{{ANAT2341Project2014header}}
=Musculoskeletal=
This webpage will be focusing on fetal muscular development.

==Muscle development General Timeline==

Primary myofibers develop in first trimester, Secondary myofibers develop during Second and Third Trimester
We should do a brief overview of primary myofiber formation, and extensive overview of secondary myofiber formation)

==Background Early Embryonic development==
Mesenchymal progenitor cells from somites(occiptal, cervical, thoracic, lumbar, sacral), undergo multiple differentiation stages to create muscle fibers.

http://www.mdconsult.com/books/figure.do?figure=true&eid=4-u1.0-B978-1-4377-2002-0..00015-1--f0025&sectionEid=4-u1.0-B978-1-4377-2002-0..00015-1&isbn=978-1-4377-2002-0&uniqId=464007141-2 (demonstration of myotomes in week 6 and 8)

Skeletal Muscle develops from a process known as myogenesis. Mesenchymal cells differentiates into embryonic muscle cells, myoblasts. Myoblasts which have single nuclei fuse and elongate to form myotubes which are multinucleated and cylindrical.

The neural tube and notochord release signaling molecules like Shh, Wnts and [BMP]-4. These signaling molecules act on transcription factors of the MyoD family and Pax <ref name="PMID10809386"><pubmed>10809386</pubmed></ref>.The MyoD or MrF family includes MyoD, Myf-5 , myogenin and Myrf4 <ref name="PMID7748174"><pubmed>7748174</pubmed></ref>. The MyoD family of transcription factors, like MyoD are myogenic bHLH (basic helix loop helix) transcription factors. Pax-3 and the MyoD induce myogenesis, formation of myoblasts. Pax-3 also acts on c-met which is a migratory peptide.

Skeletal muscle is derived from the somites. Paraxial mesoderm segments into somite structures on both sides of the notochord and neural tube.

[[Image: Mesoderm-cartoon3.jpg]]

Somites are mesodermal structures where the dorsal most end of the somite, which is known as the dermomyotome, becomes skeletal muscle and dermis <ref name="PMID9094722"><pubmed> 9094722</pubmed></ref>. A small anterior portion of the paraxial mesoderm remains un-segmented and eventually forms some muscles of the head <ref name="PMID12587921"><pubmed> 12587921</pubmed></ref>.

The myotome lies in-between the scleratome which is ventrally located. The scleratome forms the cartilage and bone of the axial skeleton of the embryo. The dermomyotome is located dorsally and forms the first skeletal muscle in the embryo.
The medial part of the dermomyotome forms the dorsal and intecostal muscles whilst the lateral part of the dermomyotome forms the limb and ventral muscles <ref name="PMID9094722"/>.

[[Image: Somite_cartoon5.png]]

Myogenesis occurs in two phases; primary and secondary which occur in embryonic and fetal periods Primary myotubes express MHC slow myosin heavy chains <ref name="PMID21204650"><pubmed> 21204650</pubmed></ref>. The primary myotubes form the structure and scaffold upon which secondary myotubes form during secondary myogenesis which occurs in the fetal period <ref name="PMID21204650"/>.

==Molecular and Cellular regulation of fetal myogenesis==

Skeletal myofiber number is set at birth <ref name="PMID5804561"><pubmed>5804561</pubmed></ref>. This was found using experiments on mice and pigs. Similar trends have been observed in humans as found by Widdowson et al (1972) where a huge increase and then levelling of gastrocnemius myofibers were found in the gestational period <ref name="PMID5046781"><pubmed>5046781</pubmed></ref>.

Skeletal muscle development
Multipotent mesenchymal cells (MSC) form myoblasts as well as adipocytes and fibroblasts <ref name="PMID10102814"><pubmed>10102814</pubmed></ref>. Therefore specific the nutrients and growth are important in directing the growth of these MSC into either adipocytes or myoblasts <ref name="PMID23100595"><pubmed>23100595</pubmed></ref>.
Myoblasts differentiate form the embryonic, fetal and adult skeletal muscle and in fact myoblasts differentiate to form either three early on. Myoblasts fuse and form primary myoblast or myotubes. As discussed previously the primary myotubes form the scaffolding for which the fetal myoblasts will differentiate into secondary myotubes and add too <ref name="PMID640968 "><pubmed>640968 </pubmed></ref>. There are a greater deal of secondary myofibers than primary and are receptive to nutrients and growth factors.
Muscle regulatory factors (MRFs) control the proliferation of secondary myoblasts. MRF’s are helix-loop-helix transcription factors.
Myoblasts early on express PAX7 and MYF5. PAX7 and MYF5 act on regulatory proteins of the cell cycle like CDK4 and Cyclin D1. CDK4 and Cyclin D1 dephosphorylate RB. RB when phosphorylated is active and inhibits the cell-cell progression in the cell cycle and hence inhibits proliferation. Therefore Cyclin and CDK4 in this case induces cell proliferation. <ref name="PMID24532817 "><pubmed>24532817</pubmed></ref>; <ref name="PMID22445545 "><pubmed>22445545</pubmed></ref>, <ref name="PMID12242286 "><pubmed>12242286</pubmed></ref> These creates more fetal myoblasts committed which are committed or programmed to form fetal or secondary myofibers later on. These committed myoblasts express MRF, MYOD. MYOD acts on myostatin to take the myoblast out of the cell cycle and stop it from proliferating <ref name="PMID24532817 "><pubmed>24532817</pubmed></ref>; <ref name="PMID12242286 "><pubmed>12242286</pubmed></ref>.Now the myoblast is ready for differentiation. The phosphorylation of RB at this period helps to slow the activity of the cell cycle. The myoblasts differentiate and fuse to become multinucleated myotubes as discussed previously. In the formed myotubes MRFs like Myogenin and MRF4 act to inhibit cell cycle and proliferation by acting regulatory proteins like P21 to stop the cell cycle <ref name="PMID24532817"/>; <ref name="PMID10733231 "><pubmed>10733231</pubmed></ref> This allows the conversion of the myotube into a myofiber. The myofiber can express desmin an intermediate filament that is increasingly seen as gestation progresses. Growth factors, amino acids and stretch/load activity act on myofibers to affect subsequent hypertrophy maturation of myofibers <ref name="PMID 24532817 "/>

[[File:Mouse limb tissue development.jpg|border|right|600px]]

Myofiber hypertrophy is occurs when protein synthesis is greater than protein degradation. The resulting accumulation of protein results in hypertrophy. Therefore maintaining protein levels is important in the hypertrophy of myofibers. This is regulated by nutrients and growth factors (GFs).
Growth factors
There are a large number of growth factors that affect fetal myogenesis. Some of the main GFs as Brown (2014) describes in her article “IGF1, insulin, basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF- β)” <ref name="PMID 24532817"/>; <ref name="PMID2190237 "><pubmed> 2190237</pubmed></ref>, <ref name="PMID22682632"><pubmed> 22682632</pubmed></ref>. Experiments have shown that if IGF1 is removed reduced muscle mass and hypoplasia has resulted. And increased IFG1 expression has resulted in hyperplasia and increased skeletal muscle. IFG1 has also been found to enhance protein synthesis. <ref name="PMID 24532817"/>; <ref name="PMID3546571"><pubmed> 3546571</pubmed></ref>, <ref name="PMID 7744859"><pubmed> 7744859</pubmed></ref>. Insulin similarly has been found to promote fetal muscle growth and protein synthesis.
Basic fibroblast growth factor (bFGF) and transforming growth factor-β (TGF- β) induce proliferation and myogenesis by upregulating cyclin D.

Nutrients
Being the building block of proteins, amino acids are important in muscle protein synthesis. But they are more important in adult muscle protein synthesis than fetal muscle protein synthesis. Experiments have shown for example an amino acid infusion did not always result in a fetal muscle growth, only when there was a rise in the insulin levels. And little is known of the interaction of amino acids and growth factors in the context of fetal myogenesis.
Research has shown that fetal or secondary myofibers are more prone to suffer from nutrient deficiency than primary myofibers in pigs and sheep. <ref name="PMID 2041547"><pubmed> 2041547</pubmed></ref>, <ref name="PMID 8014156"><pubmed> 8014156</pubmed></ref>, <ref name="PMID 15317692"><pubmed> 15317692</pubmed></ref>.

Fetal myofiber number have been observed to decrease with detal nutrient deficiency. This is concerning as myofiber numbers are set at birth.
Nutrients are also important for fetal myofiber hypertrophy.
Stretch and loading
Stretch and loading also affect hypertrophy. <ref name="PMID23629510"><pubmed>23629510</pubmed></ref>.

==Tendon Development==

Tendons are connective tissue which join muscle and bone allowing the transmission of force. Tendons primary embryonic structure originate from mesenchymal progenitor somite cells with further contributions of Neural Crest and Lateral plate mesoderm.

Appearance of tendons begins in the 20th Carnegie stage and marks the beginning of fibrillogenesis. This process is initiated by fibroblasts in series of extracellular compartments; they enlarge the cells domain into extracellular space. Channels deep in the cytoplasm drive the process of elongation, these channels location is associated with Golgi bodies.

First type of compartments are formed by collagen containing secretory vacuoles which fuse with surrounding cell membranes. Initial fibrillogenesis is mediated my macromolecular interactions based on vacuole content, with a lesser input from receptor membrane interactions. Fibril groups as fibres close to the cell surface and Secondary extracellular compartments form; at this stage fibroblast are arranged adjacently. Third level of compartmentalization forms later when fibroblast are adjacent with 2 or more other fibroblasts. As the tendon matures fibres coalesce invading each other with interdigitating processes. Secretory vacuole persist, laterally aggregating with further growth preserving sites for fibril deposit.
Collagen fiber assembly branch creating fibre networks along fascicles. Fibres branch within tendon fascicle

The main regulatory factors of tendon fibrillogenesis are Leucine-rich repeat proteoglycans.

