Limb Formation

• Day 24: appearance of upper limb bud
• Day 26: appearance of lower limb bud
• Week 5: tail segment present between lower limbs → gradually disappears
• Limb bud: 
  • Location: ventrolateral aspect of the body
  • Coming from lateral plate mesoderm initiated by fibroblast growth factor 10 (FGF10)
  • Covered by ectoderm Ectoderm The outer of the three germ layers of an embryo. Gastrulation and Neurulation
  • Has a core of denser condensed mesenchyme
  • At the tip: apical ectodermal ridge (AER)
  • Position:
    • Upper limb bud: opposite to caudal cervical segment
    • Lower limb bud: opposite to lumbar and sacral segments
• AER:
  • Part of the ectoderm Ectoderm The outer of the three germ layers of an embryo. Gastrulation and Neurulation that underlies the limb bud
  • Produces FGF8 to maintain elongation of the limbs
• AER exerts influence on surrounding tissue to keep it undifferentiated:
  • As AER migrates away, more proximal tissue differentiates into cartilage Cartilage Cartilage is a type of connective tissue derived from embryonic mesenchyme that is responsible for structural support, resilience, and the smoothness of physical actions. Perichondrium (connective tissue membrane surrounding cartilage) compensates for the absence of vasculature in cartilage by providing nutrition and support. Cartilage and muscle.
  • Limbs form proximally to distally in segments:
    • Stylopod: humerus and femur
    • Zeugopod: radius/ulna and tibia/fibula
    • Autopod: carpals Carpals The eight bones of the wrist: scaphoid bone; lunate bone; triquetrum bone; pisiform bone; trapezium bone; trapezoid bone; capitate bone; and hamate bone. Wrist Joint, metacarpals Metacarpals The five cylindrical bones of the metacarpus, articulating with the carpal bones proximally and the phalanges of fingers distally. Wrist Joint, digits/tarsals, digits/metatarsals
• Hand Hand The hand constitutes the distal part of the upper limb and provides the fine, precise movements needed in activities of daily living. It consists of 5 metacarpal bones and 14 phalanges, as well as numerous muscles innervated by the median and ulnar nerves. Hand and foot Foot The foot is the terminal portion of the lower limb, whose primary function is to bear weight and facilitate locomotion. The foot comprises 26 bones, including the tarsal bones, metatarsal bones, and phalanges. The bones of the foot form longitudinal and transverse arches and are supported by various muscles, ligaments, and tendons. Foot plates form as upper and lower limbs extend:
  • Initially appear like fused paddles around 6th week
  • Digital rays: appear as webbed tissue between fingers begins to undergo apoptosis
  • Day 52–56: Fingers and toes appear.

Mnemonic:

To quickly recall what happens during the 4th week, remember: 4 weeks → 4 limbs, 4 heart chambers

Rotation of the Limbs

• As the embryo Embryo The entity of a developing mammal, generally from the cleavage of a zygote to the end of embryonic differentiation of basic structures. For the human embryo, this represents the first two months of intrauterine development preceding the stages of the fetus. Fertilization and First Week changes from a flat disk to a 3-dimensional structure, the limbs rotate.
• Upper limb:
  • Week 4: Limb buds emerge from coronal plane.
  • Week 6: Limb buds move toward sagittal plane.
  • Weeks 6–8: lateral 90-degree rotation: 
    • Flexor compartments shifted anteriorly
    • Elbows located posteriorly
    • Proper allocation of dermatome innervation
• Lower limb:
  • Week 4: Limb buds emerge from coronal plane.
  • Week 6: Limb buds move toward sagittal plane.
  • Weeks 6–8: medial 90-degree rotation: 
    • Flexor compartments located posteriorly
    • Extensor compartments located anteriorly

Formation of Musculature

Overview

Muscle comes from myotome: ventrolateral cells originating from somites located on either side of neural tube that migrate to form muscles. As myotomes migrate, they form:

• Epimere (epiaxial):
  • Cluster of developing muscle from the myotome
  • Goes into the back → future intrinsic muscles of the back Muscles of the back The back is composed of several muscles of varying sizes and functions, which are grouped into intrinsic (or primary) back muscles and extrinsic (or secondary) back muscles. The extrinsic muscles comprise the superficial and intermediate muscle groups, while the intrinsic muscles comprise the deep muscles. Muscles of the Back
• Hypomere (hypoaxial):
  • Travels into the torso → anterior thoracic wall → abdominal wall → limbs
  • Extends ventrolaterally
  • Forms body-wall and limb muscle
  • After migration to the limb buds, they separate again:
    • Posterior mass (dorsal) → extensor muscles
    • Anterior mass (ventral) → flexor muscles

Upper limbs

• Somites give rise to the musculature of levels C4–C8, T1–T2.
• Posterior condensation gives rise to extensor and supinator muscles.
• Anterior condensation gives rise to flexor and pronator muscles.

