Morphogenesis of the telencephalic commissure requires scaffold protein JNK-interacting protein 3 (JIP3)

Abstract

The murine JNK-interacting protein 3 (JIP3) protein (also known as JSAP1) is expressed exclusively in neurons and has been identified as a scaffold protein for the c-Jun NH2-terminal kinase (JNK) signaling pathway and as an adapter protein for cargo transport by the microtubule motor protein kinesin. To investigate the physiological function of JIP3, we examined the effect of Jip3 gene disruption in mice. The Jip3-/- mice were unable to breathe and died shortly after birth. Microscopic analysis demonstrated that Jip3 gene disruption causes severe defects in the morphogenesis of the telencephalon. Jip3-/- mice lack the telencephalic commissure, a major connection between the left and right hemispheres of the brain. The central nervous system abnormalities of Jip3-/- mice may be accounted for in part by a reduction in signal transduction by RhoA and its effector ROCK.

Fig. 1.

Targeted disruption of the murine Jip3 gene. (A) Schematic illustration of the strategy used for disruption of the Jip3 gene. A fragment of the wild-type gene that includes exons 1–3 is shown. The targeting vector was designed to replace exon 1 with a Neo^R cassette. The diagram also indicates the probe used for distinguishing the wild-type and disrupted alleles by Southern blot analysis. TK, thymidine kinase. (B) Mice with homozygous and heterozygous disruptions of the Jip3 gene were identified by Southern blot analysis. WT, wild type; KO, knockout. (C) Total RNA isolated from the brains of E18.5 mice was examined by Northern blot analysis by using probes for GAPDH, actin, JIP1, JIP2, JIP3, and JIP4. Equal loading of RNA isolated from wild-type, Jip3^–/+, and Jip3^–/– embryos was confirmed by staining of ribosomal RNA with ethidium bromide. (D) E18.5 embryonic brains from wild-type, Jip3^–/+, and Jip3^–/– mice were examined by immunoblot analysis. Control experiments were performed by probing the blots with an antibody to tubulin. The increased expression of kinesin light chain in Jip3^–/– mice shown in the figure was not reproducibly observed in independent experiments. KLC1, kinesin light chain 1; KHC, kinesin heavy chain.

Fig. 2.

Jip3 gene disruption causes defective lung development. (A) Newborn wild-type and Jip3^–/– littermates obtained immediately after birth. The mutant mice appear to be morphologically normal but exhibit symptoms of severe anoxia and die shortly after birth. (B) The lungs of newborn wild-type and Jip3^–/– littermates were dissected and placed in PBS. The wild-type and mutant lungs are indicated. (C) Microscopic comparison of lungs from newborn wild-type and Jip3^–/– littermates. Wild-type alveolar spaces are expanded, indicating respiration; however, Jip3^–/– alveoli are still collapsed, and the septae are hypercellular. The sections were stained with hematoxylin/eosin.

Fig. 3.

Effect of Jip3 and Jnk gene disruption on protein expression in the brain. (A) Brain extracts were examined by immunoblot analysis by using antibodies to JNK, Munc18-1, and tubulin. The results of immunoblot analysis of wild-type (WT), Jnk1^–/– (J1–/–), Jnk2^–/– (J2–/–), and Jnk1^–/–Jnk2^–/+ (J1–/–J2–/+) mice are shown. The compound mutant Jnk1^–/–Jnk2^–/– was not examined because these mice die during early embryonic development. (B) E18.5 embryonic brains from wild-type, Jip3^–/+, and Jip3^–/– mice were examined by immunoblot analysis. Control experiments were performed by probing the blots with an antibody to tubulin.

Fig. 4.

Jip3 gene disruption causes defects in the formation of the telencephalon. (A) The wild-type E18.5 brain has compact neuronal layers (N) surrounding the lateral ventricles (LV) and a well demarcated thick band of axonal fibers [telencephalic commissure (TC)] connecting the hemispheres at the level of the lateral ventricles. (Magnification, ×5.) The Jip3^–/– brain has a more diffuse, less compact zone of neurons (N) surrounding each dilated lateral ventricle, and bilateral white tract (WT) areas in each hemisphere have no connection to the opposite side. 3V, third ventricle. (B) An organized zone of axons connecting the hemispheres of wild-type brains. (Magnification, ×20.) In contrast, no white matter intrahemispheric connection in the Jip3^–/– brain was observed. (C) Compact neuronal layers (N) and a well demarcated thick band of axonal fibers [white-matter tracts (WT)] connecting the hemispheres in the wild-type brain. (Magnification, ×40.) The Jip3^–/– brain has more randomly distributed neurons (N) with scattered debris (D) and disorganized white tracts with frequent vacuoles (V). All slides were stained with hematoxylin/eosin.