{| class="wikitable"
|-
! Non-fibrillar components!! Molecular Characteristics!! Function
|-
| Fibromodulin || Keratin sulphate, proteoglycan || Fibromodulin modulates the site-specific cross-linking ultrastructure of collagen, ensuring mechanical strength. Control the pattern of lysyl oxidase-mediated collagen cross-linking by reducing access of the enzyme to telopeptides, by binding to the collagen. <ref name="PMID 24849606"><pubmed>24849606</pubmed></ref>
|-
| Decorin || Chondoirin sulphate, proteoglycan || Regulates expression of multiple leucine-rich proteoglycansins(SLRP) during tendon fibrillogenesis, via Class I and II Small(SLRP). Competes with Biglycan for binding sights on collagen types I-VI . Concentration increases as fetal development continues. <ref name="PMID 16518859"><pubmed>16518859</pubmed></ref>
|-
| Biglycan ||Chrondroitin sulphate and Dermatan sulphate, proteoglycan|| Process of regulation closely mimic Decorin though concentration is maximal expression at day 16-18 during embryonic development, reducing after this point. Competes with Decorin for bonding sights on collagen I-VI. <ref name="PMID 16810681"><pubmed>16810681</pubmed></ref>
|}

==Historical findings==

No studies focusing on the fetal development of muscle existed before the 1970's. During the 70's a majority of research in the field focused on the histologist differences and development of differing fiber types. Research on morphological development was very limited with eh exception of two Czechoslovakian studies in the late 1980's which displayed muscle formation for multiple primordia and sexual differentiation.

The differing developments of alpha and beta fibers was revealed in a study by University of California published in 1972 using lamb fetuses as an experimental model. Beta muscle fibers are formed during the first stages of fusion, the individual Beta fibers create a network for the alpha fibers to develop on. Red muscle fasciculi are formed by merging of small fiber bundles. White muscles are formed by ongoing addition of alpha fibers. Additionally it was concluded that fetal muscle contraction didn't significantly effect number of fibers present. Most fibers had already been formed by the 20th week, during this period the limit muscle contractions result in little mechanical tension.

===Gluteus Maximus muscle Morphogenesis===

A 1985 Czechoslovakian study investigated the development of Gluteus Maximus during the embryonic and fetal periods. Pelvic micro-dissection of human embryos and foetuses with crown length varying from 22-215mm were compared with newborns and adults. The presence of a muscle not present in post-natal adults<ref name="PMID 5043313"><pubmed>5043313</pubmed></ref> was discovered called the coccygeofemoralis or pars coccygea; this muscle originates from sides of coccyx and inserts onto the gluteal tuberosity. In the adult human this muscle fuses with the larger pars sacroiliac or fetal gluteus maximus to create the adult gluteus maximus. Pars sacroiliac originates from ilium and sacrum and inserts onto the gluteal tuberosity<ref name="PMID 4083527"><pubmed>4083527</pubmed></ref>.

In fetus with crown length of less than 45 mm the two muscle primordia are separated by small amount of loose connective tissue. This point onwards the muscles become fused by a small furrow which persists until 215mm crown length. By the time of birth the furrow is absence and the muscles are entirely fused <ref name="PMID 4083527"><pubmed>4083527</pubmed></ref>. The coccygeofemoralis in long tailed mammals remains separate from the gluteus maximus, known as the caudofemeralis muscle <ref name="PMID 1255730"><pubmed>1255730</pubmed></ref>. In these animals its function is lateral flexion of the tail <ref name="PMID 8843689"><pubmed>8843689</pubmed></ref>. This is the sole morphogenesis study on any of the large muscles; it is likely other muscles with multiple origins and insertion have separate fetal muscle primordia.

===Morphogenesis of human sphincter urethrae muscle===

This study was completed in 1989 at the same Czechoslovakian institute of that of Gluteus maximus morphogenesis. . Study displayed three developmental phasess differentiating them by morphogenesis, histology and sexual dimorphism. External urethral sphincter in embryos and fetuses with crown length varying 18-320mm, neonates, children and adults was fixed in formaldehyde and embedded in paraplast. Then cut in series and stained with hematoxylin and eosin for histological analysis. Morphogenesis and Sexual dimorphism specimens were micro-dissected using a stereomicroscope.
{| class="wikitable"
|-
! Phase!! Time Period!! Characteristics
|-
| Indifferent Phase || Before 10th week || Muscle primordia discernible from neighbouring muscle by week 8. Grow to forms a shallow arch; connecting the urethra and urogenital diaphragm. Consists of condensation of myoblasts up to 9.5 weeks, past this point myotubes and muscle fibres appear.
|-
| Sexual dimorphic Phase || 10th week to Birth || Associated with the development of prostate and vagina. Primordia spread along urethra wall posteriorly.
In Males: Spreads to create the infraprostatic part of external urethral sphincter. Arches anteriorly to join prostate and urethra
In Female: Spreads to create upper part of external urethral sphincter. Lower sixth of sphincter connects anterior and lateral urethral walls, additionally projects to lateral vaginal walls.
|-
| Definite Structuring Phase ||After birth|| Position of urethral sphincter does not alter in relation with prostate and inferior part of vagina. Infraprostatic region in males and upper part in females grow to form a complete ring.
|}

==Current Research and Findings==

===Intermediate tendon of Human Digastricus and Omohyoideus===

Early Fetal Development of the Intermediate Tendon of the Human Digastricus and Omohyoideus Muscles: A Critical Difference in Histogenesis
Katori et al (2011) did a study to understand the development of the intermediate tendon within digastricus and omohyoideus muscles. Both digastricus and omohyoideus have two bellies of muscle with an intermediate tendon. Digastricus has an anterior and posterior belly whilst omohyoideus has a superior and inferior belly. Omohyoideus is supplied by ansa cervicalis and digastricus is supplied by cranial nerves; trigeminal and facial. The difference in innervation gives reason to believe that these muscles are different developmentally. Katori et al (2011) investigate the differences in the formation of the intermediate tendon between these two muscles.
Katori et al (2011) observed in week 7-9 the posterior belly of digastricus was developing the intramuscular tendon, intermediate tendon, with a bulb like terminal tendon. The anterior belly however did not have any intramuscular tendon present within it. The anterior bellies of 50% of specimens moved towards and attached on the bulb like terminal part of tendon. Instead of finding its insertion on reichart’s cartilage or hyoid bone it attaches to terminal pat of posterior tendon. Katori et al hypothesize that this is due impediment such as stylohyoideus, vascular arteries and hypoglossal nerve. How these structures may affect digastricus is for example the thicker hypoglossal nerve separating the posterior tendon from attaching the hyoid bone. Also the stylohoideus’ caudal end may form from mesenchymal condensation near the caudel end of Reichart’s cartilage inhibiting attachment by digastricus. This shows how timing of muscle development affects topography. But eventually muscle fibers in posterior belly intramuscular tendon get converted. The caudal and bulb end of the posterior belly becomes into a tight intermediate tendon at 15 weeks. In half of the specimens in week 15 and week 18 it was observed by desmin immunohistochemistry that anterior belly muscle fibers were integrated into bulb-like and caudal end of posterior tendon.
The omohyoid at 7-9 weeks had a nearly straight and superior-inferior path. Omohyoid becomes angulated by the lateral expansion of the clavicle and shoulder joint (Katori et al, 2011). Omohyoid is tightly fitted in-between think sternocleidomastoid, scalene muscles. The fascia and lymphatic tissue also press the omohyoid towards the sternocleidomastoid. At this time during 7-9 weeks unlike digastricus, omohyoid is a single muscle belly. It reaches its greatness thickness at week 15 at 0.5mm then at the point where tendon develops the thickness decreases. In omohyoideus the intermediate tendon forms secondarily. From week 18 to week 20 the intermediate tendon muscle fibers are replaced by collagen fibers most likely after the tendon has fully shaped itself. Cells along the medial margin of omohyoid were found to be vimentin positive. As vimentin is a intermediate filament its presence is an indication mechanical or osmotic stress (Pekny and Lane, 2007).

===Stapedius===

Rodri´guez-va´ zquez et al (2010) in order to understand the isolated case of a unilateral agenesia of the stapedius tendonduring week 14 of post-conception development (PCd), have tried to understand the way the stapedius muscle develops. The stapedius is essentially formed by two anlagen; one anlagen forms the tendon of the stapedius and the other forms the muscle belly of stapedius. The anlagen forming the tendon are derived from the internal segment of interhyale. The anlagen forming the muscle belly are derived from the 2nd pharyngeal arch near the interhyale, medial to the facial nerve. Interhyale is the internal part of the second branchial arch and it develops into stapedius’ tendon (http://www.drugs.com/dict/interhyale.html ). The observed unilateral agenesia of the tendon of stapedius was found by Rodriguez-va et al (2010) to be due to the internal segment of the interhyale regression. Instead the belly of stapedius was accompanied with a pseudo tendon formed by the external segment of the interhyale. To come to this conclusion the formation of the stapedius and pyramidal eminence was tracked.
Interhyale was observed as a mesenchymal condensation formed at O’Rahilly stage 16 at cranial component of the second branchial arch. The stapes and reichert cartilage are eventually differentiated from the interhyale. At Stage 18 and 19 a mesenchymal bridge forms by the interhyale which bridges stapes and cranial part laterohyale (Reichert’s cartilage). And by stage O’Rahilly stages 20 and 21 the interhyale fully develops.
Rodriguez-va et al (2010) found the belly of stapedius to derive from a blastema which develops adjacent the interhyale to form its own anlage. In O’Rahilly stage 22 Rodriguez-va et al (2010) found that the interhyale began to take on an angular shape and the anlage of the stapedius belly connected with the vertex of the now angular interhyale.
Interhyale developed two segments; internal thick segment and an external thin segment. The thick internal segment as discussed contributes to the formation of the tendon of the stapedius muscle. The external segment begins to regress in the embryonic period and by the fetal period, week 9, the external segment was observed to be fully regressed.
In weeks 10-11 a conical belly continuous with the stapedius tendon was observed and the stapedius tendon attached at the back of the stapes head.The anlage of the pyramidal eminence formed around week 12-14 and around the stapedius muscle belly. The mesenchymal condensation forming the pyramidal eminence grew until it was inhibited around week 15-17.
After week 9 there was not much morphological change of the stapedius. From this study we can see how the stapedius muscle develops its shape and attachments in the embryonic and fetal periods.

