2014 Group Project 8 part 2

From Embryology

Current Research and Findings

Research Models

Current research models used to explore fetal myogensis include both human and animal models. Below is a list of a few different models;

  • Rats and Mice as Langlois et al (2014) use in their experiment to do with Pannexin 1 and Pannexin 3 Channels and Skeletal muscle. [1]
  • Pigs as Yang et al (2014) use in their experiment in comparing lean and obese pigs’ genes and muscle development[2]
  • Sheep as Duckett et al (2014) use in their experiment on the effects of ergot alkaloids on fetal growth in sheep. [3]
  • Humans as Langlois et al (2014) use in as Langlois et al (2014) use in their experiment to do with Pannexin 1 and Pannexin 3 Channels and Skeletal muscle. [4]

Intermediate tendon of Human Digastricus and Omohyoideus

Katori et al (2011) did a study to understand the development of the intermediate tendon within digastricus and omohyoideus muscles and their differences and similarities.

Digastricus Omohyoideus
  • 2 bellies of muscle with an intermediate tendon[5]
  • Week 7-9 posterior belly develops intermediate tendon with a bulb like terminal part and anterior belly doesn’t. [5]
  • Anterior belly moves towards and attaches to terminal bulb part of posterior belly’s intermediate tendon. [5]
  • Anterior belly hypothesized not to attach to cartilage and attach to intermediate tendon because of stylohyoideus, vascular arteries and hypoglossal nerve which blocked it from doing this. [5]
  • 2 bellies of muscle with an intermediate tendon. [5]
  • Week 7-9 omohyoideus takes a straight and superior-inferior path and is a single muscle belly. [5]
  • Omohyoideus becomes angulated by the lateral expansion of the clavicle and the shoulder. [5]
  • Omohyoid is tightly fitted in-between sternocleidomastoid and scalene muscles. [5]
  • Omohyoideus reaches greatest thickness (0.5mm) and intermediate tendon develops secondarily. [5]
  • Week 18-20 muscle fibers of intermediate tendon are converted to collagen fibers. [5]


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. 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.[6]

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.[6]

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.[6]

Two studies on Triceps Brachii and Biceps Brachii and their parameters

Grzonkowska et al (2014) studied 30 fetuses from the age of 12-29 weeks and observed that with an increase in fetal age there was an increase in fetal triceps brachii parameters. [7] Similarly Szpinda et al (2013) studied the anatomical parameters of biceps brachii of 30 fetuses aged 17-30 weeks. [8]

Triceps Brachii Biceps Brachii
  • Triceps has three heads; long, lateral and medial head.[9]
  • The long head was observed to be the longest with a mean length of 43.36mm when compared with 37.06mm and 33.24 for lateral and medial heads respectively. [9]
  • The lateral heads muscle mean width was the greatest at 5.34mm with the long head and medial head’s mean width observed to be 3.74mm and 4.42mm respectively. [9]
  • The formation of the widest belly as the lateral head’s and the thinnest is the long head’s muscle belly. [9]
  • no variability due to sex or laterality. [9]
  • proportionate increase with fetal age of these parameters. [9]
  • Biceps have two heads; long and short heads. [8]
  • The long head of biceps brachii’s mean length was 5.68mm and short head’s mean length was 5.93mm. [8]
  • The long head of biceps brachii’s mean length was 5.68mm and its mean width at mid-length was 0.60mm. Whilst the short head had a mean width at mid-length of 0.65 and mean width at widest part of 0.72. [8]
  • Therefore in the fetus the belly of the long head is shorter and thinner than the short head’s. [8]
  • No variability due to sex or laterality. [8]
  • Proportionate increase with fetal age of these parameters. [8]
  • Szpinda et al (2013) observed a linear pattern of growth of biceps brachii. For example the long head of the biceps brachii’s length from 3.26-8.84mm which Szpinda et al (2013) found to follow the linear relationship; y = –0.801 + 0.276 × Age (R^2 = 0.591). [8]

The importance of these studies is that they measured the parameters of these developing muscles which were not done before. And they found that the individual components of these muscles developed proportionately as they are seen when they are fully developed.

Pannexin 1 and Pannexin 3 and Myoblast differentiation and proliferation

Langlois et al (2014) investigate the role of these Panx1 and Panx3 in the skeletal muscle of rodents and humans and their expression in the skeletal muscle of fetal and adult life.[10]

Panx1 and Panx3 are part of the pannexin channel protein family. Panx1 is known to play many roles, a few of which are vasodilation and inflammatory responses.[11] Panx3 is known for fewer functions such as carcinogenesis and osteoprogenitor cell proliferation. [12] [13] However recent studies have shown the potential role of Panx1 and Panx3 in skeletal muscle development.