Lower limbs

Nerve innervation

• Somites give rise to the musculature of levels L1–L5, S1–S2.
• Posterior condensation gives rise to extensor and abductor muscles.
• Anterior condensation gives rise to flexor and adductor muscles.
• Upper limbs: C4–T2
• Lower limbs: L4–S3
• Nerves get stretched from the spinal cord Spinal cord The spinal cord is the major conduction pathway connecting the brain to the body; it is part of the CNS. In cross section, the spinal cord is divided into an H-shaped area of gray matter (consisting of synapsing neuronal cell bodies) and a surrounding area of white matter (consisting of ascending and descending tracts of myelinated axons). Spinal Cord as upper and lower limbs develop: Brachial and lumbosacral plexus are made.

Development of Vasculature

• Blood vessels invade developing musculature around 35–36 days of gestation.
• Upper limb: segmental artery → axillary artery
• Lower limb: segmental artery → femoral artery
• Initially, arteries Arteries Arteries are tubular collections of cells that transport oxygenated blood and nutrients from the heart to the tissues of the body. The blood passes through the arteries in order of decreasing luminal diameter, starting in the largest artery (the aorta) and ending in the small arterioles. Arteries are classified into 3 types: large elastic arteries, medium muscular arteries, and small arteries and arterioles. Arteries go to the limb buds → enlarge but stay deep → capillaries Capillaries Capillaries are the primary structures in the circulatory system that allow the exchange of gas, nutrients, and other materials between the blood and the extracellular fluid (ECF). Capillaries are the smallest of the blood vessels. Because a capillary diameter is so small, only 1 RBC may pass through at a time. Capillaries form
• Venous drainage is more superficial.
• No superficial arteries Arteries Arteries are tubular collections of cells that transport oxygenated blood and nutrients from the heart to the tissues of the body. The blood passes through the arteries in order of decreasing luminal diameter, starting in the largest artery (the aorta) and ending in the small arterioles. Arteries are classified into 3 types: large elastic arteries, medium muscular arteries, and small arteries and arterioles. Arteries at the core of the limbs

