Disruption of the mouse Jhy gene causes abnormal ciliary microtubule patterning and juvenile hydrocephalus

Abstract

Congenital hydrocephalus, the accumulation of excess cerebrospinal fluid (CSF) in the ventricles of the brain, affects one of every 1000 children born today, making it one of the most common human developmental disorders. Genetic causes of hydrocephalus are poorly understood in humans, but animal models suggest a broad genetic program underlying the regulation of CSF balance. In this study, the random integration of a transgene into the mouse genome led to the development of an early onset and rapidly progressive hydrocephalus. Juvenile hydrocephalus transgenic mice (Jhy(lacZ)) inherit communicating hydrocephalus in an autosomal recessive fashion with dilation of the lateral ventricles observed as early as postnatal day 1.5. Ventricular dilation increases in severity over time, becoming fatal at 4-8 weeks of age. The ependymal cilia lining the lateral ventricles are morphologically abnormal and reduced in number in Jhy(lacZ/lacZ) brains, and ultrastructural analysis revealed disorganization of the expected 9+2 microtubule pattern. Rather, the majority of Jhy(lacZ/lacZ) cilia develop axonemes with 9+0 or 8+2 microtubule structures. Disruption of an unstudied gene, 4931429I11Rik (now named Jhy) appears to underlie the hydrocephalus of Jhy(lacZ/lacZ) mice, and the Jhy transcript and protein are decreased in Jhy(lacZ/lacZ) mice. Partial phenotypic rescue was achieved in Jhy(lacZ/lacZ) mice by the introduction of a bacterial artificial chromosome (BAC) carrying 60-70% of the JHY protein coding sequence. Jhy is evolutionarily conserved from humans to basal vertebrates, but the predicted JHY protein lacks identifiable functional domains. Ongoing studies are directed at uncovering the physiological function of JHY and its role in CSF homeostasis.

Keywords: 4931429I11Rik; Cilia; Hydrocephalus; Jhy; Mouse.

Figure 1. Jhy^lacZ/lacZ mice develop hydrocephalus

A) Jhy^lacZ/lacZ embryo at e11.5 showing lacZ expression in the epi physis of the diencephalon. B) Enlarged view of lacZ expression in A. C) Saggital section of diencephalon lacZ expression in e11.5 Jhy^lacZ/lacZ embryo. D) Jhy^+/+ (left) and Jhy^lacZ/lacZ littermates (right) at 3 weeks of age, showing doming of the skull and ataxia in the Jhy^lacZ/lacZ mouse. E) Hemorrhaging beneath the skull is visible in a 3 week old Jhy^lacZ/lacZ mouse (top) compared to a Jhy^+/+ littermate (bottom). F, G) Hematoxylin/eosin stained sections of Jhy^+/+ (F) and Jhy^lacZ/lacZ (G) brains at P5, showing ventricular dilation.

Figure 2. Jhy^lacZ/lacZ hydrocephalus is rapid and progressive

A) Jhy^lacZ/lacZ mice (squares) develop outwardly visible hydrocephalus with a penetrance of 74%, with most mice affected by 5 weeks of age. No Jhy^lacZ/+ mice (diamonds) developed hydrocephalus or died during the 12 week monitoring period. B) Most Jhy^lacZ/lacZ mice die by 4-8 weeks of age. C) Early onset hydrocephalus occurs in 63% of Jhy^lacZ/lacZ mice, with an additional 11% developing a late onset milder form of the disease.

Figure 3. Jhy^lacZ/lacZ brains show normal development and patterning

A-H) Coronal sections of Jhy^+/+ and Jhy^lacZ/lacZ brains at P0.5 show normal morphology and patterning. I) Transverse section of Jhy^lacZ/lacZ brain at P0.5 shows a patent aqueduct. The inset shows an enlarged image of the aqueduct. J-M) Magnetic resonance imaging of Jhy^+/+ and Jhy^lacZ/lacZ brains. J, K) MRI at P6 shows significant dilation of the lateral ventricles of the Jhy^lacZ/lacZ mouse (K) as compared to a Jhy^+/+ littermate (J). L, M) MRI at P13 shows more pronounced ventricular dilation with loss of brain tissue. N-Q) Adult testis (N, O) shows reduced numbers of mature sperm, while kidney (P, Q) shows normal development.

Figure 4. Jhy^lacZ transge ne reporter expression in the brain

Jhy^lacZ transgene expression was examined in Jhy^+/+ and Jhy^lacZ/lacZ mice at P0.5 and P5. A-D) Expression of the lacZ reporter is observed in the pineal gland and hypothalamus at P0.5 and P5. Dilation of the lateral ventricles can be seen in Jhy^lacZ/lacZ mice at P5. E-H) Higher magnification of the pineal gland lacZ expression, and lack of expression in the subcommissural organ. I-L) Expression of lacZ is seen in the choroid plexus of the third ventricle at P5, but not in the choroid plexus or ependyma of the lateral ventricles. At P5 dilation of the third ventricle is seen in Jhy^lacZ/lacZ mice. M-P) Higher magnification of the hypothalamus lacZ expression. Q-T) Expression of lacZ in the ependyma lining the aqueduct of Sylvius. Structures are indicated as follows: A, aqueduct of Sylvius; C, choroid plexus; H, hypothalamus; L, lateral ventricle; P, pineal gland; S, subcommissural organ; 3, third ventricle.

