gould, 2017; Kuo et al., 2012; Gould et al., 2005; Gould et al., 2007). If NCCs need to travel using collagen α1α1α2(IV) as a substrate and/or if they themselves need to produce collagen α1α1α2(IV) to move is not known. Thus, which cell population secretes collagen α1α1α2(IV) and if/how they crosstalk to lead to impaired NCCs migration in Col4a1 mutant mice remain open questions. Detailed lineage tracing strategies using membrane-tagged fluorophores and/or reporters are needed to start uncovering these mechanisms. Such strategies will also allow a detailed analysis of size and shape of NCCs to address if and how Col4a1 mutations affects these parameters, that are at the basis of cell migration.

Development of the anterior segment is a complex process involving tissues of different embryonic origins. Surface ectoderm gives rise to the lens and corneal epithelium, while the neural ectoderm gives rise to the retina and pigmented epithelia of the iris and ciliary body. The POM gives rise to most of the anterior segment structures including corneal stroma and endothelium, iris and ciliary stroma, and ocular drainage structures (Cvekl and Tamm, 2004; Hay et al., 1979). Col4a1 mutant mice show reduced corneal thickness that persists through adulthood due to corneal stromal and endothelial abnormalities (Mao et al., 2022). Together with our data, this suggests potential defects in POM differentiation. Future lineage tracing analysis of NCC- and mesodermal-derived POM cells and their derivatives combined with single- cell RNA sequencing experiments will clearly assess their differentiation potential in the context of Gould syndrome. FOXC1 and PITX2 are transcription factors critical for POM development, and FOXC1 and PITX2 mutations are among the most common causes of ASD and developmental glaucoma (Ito and Walter, 2014). Interestingly, animal models also highlight FOXC1 requirement for proper cerebral vascular development, and patients with FOXC1 and PITX2 mutations have cerebral small vessel disease (Silla et al., 2014; Smith et al., 2000; Souzeau et al., 2021; Skarie and Link, 2009; Prem Senthil et al., 2022), suggesting that COL4A1, FOXC1, and PITX2 may participate in overlapping pathways. Single-cell RNA sequencing experiments would shed light on these possible interactions.

Although analyses presented in this study focused on ocular development, compromised NCCs behaviors may also contribute to progressive ocular defects and cerebrovascular disease observed in Col4a1 mutant mice. Col4a1 mutations cause glaucoma in a subset of individuals with Gould syndrome and glaucoma-like phenotypes in mouse models (Jeanne and Gould, 2017; Labelle-Dumais et al., 2011; Mao et al., 2022; Weng et al., 2012; Branyan et al., 2022; Boyce et al., 2021). It is possible that altered NCCs biology could also impair differentiation of ocular drainage tissues leading to progressive intraocular pressure elevation and glaucoma. Notably, many tissues affected by Gould syndrome are derived from two distinct embryonic origins: the NCCs and the mesoderm germ layer. For example, components of the neurovascular unit, such as pericytes and smooth muscle cells, are NCCs derivatives and so it is possible that impaired NCCs migration and/or differentiation could contribute to other aspects related to Gould syndrome pathology.

5. Conclusion

Homozygosity for the Col4a1Δex41 allele is embryonically lethal (Gould et al., 2005) and here we report that Col4a1Δex41/Δex41 die between E9.5 and E10.5. These data also highlight the increased developmental delay in mutant offspring of Col4a1 mutant mothers, suggesting a possible detrimental effect of the mutant uterine environment on embryo development and maternal effect involvement in Gould syndrome as already proposed in a previous human study (Zagaglia et al., 2018). Collectively, the findings presented in this study suggest a role for type IV collagen in NCCs migration regulation that could contribute to ocular dysgenesis and a potential connection between ocular and cerebrovascular developmental defects in Col4a1 mutant mice. Further examinations are needed to understand both the molecular causes and consequences as well as the potential for a broader contribution of NCCs and mesodermal differentiation defects caused by Col4a1 mutations.

Supplementary Material

Supplemental material

Acknowledgments

We thank Dr. Cassandre Labelle-Dumais for critical review of the manuscript, and Dr. Yien-Ming Kuo for support with the UCSF Ophthalmology department histology core.

Funding

This project was supported by the National Institutes of Health (NIH) under Award Numbers R01EY019887 (DBG), Knights Templar Eye Foundation (CC) and All May See Foundation (CC), and in part by the UCSF Vision Core shared resource of the NIH/NEI P30 EY002162, and by an unrestricted grant from Research to Prevent Blindness, New York, NY. The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or other funding agencies.

Footnotes

Declaration of competing interest

None.

CRediT authorship contribution statement

Corinna Cozzitorto: Writing – review & editing, Writing – original draft, Visualization, Validation, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Zoe Peltz: Investigation. Lourdes M. Flores: Investigation. Luca Della Santina: Software, Resources, Methodology. Mao Mao: Writing – review & editing, Conceptualization. Douglas B. Gould: Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.cdev.2024.203926.

Data availability

Data will be made available on request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental material
NIHMS2069909-supplement-Supplemental_material.docx (64.2MB, docx)

Data Availability Statement

Data will be made available on request.

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