Pannexins are known to be present within skeletal muscle. Pannexins were found in mRNA of skeletal muscle [14]. And Panx1 in sarcolemma of rodent skeletal muscle [15] Pannexins have also been observed to play certain roles in contraction as studies showed that Panx channel blockers reduced ATP release from electrical stimulation of myotubes [15][16] The role however of pannexins in cell differentiation and proliferation is unknown and what Langlois et al (2014) investigate in this experiment. [11] HSMM (primary Human Skeletal Muscle Myoblasts) was obtained from post quadriceps and psoas major. And SkMC (primary Human Skeletal Muscle cells) was got from upper arm or leg. [11] Western Blot analysis confirmed the presence of Panx1, Panx2 and Panx3 in the human, rat and mouse skeletal muscle tissue samples. Pan1 and Panx3 was detected in the tissue samples but Panx2 wasn’t. Panx1 and Panx2 stained differently within skeletal muscle and hinted that they had different functions. [11] It is known that in the fetal stage there is lots of proliferation whilst in the adult stages there is decreased proliferation. Decreased proliferation was indicated by decreased proliferating cell nuclear antigen (PCNA). Whilst increased differentiation resulted in increased myosin heavy chains (MHC). [11] Panx1 of higher molecular weight is the main pannexin found in skeletal muscle, more so in fetal than adult cells. Lower molecular forms of both Panx1 and Panx3 are more increased in adult forms and the higher weight forms are more increased in fetal forms. This shift possibly suggests that Panx1 and Panx3 plays a role in skeletal muscle development. [11] Langlois et al (2014) also found that Panx1 and Panx3 had a role to play in the sophisticated process of myogenesis, in particular myoblast proliferation and differentiation. Panx1 was low in in undifferentiated skeletal muscle cells and myoblasts but promoted myoblast differentiation and were thereafter more abundant. Panx3 of low molecular weight (~ 43 kDa) acted differently to higher weight species (~ 70 kDa). Expression of low molecular weight Panx3 promoted myoblast differentiation and repressed proliferation. Whilst expression of high molecular weight Panx3 coincided with proliferation and subsided with differentiation of myoblasts. Low molecular weight Panx3 were low in differentiated and undifferentiated HSMM but was expressed in skeletal muscle tissue which may indicate that lower molecular weight Panx3 plays a role more later on in differentiation. [11] Langlois et al (2014) used 2 Panx3 shRNAs to reduce Panx3 expression and they observed an inhibition of Proliferation whilst not initiating differentiation. Other impacts on Panx were studied such as their glycosylation, phosphorylation and being sialylated are important in their functioning as membrane channels. Post-transcriptional modifications and molecular interactions of Panx are thus important in regulating Panx channel function and thus important in myoblast differentiation and proliferation. [11] All evidences led to the conclusion that both Panx1 and Panx3 are expressed in skeletal muscle cells and participate in proliferation and differentiation of myoblast muscle cells.

Dll1 signaling and craniofacial myogenesis

Craniofacial skeletal muscle is derived from cranial mesoderm and trunk skeletal muscle is derived from somatic mesoderm. Hence craniofacial muscles are developmentally contrasting from trunk muscle. It is known that Notch signalling plays a role in myogenesis in trunk muscles. However Notch signalling role in myogenesis in craniofacial muscles has not been investigated that much, and is the focus of Czajkowski et al (2014) experiment. [17]

The normal myogenic regulatory factors (MyoD, Myf5, Mrf4) act in myogenesis and all three are necessary for myogenesis to occur. However there are other regulatory factors which act upstream to affect this standard myogenic pathway. This is another area where trunk and cranial muscle differs in regulation as Pax3/7 act upstream of the normal myogenic regulatory factors. However in craniofacial muscle development Pitx2 and Tbx1 play this role.[18]

The role of Notch signalling is not well known for craniofacial muscles. In mice Notch signalling is normally the result of a ligand such as DII1 or DII3 binding to a Notch receptor1-4. Subsequently this is followed by translocation into the nucleus and activation of target genes. In the context of myogenesis Notch signalling was observed repress MyoD and induce MyoR expression in C2C12 cells. [18]

Fetal Skeletal muscle contains a renewing pool of progenitor cells. Notch signalling results in the regulation of myogenic differentiation. Therefore when notch signalling is lost or mutated uncontrolled myogenic differentiation results and the formation of muscle. Things that repress notch signalling like DII1 and Rbpj impair muscle growth. Therefore mutations in DII1 and Rbpj results in muscle growth. Therefore Notch signalling plays role in removing MyoD and compensating for mutations in DII1 or Rbpj, and maintaining progenitor muscle pool. Notch signalling therefore suppresses myogenic differentiation in trunk muscles. But not much is known of its role in craniofacial muscles. [18]

Czajkowski et al (2014) found that in myogenic progenitor cells and muscle growth are deficient during early fetal development. Czajkowski et al (2014) observed that in the absence of these myogenic progenitor cells supernumerary myoblasts temporarily come and express MyoD. The overexpression of MyoD is in this case detrimental to the limited craniofacial progenitor cell in the mutant mice. Therefore the mutation and repression of MyoD which Czajkowski et al (2014) propose that Notch facilitates, allows for the craniofacial myogenesis. [18]

Czajkowski et al (2014) finds out that Notch signalling acts in a similar as it does in the trunk muscles in the craniofacial muscles. It does this by regulating progenitor cell differentiation by controlling MyoD expression. The difference however between Notch signalling between craniofacial and trunk muscles is in its other functions such as homng of satellite cells and the independence of Pax7 expression from DII1 signalling in craniofacial muscles. [18]


Duchenne Muscular Dystrophy(DMD)

Progressive myofiber replacement by fibrotic and fat tissue in Duchenne Muscular Dystrophy rats [19]

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. [20] The most common approach is Corticosteroid based medication, which significantly improves muscle strength and function over a 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. [21] Additionally assisted ventilation, supportive equipment, proton-pump inhibitors, increased excercise, beta-blockers and diuretics culminate to reduce symptoms and increase sufferers quality of life. [22] [23] [24] [25]


Partial Contracture of Hands, typical manifestation of 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[26]. 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. [27] 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. [26]

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[28]. 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. In total 6 genes have been associated with the formation of nemaline myopathy with the NEB and ACTA1 genes being the most prominent[29] [30]. Over 60 NEB mutations have been discovered which result in nemaline myopathy, it results in the decreased production and decreased length of the nebulin protein, half of all NM cases are associated with NEB mutations[29]. About 140 mutations to ACTA1 can lead to formation of nemaline myopathy, this results in either aggregation of α-actin fibres preventing functional muscle contraction or total absence of α-actin production[30].


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