Embryo Take Home Exam

Karyl Sabrina I. Javellana

BS Biology 4

October 16, 2006

Given an organ derivative of the 3 germ layers Somatic Muscles. Discuss the following: 1. A(n) experiment(s), including the procedure, results and implications to ontogenetic and/or phylogenetic development that demonstrate the inductive influences governing the differentiation of this organ. Experiment 1: MyoD or Myf-5 is required for the formation of skeletal muscle -Rudnicki MA, Schnegelsberg PN, Stead RH, Braun T, Arnold HH, Jaenisch R Mice carrying null mutations in the myogenic regulatory factors Myf-5 or MyoD have apparently normal skeletal muscle. To address whether these two factors functionally substitute for one another in myogenesis, mice carrying mutant Myf-5 and MyoD genes were interbred. While mice lacking both MyoD and Myf-5 were born alive, they were immobile and died soon after birth. Northern blot and S1 nuclease analyses indicated that Myf-5(-1-);MyoD(-1-) mice expressed no detectable skeletal muscle-specific mRNAs. Histological examination of these mice revealed a complete absence of skeletal muscle. Immunohistochemical analysis indicated an absence of desminexpressing myoblast-like cells. These observations suggest that either Myf-5 or MyoD is required for the determination of skeletal myoblasts, their propagation, or both during embryonic development and indicate that these factors play, at least in part, functionally redundant roles in myogenesis. Experiment 2: Mouse limb muscle is determined in the absence of the earliest myogenic factor myf-5 -Shahragim Tajbakhsh and Margaret E. Buckingham myf-5 is the only member of the MyoD family of myogenic regulatory genes to be expressed in the mouse embryo prior to muscle cell differentiation. We have used the developing limb as a model in which to follow the formation of peripheral muscle, to address the question of whether myogenic precursor cells are already present in the limb bud before expression of myf-5. The lacZ reporter gene has been introduced into the myf-5 gene by homologous recombination so that its expression is under the control of the endogenous myf-5 locus. , β-Galactosidase (β -gal) coloration provides a sensitive assay for myf-5+ cells. Embryos were generated from embryonic stem cells carrying this mutation, and the appearance of β -gal+ (myf-5+) cells was followed during limb development in vivo. Limb buds, at a stage when they are β -gal- , were cultured in vitro. After several days, β -gal+ cells accumulated in the premuscle mass. We conclude that determined muscle precursor cells in the limb bud do not initially express any member of the MyoD family. Furthermore, myogenic precursor cells in the somite, which, according to the avian model, migrate from the ventral/lateral edge of the dermomyotome to form peripheral muscle masses, are also negative for these factors. myf-5-targeted ES cells carrying the lacZ reporter gene under the control of the endogenous myf-5 gene were used to generate chimeric embryos. These embryos faithfully reproduce the predicted pattern of myf-5 expression (S.T. and M.E.B., unpublished results), indicating that the introduction of the lacZ and neomycin-resistance genes into the coding region of the first exon has probably not perturbed myf-5 regulatory sequences. In 24- to 27-somite embryos (about 9-9.5 d), caudal myotomes were strongly positive for, β -gal labeling, whereas the forelimb buds were β -gal-. By the 35- to 37-somite stage (about 10-10.5 d), forelimb buds

contain , β -gal+ cells, whereas hindlimb buds are still negative. Forelimb and hindlimb buds from 24- to 27- and 35- to37-somite embryos, respectively, were removed from the embryos and cultured in vitro. Forelimbs of the latter age group served as controls. After dissection the contralateral limb buds were stained to confirm that they were β -gal-.. β -gal+ cells are clearly detectable in the forelimb but not the hindlimb bud at this stage. There is an accumulation of , β -gal+ cells in a hindlimb explant where the contralateral bud was β -gal-. This result indicates the presence of myogenic precursors in the limb bud that are initially β -gal-. In these experiments, we were able to control for contaminating myotomal cells, which proliferate extensively to form a β -gal+ layer. Another important technical point concerns the level of sensitivity of the P-gal marker. At the cellular level, singlepositive nuclei are detectable in the limb bud by whole-mount coloration. Quantitative considerations clearly indicate that the β -gal+ cells must have been generated from a population of cells that were initially β -gal- and not one or two labeled cells that may have escaped detection. These studies were carried out with chimeric embryos, and it is only the population of ES cell origin that was monitored. However, the degree of chimerism obtained was high, and our observations are representative of the entire muscle precursor cell population in the limb. To verify that β -gal- limb buds did not contain myf-5 transcripts, we carried out a reverse transcriptase-PCR analysis on normal 37-somite embryos. No myf-5 transcripts were found in hindlimb buds after 30 cycles of amplification and hybridization with a myf-5 specific probe (19), whereas forelimb buds at this stage were clearly positive. In forelimbs but not hindlimbs, myogenin transcripts were detectable, as expected (10). We conclude from these experiments that myogenic precursor cells initially present in the limb bud do not express the myf-5 gene. This population subsequently becomes positive after culturing. The β -gal protein is relatively stable and can therefore be used in chase experiments to track cells that previously expressed the marked gene (20). Since no β -gal+ cells were present initially in the cultured limb buds, we conclude that the myf-S gene was not expressed in limb precursor cells in the somite. Furthermore, in embryos at earlier stages, no myf-5-expressing cells are detected in the field between somite and limb bud or concentrated in the ventral lateral region of the dermomyotome from which migratory muscle precursors originate in the chicken. The onset of myf-5 expression in the immature somite is seen in the more rostral region. This initial site of expression is consistent with observations in the chicken, where the earliest myotomal cells were localized to this compartment of the immature somite. This labeling then continues to be restricted to the medial half of the somite along the cranial-caudal axis. It is clear that the dorsal/medial part of the somite, mainly dermomyotome at this stage, has a concentration of , β -gal+ cells, whereas no such labeling is evident in the ventral/lateral part. If myf-5 were expressed transiently in these latter cells, we would have expected to see β -gal+ cells in the early limb bud, due to the stability of this reporter since the events in question occur in a matter of hours. This was not the case. Therefore our data is consistent with the proposition that precursor cells that will migrate to form peripheral muscle do not express myf-5 in the somite. Experiment 3: Pax-3 is necessary for migration but not differentiation of limb muscle precursors in the mouse -G Daston, E Lamar, M Olivier and M Goulding

