Defective neural tube closure and anteroposterior patterning in mice lacking the LIM protein LMO4 or its interacting partner Deaf-1

Abstract

LMO4 belongs to a family of transcriptional regulators that comprises two zinc-binding LIM domains. LIM-only (LMO) proteins appear to function as docking sites for other factors, leading to the assembly of multiprotein complexes. The transcription factor Deaf-1/NUDR has been identified as one partner protein of LMO4. We have disrupted the Lmo4 and Deaf-1 genes in mice to define their biological function in vivo. All Lmo4 mutants died shortly after birth and showed defects within the presphenoid bone, with 50% of mice also exhibiting exencephaly. Homeotic transformations were observed in Lmo4-null embryos and newborn mice, but with incomplete penetrance. These included skeletal defects in cervical vertebrae and the rib cage. Furthermore, fusions of cranial nerves IX and X and defects in cranial nerve V were apparent in some Lmo4(-/-) and Lmo4(+/-) mice. Remarkably, Deaf-1 mutants displayed phenotypic abnormalities similar to those observed in Lmo4 mutants. These included exencephaly, transformation of cervical segments, and rib cage abnormalities. In contrast to Lmo4 nullizygous mice, nonexencephalic Deaf-1 mutants remained healthy. No defects in the sphenoid bone or cranial nerves were apparent. Thus, Lmo4 and Deaf-1 mutant mice exhibit overlapping as well as distinct phenotypes. Our data indicate an important role for these two transcriptional regulators in pathways affecting neural tube closure and skeletal patterning, most likely reflecting their presence in a functional complex in vivo.

FIG. 1.

Targeted disruption of the mouse Lmo4 gene. (A) Partial restriction map of the mouse Lmo4 gene (top) and structure of the Lmo4 targeting vector (bottom), which contains the thymidine kinase, cytosine deaminase, and neomycin resistance (neo^r) genes, all under the control of the mouse PGK promoter. The location of the three loxP sites is given. Homologous recombination results in disruption of the coding sequence of Lmo4 and removal of exon 2, which encodes the first LIM domain. The filled boxes represent coding exons. The flanking probe used for Southern blot analysis is shown as a black bar. (B) Structure of the Lmo4:LacZ knockin construct. The construct contains the neo^r and thymidine kinase genes under the control of the mouse PGK promoter. Homologous recombination results in removal of the majority of exon 2 and disruption of the coding sequence of Lmo4. The LacZ gene (shaded) was fused in frame with the translation initiation codon located at the beginning of exon 2. Exons are indicated as filled boxes. Abbreviations for restriction sites: B, BamHI; RI, EcoRI; H, HindIII; X, XhoI; C, ClaI. (C) Generation of mice bearing either a constitutive null Lmo4 allele or a floxed allele. Chimeras were crossed with Gata1-cre transgenic females to obtain F₁ progeny carrying either the floxed Lmo4 or knockout allele. Wild-type (11-kb), knockout (10-kb; both the neo^r cassette and floxed alleles excised), floxed (1.6-kb; only the neo cassette excised), and wild-type but modified (2.8-kb; targeted but neo^r cassette present) alleles are indicated. Tail DNA was digested with XhoI and EcoRV, and Southern blot analysis of F₂ progeny was performed using a 600-bp XhoI-HindIII probe (A). (D) Lmo4:LacZ knockin mice were generated by breeding chimeras with Gata1-cre transgenic mice. Southern blot analysis of F₁ progeny was performed using DNA digested with EcoRV and XhoI and a 600-bp XhoI-HindIII probe. (E) Protein lysates (30 μg) from either Lmo4^−/− or wild-type embryos at E16.5 were analyzed by Western blotting using a monoclonal antibody specific for the second LIM domain of Lmo4. No truncated Lmo4 protein was detected in Lmo4^−/− embryos. Immunoblotting with an antitubulin antibody (α-tubulin) confirmed equal loading of protein.

FIG. 2.

Failure of neural tube closure in Lmo4 mutants. Exencephaly was observed in approximately 50% of Lmo4 mutants. (A to C) Wild-type (A) and mutant Lmo4^−/− (B and C) embryos at E9.0 with exencephaly. (D to F) Lmo4 expression in embryos at E8.5 (D and E) and E9.5 (F), as detected by LacZ staining of Lmo4:LacZ knockin embryos.

FIG. 3.