==Abnormalities==

===Duchenne Muscular Dystrophy(DMD)===

Caused by a mutation of the dystrophin protein on locus Xp21; this protein complex connects the cytoskeleton of muscle fibres to the extracellular matrix. Abnormal dystrophin results in a degradation of cellular integrity, excessive penetration of sarcolemma by calcium and water entering mitochondria increasing pressure and bursting. The lack of significant load during fetal development results in minimal wasting, it is detected in postnatal babies at 3-5 years old with muscles resisting gravity being the first to waste. Patients are restricted to wheelchairs by their early teens and have a life expectancy of 25 years. The incidence in male infants is 1 in 36,000.
Typically males are affected while females are carriers. In the offspring of a carrier mother and unaffected father; sons have 50% chance of affected and daughter’s 50% chance of becoming carriers. Since the disease is a terminal illness killing in mid-twenties; affected fathers are not considered in the situation. Absence of affected fathers means it is very unlikely for daughters to be affected.

Duchenne Muscular Dystrophy has no present cure, treatment is aimed at altering onset of symptoms and maximising quality of life, these include: <ref name="PMID 25187493"><pubmed> 25187493</pubmed></ref>

*Corticosteroid based medication significantly improves muscle strength and function over the short term. Patients using the drugs long term gradually have reduced doses to avoid severe side effects which can include; weight gain, behavioural disorders and osteoporosis. Most effective corticosteroids are prednisolone, and deflazacort. Theories on how the steroids work include; include activation of T-Cell pathways, directly reducing muscle regeneration, modulating cell inflammation and enhancement of myogenic precursors. <ref name="PMID 17541998"><pubmed> 17541998</pubmed></ref>

===Amyoplasia===

Amyoplasia is characterised by replacement of newborn muscle tissue with fat and dense fibrous tissue. Is the most common of Arthrogryposis multiplex congetia disorders; these result in multiple joint contractures. Affected limbs have significantly altered positioning, typically clubfoot is present and elbows are extended<ref name="PMID 24459070"><pubmed>24459070</pubmed></ref>. Primary cause is limited fetal movement, muscle is replaced by dense fibrous tissue. Conditions which limit fetal movement include abnormal uterus morphology and reduced amniotic fluid. Presently no genes have been linked to the deformity. <ref name="PMID 9260643"><pubmed>9260643</pubmed></ref>
85-90% of newborns undergo surgery within days of birth primarily on the legs and hips, releasing tendons from contractures. Motion can be improved by casting and splinting, lower limbs are typically cast and upper limbs are typically splinted.
<ref name="PMID 24459070"><pubmed>24459070</pubmed></ref>

{| class="wikitable"
|-
! Involvement subtype!!Percentage of Case
|-
| Four-limb symmetrical || 55%
|-
| Three limb || 5%
|-
| Upper limb only || 17%
|-
| Lower limb only ||16%
|}

===Nemaline Rod Myopathy===

Nemaline rod myopathy is congenital non-dystrophic muscle disease. Sufferers experience general muscle weakness, weakness is most severe in face, neck and limbs. Further clinical manifestations include feeding problems, scoliosis of spine, foot deformities and respiration difficulties. The disease is an inherited both autosomal dominant and recessive, 30% autosomal dominant, 20% autosomal recessive and 50% simplex.

Mutations in six different genes are associated with nemaline myopathy, with nebulin mutations being the most common. No treatments or disease-modifying therapies have been identified for this disease. One of the major barriers to treatment development is the lack of models amenable to rapid and coordinated testing of potential therapeutic strategies. To overcome this barrier, we have characterized the first zebrafish model of nemaline myopathy. This model, termed neb, harbors a recessive mutation in the nebulin gene that results in decreased Nebulin protein levels, a severe motor phenotype and premature lethality. In addition to impaired motor function, neb zebrafish exhibit many of the features associated with human nemaline myopathy. These include impaired force generation, altered thin filament length and the presence of specific histopathological changes, including the formation of nemaline bodies. In summary, neb zebrafish mirror the genetic, clinical and pathological aspects of nemaline myopathy due to NEB mutation, and thus are an excellent model for future therapy development for this devastating disorder.

==References==
Anatomy and variations of palmaris longus in fetuses.<ref><pubmed> 23529313</pubmed>| [http://www.ncbi.nlm.nih.gov/pubmed/23529313]</ref>

Development of the rectus abdominis and its sheath in the human fetus.<ref><pubmed> 22869489</pubmed>| [http://www.ncbi.nlm.nih.gov/pubmed/22869489]</ref>

Sonic hedgehog acts cell-autonomously on muscle precursor cells to generate limb muscle diversity.<ref><pubmed> 22987640</pubmed>| [http://www.ncbi.nlm.nih.gov/pubmed/22987640]</ref>

The normal growth of the biceps brachii muscle in human fetuses.<ref><pubmed>23468258</pubmed>| [http://www.ncbi.nlm.nih.gov/pubmed/23468258]</ref>

<references/>

References to format in later

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Birk, D. and Trelstad, R. (1986). Extracellular compartments in tendon morphogenesis: collagen fibril, bundle, and macroaggregate formation. The Journal of cell biology, 103(1), pp.231--240.

Deries, M., Gon\ccalves, A., Vaz, R., Martins, G., Rodrigues, G. and Thorsteinsd\'ottir, S. (2012). Extracellular matrix remodeling accompanies axial muscle development and morphogenesis in the mouse.Developmental Dynamics, 241(2), pp.350--364.

Dom\`enech-Mateu, J., Mart\'\inez-Pozo, A. and Arn\'o-Palau, A. (1994). Development of the tendon of todaro during the human embryonic and fetal periods. The Anatomical Record, 238(3), pp.374--382.
Harel, I., Maezawa, Y., Avraham, R., Rinon, A., Ma, H., Cross, J., Leviatan, N., Hegesh, J., Roy, A., Jacob-Hirsch, J. and others, (2012). Pharyngeal mesoderm regulatory network controls cardiac and head muscle morphogenesis. Proceedings of the National Academy of Sciences, 109(46), pp.18839--18844.

Herchenhan, A., Bayer, M., Svensson, R., Magnusson, S. and Kj\aer, M. (2013). In vitro tendon tissue development from human fibroblasts demonstrates collagen fibril diameter growth associated with a rise in mechanical strength. Developmental Dynamics, 242(1), pp.2--8.

Hoffman, E., Brown Jr, R. and Kunkel, L. (1987). Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell, 51(6), pp.919--928.

Katori, Y., Hyun Kim, J., Rodr\'\iguez-V\'azquez, J., Kawase, T., Murakami, G. and Hwan Cho, B. (2011). Early fetal development of the intermediate tendon of the human digastricus and omohyoideus muscles: a critical difference in histogenesis. Clinical Anatomy, 24(7), pp.843--852.

KINMONT, P. (2008). development of the human achilles tendon enthesis organ. Journal of Anatomy.

Nichol, P., Corliss, R., Yamada, S., Shiota, K. and Saijoh, Y. (2012). Muscle Patterning in Mouse and Human Abdominal Wall Development and Omphalocele Specimens of Humans. The Anatomical Record, 295(12), pp.2129--2140.

Rodriguez-Guzman, M., Montero, J., Santesteban, E., Ga\~nan, Y., Macias, D. and Hurle, J. (2007). Tendon-muscle crosstalk controls muscle bellies morphogenesis, which is mediated by cell death and retinoic acid signaling. Developmental biology, 302(1), pp.267--280.

Rodr\'\iguez-V\'azquez, J., M\'erida-Velasco, J. and Verdugo-L\'opez, S. (2010). Development of the Stapedius Muscle and Unilateral Agenesia of the Tendon of the Stapedius Muscle in a Human Fetus. The anatomical record, 293(1), pp.25--31.

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2014 Group Project 8

2014-10-22T03:39:53Z

Z3418779: /* Molecular and Cellular regulation of fetal myogenesis */

{{ANAT2341Project2014header}}
=Musculoskeletal=
This webpage will be focusing on fetal muscular development.

==Muscle development General Timeline==

Primary myofibers develop in first trimester, Secondary myofibers develop during Second and Third Trimester
We should do a brief overview of primary myofiber formation, and extensive overview of secondary myofiber formation)

==Background Early Embryonic development==
Mesenchymal progenitor cells from somites(occiptal, cervical, thoracic, lumbar, sacral), undergo multiple differentiation stages to create muscle fibers.

http://www.mdconsult.com/books/figure.do?figure=true&eid=4-u1.0-B978-1-4377-2002-0..00015-1--f0025&sectionEid=4-u1.0-B978-1-4377-2002-0..00015-1&isbn=978-1-4377-2002-0&uniqId=464007141-2 (demonstration of myotomes in week 6 and 8)

Skeletal Muscle develops from a process known as myogenesis. Mesenchymal cells differentiates into embryonic muscle cells, myoblasts. Myoblasts which have single nuclei fuse and elongate to form myotubes which are multinucleated and cylindrical.

The neural tube and notochord release signaling molecules like Shh, Wnts and [BMP]-4. These signaling molecules act on transcription factors of the MyoD family and Pax <ref name="PMID10809386"><pubmed>10809386</pubmed></ref>.The MyoD or MrF family includes MyoD, Myf-5 , myogenin and Myrf4 <ref name="PMID7748174"><pubmed>7748174</pubmed></ref>. The MyoD family of transcription factors, like MyoD are myogenic bHLH (basic helix loop helix) transcription factors. Pax-3 and the MyoD induce myogenesis, formation of myoblasts. Pax-3 also acts on c-met which is a migratory peptide.

Skeletal muscle is derived from the somites. Paraxial mesoderm segments into somite structures on both sides of the notochord and neural tube.

[[Image: Mesoderm-cartoon3.jpg]]

Somites are mesodermal structures where the dorsal most end of the somite, which is known as the dermomyotome, becomes skeletal muscle and dermis <ref name="PMID9094722"><pubmed> 9094722</pubmed></ref>. A small anterior portion of the paraxial mesoderm remains un-segmented and eventually forms some muscles of the head <ref name="PMID12587921"><pubmed> 12587921</pubmed></ref>.

The myotome lies in-between the scleratome which is ventrally located. The scleratome forms the cartilage and bone of the axial skeleton of the embryo. The dermomyotome is located dorsally and forms the first skeletal muscle in the embryo.
The medial part of the dermomyotome forms the dorsal and intecostal muscles whilst the lateral part of the dermomyotome forms the limb and ventral muscles <ref name="PMID9094722"/>.