The limb muscles of vertebrates are derived from precursor cells that migrate from the lateral edge of the dermomyotome into the limb bud. Previous studies have shown that the paired domain-containing transcription factor Pax-3 is expressed in

the limb in cells that are precursors for limb muscles (Williams, B. and Ordahl, C.P. (1994) Development 120, 785-796). In splotch (Pax-3-) embryos, the limb muscles fail to develop and cells expressing Pax-3 are no longer found in the limb. In this paper we have analyzed the role of Pax-3 in the migration and subsequent differentiation of limb muscle precursors. By labeling somites adjacent to the prospective forelimb with the lipophilic dye DiI, we have shown that cells derived from these somites do not migrate into the limbs of splotch mice. The failure of limb muscle precursors to invade the limb in splotch mice is associated with the absence of c-met expression in premigratory cells, together with a change in the morphology of the ventral dermomyotome. In addition, we have shown the lateral half of somites derived from day E9.25 splotch embryos can undergo muscle differentiation when grafted into the limb bud stage 20 chick host embryos. Our results indicate that Pax3 regulates the migration of limb muscle precursors into the limb and is not required for cells in the lateral somite to differentiate into muscle. 2. Genetic systems that explain these experimental results. Trace the sequence of these inductions by describing the shifts in molecular indicators expressed in the course of differentiation. Determination and Differentiation of Skeletal Muscle: •

Myogenic cells (in the myotome of the somite) pass through several additional mitotic divisions before completing a terminal mitotic division and becoming postmitotic myoblasts.

•

Proliferating myogenic cells are kept in the cell cycle through the action of growth factors, such as fibroblast growth factor (FGF) and transforming growth factor-β.

•

With the accumulation of myogenic regulatory factors, myogenic cells upregulate the synthesis of the cell cycle protein p21, which irreversibly removes them from the cell cycle.

•

Then, under the influence of other growth factors, such as insulin-like growth factor, the postmitotic myoblasts begin to transcribe the mRNAs for the major contractile proteins actin and myosin.

•

A postmitotic myoblast fuses with other similar cells into a multinucleated myotube.

•

Myotubes are intensively involved in mRNA and protein synthesis, which include the regulatory proteins of muscle contraction- troponin and tropomyosin. These proteins assemble into myofibrils, which are precisely arranged aggregates of functional contractile units called sarcomeres. As the myotubes fill with myofibrils, their nuclei, which had been arranged in regular central chains, migrate to the periphery of the myotube.

•

The peripheral migration of the nuclei of the myotube then takes place. The nuclei (myonuclei) of a multinucleated muscle fiber are no longer able to proliferate, but the muscle fiber must continue to grow in proportion to the rapid growth of the fetus and then the infant.

•

Satellite cells accomplish muscle fiber growth through the up take of positions between the muscle fiber and the basal lamina in which each muscle fiber encases itself. Operating under a poorly understood control mechanism, satellite cells divide slowly during the growth of an individual. After muscle fiber damage, satellite cells proliferate and fuse to form regenerating muscle fibers.