Malformation of the presphenoid bone in Lmo4 mutants. (A to F) Comparison of the internal skull base (ventral view) of wild-type mice (A and D) and newborn Lmo4 mutants without exencephaly (B and E) and with exencephaly (C and F), following staining for bone and cartilage using alizarin red and Alcian blue. The region surrounding the sphenoid bone is shown at higher magnification in D, E, and F. The presphenoid (PS) and the basi-sphenoid (BS) bodies are fully ossified (red stain). (D) The arrow depicts the lateroposterior processes protruding from the presphenoid body; these are missing in panels E and F. (G and H) Skull bases of E16.5 wild-type and Lmo4^−/− embryos without exencephaly, respectively. Arrows point to the region where the lateroposterior processes lie. These are missing in panel H. (I to K) Skull bases of wild-type (I), Lmo4:LacZ knockin heterozygote (J), and ROSA26:LacZ:Wnt1-cre (K) embryos at E18.5 were stained for LacZ activity (ventral view). The lateroposterior processes present in panel J are missing in panel K. The blue staining in panel K, in the area where the processes lie, reflects background staining from the underlying tissue. ROSA26:LacZ:Wnt1-cre reporter embryos were generated by crossing Rosa26:LacZ reporter mice with Wnt1-cre transgenic mice. (L) Alizarin red and Alcian blue staining of bone and cartilage in the presphenoid bone in Lmo4^fl/fl:Wnt1-cre newborn mice. The arrows mark the region where posterolateral processes protrude from the presphenoid body.

FIG. 4.

Skeletal abnormalities in Lmo4 mutant mice. Bone and cartilage were stained with alizarin red and Alcian blue, respectively. (A to D) Ventral view of sternum and ribcage in wild-type mice (A and C) and Lmo4 mutants (B and D). (B) Aberrant attachment of the eighth rib to the sternum is indicated by a solid arrow. (D) Asymmetric alignment of the ribs is shown. (C) Symmetric alignment occurs in wild-type mice. In some Lmo4 mutants (F), anterior tubercules (AT) were attached to C7 (F) instead of C6, as occurs in wild-type mice (E). (F) One AT appears to be attached to both C6 and C7. (G) In another Lmo4 mutant, partial fusion of C2 and C3 was observed. AT stained with Alcian blue are indicated by white arrows. Vertebrae C2 to C7 are indicated by arrows.

FIG. 5.

Cranial nerve malformation in Lmo4 mutant embryos. Wild-type (A), heterozygous (B), and homozygous Lmo4 (C and D) embryos at E9.5 were stained for neurofilaments using antibody 2H2. Fusion of cranial nerves IX and X, which occurs in 50% of Lmo4^−/− and 25% of Lmo4^+/− embryos, is indicated by arrows. In 2 out of 10 Lmo4^−/− embryos, abnormal staining of axons surrounding ganglion V was observed (arrowhead in panel D). Cranial nerves V, VII, IX, and X and branchial arch II (BAII) are indicated.

FIG. 6.

Targeted disruption of the mouse Deaf-1 gene. (A) Partial restriction map of the mouse Deaf-1 gene (top) and structure of the Deaf-1 targeting vector (bottom), which contains the thymidine kinase and neomycin resistance (neo^r) genes, both under the control of the mouse PGK promoter. Homologous recombination results in disruption of two exons encoding the SAND domain, represented as open boxes. The flanking probe used for Southern blot analysis is shown as a black bar. Abbreviations for restriction sites: Bg, BglII; B, BamHI; K, Asp718; R1, EcoRI; X, XhoI. (B) Generation of Deaf-1^+/− mice. Deaf-1 chimeras were crossed with Gata1-cre transgenic mice to generate F₁ progeny heterozygous for the targeted Deaf-1 allele. DNA was digested with EcoRI, and Southern blot analysis was performed using the 1.2-kb BglII-EcoRV probe indicated in panel A. (C) Protein lysates (30 μg) derived from E16.5 embryos were analyzed by Western blotting using a polyclonal antibody raised against Deaf-1. No Deaf-1 protein was observed in Deaf-1^−/− embryos. The faint band at 46 kDa appears in both wild-type and −/− embryonic extracts and represents a nonspecific cross-reactive product. 293T cells transfected with an expression vector encoding hemagglutinin-tagged Deaf-1 served as a control. Western blot analysis using antitubulin antibody (α-tubulin) confirmed equal loading.

FIG. 7.

Defective neural tube closure and skeletal abnormalities in Deaf-1-deficient embryos. Deaf-1 mutant embryos (B) fail to close their neural tube by E10.5 and show outgrowth of the mid-hindbrain region compared to wild-type embryos (A). The penetrance of this phenotype on a mixed genetic background was 80%. (C to G) Bone and cartilage of wild-type (C) and Deaf-1 mutants (D to G) were stained with alizarin red and Alcian blue to analyze potential skeletal defects. Five out of twelve Deaf-1 mutants showed attachment of the eighth rib to the sternum (D) as seen in Lmo4 mutants. Two out of 35 Deaf-1^+/− (E and G) and 2 out of 12 Deaf-1^−/− (F) embryos showed bifurcation or fusion of ribs. The eighth and ninth ribs were bifurcated (E), while the first and second ribs appeared to be fused, as indicated by the arrows (F and G). Only one Deaf-1^−/− embryo (F) showed fusion of the first and second ribs, and the anterior tubercule (AT) was attached to C7 instead of C6, as indicated by the arrow. In a Deaf-1 heterozygote (G), the first rib was attached to C7 instead of T1 and the AT was attached to C5 instead of C6.