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Vax2 inactivation in mouse determines alteration of the eye dorsal-ventral axis, misrouting of the optic fibres and eye coloboma

Anna Maria Barbieri1,*, Vania Broccoli1,{dagger}, Paola Bovolenta2, Giovanna Alfano1, Anna Marchitiello1, Cristina Mocchetti1, Luca Crippa3, Alessandro Bulfone1,*, Valeria Marigo1, Andrea Ballabio1,4 and Sandro Banfi1,{ddagger}

1 Telethon Institute of Genetics and Medicine (TIGEM), Via Pietro Castellino 111, Naples, Italy
2 Departamento de Neurobiologia del Desarrollo, Instituto Cajal, CSIC, Madrid, Spain
3 Novuspharma, Monza, Italy
4 Istituto di Patologia Generale ed Oncologia, Facolta’ di Medicina e Chirurgia, Seconda Universita degli Studi di Napoli, Naples, Italy
* Present address: Molecular Histology Unit, Department of Biological and Technological Research (DIBIT), San Raffaele Biomedical Science Park, Milan, Italy
{dagger} Present address: Stem Cell Research Institute (SCRI), Department of Biological and Technological Research (DIBIT), San Raffaele Biomedical Science Park, Milan, Italy



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  		Fig. 1. Generation of Vax2 mutant mice. (A) Targeted deletion of exon 2 (EX2) of the Vax2 gene by homologous recombination in ES cells. The PGK-Neo expression cassette introduces two novel HindIII sites and removes two BamHI restriction sites. The probes used for Southern-blot analysis are also shown (hatched boxes). Tk, thymidine kinase; B, BamHI; H, HindIII; RI, EcoRI; X, XbaI. (B) Southern blot analysis of genomic DNA from wild-type (+/+), heterozygous (–/+) and homozygous (–/–) mutant mice, hybridised with the 3' probe indicated in A. (C) RT-PCR analysis of total retina RNA from wild-type and Vax2–/– mice using primers 32F and 7R located, respectively, in the first and in the third coding exons of the Vax2 gene. The amplified RT-PCR fragment in the Vax2–/– lane does not contain exon 2 (see the text for more details). Lanes 1 and 2, molecular weight markers.

 


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  		Fig. 2. Homozygous Vax2 mutant mice show coloboma in a significant percentage of cases. (A,B) Whole-mount eyes dissected from E16.5 foetuses. (A,B) Frontal views of wild-type (A) and mutant (B) eyes. The open optic fissure in the mutant is indicated by arrows in B. (C,D) Detection of laminin by immunohistochemistry in order to identify the basal lamina in frontal sections of wild-type (C) and Vax2–/– (D) E12.5 embryos. Note how the basal lamina still persists in the mutant eye in the contact regions of the converging lips of the optic fissure (arrowheads), while in the wild-type eye it has completely dissolved. (E-H) Frontal sections, stained with Haematoxylin and Eosin, of wild-type (E,G) and Vax2 mutant (F, H) eyes at E16.5 (E,F) and P7 (G,H). No differences, apart from the presence of coloboma (arrowheads) can be detected in Vax2 mutant mice compared with wild-type animals in the organisation of retinal cell layers. re, retina; le, lens.

 


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  		Fig. 3. Effects of Vax2 inactivation on the expression of markers of DV and of temporo-nasal asymmetry in the eye, as determined by RNA in situ hybridisation. Pax2 expression at E12.5, which is normally restricted (within the optic cup) to the lips of the optic fissure, was the same in wild-type (A) and Vax2 mutant (B) mice. The white arrow in B indicates the persistence of the optic fissure in Vax2–/– mice. Similarly, the expression of Tbx5, a marker of the dorsal retina, was comparable between normal mice at E12.5 (C) and E16.5 (E), and mutant animals at the same stages (D,F, respectively). (G,H) Frontal sections of a wild-type (G) and of a Vax2–/– (H) mouse embryos at E16.5 hybridised with the probe for Efnb2 (ephrin B2) probe. Expression of Efnb2, which is normally restricted to the dorsal retina (G), is extended to the ventral retina of Vax2–/– mice. (I,J) Frontal sections of a wild-type (I) and of a Vax2–/– (J) mouse embryos at E16.5 hybridised with the probe for Ephb2. The Ephb2 mRNA at E16.5 is expressed with a high ventral to low dorsal gradient in wild-type mice, while in Vax2–/– mice, it is expressed uniformly, and at lower levels, throughout the retina. (K-N) Sagittal sections of wild-type (K,M) and of Vax2–/– (L,N) mouse embryos at E16.5 hybridised with the probes for Epha5 (K,L) and Efna5 probes (M,N). No differences could be detected in the expression of the latter marker between wild-type and Vax2–/– mice. The white arrows in F,H indicate the presence of coloboma in Vax2–/– mice. le, lens.

 


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  		Fig. 4. Retinal ganglion cell axon pathway and projections are altered in Vax2 mutant mice. The ventral retina (C,D) or the right optic nerve head (A,B; E,H) of new-born wild-type (WT) or Vax2 mutant mice were filled with DiI to label the retinal ganglion cell axon trajectory. (A-D) Ventral views of the brain at the chiasm region (dotted lines indicate the midline). (E,H) Coronal sections through the contralateral (E,F) and ipsilateral superior colliculus (G,H). All sections are oriented rostral (R) towards the top and lateral (L) towards the right, as indicated in E. Note how retinal ganglion cell axons normally projecting in the ipsilateral optic tract (arrows in A,C) are totally absent in the mutant mice (B,D). Furthermore, note how, in mutant mice, projections to ipsilateral superior colliculus are absent (compare G with H), while those to the contralateral side terminate predominantly in the lateral region (compare E with F), where normally the majority of axons arising from the dorsal retina project.

 


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  		Fig. 5. Visual projections are altered in adult mutant mice. Frontal sections of adult wild type (A,B) and Vax2–/– (C,D) brains at the mid-superior colliculus. (A,C) Phase contrast views of the sections presented in B,D, respectively. DiI was injected into the ventral retina of adult wild-type and mutant mice. (B) In wild-type mice, DiI-labelled axons project to the entire medial region (thick arrow) of the contralateral superior colliculus and to patches of the stratum opticum of the ipsilateral superior colliculus (thin arrows). (D) Note the absence of the ipsilateral and most medial contralateral projections in the superior colliculus of mutant mice. SC, superior colliculus. Scale bar: 150 µm.

 

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