Development of Limb Bones and Joints

Limb bones

• Limb buds condense → Models of hyaline cartilage Cartilage Cartilage is a type of connective tissue derived from embryonic mesenchyme that is responsible for structural support, resilience, and the smoothness of physical actions. Perichondrium (connective tissue membrane surrounding cartilage) compensates for the absence of vasculature in cartilage by providing nutrition and support. Cartilage begin to form.
• Endochondral ossification: mesenchyme → cartilage Cartilage Cartilage is a type of connective tissue derived from embryonic mesenchyme that is responsible for structural support, resilience, and the smoothness of physical actions. Perichondrium (connective tissue membrane surrounding cartilage) compensates for the absence of vasculature in cartilage by providing nutrition and support. Cartilage → bone Bone Bone is a compact type of hardened connective tissue composed of bone cells, membranes, an extracellular mineralized matrix, and central bone marrow. The 2 primary types of bone are compact and spongy. Structure of Bones:
  • Mesenchyme: loose, undifferentiated connective tissue Connective tissue Connective tissues originate from embryonic mesenchyme and are present throughout the body except inside the brain and spinal cord. The main function of connective tissues is to provide structural support to organs. Connective tissues consist of cells and an extracellular matrix. Connective Tissue
  • Cartilage: tougher, avascular; relies on diffusion
• Bone collar:
  • Layer of periosteum that surrounds the diaphysis Diaphysis The shaft of long bones. Structure of Bones
  • Formed by osteoblasts
• Primary ossification center:
  • Forms diaphysis Diaphysis The shaft of long bones. Structure of Bones—shaft of the bone Bone Bone is a compact type of hardened connective tissue composed of bone cells, membranes, an extracellular mineralized matrix, and central bone marrow. The 2 primary types of bone are compact and spongy. Structure of Bones containing bone marrow Bone marrow Bone marrow, the primary site of hematopoiesis, is found in the cavities of cancellous bones and the medullary canals of long bones. There are 2 types: red marrow (hematopoietic with abundant blood cells) and yellow marrow (predominantly filled with adipocytes). Composition of Bone Marrow cavity
  • Artery invades middle of the cartilage Cartilage Cartilage is a type of connective tissue derived from embryonic mesenchyme that is responsible for structural support, resilience, and the smoothness of physical actions. Perichondrium (connective tissue membrane surrounding cartilage) compensates for the absence of vasculature in cartilage by providing nutrition and support. Cartilage → brings osteoprogenitor cells
  • Bone formation proceeds outward
• Secondary ossification center:
  • Forms epiphysis Epiphysis The head of a long bone that is separated from the shaft by the epiphyseal plate until bone growth stops. At that time, the plate disappears and the head and shaft are united. Structure of Bones
  • At the bone Bone Bone is a compact type of hardened connective tissue composed of bone cells, membranes, an extracellular mineralized matrix, and central bone marrow. The 2 primary types of bone are compact and spongy. Structure of Bones extremities
  • Separated from primary ossification center by metaphysis
• Alkaline phosphatase: 
  • Produced by chondrocytes
  • Responsible for mineral deposition on bones
• Primary and secondary ossification centers get closer → Epiphyseal growth plate tries to proliferate and push them away.
• Epiphyseal plates remain open until the early 20s → bone Bone Bone is a compact type of hardened connective tissue composed of bone cells, membranes, an extracellular mineralized matrix, and central bone marrow. The 2 primary types of bone are compact and spongy. Structure of Bones can no longer elongate, and growth arrests
• Medullary cavity: 
  • Formed by the action of osteoclasts
  • Core of diaphysis Diaphysis The shaft of long bones. Structure of Bones
  • Major site of hematopoiesis

Joints

• Mesenchyme between the bones stays as dense irregular connective tissue Connective tissue Connective tissues originate from embryonic mesenchyme and are present throughout the body except inside the brain and spinal cord. The main function of connective tissues is to provide structural support to organs. Connective tissues consist of cells and an extracellular matrix. Connective Tissue.
• Synovial spaces form as the bones grow through the complex process of apoptosis.
• Intracapsular ligaments (anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL)):
  • Remnants of connective tissue Connective tissue Connective tissues originate from embryonic mesenchyme and are present throughout the body except inside the brain and spinal cord. The main function of connective tissues is to provide structural support to organs. Connective tissues consist of cells and an extracellular matrix. Connective Tissue
  • Dense and regular

Genetic and Molecular Patterning of Limbs

Positioning, orientation, and growth of limbs in the developing embryo Embryo The entity of a developing mammal, generally from the cleavage of a zygote to the end of embryonic differentiation of basic structures. For the human embryo, this represents the first two months of intrauterine development preceding the stages of the fetus. Fertilization and First Week is determined genetically and by the expression of specific timed chemical signals.

Craniocaudal axis

Positioning of limbs along the craniocaudal axis is determined by the homeobox (HOX) genes.

• This segment of genes (60 amino acids long) encodes for DNA DNA The molecule DNA is the repository of heritable genetic information. In humans, DNA is contained in 23 chromosome pairs within the nucleus. The molecule provides the basic template for replication of genetic information, RNA transcription, and protein biosynthesis to promote cellular function and survival. DNA Types and Structure-binding proteins, which regulate gene expression.
• The HOX-containing genes regulate multiple aspects of cell growth and differentiation. 
• These genes are arranged sequentially in a cranial-to-caudal manner, and they determine the overall pattern and shape of the developing embryo Embryo The entity of a developing mammal, generally from the cleavage of a zygote to the end of embryonic differentiation of basic structures. For the human embryo, this represents the first two months of intrauterine development preceding the stages of the fetus. Fertilization and First Week. 
• For example:
  • HOXB8 expressed at cranial border of forelimb
  • Mutations in this gene alter the position of these limbs. 
  • Forelimb positioning is determined by the transcription Transcription Transcription of genetic information is the first step in gene expression. Transcription is the process by which DNA is used as a template to make mRNA. This process is divided into 3 stages: initiation, elongation, and termination. Stages of Transcription factor T-box transcription Transcription Transcription of genetic information is the first step in gene expression. Transcription is the process by which DNA is used as a template to make mRNA. This process is divided into 3 stages: initiation, elongation, and termination. Stages of Transcription factor 5 (TBX5); hindlimb positioning is determined by TBX4.