[[Image: Somite_cartoon5.png]]

Myogenesis occurs in two phases; primary and secondary which occur in embryonic and fetal periods Primary myotubes express MHC slow myosin heavy chains <ref name="PMID21204650"><pubmed> 21204650</pubmed></ref>. The primary myotubes form the structure and scaffold upon which secondary myotubes form during secondary myogenesis which occurs in the fetal period <ref name="PMID21204650"/>.

==Molecular and Cellular regulation of fetal myogenesis==

Skeletal myofiber number is set at birth <ref name="PMID5804561"><pubmed>5804561</pubmed></ref>. This was found using experiments on mice and pigs. Similar trends have been observed in humans as found by Widdowson et al (1972) where a huge increase and then levelling of gastrocnemius myofibers were found in the gestational period <ref name="PMID5046781"><pubmed>5046781</pubmed></ref>.

Skeletal muscle development
Multipotent mesenchymal cells (MSC) form myoblasts as well as adipocytes and fibroblasts <ref name="PMID10102814"><pubmed>10102814</pubmed></ref>. Therefore specific the nutrients and growth are important in directing the growth of these MSC into either adipocytes or myoblasts <ref name="PMID23100595"><pubmed>23100595</pubmed></ref>.
Myoblasts differentiate form the embryonic, fetal and adult skeletal muscle and in fact myoblasts differentiate to form either three early on. Myoblasts fuse and form primary myoblast or myotubes. As discussed previously the primary myotubes form the scaffolding for which the fetal myoblasts will differentiate into secondary myotubes and add too <ref name="PMID640968 "><pubmed>640968 </pubmed></ref>. There are a greater deal of secondary myofibers than primary and are receptive to nutrients and growth factors.
Muscle regulatory factors (MRFs) control the proliferation of secondary myoblasts. MRF’s are helix-loop-helix transcription factors.
Myoblasts early on express PAX7 and MYF5. PAX7 and MYF5 act on regulatory proteins of the cell cycle like CDK4 and Cyclin D1. CDK4 and Cyclin D1 dephosphorylate RB. RB when phosphorylated is active and inhibits the cell-cell progression in the cell cycle and hence inhibits proliferation. Therefore Cyclin and CDK4 in this case induces cell proliferation. <ref name="PMID24532817 "><pubmed>24532817</pubmed></ref>; <ref name="PMID22445545 "><pubmed>22445545</pubmed></ref>, <ref name="PMID12242286 "><pubmed>12242286</pubmed></ref> These creates more fetal myoblasts committed which are committed or programmed to form fetal or secondary myofibers later on. These committed myoblasts express MRF, MYOD. MYOD acts on myostatin to take the myoblast out of the cell cycle and stop it from proliferating <ref name="PMID24532817 "><pubmed>24532817</pubmed></ref>; <ref name="PMID12242286 "><pubmed>12242286</pubmed></ref>.Now the myoblast is ready for differentiation. The phosphorylation of RB at this period helps to slow the activity of the cell cycle. The myoblasts differentiate and fuse to become multinucleated myotubes as discussed previously. In the formed myotubes MRFs like Myogenin and MRF4 act to inhibit cell cycle and proliferation by acting regulatory proteins like P21 to stop the cell cycle <ref name="PMID24532817"/>; <ref name="PMID10733231 "><pubmed>10733231</pubmed></ref> This allows the conversion of the myotube into a myofiber. The myofiber can express desmin an intermediate filament that is increasingly seen as gestation progresses. Growth factors, amino acids and stretch/load activity act on myofibers to affect subsequent hypertrophy maturation of myofibers <ref name="PMID 24532817 "/>

[[File:Mouse limb tissue development.jpg|border|right|300px]]

Myofiber hypertrophy is occurs when protein synthesis is greater than protein degradation. The resulting accumulation of protein results in hypertrophy. Therefore maintaining protein levels is important in the hypertrophy of myofibers. This is regulated by nutrients and growth factors (GFs).
Growth factors
There are a large number of growth factors that affect fetal myogenesis. Some of the main GFs as Brown (2014) describes in her article “IGF1, insulin, basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF- β)” <ref name="PMID 24532817"/>; <ref name="PMID2190237 "><pubmed> 2190237</pubmed></ref>, <ref name="PMID22682632"><pubmed> 22682632</pubmed></ref>. Experiments have shown that if IGF1 is removed reduced muscle mass and hypoplasia has resulted. And increased IFG1 expression has resulted in hyperplasia and increased skeletal muscle. IFG1 has also been found to enhance protein synthesis. <ref name="PMID 24532817"/>; <ref name="PMID3546571"><pubmed> 3546571</pubmed></ref>, <ref name="PMID 7744859"><pubmed> 7744859</pubmed></ref>. Insulin similarly has been found to promote fetal muscle growth and protein synthesis.
Basic fibroblast growth factor (bFGF) and transforming growth factor-β (TGF- β) induce proliferation and myogenesis by upregulating cyclin D.

Nutrients
Being the building block of proteins, amino acids are important in muscle protein synthesis. But they are more important in adult muscle protein synthesis than fetal muscle protein synthesis. Experiments have shown for example an amino acid infusion did not always result in a fetal muscle growth, only when there was a rise in the insulin levels. And little is known of the interaction of amino acids and growth factors in the context of fetal myogenesis.
Research has shown that fetal or secondary myofibers are more prone to suffer from nutrient deficiency than primary myofibers in pigs and sheep. <ref name="PMID 2041547"><pubmed> 2041547</pubmed></ref>, <ref name="PMID 8014156"><pubmed> 8014156</pubmed></ref>, <ref name="PMID 15317692"><pubmed> 15317692</pubmed></ref>.

Fetal myofiber number have been observed to decrease with detal nutrient deficiency. This is concerning as myofiber numbers are set at birth.
Nutrients are also important for fetal myofiber hypertrophy.
Stretch and loading
Stretch and loading also affect hypertrophy. <ref name="PMID23629510"><pubmed>23629510</pubmed></ref>.

==Tendon Development==

Tendons are connective tissue which join muscle and bone allowing the transmission of force. Tendons primary embryonic structure originate from mesenchymal progenitor somite cells with further contributions of Neural Crest and Lateral plate mesoderm.

Appearance of tendons begins in the 20th Carnegie stage and marks the beginning of fibrillogenesis. This process is initiated by fibroblasts in series of extracellular compartments; they enlarge the cells domain into extracellular space. Channels deep in the cytoplasm drive the process of elongation, these channels location is associated with Golgi bodies.

First type of compartments are formed by collagen containing secretory vacuoles which fuse with surrounding cell membranes. Initial fibrillogenesis is mediated my macromolecular interactions based on vacuole content, with a lesser input from receptor membrane interactions. Fibril groups as fibres close to the cell surface and Secondary extracellular compartments form; at this stage fibroblast are arranged adjacently. Third level of compartmentalization forms later when fibroblast are adjacent with 2 or more other fibroblasts. As the tendon matures fibres coalesce invading each other with interdigitating processes. Secretory vacuole persist, laterally aggregating with further growth preserving sites for fibril deposit.
Collagen fiber assembly branch creating fibre networks along fascicles. Fibres branch within tendon fascicle

The main regulatory factors of tendon fibrillogenesis are Leucine-rich repeat proteoglycans.

{| class="wikitable"
|-
! Non-fibrillar components!! Molecular Characteristics!! Function
|-
| Fibromodulin || Keratin sulphate, proteoglycan || Fibromodulin modulates the site-specific cross-linking ultrastructure of collagen, ensuring mechanical strength. Control the pattern of lysyl oxidase-mediated collagen cross-linking by reducing access of the enzyme to telopeptides, by binding to the collagen. <ref name="PMID 24849606"><pubmed>24849606</pubmed></ref>
|-
| Decorin || Chondoirin sulphate, proteoglycan || Regulates expression of multiple leucine-rich proteoglycansins(SLRP) during tendon fibrillogenesis, via Class I and II Small(SLRP). Competes with Biglycan for binding sights on collagen types I-VI . Concentration increases as fetal development continues. <ref name="PMID 16518859"><pubmed>16518859</pubmed></ref>
|-
| Biglycan ||Chrondroitin sulphate and Dermatan sulphate, proteoglycan|| Process of regulation closely mimic Decorin though concentration is maximal expression at day 16-18 during embryonic development, reducing after this point. Competes with Decorin for bonding sights on collagen I-VI. <ref name="PMID 16810681"><pubmed>16810681</pubmed></ref>
|}

==Historical findings==

No studies focusing on the fetal development of muscle existed before the 1970's. During the 70's a majority of research in the field focused on the histologist differences and development of differing fiber types. Research on morphological development was very limited with eh exception of two Czechoslovakian studies in the late 1980's which displayed muscle formation for multiple primordia and sexual differentiation.

The differing developments of alpha and beta fibers was revealed in a study by University of California published in 1972 using lamb fetuses as an experimental model. Beta muscle fibers are formed during the first stages of fusion, the individual Beta fibers create a network for the alpha fibers to develop on. Red muscle fasciculi are formed by merging of small fiber bundles. White muscles are formed by ongoing addition of alpha fibers. Additionally it was concluded that fetal muscle contraction didn't significantly effect number of fibers present. Most fibers had already been formed by the 20th week, during this period the limit muscle contractions result in little mechanical tension.

===Gluteus Maximus muscle Morphogenesis===

A 1985 Czechoslovakian study investigated the development of Gluteus Maximus during the embryonic and fetal periods. Pelvic micro-dissection of human embryos and foetuses with crown length varying from 22-215mm were compared with newborns and adults. The presence of a muscle not present in post-natal adults<ref name="PMID 5043313"><pubmed>5043313</pubmed></ref> was discovered called the coccygeofemoralis or pars coccygea; this muscle originates from sides of coccyx and inserts onto the gluteal tuberosity. In the adult human this muscle fuses with the larger pars sacroiliac or fetal gluteus maximus to create the adult gluteus maximus. Pars sacroiliac originates from ilium and sacrum and inserts onto the gluteal tuberosity<ref name="PMID 4083527"><pubmed>4083527</pubmed></ref>.

