Left-right asymmetry and kinesin superfamily protein KIF3A: new insights in determination of laterality and mesoderm induction by kif3A-/- mice analysis

Abstract

KIF3A is a classical member of the kinesin superfamily proteins (KIFs), ubiquitously expressed although predominantly in neural tissues, and which forms a heterotrimeric KIF3 complex with KIF3B or KIF3C and an associated protein, KAP3. To elucidate the function of the kif3A gene in vivo, we made kif3A knockout mice. kif3A-/- embryos displayed severe developmental abnormalities characterized by neural tube degeneration and mesodermal and caudal dysgenesis and died during the midgestational period at approximately 10.5 dpc (days post coitum), possibly resulting from cardiovascular insufficiency. Whole mount in situ hybridization of Pax6 revealed a normal pattern while staining by sonic hedgehog (shh) and Brachyury (T) exhibited abnormal patterns in the anterior-posterior (A-P) direction at both mesencephalic and thoracic levels. These results suggest that KIF3A might be involved in mesodermal patterning and in turn neurogenesis.

Figure 1

Gene targeting strategy adopted in this study. (A) Gene targeting vector. The anterior half of the P-loop exon is replaced with a PGK-neo selection marker. We placed DT-A negative selection marker immediately upstream of the short arm consensus region. (B) Screening of the homologous recombinants was carried out by using three probes (neo, external and internal). Only external probe of ∼0.4 kb is indicated as a hatched box. In the wild-type locus, enzymatic digestion by EcoRI gave rise to a single 3.2-kb band whereas a 2.1-kb band was observed in the case of the knocked-out allele. (C) Western blotting of the whole embryos killed at 9.5 dpc revealed no KIF3A band in homozygotes while KIF3B was still expressed. (D) PCR analysis of littermates. Null mutant homozygous embryos do not generate any bands by primer set #2 while #1 (neo) produced a single distinct band.

Figure 1

Gene targeting strategy adopted in this study. (A) Gene targeting vector. The anterior half of the P-loop exon is replaced with a PGK-neo selection marker. We placed DT-A negative selection marker immediately upstream of the short arm consensus region. (B) Screening of the homologous recombinants was carried out by using three probes (neo, external and internal). Only external probe of ∼0.4 kb is indicated as a hatched box. In the wild-type locus, enzymatic digestion by EcoRI gave rise to a single 3.2-kb band whereas a 2.1-kb band was observed in the case of the knocked-out allele. (C) Western blotting of the whole embryos killed at 9.5 dpc revealed no KIF3A band in homozygotes while KIF3B was still expressed. (D) PCR analysis of littermates. Null mutant homozygous embryos do not generate any bands by primer set #2 while #1 (neo) produced a single distinct band.

Figure 1

Gene targeting strategy adopted in this study. (A) Gene targeting vector. The anterior half of the P-loop exon is replaced with a PGK-neo selection marker. We placed DT-A negative selection marker immediately upstream of the short arm consensus region. (B) Screening of the homologous recombinants was carried out by using three probes (neo, external and internal). Only external probe of ∼0.4 kb is indicated as a hatched box. In the wild-type locus, enzymatic digestion by EcoRI gave rise to a single 3.2-kb band whereas a 2.1-kb band was observed in the case of the knocked-out allele. (C) Western blotting of the whole embryos killed at 9.5 dpc revealed no KIF3A band in homozygotes while KIF3B was still expressed. (D) PCR analysis of littermates. Null mutant homozygous embryos do not generate any bands by primer set #2 while #1 (neo) produced a single distinct band.

Figure 1

Gene targeting strategy adopted in this study. (A) Gene targeting vector. The anterior half of the P-loop exon is replaced with a PGK-neo selection marker. We placed DT-A negative selection marker immediately upstream of the short arm consensus region. (B) Screening of the homologous recombinants was carried out by using three probes (neo, external and internal). Only external probe of ∼0.4 kb is indicated as a hatched box. In the wild-type locus, enzymatic digestion by EcoRI gave rise to a single 3.2-kb band whereas a 2.1-kb band was observed in the case of the knocked-out allele. (C) Western blotting of the whole embryos killed at 9.5 dpc revealed no KIF3A band in homozygotes while KIF3B was still expressed. (D) PCR analysis of littermates. Null mutant homozygous embryos do not generate any bands by primer set #2 while #1 (neo) produced a single distinct band.

Figure 2

Macroscopic overview of both wild-type and homozygous embryos at 9.5 dpc. (A) Lateral view of a wild-type embryo. Normal development of somites and the nervous system can be observed. (B) Same view of a homozygous embryo. Note the severe degeneration of the lower half of the body accompanied by thin neural tube wall. (C) Dorsal view of a homozygous embryo, exhibiting a staggered midline structure with disorganized somites. (D) Extensive pericardial effusion (arrow) resulting in circulatory insufficiency. Bar, 1 mm.