Proximodistal axis

• Proximodistal patterning is regulated by maintaining the AER distally as the embryo Embryo The entity of a developing mammal, generally from the cleavage of a zygote to the end of embryonic differentiation of basic structures. For the human embryo, this represents the first two months of intrauterine development preceding the stages of the fetus. Fertilization and First Week develops.
• Radical fringe is a signaling factor expressed in the dorsal ½ of the limb ectoderm Ectoderm The outer of the three germ layers of an embryo. Gastrulation and Neurulation:
  • Induces the expression SER2 at the border between cells that are expressing radical fringe and those that are not
  • AER is localized to that border.
• Once AER is developed:
  • Expresses multiple classes of FGF 
  • Maintains the undifferentiated area of profusely replicating mesenchyme cells immediately proximal to the AER
  • As the cells duplicate, the limb grows distally.
  • The further the mesenchymal cells are from the AER, the less FGF can influence the mesenchymal cells, which can then differentiate thanks to the influence of other signaling molecules:
    • Retinoic acid: synthesized by flank mesenchyme, causes differentiation of tissue into stylopod 
    • Sonic hedgehog (SHH): expression of this gene leads to differentiation of the zeugopod and autopod

Anteroposterior axis

• Regulated by zone of polarizing activity (ZPA)
• A cluster of mesenchymal cells in the posterior portion of the limb, near the AER
• Secrete the signaling factor SHH
• The expression of SHH determines the correct anteroposterior orientation of limbs; e.g., misexpression of SHH in the anterior portion of the hand can lead to digit duplication.

Dorsoventral axis

• Ventral ectoderm Ectoderm The outer of the three germ layers of an embryo. Gastrulation and Neurulation expresses the transcription Transcription Transcription of genetic information is the first step in gene expression. Transcription is the process by which DNA is used as a template to make mRNA. This process is divided into 3 stages: initiation, elongation, and termination. Stages of Transcription factor EN1.
• EN1 blocks the secretion of WNT7a:
  • WNT7a is therefore localized to the dorsal ectoderm Ectoderm The outer of the three germ layers of an embryo. Gastrulation and Neurulation.
  • Induces expression of LMX1 → a transcription Transcription Transcription of genetic information is the first step in gene expression. Transcription is the process by which DNA is used as a template to make mRNA. This process is divided into 3 stages: initiation, elongation, and termination. Stages of Transcription factor that specifies “dorsal” orientation

Clinical Relevance

• Achondroplasia: mutation Mutation Genetic mutations are errors in DNA that can cause protein misfolding and dysfunction. There are various types of mutations, including chromosomal, point, frameshift, and expansion mutations. Types of Mutations in FGFR3 gene and corresponding protein that has a negative effect on endochondral ossification. Mutation causes overactivity of the receptor signaling pathway, which results in impaired growth and proliferation of chondrocytes. Affected individuals tend to have shortened limb bones, and facial bones do not elongate.
• Sirenomelia: also called mermaid syndrome. Sirenomelia is a rare congenital deformity in which the legs are fused together. The disorder is mainly characterized by the fusion of the legs with rotation of the fibula. Sirenomelia may also include absence of the lower spine and abnormalities of the pelvis Pelvis The pelvis consists of the bony pelvic girdle, the muscular and ligamentous pelvic floor, and the pelvic cavity, which contains viscera, vessels, and multiple nerves and muscles. The pelvic girdle, composed of 2 "hip" bones and the sacrum, is a ring-like bony structure of the axial skeleton that links the vertebral column with the lower extremities. Pelvis and renal organs. Sirenomelia has a poor prognosis, with most infants not surviving the 1st year of life.
• Syndactyly: Cutaneous syndactyly is webbing between the digits. Syndactyly occurs because of the failure of apoptosis, which is responsible for the separation of the digits. Osseous syndactyly consists of fusions of the bones of the digits.
• Amelia: absence of 1 or both limbs, which occurs because of suspension in development during the 4th week.
• Meromelia: condition in which a part of the limb is missing. Meromelia occurs because of a disturbance in development during the 5th week of gestation. Examples include hemimelia (absence of fibula) and phocomelia (hands and feet attached directly to the body).