The bHLH regulator pMesogenin1 is required for maturation and segmentation of paraxial mesoderm

Abstract

Paraxial mesoderm in vertebrates gives rise to all trunk and limb skeletal muscles, the trunk skeleton, and portions of the trunk dermis and vasculature. We show here that germline deletion of mouse pMesogenin1, a bHLH class gene specifically expressed in developmentally immature unsegmented paraxial mesoderm, causes complete failure of somite formation and segmentation of the body trunk and tail. At the molecular level, the phenotype features dramatic loss of expression within the presomitic mesoderm of Notch/Delta pathway components and oscillating somitic clock genes that are thought to control segmentation and somitogenesis. Subsequent patterning and specification steps for paraxial mesoderm also fail, leading to a complete absence of all trunk paraxial mesoderm derivatives, which include skeletal muscle, vertebrae, and ribs. We infer that pMesogenin1 is an essential upstream regulator of trunk paraxial mesoderm development and segmentation.

Figure 1

Targeted disruption of mouse pMesogenin1 gene. (A) In the targeting vector, the coding sequence of pMesogenin1 gene was replaced with a nuclear β-galactosidase gene of E. coli in frame at the first methionine. A neomycin-resistance gene flanked by two loxP sites (Cre-recombinase recognition sites, gray colored circles), and thymidine kinase gene were used for positive and negative selections, respectively. Arrowheads indicate the locations of PCR primers. The bars below the mutated allele map indicate the expected band size of both wild type (7 kb) and mutated alleles (2.9 kb) in Southern blot analysis of XbaI-digested DNA. (B) Southern blot analysis of DNA isolated from transfected and selected ES (embryonic stem) cell clones. (C) Genotype of embryos or fetuses are determined by PCR (polymerase chain reaction) using P1, P2, and Z primers as indicated in panel A. 344 bp and 218 bp PCR products are produced from wild type and the mutated alleles, respectively. The presence of the neo selection cassette after Cre recombination was monitored by PCR using N1 and N2 primers (see Materials and Methods for the primer sequences).

Figure 1

Targeted disruption of mouse pMesogenin1 gene. (A) In the targeting vector, the coding sequence of pMesogenin1 gene was replaced with a nuclear β-galactosidase gene of E. coli in frame at the first methionine. A neomycin-resistance gene flanked by two loxP sites (Cre-recombinase recognition sites, gray colored circles), and thymidine kinase gene were used for positive and negative selections, respectively. Arrowheads indicate the locations of PCR primers. The bars below the mutated allele map indicate the expected band size of both wild type (7 kb) and mutated alleles (2.9 kb) in Southern blot analysis of XbaI-digested DNA. (B) Southern blot analysis of DNA isolated from transfected and selected ES (embryonic stem) cell clones. (C) Genotype of embryos or fetuses are determined by PCR (polymerase chain reaction) using P1, P2, and Z primers as indicated in panel A. 344 bp and 218 bp PCR products are produced from wild type and the mutated alleles, respectively. The presence of the neo selection cassette after Cre recombination was monitored by PCR using N1 and N2 primers (see Materials and Methods for the primer sequences).

Figure 2

Disruption of pMesogenin1 results in defects of paraxial mesoderm formation. (A) Whole-mount in situ hybridization of pMesogenin1 RNA in wild type embryo of 9 dpc. (A′). Both pMesogenin1 (reddish brown) and MesP2 (dark blue) RNAs were detected simultaneously in 9 dpc embryo by two-color whole-mount in situ hybridization. Expression of pMesogenin1 and MesP2 in PSM is mutually exclusive. This photo shows a lateral view of tailbud region. The rostral (r) and caudal (c) side of embryo is indicated. (B) Whole-mount β-galactosidase histochemical staining in pMesogenin1 null embryo of 9 dpc shows LacZ-positive cells are largely localized to the enlarged tailbud. (C) pMesogenin1 null embryo of 10.5 dpc shows no detectable segmented somites in the interlimb region indicated by two arrowheads. The null mutant also develops an enlarged tailbud and ectopic blood pools (asterisk). (D) At 14.5 dpc, the null mutant fetus lacks a tail (arrow), but possesses grossly normal limbs. (E,F) Transverse sections of both control heterozygous (E) and homozygous mutant embryos (F) of 9.5 dpc were stained with hematoxylin and eosin (H/E). Arrows indicate the boundary between paraxial mesoderm and lateral mesoderm. Paraxial mesoderm is absent in the null mutant (F). (G,H) H/E staining of parasagittal sections at interlimb region from 11.5 dpc embryos also show lack of myotomes and presence of unsegmented DRG in homozygous mutant embryos (H), but not in control heterozygous mutant embryos (G). Abbreviations: d, dorsal root ganglia; m, myotome; and nt, neural tube. (I–L) Increased apoptosis was observed in the tailbud of pMesogenin1 null embryos of 9.5 dpc. Cryosections of both control (I,K) and null mutant embryos (J,L) were subjected to TUNEL (K,L) in the presence of fluorescein-conjugated dT and costained with DAPI (I,J).