Figure 5

Histological analyses at 9.5 dpc stained by the HE method. Sagittal section of a wild-type (A) and a homozygous (B) embryo. Systemic hypoplasia, extended pericardium (arrows) and thinning of neural tube wall (arrowheads) are characteristic to the homozygotes. Frontal sections of both type of embryos (C, wild-type; D, homozygote) display striking differences as described in the sagittal sections. Cardiac insufficiency in homozygotes is partly explained by the hypoplastic myocardium (F). Note the well-developed myocardial wall in the case of wild-type (E) embryos. Bars, 1 mm.

Figure 4

Expression pattern of KIF3A molecule in 9.5 dpc embryos (B). Expression of the KIF3A molecule is almost ubiquitous although we can recognize relatively strong accent in the neural tube (Nt) and the heart (Cor). Orl, oral cavity; Caud, caudal region. (A) Control panel stained with preimmune serum. Bar, 1 mm.

Figure 3

Scanning electron microscopy of the embryos at 8.5 dpc. Wild-type embryo displays normal body turning (A) and heart loop (D, D-type) while homozygotes (B and C) did not make turning. Some homozygotes exhibited inversed heart looping (F, L-type). In addition, seemingly normal heart of homozygote (E) display irregular cardiac surface as seen in F. Bars: (A–C) 500 μm; (D–F) 100 μm.

Figure 6

Whole mount in situ hybridization of embryos probed with lefty-2. As represented in A, almost all wild-type and heterozygous embryos expressed lefty-2 mRNA at the left paraxial mesoderm. In homozygotes (B), certain extent of embryos exhibited lefty-2 on both sides while there still exist embryos expressing it only on the leftside. Bar, 1 mm.

Figure 7

Scanning electron microscopy of the node (N) of a wild-type embryo (A) and a homozygote (B). Single cilium (arrow) on each nodal cells can be observed in the wild-type embryo (C) while no or extremely short cilia were present in the homozygote (D). Bars: (A and B) 50 μm; (C and D) 5 μm.

Figure 8

Ciliary movement assay. (A) Leftward nodal flow was observed in the case of wild-type embryos (n = 3). (B) In kif3A null mutants (n = 5), only Brownian movement of fluorescent beads was observed, which was markedly different from regular flow. Bars, 5 μm.

Figure 9

Presence of axonemal dynein within monocilia. (B, D, F, and H) Immunostaining of nodal cilia at 7.5 dpc with anti–α-tubulin antibody. Nodal cilia (arrowheads) were identified and double labeled with anti-KIF3A antibody (A). These cilia also contain the axonemal outer arm dynein (C, arrows) (AD2) and intermediate chain (IC140, E) while negative control (G) did not stain the monocilia. By transmission electron microscopy (I and J), both inner and outer arm dynein localize on the surface of MT doublets (arrowheads). Bars: (A–H) 5 μm; (I and J) 100 nm.

Figure 9

Presence of axonemal dynein within monocilia. (B, D, F, and H) Immunostaining of nodal cilia at 7.5 dpc with anti–α-tubulin antibody. Nodal cilia (arrowheads) were identified and double labeled with anti-KIF3A antibody (A). These cilia also contain the axonemal outer arm dynein (C, arrows) (AD2) and intermediate chain (IC140, E) while negative control (G) did not stain the monocilia. By transmission electron microscopy (I and J), both inner and outer arm dynein localize on the surface of MT doublets (arrowheads). Bars: (A–H) 5 μm; (I and J) 100 nm.

Figure 10

Whole mount in situ hybridization. The wild-type embryos (A and C) and homozygous embryos (B and D) were probed with Pax6 (A and B) and Brachyury (C and D) at 9.5 dpc. In the case of sonic hedgehog, lateral view (E) and dorsal view (F) of both wild-type embryos (upper embryos) and homozygous embryos (lower embryos) are presented at 8.5 dpc. Although normal expression pattern of Pax6 was almost preserved in kif3A-deficient embryos while its expression level was downregulated (see text). Expression patterns of shh and T were significantly altered. Note the absence of shh at the mesencephalic region (arrowhead). Some nonspecific deposition of signals can be observed in the head region of the panel (D), which might result from precipitate flow through the distended canalis centralis in mutants. At 7.5 dpc (G), shh expression is dramatically reduced without strong localization within node (arrow) compared with wild-type embryo (Node). Bars: (A–F) 1 mm; (G) 150 μm.

Figure 10

Whole mount in situ hybridization. The wild-type embryos (A and C) and homozygous embryos (B and D) were probed with Pax6 (A and B) and Brachyury (C and D) at 9.5 dpc. In the case of sonic hedgehog, lateral view (E) and dorsal view (F) of both wild-type embryos (upper embryos) and homozygous embryos (lower embryos) are presented at 8.5 dpc. Although normal expression pattern of Pax6 was almost preserved in kif3A-deficient embryos while its expression level was downregulated (see text). Expression patterns of shh and T were significantly altered. Note the absence of shh at the mesencephalic region (arrowhead). Some nonspecific deposition of signals can be observed in the head region of the panel (D), which might result from precipitate flow through the distended canalis centralis in mutants. At 7.5 dpc (G), shh expression is dramatically reduced without strong localization within node (arrow) compared with wild-type embryo (Node). Bars: (A–F) 1 mm; (G) 150 μm.