Vertebrate skeletogenesis

Abstract

Vertebrate skeletogenesis consists in elaborating an edifice of more than 200 pieces of bone and cartilage. Each skeletal piece is crafted at a distinct location in the body, is articulated with others, and reaches a specific size, shape, and tissue composition according to both species instructions and individual determinants. This complex, customized body frame fulfills multiple essential tasks. It confers morphological features, allows controlled postures and movements, protects vital organs, houses hematopoiesis, stores minerals, and adsorbs toxins. This review provides an overview of the multiple facets of this ingenious process for experts as well as nonexperts of skeletogenesis. We explain how the developing vertebrate uses both specific and ubiquitously expressed genes to generate multipotent mesenchymal cells, specify them to a skeletogenic fate, control their survival and proliferation, and direct their differentiation into cartilage, bone, and joint cells. We review milestone discoveries made toward uncovering the intricate networks of regulatory factors that are involved in these processes, with an emphasis on signaling pathways and transcription factors. We describe numerous skeletal malformation and degeneration diseases that occur in humans as a result of mutations in regulatory genes, and explain how these diseases both help and motivate us to further decipher skeletogenic processes. Upon discussing current knowledge and gaps in knowledge in the control of skeletogenesis, we highlight ultimate research goals and propose research priorities and approaches for future endeavors.

Figure 1. Origin of skeletal cells in the vertebrate embryo

A. Schematic of a cross section through a mouse embryo soon after gastrulation at day 8 of development (equivalent to gestation day 17 in humans). The three germ layers are seen: ectoderm, endoderm, and mesoderm. Ectoderm-derived neural folds are rising. The mesoderm has formed the notochord and is starting to form lateral plate and paraxial derivatives on either sides of the midline. B. Schematic showing the delamination of neural crest cells from the neural folds at the time of neural tube closure. These cells are starting to migrate inside the embryo (blue arrows), where they will participate in the formation of various structures. C. Schematic showing the contribution of the neural crest, lateral plate mesoderm, paraxial mesoderm, and notochord to the three major parts of the skeleton. D. Mid-sagittal sections through the notochord of mouse embryos at the gestation days 12.5 (E12.5, top) and E15.5 (bottom). The E12.5 notochord is a rod-like structure that becomes surrounded by the mesenchymal cell condensations of the prospective vertebral bodies (VB) and intervertebral discs (IVD). E15.5 vertebral bodies are cartilaginous and notochord cells have migrated into the intervertebral disc spaces, where they have formed nuclei pulposi (NP). Sections are stained with nuclear fast red and with Alcian blue, which is specific of the notochord and cartilage extracellular matrix.

Figure 2

Fate and molecular control of skeletogenic mesenchymal cells.

Figure 3. Chondrocyte early differentiation and development of cartilage primordia

A. Alcian blue staining of a mouse embryo at E14.5 demonstrates that chondrocyte differentiation of skeletogenic cells leads to the formation of a primary skeleton that is entirely cartilaginous. B. Sections through the developing paws of mouse embryos illustrate the major steps of early chondrogenesis. At E10.5, the limb bud is filled with skeletogenic cells. By E12.5, some of these cells have formed precartilaginous condensations that prefigure the future digits. By E14.5, condensed prechondrocytes have undergone chondrocyte early differentiation. The sections are stained with Alcian blue and nuclear fast red.

Figure 4. Chondrocyte maturation and development of cartilage growth plates

A. Sections through a mouse embryo tibia illustrate the development of growth plates and endochondral bone. At E13.5, early chondrocytes in the center of cartilage primordia undergo prehypertrophic and hypertrophic maturation. They reach terminal maturation and are replaced by endochondral bone by E15.5. Later on, growth plates maintain themselves and elongate developing bones. Chondrocytes keep proliferating and give rise, layer by layer, to maturing chondrocytes. These cells which eventually die and are replaced by bone. The sections are stained with Alcian blue and nuclear fast red. B. Schematic of the molecular control of growth plate chondrocytes.

Figure 5. Osteoblast differentiation and intramembranous and endochondral ossification

A. Sections through an endochondral bone in a newborn mouse show the replacement of cartilage by bone. The left section is stained with Alcian blue and the right one with the von Kossa reagent, which leaves a brown precipitate on the mineralized bone matrix. B. Schematic showing how growth plate chondrocytes and bone-forming cells interact with each other to achieve endochondral ossification. C. Coronal sections of a newborn mouse head. In the suture linking the two frontal bones (top panel), osteoblast precursors are surrounded by an abundant collagenous matrix. Further away (bottom panel), osteoblasts mature and deposit a mineralized bone matrix. This matrix is stained with the von Kossa reagent. D. Schematic of the molecular control of osteoblast differentiation.

Figure 6. Synovial joint development

A. Sections through the mouse knee joint at various stages of development. At E12, the presumptive joint region (arrow) is not distinguishable from the femur (F) and tibia (T) precartilaginous condensations. At E13.5, this region becomes distinguishable as surrounding cartilage primordia are overtly developing. At E16.5, joint morphogenesis is well advanced. The joint cavity has formed between the patella (P) and femur. Cruciate ligaments (CL) and fat pad (FP), lined with synovial tissue, are developed. At the postnatal day 19, the knee joint is mature. The articular cartilage (AC) is separated from the epiphyseal growth plate (GP) by a secondary center of ossification. The sections are stained with Alcian blue and nuclear fast red. B. Schematic of the molecular control of synovial joint cell differentiation.