In fetus with crown length of less than 45 mm the two muscle primordia are separated by small amount of loose connective tissue. This point onwards the muscles become fused by a small furrow which persists until 215mm crown length. By the time of birth the furrow is absence and the muscles are entirely fused <ref name="PMID 4083527"><pubmed>4083527</pubmed></ref>. The coccygeofemoralis in long tailed mammals remains separate from the gluteus maximus, known as the caudofemeralis muscle <ref name="PMID 1255730"><pubmed>1255730</pubmed></ref>. In these animals its function is lateral flexion of the tail <ref name="PMID 8843689"><pubmed>8843689</pubmed></ref>. This is the sole morphogenesis study on any of the large muscles; it is likely other muscles with multiple origins and insertion have separate fetal muscle primordia.

===Morphogenesis of human sphincter urethrae muscle===

This study was completed in 1989 at the same Czechoslovakian institute of that of Gluteus maximus morphogenesis. . Study displayed three developmental phasess differentiating them by morphogenesis, histology and sexual dimorphism. External urethral sphincter in embryos and fetuses with crown length varying 18-320mm, neonates, children and adults was fixed in formaldehyde and embedded in paraplast. Then cut in series and stained with hematoxylin and eosin for histological analysis. Morphogenesis and Sexual dimorphism specimens were micro-dissected using a stereomicroscope.
{| class="wikitable"
|-
! Phase!! Time Period!! Characteristics
|-
| Indifferent Phase || Before 10th week || Muscle primordia discernible from neighbouring muscle by week 8. Grow to forms a shallow arch; connecting the urethra and urogenital diaphragm. Consists of condensation of myoblasts up to 9.5 weeks, past this point myotubes and muscle fibres appear.
|-
| Sexual dimorphic Phase || 10th week to Birth || Associated with the development of prostate and vagina. Primordia spread along urethra wall posteriorly.
In Males: Spreads to create the infraprostatic part of external urethral sphincter. Arches anteriorly to join prostate and urethra
In Female: Spreads to create upper part of external urethral sphincter. Lower sixth of sphincter connects anterior and lateral urethral walls, additionally projects to lateral vaginal walls.
|-
| Definite Structuring Phase ||After birth|| Position of urethral sphincter does not alter in relation with prostate and inferior part of vagina. Infraprostatic region in males and upper part in females grow to form a complete ring.
|}

==Current Research and Findings==

===Intermediate tendon of Human Digastricus and Omohyoideus===

Early Fetal Development of the Intermediate Tendon of the Human Digastricus and Omohyoideus Muscles: A Critical Difference in Histogenesis
Katori et al (2011) did a study to understand the development of the intermediate tendon within digastricus and omohyoideus muscles. Both digastricus and omohyoideus have two bellies of muscle with an intermediate tendon. Digastricus has an anterior and posterior belly whilst omohyoideus has a superior and inferior belly. Omohyoideus is supplied by ansa cervicalis and digastricus is supplied by cranial nerves; trigeminal and facial. The difference in innervation gives reason to believe that these muscles are different developmentally. Katori et al (2011) investigate the differences in the formation of the intermediate tendon between these two muscles.
Katori et al (2011) observed in week 7-9 the posterior belly of digastricus was developing the intramuscular tendon, intermediate tendon, with a bulb like terminal tendon. The anterior belly however did not have any intramuscular tendon present within it. The anterior bellies of 50% of specimens moved towards and attached on the bulb like terminal part of tendon. Instead of finding its insertion on reichart’s cartilage or hyoid bone it attaches to terminal pat of posterior tendon. Katori et al hypothesize that this is due impediment such as stylohyoideus, vascular arteries and hypoglossal nerve. How these structures may affect digastricus is for example the thicker hypoglossal nerve separating the posterior tendon from attaching the hyoid bone. Also the stylohoideus’ caudal end may form from mesenchymal condensation near the caudel end of Reichart’s cartilage inhibiting attachment by digastricus. This shows how timing of muscle development affects topography. But eventually muscle fibers in posterior belly intramuscular tendon get converted. The caudal and bulb end of the posterior belly becomes into a tight intermediate tendon at 15 weeks. In half of the specimens in week 15 and week 18 it was observed by desmin immunohistochemistry that anterior belly muscle fibers were integrated into bulb-like and caudal end of posterior tendon.
The omohyoid at 7-9 weeks had a nearly straight and superior-inferior path. Omohyoid becomes angulated by the lateral expansion of the clavicle and shoulder joint (Katori et al, 2011). Omohyoid is tightly fitted in-between think sternocleidomastoid, scalene muscles. The fascia and lymphatic tissue also press the omohyoid towards the sternocleidomastoid. At this time during 7-9 weeks unlike digastricus, omohyoid is a single muscle belly. It reaches its greatness thickness at week 15 at 0.5mm then at the point where tendon develops the thickness decreases. In omohyoideus the intermediate tendon forms secondarily. From week 18 to week 20 the intermediate tendon muscle fibers are replaced by collagen fibers most likely after the tendon has fully shaped itself. Cells along the medial margin of omohyoid were found to be vimentin positive. As vimentin is a intermediate filament its presence is an indication mechanical or osmotic stress (Pekny and Lane, 2007).

===Stapedius===

Rodri´guez-va´ zquez et al (2010) in order to understand the isolated case of a unilateral agenesia of the stapedius tendonduring week 14 of post-conception development (PCd), have tried to understand the way the stapedius muscle develops. The stapedius is essentially formed by two anlagen; one anlagen forms the tendon of the stapedius and the other forms the muscle belly of stapedius. The anlagen forming the tendon are derived from the internal segment of interhyale. The anlagen forming the muscle belly are derived from the 2nd pharyngeal arch near the interhyale, medial to the facial nerve. Interhyale is the internal part of the second branchial arch and it develops into stapedius’ tendon (http://www.drugs.com/dict/interhyale.html ). The observed unilateral agenesia of the tendon of stapedius was found by Rodriguez-va et al (2010) to be due to the internal segment of the interhyale regression. Instead the belly of stapedius was accompanied with a pseudo tendon formed by the external segment of the interhyale. To come to this conclusion the formation of the stapedius and pyramidal eminence was tracked.
Interhyale was observed as a mesenchymal condensation formed at O’Rahilly stage 16 at cranial component of the second branchial arch. The stapes and reichert cartilage are eventually differentiated from the interhyale. At Stage 18 and 19 a mesenchymal bridge forms by the interhyale which bridges stapes and cranial part laterohyale (Reichert’s cartilage). And by stage O’Rahilly stages 20 and 21 the interhyale fully develops.
Rodriguez-va et al (2010) found the belly of stapedius to derive from a blastema which develops adjacent the interhyale to form its own anlage. In O’Rahilly stage 22 Rodriguez-va et al (2010) found that the interhyale began to take on an angular shape and the anlage of the stapedius belly connected with the vertex of the now angular interhyale.
Interhyale developed two segments; internal thick segment and an external thin segment. The thick internal segment as discussed contributes to the formation of the tendon of the stapedius muscle. The external segment begins to regress in the embryonic period and by the fetal period, week 9, the external segment was observed to be fully regressed.
In weeks 10-11 a conical belly continuous with the stapedius tendon was observed and the stapedius tendon attached at the back of the stapes head.The anlage of the pyramidal eminence formed around week 12-14 and around the stapedius muscle belly. The mesenchymal condensation forming the pyramidal eminence grew until it was inhibited around week 15-17.
After week 9 there was not much morphological change of the stapedius. From this study we can see how the stapedius muscle develops its shape and attachments in the embryonic and fetal periods.

==Abnormalities==

===Duchenne Muscular Dystrophy(DMD)===

Caused by a mutation of the dystrophin protein on locus Xp21; this protein complex connects the cytoskeleton of muscle fibres to the extracellular matrix. Abnormal dystrophin results in a degradation of cellular integrity, excessive penetration of sarcolemma by calcium and water entering mitochondria increasing pressure and bursting. The lack of significant load during fetal development results in minimal wasting, it is detected in postnatal babies at 3-5 years old with muscles resisting gravity being the first to waste. Patients are restricted to wheelchairs by their early teens and have a life expectancy of 25 years. The incidence in male infants is 1 in 36,000.
Typically males are affected while females are carriers. In the offspring of a carrier mother and unaffected father; sons have 50% chance of affected and daughter’s 50% chance of becoming carriers. Since the disease is a terminal illness killing in mid-twenties; affected fathers are not considered in the situation. Absence of affected fathers means it is very unlikely for daughters to be affected.

Duchenne Muscular Dystrophy has no present cure, treatment is aimed at altering onset of symptoms and maximising quality of life, these include: <ref name="PMID 25187493"><pubmed> 25187493</pubmed></ref>

*Corticosteroid based medication significantly improves muscle strength and function over the short term. Patients using the drugs long term gradually have reduced doses to avoid severe side effects which can include; weight gain, behavioural disorders and osteoporosis. Most effective corticosteroids are prednisolone, and deflazacort. Theories on how the steroids work include; include activation of T-Cell pathways, directly reducing muscle regeneration, modulating cell inflammation and enhancement of myogenic precursors. <ref name="PMID 17541998"><pubmed> 17541998</pubmed></ref>

===Amyoplasia===

Amyoplasia is characterised by replacement of newborn muscle tissue with fat and dense fibrous tissue. Is the most common of Arthrogryposis multiplex congetia disorders; these result in multiple joint contractures. Affected limbs have significantly altered positioning, typically clubfoot is present and elbows are extended<ref name="PMID 24459070"><pubmed>24459070</pubmed></ref>. Primary cause is limited fetal movement, muscle is replaced by dense fibrous tissue. Conditions which limit fetal movement include abnormal uterus morphology and reduced amniotic fluid. Presently no genes have been linked to the deformity. <ref name="PMID 9260643"><pubmed>9260643</pubmed></ref>
85-90% of newborns undergo surgery within days of birth primarily on the legs and hips, releasing tendons from contractures. Motion can be improved by casting and splinting, lower limbs are typically cast and upper limbs are typically splinted.
<ref name="PMID 24459070"><pubmed>24459070</pubmed></ref>

{| class="wikitable"
|-
! Involvement subtype!!Percentage of Case
|-
| Four-limb symmetrical || 55%
|-
| Three limb || 5%
|-
| Upper limb only || 17%
|-
| Lower limb only ||16%
|}

===Nemaline Rod Myopathy===

Nemaline rod myopathy is congenital non-dystrophic muscle disease. Sufferers experience general muscle weakness, weakness is most severe in face, neck and limbs. Further clinical manifestations include feeding problems, scoliosis of spine, foot deformities and respiration difficulties. The disease is an inherited both autosomal dominant and recessive, 30% autosomal dominant, 20% autosomal recessive and 50% simplex.