Figure 5. Jhy^lacZ/lacZ ependymal cilia exhibit structural abnormalities

A-B) SEM was used to visualize the ultrastructure of the ependymal cilia in P5 Jhy^+/+ (A) and Jhy^lacZ/lacZ (B) lateral ventricles. Caudal is to the left. C-D) Higher magnification images of the ependymal cilia in P5 Jhy^+/+ (C) and Jhy^lacZ/lacZ (D) lateral ventricles. E-F) High magnification images of the ependymal cilia in P5 Jhy^+/+ (E) and Jhy^lacZ/lacZ (F) lateral ventricles. G-I) Representative TEM images of Jhy^+/+ 9+2 (G), and Jhy^lacZ/lacZ 9+0 (H) and 8+2 (I) axonemes. J) TEM image showing partial displacement of a peripheral ring microtubule doublet towards the center of the axoneme in a Jhy^lacZ/lacZ brain. Magnification for all TEM images is 100,000X. Corresponding regions of the lateral ventricle were examined in each mouse (n = 3 each for Jhy^+/+ and Jhy^lacZ/lacZ), and representative images were taken with scale bars embedded at 75× (A, B), 2,000× (C, D), and 8,000× (E, F).

Figure 6. The Jhy^lacZ transgene disrupts the uncharacterized gene 4931429I11Rik (Jhy)

A) The Jhy^lacZ transgene integrated within the Jhy gene on mouse chromosome 9. Boxes denote the Jhy gene and flanking genes Bsx and Crtam, the arrowhead indicates the Jhy^lacZ integration, the arrows indicate the direction of transcription and the primers used for qRT-PCR are denoted with half-arrows. B) The Jhy^lacZ transgene integrated at the beginning of Jhy exon 5, generating a deletion that removes 425 bp of intron 4 and the first 88 bp of exon 5. The black boxes represent Jhy exons, the grey box the Jhy^lacZ transgene, the dotted line the intronic sequence deleted, and the bracket indicates the entire deletion. (In this view the chromosomal orientation of the Jhy gene has been rotated for clarity.) C) Jhy exons 4 and 5 are juxtaposed to show the exon 5 sequence deleted. D) qRT-PCR analysis of the expression levels of Jhy (white bars), Bsx (black bars), and Crtam (hatched bars) in P0.5 Jhy^lacZ/+ and Jhy^lacZ/lacZ brain in comparison to Jhy^+/+ levels (n ≥ 7 for all genotypes), * denotes p ≤ 0.05.

Figure 7. Jhy^lacZ/lacZ mice express reduced levels of JHY protein

A) JHY protein localizes to the ependymal cells of the lateral ventricle in Jhy^+/+ P5 mouse brain using an antibody directed against the human JHY homolog C11ORF63. B) Jhy^lacZ/lacZ brain shows significantly reduced levels of JHY protein.

Figure 8. Transgenic rescue of Jhy^lacZ/lacZ mice

A) The RP23-273J15 BAC clone spans the region 80 kb proximal to 20 kb distal to the Jhy gene, while the integrated Tg^BAC has been truncated to contain 60-70% of the protein coding region of Jhy. The white boxes represent the Bsx, Jhy and Crtam genes, the arrowhead indicates the Jhy^lacZ integration, the arrows indicate the direction of gene transcription, the grey box indicates the end of the truncated portion of Tg^BAC that cannot be defined due to a lack of SNPs. B) The incidence of hydrocephalus is reduced from 74% in Jhy^lacZ/lacZ mice (diamonds) to 37% in Tg^BAC/+;Jhy^lacZ/lacZ mice (squares). C) Image of Jhy^lacZ/lacZ (left) and Tg^BAC/+;Jhy^lacZ/lacZ (right) littermates showing phenotypic rescue by Tg^BAC. D) Quantification of TEM analysis of ependymal ciliary microtubule arrangement in Jhy^+/+ (black bars), Jhy^lacZ/lacZ (white bars) and Tg^BAC/+;Jhy^lacZ/lacZ (grey bars) at P5 (n=620 for Jhy^+/+, 147 for Jhy^lacZ/lacZ, and 117 for Tg^BAC/+;Jhy^lacZ/lacZ), * denotes p ≤ 0.05 relative to Jhy^+/+, ** denotes p ≤ 0.05 relative to Jhy^lacZ/lacZ.