Figure 3

(A–F) Molecular components of segmentation including the Notch/Delta pathway were severely disrupted in pMesogenin1 null mutants. Expression of Notch1 (A), Notch 2 (B), Dll-1 (C), Dll-3 (D), Lunatic fringe (E), Hes1 (F), and MesP2 (G) were examined in both heterozygous and homozygous mutant embryos of 9.5 dpc. (H) Expression of pMesogenin1 in Notch1 null mutant embryos of 9 dpc. In panels A to H, the heterozygous embryos are positioned at the left side and the homozygous embryo at the right side. The PSM of control embryos and the corresponding presumptive PSM in mutant embryos are marked by black brackets. In some cases, the head of the embryo has been removed to facilitate photography. In panel A, the rostral half of the trunk of the homozygous mutant embryo is in a twisted position. The consequence is that the dorsal side of the embryo at the mid trunk level faces outward toward the reader.

Figure 4

Whole-mount in situ analysis of gene expression. In each panel, the control heterozygous embryo is located on the left and the null mutant embryo on the right. (A) Early myogenic marker Myf-5 expression in mutant embryo was normal in rostral somites, but was not detected in the trunk domain posterior to the forelimbs, where somites are normally present. The arrow indicates the boundary between Myf-5 positive and negative domains. (B) Paraxis expression was also affected in the domain posterior to forelimbs in the pMesogenin1 homozygous mutant embryo. The arrow represents the boundary between paraxis positive and negative domains. (C) Brachyury T expression in caudal paraxial mesoderm was upregulated in the null embryos of 9.5 dpc, but the expression in notochord was grossly unaffected. Arrows indicate the Brachyury T staining area where the notochord is kinked. (D) The expression of Tbx6 was not affected in the 9.5 dpc pMesogenin1 null mutants.

Figure 5

Absence of differentiated muscles in pMesogenin1 null mutants. (A–F) Expression of differentiated skeletal muscle marker α-actinin. Immunofluorescent staining of anti α-actinin antibodies on transverse sections at cervical level (A,B) and hindlimb level (E,F), and parasagittal section of interlimb region (C,D) of 11.5 dpc embryos. Myotomal staining of α-actinin in the interlimb and the hindlimb region was absent in the homozygous mutant embryos (D,F), whereas expression of α-actinin at the cervical level was unaffected in the null embryo (A,B). Arrows (panels C,D) and arrowheads (panels E,F) indicate the position of myotomes (panels C,E) in the control animals and the corresponding location in the homozygous mutants (panels D,F). An asterisk in panel F indicates a weak background staining from blood cells found in the ectopic blood pool.

Figure 6

(A–F) Whole-mount skeleton preparation of heterozygous (A,C,E) and homozygous mutants (B,D,F) of 14.5 dpc (A,B) and 17.5 dpc (C–F). Bones and cartilage stained red and blue by Alizarin Red S and Alcian Blue, respectively. All of the trunk skeleton posterior to the forelimbs (thoracic to downward) was missing in the null mutants (B,D,F), whereas head skeleton, cervical vertebrae, and limb skeleton were largely normal. Some cervical vertebrae in the null mutants were fused and malformed, indicating a milder defect than that observed in the trunk and tail domains. Abbreviations: Cn, cervical vertebrae #n; R1, rib #1; S, sternum; and T1, thoracic vertebrae #1.