Mutations in six different genes are associated with nemaline myopathy, with nebulin mutations being the most common. No treatments or disease-modifying therapies have been identified for this disease. One of the major barriers to treatment development is the lack of models amenable to rapid and coordinated testing of potential therapeutic strategies. To overcome this barrier, we have characterized the first zebrafish model of nemaline myopathy. This model, termed neb, harbors a recessive mutation in the nebulin gene that results in decreased Nebulin protein levels, a severe motor phenotype and premature lethality. In addition to impaired motor function, neb zebrafish exhibit many of the features associated with human nemaline myopathy. These include impaired force generation, altered thin filament length and the presence of specific histopathological changes, including the formation of nemaline bodies. In summary, neb zebrafish mirror the genetic, clinical and pathological aspects of nemaline myopathy due to NEB mutation, and thus are an excellent model for future therapy development for this devastating disorder.

==References==
Anatomy and variations of palmaris longus in fetuses.<ref><pubmed> 23529313</pubmed>| [http://www.ncbi.nlm.nih.gov/pubmed/23529313]</ref>

Development of the rectus abdominis and its sheath in the human fetus.<ref><pubmed> 22869489</pubmed>| [http://www.ncbi.nlm.nih.gov/pubmed/22869489]</ref>

Sonic hedgehog acts cell-autonomously on muscle precursor cells to generate limb muscle diversity.<ref><pubmed> 22987640</pubmed>| [http://www.ncbi.nlm.nih.gov/pubmed/22987640]</ref>

The normal growth of the biceps brachii muscle in human fetuses.<ref><pubmed>23468258</pubmed>| [http://www.ncbi.nlm.nih.gov/pubmed/23468258]</ref>

<references/>

References to format in later

Biggar, W. (2006). Duchenne muscular dystrophy. Pediatrics in Review, 27(3), pp.83--88.

Birk, D. and Trelstad, R. (1986). Extracellular compartments in tendon morphogenesis: collagen fibril, bundle, and macroaggregate formation. The Journal of cell biology, 103(1), pp.231--240.

Deries, M., Gon\ccalves, A., Vaz, R., Martins, G., Rodrigues, G. and Thorsteinsd\'ottir, S. (2012). Extracellular matrix remodeling accompanies axial muscle development and morphogenesis in the mouse.Developmental Dynamics, 241(2), pp.350--364.

Dom\`enech-Mateu, J., Mart\'\inez-Pozo, A. and Arn\'o-Palau, A. (1994). Development of the tendon of todaro during the human embryonic and fetal periods. The Anatomical Record, 238(3), pp.374--382.
Harel, I., Maezawa, Y., Avraham, R., Rinon, A., Ma, H., Cross, J., Leviatan, N., Hegesh, J., Roy, A., Jacob-Hirsch, J. and others, (2012). Pharyngeal mesoderm regulatory network controls cardiac and head muscle morphogenesis. Proceedings of the National Academy of Sciences, 109(46), pp.18839--18844.

Herchenhan, A., Bayer, M., Svensson, R., Magnusson, S. and Kj\aer, M. (2013). In vitro tendon tissue development from human fibroblasts demonstrates collagen fibril diameter growth associated with a rise in mechanical strength. Developmental Dynamics, 242(1), pp.2--8.

Hoffman, E., Brown Jr, R. and Kunkel, L. (1987). Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell, 51(6), pp.919--928.

Katori, Y., Hyun Kim, J., Rodr\'\iguez-V\'azquez, J., Kawase, T., Murakami, G. and Hwan Cho, B. (2011). Early fetal development of the intermediate tendon of the human digastricus and omohyoideus muscles: a critical difference in histogenesis. Clinical Anatomy, 24(7), pp.843--852.

KINMONT, P. (2008). development of the human achilles tendon enthesis organ. Journal of Anatomy.

Nichol, P., Corliss, R., Yamada, S., Shiota, K. and Saijoh, Y. (2012). Muscle Patterning in Mouse and Human Abdominal Wall Development and Omphalocele Specimens of Humans. The Anatomical Record, 295(12), pp.2129--2140.

Rodriguez-Guzman, M., Montero, J., Santesteban, E., Ga\~nan, Y., Macias, D. and Hurle, J. (2007). Tendon-muscle crosstalk controls muscle bellies morphogenesis, which is mediated by cell death and retinoic acid signaling. Developmental biology, 302(1), pp.267--280.

Rodr\'\iguez-V\'azquez, J., M\'erida-Velasco, J. and Verdugo-L\'opez, S. (2010). Development of the Stapedius Muscle and Unilateral Agenesia of the Tendon of the Stapedius Muscle in a Human Fetus. The anatomical record, 293(1), pp.25--31.

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Tich\`y, M. and Grim, M. (1985). Morphogenesis of the human gluteus maximus muscle arising from two muscle primordia. Anatomy and embryology, 173(2), pp.275--277.

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2014 Group Project 8

2014-10-22T03:36:51Z

Z3418779: /* Molecular and Cellular regulation of fetal myogenesis */

{{ANAT2341Project2014header}}
=Musculoskeletal=
This webpage will be focusing on fetal muscular development.

==Muscle development General Timeline==

Primary myofibers develop in first trimester, Secondary myofibers develop during Second and Third Trimester
We should do a brief overview of primary myofiber formation, and extensive overview of secondary myofiber formation)

==Background Early Embryonic development==
Mesenchymal progenitor cells from somites(occiptal, cervical, thoracic, lumbar, sacral), undergo multiple differentiation stages to create muscle fibers.

http://www.mdconsult.com/books/figure.do?figure=true&eid=4-u1.0-B978-1-4377-2002-0..00015-1--f0025&sectionEid=4-u1.0-B978-1-4377-2002-0..00015-1&isbn=978-1-4377-2002-0&uniqId=464007141-2 (demonstration of myotomes in week 6 and 8)

Skeletal Muscle develops from a process known as myogenesis. Mesenchymal cells differentiates into embryonic muscle cells, myoblasts. Myoblasts which have single nuclei fuse and elongate to form myotubes which are multinucleated and cylindrical.

The neural tube and notochord release signaling molecules like Shh, Wnts and [BMP]-4. These signaling molecules act on transcription factors of the MyoD family and Pax <ref name="PMID10809386"><pubmed>10809386</pubmed></ref>.The MyoD or MrF family includes MyoD, Myf-5 , myogenin and Myrf4 <ref name="PMID7748174"><pubmed>7748174</pubmed></ref>. The MyoD family of transcription factors, like MyoD are myogenic bHLH (basic helix loop helix) transcription factors. Pax-3 and the MyoD induce myogenesis, formation of myoblasts. Pax-3 also acts on c-met which is a migratory peptide.

Skeletal muscle is derived from the somites. Paraxial mesoderm segments into somite structures on both sides of the notochord and neural tube.

[[Image: Mesoderm-cartoon3.jpg]]

Somites are mesodermal structures where the dorsal most end of the somite, which is known as the dermomyotome, becomes skeletal muscle and dermis <ref name="PMID9094722"><pubmed> 9094722</pubmed></ref>. A small anterior portion of the paraxial mesoderm remains un-segmented and eventually forms some muscles of the head <ref name="PMID12587921"><pubmed> 12587921</pubmed></ref>.

The myotome lies in-between the scleratome which is ventrally located. The scleratome forms the cartilage and bone of the axial skeleton of the embryo. The dermomyotome is located dorsally and forms the first skeletal muscle in the embryo.
The medial part of the dermomyotome forms the dorsal and intecostal muscles whilst the lateral part of the dermomyotome forms the limb and ventral muscles <ref name="PMID9094722"/>.

[[Image: Somite_cartoon5.png]]

Myogenesis occurs in two phases; primary and secondary which occur in embryonic and fetal periods Primary myotubes express MHC slow myosin heavy chains <ref name="PMID21204650"><pubmed> 21204650</pubmed></ref>. The primary myotubes form the structure and scaffold upon which secondary myotubes form during secondary myogenesis which occurs in the fetal period <ref name="PMID21204650"/>.

==Molecular and Cellular regulation of fetal myogenesis==

Skeletal myofiber number is set at birth <ref name="PMID5804561"><pubmed>5804561</pubmed></ref>. This was found using experiments on mice and pigs. Similar trends have been observed in humans as found by Widdowson et al (1972) where a huge increase and then levelling of gastrocnemius myofibers were found in the gestational period <ref name="PMID5046781"><pubmed>5046781</pubmed></ref>.

