Unique Features of River Lamprey (Lampetra fluviatilis) Myogenesis

Abstract

The river lamprey (L. fluviatilis) is a representative of the ancestral jawless vertebrate group. We performed a histological analysis of trunk muscle fiber differentiation during embryonal, larval, and adult musculature development in this previously unstudied species. Investigation using light, transmission electron (TEM), and confocal microscopy revealed that embryonal and larval musculature differs from adult muscle mass. Here, we present the morphological analysis of L. fluviatilis myogenesis, from unsegmented mesoderm through somite formation, and their differentiation into multinucleated muscle lamellae. Our analysis also revealed the presence of myogenic factors LfPax3/7 and Myf5 in the dermomyotome. In the next stages of development, two types of muscle lamellae can be distinguished: central surrounded by parietal. This pattern is maintained until adulthood, when parietal muscle fibers surround the central muscles on both sides. The two types show different morphological characteristics. Although lampreys are phylogenetically distant from jawed vertebrates, somite morphology, especially dermomyotome function, shows similarity. Here we demonstrate that somitogenesis is a conservative process among all vertebrates. We conclude that river lamprey myogenesis shares features with both ancestral and higher vertebrates.

Keywords: Lampetra fluviatilis; MfPax3/7; Mrf5; embryogenesis; larval musculature; myogenesis; myogenic factors; river lamprey; somitogenesis.

Figure 1

Early stages of river lamprey (L. fluviatilis) embryogenesis. Cross (A–C) and longitudinal (E) sections of trunk muscles are stained with methylene blue. (D) Longitudinal confocal section of trunk muscles. Nuclei (blue), actin (red). Yolk platelets (white arrows), notochord (N), lipid droplets (Ld), dermomyotome (Dm), myotome (Mt), sclerotome (Sc), somite (Som). The white dashed arrow shows a posterior–anterior gradient of somitogenesis. The black dashed arrow shows the mediolateral gradient of myoblast fusion in the myotome.

Figure 2

Formation of multinucleated muscle lamellae during river lamprey (L. fluviatilis) myogenesis (stage X). (A) Cross section through the trunk muscles stained with methylene blue. (B) Ultrastructure of the embryo skeletal muscles. Inset—terminal parts of muscle lamellae. Muscle lamellae (ML, colored in blue at (B)), yolk platelets (white arrows), dermomyotome (Dm, colored in yellow), myofibrils (yellow arrows), glycogen granules (black, thin arrows), nuclei (*).

Figure 3

LfPax3/7 and Myf5 presence in the river lamprey (L. fluviatilis) dermomyotome. (A) Confocal cross section through the trunk muscles (stage XVII). LfPax3/7 (red), nuclei (blue), actin (green). (B) Confocal cross section of the trunk muscles (stage XXI). Myf5 (red), nuclei (blue), actin (green). The white arrows point out the LfPax3/7 and Myf5 signal in the nucleus. Somite (Som). Scale: 150 µm.

Figure 4

Formation of central and parietal muscle lamellae in the river lamprey (L. fluviatilis). Longitudinal (A,C) and cross sections (B) of trunk muscles stained with methylene blue. (A’–D’) Ultrastructure of skeletal muscles. (D) Confocal longitudinal section. LfPax3/7 (red), nuclei (blue), actin (green). Muscle lamellae (ML), glycogen granules (black, thin arrows), nuclei (*), parietal muscle lamellae (PML, colored in pink at (B’,C’)), central muscle lamellae (CML, colored in green at (B’,C’)), myofibrils (yellow arrows), intermyotomal space (IMS), collagen fibers (red arrows).

Figure 5

Adult musculature of river lamprey (L. fluviatilis). (A) Longitudinal sections of trunk muscles stained with methylene blue. Four central muscle fibers (CM), surrounded by groups of parietal muscle (PM) fibers. (A’) Zoomed view of CM and PM. (B) Ultrastructure of parietal muscles. (C) Ultrastructure of muscle–tendon junction. (D) Ultrastructure of muscle satellite-cell precursor (colored in pink) located along fiber. The proximity of the cell is shown in the enlarged photogram. Lipid droplets (Ld), nuclei (*), collagen fibers (red arrows), intermyotomal space (IMS), mitochondria (mt).

Figure 6

The conserved structure of river lamprey (Lampetra fluviatilis) protein LfPax3/7. (A) The aminoacidic sequences alignment of L. fluviatilis LfPax37 (GenBank: AAY90105.1, partial), Eptatretus burger paired-box protein 3/7 (GenBank: BAG11537.1), and Lethenteron camtschaticum paired-domain transcription factor Pax3/7-A (GenBank: ADP37890.1). The black frame shows the homological fragments. (B) The mouse monoclonal anti-Pax3 (Developmental Studies Hybridoma Bank, cat. # 528426) antibody epitope shows a high degree of homology with both LfPax37 orthologs present in E. burger and L. camtschaticum (indicated with a black frame). An * (asterisk) indicates positions that have a single, fully conserved residue; A: (colon) indicates conservation between groups of strongly similar properties, equivalent to scoring > 0.5 in the Gonnet PAM 250 matrix; A: (period) indicates conservation between groups of weakly similar properties, equivalent to scoring =< 0.5 and > 0 in the Gonnet PAM 250 matrix. The amino acid sequence alignment was performed using Clustal Omega, version 1.2.2, currently maintained at by Des Higgins, Fabian Sievers, David Dineen, and Andreas Wilm, Conway Institute UCD Dublin, Dublin, Ireland [56].

Figure 7

The conserved structure of river lamprey (Lampetra fluviatilis) myogenic factor Myf5. The amino acid sequences alignment of human Myf5 sequence (Gene ID: 4617) and lampreys orthologs: Petromyzon marinus factor 5-like (NCBI Reference Sequence: XP_032827047.1) and Lethenteron camtschaticum myogenic regulatory factor MRF-A (GenBank: ADP37889.1). An * (asterisk) indicates positions that have a single, fully conserved residue; A: (colon) indicates conservation between groups of strongly similar properties, equivalent to scoring > 0.5 in the Gonnet PAM 250 matrix; A: (period) indicates conservation between groups of weakly similar properties, equivalent to scoring =< 0.5 and > 0 in the Gonnet PAM 250 matrix. The amino acid sequence alignment was performed using Clustal Omega, version 1.2.2, currently maintained at by Des Higgins, Fabian Sievers, David Dineen, and Andre-as Wilm, Conway Institute UCD Dublin, Dublin, Ireland [56].