Skeletal muscle development
Multipotent mesenchymal cells (MSC) form myoblasts as well as adipocytes and fibroblasts <ref name="PMID10102814"><pubmed>10102814</pubmed></ref>. Therefore specific the nutrients and growth are important in directing the growth of these MSC into either adipocytes or myoblasts <ref name="PMID23100595"><pubmed>23100595</pubmed></ref>.
Myoblasts differentiate form the embryonic, fetal and adult skeletal muscle and in fact myoblasts differentiate to form either three early on. Myoblasts fuse and form primary myoblast or myotubes. As discussed previously the primary myotubes form the scaffolding for which the fetal myoblasts will differentiate into secondary myotubes and add too <ref name="PMID640968 "><pubmed>640968 </pubmed></ref>. There are a greater deal of secondary myofibers than primary and are receptive to nutrients and growth factors.
Muscle regulatory factors (MRFs) control the proliferation of secondary myoblasts. MRF’s are helix-loop-helix transcription factors.
Myoblasts early on express PAX7 and MYF5. PAX7 and MYF5 act on regulatory proteins of the cell cycle like CDK4 and Cyclin D1. CDK4 and Cyclin D1 dephosphorylate RB. RB when phosphorylated is active and inhibits the cell-cell progression in the cell cycle and hence inhibits proliferation. Therefore Cyclin and CDK4 in this case induces cell proliferation. <ref name="PMID24532817 "><pubmed>24532817</pubmed></ref>; <ref name="PMID22445545 "><pubmed>22445545</pubmed></ref>, <ref name="PMID12242286 "><pubmed>12242286</pubmed></ref> These creates more fetal myoblasts committed which are committed or programmed to form fetal or secondary myofibers later on. These committed myoblasts express MRF, MYOD. MYOD acts on myostatin to take the myoblast out of the cell cycle and stop it from proliferating <ref name="PMID24532817 "><pubmed>24532817</pubmed></ref>; <ref name="PMID12242286 "><pubmed>12242286</pubmed></ref>.Now the myoblast is ready for differentiation. The phosphorylation of RB at this period helps to slow the activity of the cell cycle. The myoblasts differentiate and fuse to become multinucleated myotubes as discussed previously. In the formed myotubes MRFs like Myogenin and MRF4 act to inhibit cell cycle and proliferation by acting regulatory proteins like P21 to stop the cell cycle <ref name="PMID24532817"/>; <ref name="PMID10733231 "><pubmed>10733231</pubmed></ref> This allows the conversion of the myotube into a myofiber. The myofiber can express desmin an intermediate filament that is increasingly seen as gestation progresses. Growth factors, amino acids and stretch/load activity act on myofibers to affect subsequent hypertrophy maturation of myofibers <ref name="PMID 24532817 "/>

[[File:Mouse limb tissue development.jpg|border|right|upright=0.5]]

Myofiber hypertrophy is occurs when protein synthesis is greater than protein degradation. The resulting accumulation of protein results in hypertrophy. Therefore maintaining protein levels is important in the hypertrophy of myofibers. This is regulated by nutrients and growth factors (GFs).
Growth factors
There are a large number of growth factors that affect fetal myogenesis. Some of the main GFs as Brown (2014) describes in her article “IGF1, insulin, basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF- β)” <ref name="PMID 24532817"/>; <ref name="PMID2190237 "><pubmed> 2190237</pubmed></ref>, <ref name="PMID22682632"><pubmed> 22682632</pubmed></ref>. Experiments have shown that if IGF1 is removed reduced muscle mass and hypoplasia has resulted. And increased IFG1 expression has resulted in hyperplasia and increased skeletal muscle. IFG1 has also been found to enhance protein synthesis. <ref name="PMID 24532817"/>; <ref name="PMID3546571"><pubmed> 3546571</pubmed></ref>, <ref name="PMID 7744859"><pubmed> 7744859</pubmed></ref>. Insulin similarly has been found to promote fetal muscle growth and protein synthesis.
Basic fibroblast growth factor (bFGF) and transforming growth factor-β (TGF- β) induce proliferation and myogenesis by upregulating cyclin D.

Nutrients
Being the building block of proteins, amino acids are important in muscle protein synthesis. But they are more important in adult muscle protein synthesis than fetal muscle protein synthesis. Experiments have shown for example an amino acid infusion did not always result in a fetal muscle growth, only when there was a rise in the insulin levels. And little is known of the interaction of amino acids and growth factors in the context of fetal myogenesis.
Research has shown that fetal or secondary myofibers are more prone to suffer from nutrient deficiency than primary myofibers in pigs and sheep. <ref name="PMID 2041547"><pubmed> 2041547</pubmed></ref>, <ref name="PMID 8014156"><pubmed> 8014156</pubmed></ref>, <ref name="PMID 15317692"><pubmed> 15317692</pubmed></ref>.

Fetal myofiber number have been observed to decrease with detal nutrient deficiency. This is concerning as myofiber numbers are set at birth.
Nutrients are also important for fetal myofiber hypertrophy.
Stretch and loading
Stretch and loading also affect hypertrophy. <ref name="PMID23629510"><pubmed>23629510</pubmed></ref>.

==Tendon Development==

Tendons are connective tissue which join muscle and bone allowing the transmission of force. Tendons primary embryonic structure originate from mesenchymal progenitor somite cells with further contributions of Neural Crest and Lateral plate mesoderm.

Appearance of tendons begins in the 20th Carnegie stage and marks the beginning of fibrillogenesis. This process is initiated by fibroblasts in series of extracellular compartments; they enlarge the cells domain into extracellular space. Channels deep in the cytoplasm drive the process of elongation, these channels location is associated with Golgi bodies.

First type of compartments are formed by collagen containing secretory vacuoles which fuse with surrounding cell membranes. Initial fibrillogenesis is mediated my macromolecular interactions based on vacuole content, with a lesser input from receptor membrane interactions. Fibril groups as fibres close to the cell surface and Secondary extracellular compartments form; at this stage fibroblast are arranged adjacently. Third level of compartmentalization forms later when fibroblast are adjacent with 2 or more other fibroblasts. As the tendon matures fibres coalesce invading each other with interdigitating processes. Secretory vacuole persist, laterally aggregating with further growth preserving sites for fibril deposit.
Collagen fiber assembly branch creating fibre networks along fascicles. Fibres branch within tendon fascicle

The main regulatory factors of tendon fibrillogenesis are Leucine-rich repeat proteoglycans.

{| class="wikitable"
|-
! Non-fibrillar components!! Molecular Characteristics!! Function
|-
| Fibromodulin || Keratin sulphate, proteoglycan || Fibromodulin modulates the site-specific cross-linking ultrastructure of collagen, ensuring mechanical strength. Control the pattern of lysyl oxidase-mediated collagen cross-linking by reducing access of the enzyme to telopeptides, by binding to the collagen. <ref name="PMID 24849606"><pubmed>24849606</pubmed></ref>
|-
| Decorin || Chondoirin sulphate, proteoglycan || Regulates expression of multiple leucine-rich proteoglycansins(SLRP) during tendon fibrillogenesis, via Class I and II Small(SLRP). Competes with Biglycan for binding sights on collagen types I-VI . Concentration increases as fetal development continues. <ref name="PMID 16518859"><pubmed>16518859</pubmed></ref>
|-
| Biglycan ||Chrondroitin sulphate and Dermatan sulphate, proteoglycan|| Process of regulation closely mimic Decorin though concentration is maximal expression at day 16-18 during embryonic development, reducing after this point. Competes with Decorin for bonding sights on collagen I-VI. <ref name="PMID 16810681"><pubmed>16810681</pubmed></ref>
|}

==Historical findings==

No studies focusing on the fetal development of muscle existed before the 1970's. During the 70's a majority of research in the field focused on the histologist differences and development of differing fiber types. Research on morphological development was very limited with eh exception of two Czechoslovakian studies in the late 1980's which displayed muscle formation for multiple primordia and sexual differentiation.

The differing developments of alpha and beta fibers was revealed in a study by University of California published in 1972 using lamb fetuses as an experimental model. Beta muscle fibers are formed during the first stages of fusion, the individual Beta fibers create a network for the alpha fibers to develop on. Red muscle fasciculi are formed by merging of small fiber bundles. White muscles are formed by ongoing addition of alpha fibers. Additionally it was concluded that fetal muscle contraction didn't significantly effect number of fibers present. Most fibers had already been formed by the 20th week, during this period the limit muscle contractions result in little mechanical tension.

===Gluteus Maximus muscle Morphogenesis===

A 1985 Czechoslovakian study investigated the development of Gluteus Maximus during the embryonic and fetal periods. Pelvic micro-dissection of human embryos and foetuses with crown length varying from 22-215mm were compared with newborns and adults. The presence of a muscle not present in post-natal adults<ref name="PMID 5043313"><pubmed>5043313</pubmed></ref> was discovered called the coccygeofemoralis or pars coccygea; this muscle originates from sides of coccyx and inserts onto the gluteal tuberosity. In the adult human this muscle fuses with the larger pars sacroiliac or fetal gluteus maximus to create the adult gluteus maximus. Pars sacroiliac originates from ilium and sacrum and inserts onto the gluteal tuberosity<ref name="PMID 4083527"><pubmed>4083527</pubmed></ref>.

In fetus with crown length of less than 45 mm the two muscle primordia are separated by small amount of loose connective tissue. This point onwards the muscles become fused by a small furrow which persists until 215mm crown length. By the time of birth the furrow is absence and the muscles are entirely fused <ref name="PMID 4083527"><pubmed>4083527</pubmed></ref>. The coccygeofemoralis in long tailed mammals remains separate from the gluteus maximus, known as the caudofemeralis muscle <ref name="PMID 1255730"><pubmed>1255730</pubmed></ref>. In these animals its function is lateral flexion of the tail <ref name="PMID 8843689"><pubmed>8843689</pubmed></ref>. This is the sole morphogenesis study on any of the large muscles; it is likely other muscles with multiple origins and insertion have separate fetal muscle primordia.

===Morphogenesis of human sphincter urethrae muscle===

This study was completed in 1989 at the same Czechoslovakian institute of that of Gluteus maximus morphogenesis. . Study displayed three developmental phasess differentiating them by morphogenesis, histology and sexual dimorphism. External urethral sphincter in embryos and fetuses with crown length varying 18-320mm, neonates, children and adults was fixed in formaldehyde and embedded in paraplast. Then cut in series and stained with hematoxylin and eosin for histological analysis. Morphogenesis and Sexual dimorphism specimens were micro-dissected using a stereomicroscope.
{| class="wikitable"
|-
! Phase!! Time Period!! Characteristics
|-
| Indifferent Phase || Before 10th week || Muscle primordia discernible from neighbouring muscle by week 8. Grow to forms a shallow arch; connecting the urethra and urogenital diaphragm. Consists of condensation of myoblasts up to 9.5 weeks, past this point myotubes and muscle fibres appear.
|-
| Sexual dimorphic Phase || 10th week to Birth || Associated with the development of prostate and vagina. Primordia spread along urethra wall posteriorly.
In Males: Spreads to create the infraprostatic part of external urethral sphincter. Arches anteriorly to join prostate and urethra
In Female: Spreads to create upper part of external urethral sphincter. Lower sixth of sphincter connects anterior and lateral urethral walls, additionally projects to lateral vaginal walls.
|-
| Definite Structuring Phase ||After birth|| Position of urethral sphincter does not alter in relation with prostate and inferior part of vagina. Infraprostatic region in males and upper part in females grow to form a complete ring.
|}

==Current Research and Findings==

===Intermediate tendon of Human Digastricus and Omohyoideus===

Early Fetal Development of the Intermediate Tendon of the Human Digastricus and Omohyoideus Muscles: A Critical Difference in Histogenesis
Katori et al (2011) did a study to understand the development of the intermediate tendon within digastricus and omohyoideus muscles. Both digastricus and omohyoideus have two bellies of muscle with an intermediate tendon. Digastricus has an anterior and posterior belly whilst omohyoideus has a superior and inferior belly. Omohyoideus is supplied by ansa cervicalis and digastricus is supplied by cranial nerves; trigeminal and facial. The difference in innervation gives reason to believe that these muscles are different developmentally. Katori et al (2011) investigate the differences in the formation of the intermediate tendon between these two muscles.
Katori et al (2011) observed in week 7-9 the posterior belly of digastricus was developing the intramuscular tendon, intermediate tendon, with a bulb like terminal tendon. The anterior belly however did not have any intramuscular tendon present within it. The anterior bellies of 50% of specimens moved towards and attached on the bulb like terminal part of tendon. Instead of finding its insertion on reichart’s cartilage or hyoid bone it attaches to terminal pat of posterior tendon. Katori et al hypothesize that this is due impediment such as stylohyoideus, vascular arteries and hypoglossal nerve. How these structures may affect digastricus is for example the thicker hypoglossal nerve separating the posterior tendon from attaching the hyoid bone. Also the stylohoideus’ caudal end may form from mesenchymal condensation near the caudel end of Reichart’s cartilage inhibiting attachment by digastricus. This shows how timing of muscle development affects topography. But eventually muscle fibers in posterior belly intramuscular tendon get converted. The caudal and bulb end of the posterior belly becomes into a tight intermediate tendon at 15 weeks. In half of the specimens in week 15 and week 18 it was observed by desmin immunohistochemistry that anterior belly muscle fibers were integrated into bulb-like and caudal end of posterior tendon.
The omohyoid at 7-9 weeks had a nearly straight and superior-inferior path. Omohyoid becomes angulated by the lateral expansion of the clavicle and shoulder joint (Katori et al, 2011). Omohyoid is tightly fitted in-between think sternocleidomastoid, scalene muscles. The fascia and lymphatic tissue also press the omohyoid towards the sternocleidomastoid. At this time during 7-9 weeks unlike digastricus, omohyoid is a single muscle belly. It reaches its greatness thickness at week 15 at 0.5mm then at the point where tendon develops the thickness decreases. In omohyoideus the intermediate tendon forms secondarily. From week 18 to week 20 the intermediate tendon muscle fibers are replaced by collagen fibers most likely after the tendon has fully shaped itself. Cells along the medial margin of omohyoid were found to be vimentin positive. As vimentin is a intermediate filament its presence is an indication mechanical or osmotic stress (Pekny and Lane, 2007).

===Stapedius===

Rodri´guez-va´ zquez et al (2010) in order to understand the isolated case of a unilateral agenesia of the stapedius tendonduring week 14 of post-conception development (PCd), have tried to understand the way the stapedius muscle develops. The stapedius is essentially formed by two anlagen; one anlagen forms the tendon of the stapedius and the other forms the muscle belly of stapedius. The anlagen forming the tendon are derived from the internal segment of interhyale. The anlagen forming the muscle belly are derived from the 2nd pharyngeal arch near the interhyale, medial to the facial nerve. Interhyale is the internal part of the second branchial arch and it develops into stapedius’ tendon (http://www.drugs.com/dict/interhyale.html ). The observed unilateral agenesia of the tendon of stapedius was found by Rodriguez-va et al (2010) to be due to the internal segment of the interhyale regression. Instead the belly of stapedius was accompanied with a pseudo tendon formed by the external segment of the interhyale. To come to this conclusion the formation of the stapedius and pyramidal eminence was tracked.
Interhyale was observed as a mesenchymal condensation formed at O’Rahilly stage 16 at cranial component of the second branchial arch. The stapes and reichert cartilage are eventually differentiated from the interhyale. At Stage 18 and 19 a mesenchymal bridge forms by the interhyale which bridges stapes and cranial part laterohyale (Reichert’s cartilage). And by stage O’Rahilly stages 20 and 21 the interhyale fully develops.
Rodriguez-va et al (2010) found the belly of stapedius to derive from a blastema which develops adjacent the interhyale to form its own anlage. In O’Rahilly stage 22 Rodriguez-va et al (2010) found that the interhyale began to take on an angular shape and the anlage of the stapedius belly connected with the vertex of the now angular interhyale.
Interhyale developed two segments; internal thick segment and an external thin segment. The thick internal segment as discussed contributes to the formation of the tendon of the stapedius muscle. The external segment begins to regress in the embryonic period and by the fetal period, week 9, the external segment was observed to be fully regressed.
In weeks 10-11 a conical belly continuous with the stapedius tendon was observed and the stapedius tendon attached at the back of the stapes head.The anlage of the pyramidal eminence formed around week 12-14 and around the stapedius muscle belly. The mesenchymal condensation forming the pyramidal eminence grew until it was inhibited around week 15-17.
After week 9 there was not much morphological change of the stapedius. From this study we can see how the stapedius muscle develops its shape and attachments in the embryonic and fetal periods.

==Abnormalities==

===Duchenne Muscular Dystrophy(DMD)===

Caused by a mutation of the dystrophin protein on locus Xp21; this protein complex connects the cytoskeleton of muscle fibres to the extracellular matrix. Abnormal dystrophin results in a degradation of cellular integrity, excessive penetration of sarcolemma by calcium and water entering mitochondria increasing pressure and bursting. The lack of significant load during fetal development results in minimal wasting, it is detected in postnatal babies at 3-5 years old with muscles resisting gravity being the first to waste. Patients are restricted to wheelchairs by their early teens and have a life expectancy of 25 years. The incidence in male infants is 1 in 36,000.
Typically males are affected while females are carriers. In the offspring of a carrier mother and unaffected father; sons have 50% chance of affected and daughter’s 50% chance of becoming carriers. Since the disease is a terminal illness killing in mid-twenties; affected fathers are not considered in the situation. Absence of affected fathers means it is very unlikely for daughters to be affected.

Duchenne Muscular Dystrophy has no present cure, treatment is aimed at altering onset of symptoms and maximising quality of life, these include: <ref name="PMID 25187493"><pubmed> 25187493</pubmed></ref>

*Corticosteroid based medication significantly improves muscle strength and function over the short term. Patients using the drugs long term gradually have reduced doses to avoid severe side effects which can include; weight gain, behavioural disorders and osteoporosis. Most effective corticosteroids are prednisolone, and deflazacort. Theories on how the steroids work include; include activation of T-Cell pathways, directly reducing muscle regeneration, modulating cell inflammation and enhancement of myogenic precursors. <ref name="PMID 17541998"><pubmed> 17541998</pubmed></ref>

===Amyoplasia===

Amyoplasia is characterised by replacement of newborn muscle tissue with fat and dense fibrous tissue. Is the most common of Arthrogryposis multiplex congetia disorders; these result in multiple joint contractures. Affected limbs have significantly altered positioning, typically clubfoot is present and elbows are extended<ref name="PMID 24459070"><pubmed>24459070</pubmed></ref>. Primary cause is limited fetal movement, muscle is replaced by dense fibrous tissue. Conditions which limit fetal movement include abnormal uterus morphology and reduced amniotic fluid. Presently no genes have been linked to the deformity. <ref name="PMID 9260643"><pubmed>9260643</pubmed></ref>
85-90% of newborns undergo surgery within days of birth primarily on the legs and hips, releasing tendons from contractures. Motion can be improved by casting and splinting, lower limbs are typically cast and upper limbs are typically splinted.
<ref name="PMID 24459070"><pubmed>24459070</pubmed></ref>

{| class="wikitable"
|-
! Involvement subtype!!Percentage of Case
|-
| Four-limb symmetrical || 55%
|-
| Three limb || 5%
|-
| Upper limb only || 17%
|-
| Lower limb only ||16%
|}

===Nemaline Rod Myopathy===

Nemaline rod myopathy is congenital non-dystrophic muscle disease. Sufferers experience general muscle weakness, weakness is most severe in face, neck and limbs. Further clinical manifestations include feeding problems, scoliosis of spine, foot deformities and respiration difficulties. The disease is an inherited both autosomal dominant and recessive, 30% autosomal dominant, 20% autosomal recessive and 50% simplex.

Mutations in six different genes are associated with nemaline myopathy, with nebulin mutations being the most common. No treatments or disease-modifying therapies have been identified for this disease. One of the major barriers to treatment development is the lack of models amenable to rapid and coordinated testing of potential therapeutic strategies. To overcome this barrier, we have characterized the first zebrafish model of nemaline myopathy. This model, termed neb, harbors a recessive mutation in the nebulin gene that results in decreased Nebulin protein levels, a severe motor phenotype and premature lethality. In addition to impaired motor function, neb zebrafish exhibit many of the features associated with human nemaline myopathy. These include impaired force generation, altered thin filament length and the presence of specific histopathological changes, including the formation of nemaline bodies. In summary, neb zebrafish mirror the genetic, clinical and pathological aspects of nemaline myopathy due to NEB mutation, and thus are an excellent model for future therapy development for this devastating disorder.

==References==
Anatomy and variations of palmaris longus in fetuses.<ref><pubmed> 23529313</pubmed>| [http://www.ncbi.nlm.nih.gov/pubmed/23529313]</ref>

Development of the rectus abdominis and its sheath in the human fetus.<ref><pubmed> 22869489</pubmed>| [http://www.ncbi.nlm.nih.gov/pubmed/22869489]</ref>

Sonic hedgehog acts cell-autonomously on muscle precursor cells to generate limb muscle diversity.<ref><pubmed> 22987640</pubmed>| [http://www.ncbi.nlm.nih.gov/pubmed/22987640]</ref>

The normal growth of the biceps brachii muscle in human fetuses.<ref><pubmed>23468258</pubmed>| [http://www.ncbi.nlm.nih.gov/pubmed/23468258]</ref>

<references/>

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