Muscle Transcription Factors: Myogenesis begins with a restriction event that channels a population of mesenchymal cells into a lineage of committed myogenic cells. The molecular basis for this commitment is the action of members of families of myogenic regulatory factors that, acting as master genetic regulators, turn on muscle-specific genes in the premuscle mesenchymal cells. MyoD family- The first-discovered family of myogenic regulatory factors which is composed of a group of four basic helix-loop-helix transcription factors. Muscle enhancer factor-2 (MEF-2) - another, more recently discovered regulatory factor. All of these myogenic regulatory factors are capable of converting nonmuscle cells (e.g., fibroblasts, adipocytes, chondrocytes, retinal pigment cells) to cells expressing the full range of muscle proteins. Myogenic regulatory proteins of the MyoD family form dimers and bind to a specific DNA sequence (CANNTG), called the E box, in the enhancer region of muscle-specific genes. The myogenic specificity of these proteins is encoded in the basic region. Many cells contain a transcriptional activator designated E12. When a molecule of E12 forms a heterodimer with a molecule of MyoD, the complex binds more tightly to the muscle-enhancer region of DNA than does a pure MyoD dimer. This increases the efficiency of transcription of the muscle genes. A transcriptional inhibitor called Id (inhibitor of DNA binding) can form a heterodimer with a molecule of MyoD. Id contains a loop-helix-loop region but no basic region, which is the DNA-binding part of the molecule. The Id molecule has a greater binding affinity for a MyoD molecule than another molecule of MyoD and can thus displace one of the units of a MyoD dimer, resulting in more Id-MyoD heterodimers. These bind very poorly to DNA and often fail to activate musclespecific genes. During muscle development, the myogenic regulatory factors of the MyoD family are expressed in a regular sequence. In mice: 1. Events leading to muscle formation begins in the somite 2. Pax-3 and myf-5, working through apparently separate pathways, activate MyoD which causes certain cells of the dermomyotome to become committed to forming muscle. 3. With increased levels of MyoD, the mononuclear cells withdraw from the mitotic cycle and begin to fuse into myotubes. 4. At this stage, myogenin is expressed. 5. In maturing myotubes, MRF-4 is finally expressed. In knockout mice, the absence of a single myogenic regulatory factor (e.g., myf-5, MyoD) alone does not prevent the formation of skeletal muscle (although

there may be other minor observable defects), but when myf-5 and MyoD are knocked out simultaneously, muscle fails to form. Another very instructive double knockout of Pax-3 and myf-5 produces mice that are totally lacking in muscles of the trunk and limbs, but the head musculature remains intact. This shows that in the very earliest stages of determination, different regulatory pathways are followed by muscle-forming cells of the head and trunk. The development of certain cranial muscles is controlled by a different set of transcription factors, called MyoR and capsulin. It is highly likely that a different set of transcription factors will be found to control development of the muscles of facial expression, which are derived from the second pharyngeal arch. Muscle growth is negatively controlled, as well. Myostatin, a member of the TGF-β family of signaling molecules, arrests muscle growth once a muscle has attained its normal size. In the absence of myostatin function, animals develop a grossly hypertrophic musculature. Breeds of "double-muscled" cattle are known to have mutations of the myostatin gene.

3. Abnormalities that may result from these defective gene expressions. •

Muscular dystrophy – Muscular dystrophy is a family of genetic diseases characterized by the repeated degeneration and regeneration of various groups of muscles during postnatal life. Duchenne muscular dystrophy- A disease which occurs in young boys. It is a membrane-associated protein called dystrophin is lacking from the muscle fibers. Its absence makes the muscle fibers more susceptible to damage when physically stressed. Congenital myotonic dystrophy is present at birth, almost invariably in infants whose mothers have cases of myotonic dystrophy so mild they are unaware of them. Congenital myotonic dystrophy is a rare variant of the disease with striking differences from the form it takes among adults. In the newborn period, an affected infant is profoundly weak, has difficulty which sucking and swallowing, and may have severe respiratory difficulties. Myotonia is not a feature of the condition at this stage. Motor development is usually delayed in these children, and they may show some signs of mental retardation. Generally, the condition improves through the early years but deteriorates during late childhood and adolescence, when the 'adult' features of the disease gradually emerge. Whether or not the progression is then more rapid than in cases with onset in adult life has not been established.

Pax-3- Mutations caused by Pax-3 include an absent limb muscle or no embryonic muscle formation (as observed in knockout mice), when Pax-3 and myf-5 are knocked out simultaneously. A deficiency in Pax-3 results in no formation of the limb and diaphragm muscles (as observed in splotch mice). Myf-5 – An inactivation of this gene delays the development of the myotome, paraspinal, deep back and intercostals muscles. A combined MyoD and Myf-5 knockout results in the complete absence of skeletal muscle.

Myf-6 – A gene that is also known as MRF-4 or Herculin. Its mutation may produce mild centronuclear myopathy or an exacerbate Becker muscular dystrophy. Myopathy is a neuromuscular disease in which the muscle fibers dysfunction for any one of many reasons, resulting in muscular weakness. "Myopathy" simply means disorder ("pathy" from pathology) of muscle ("myo"). This implies that the primary defect is within the muscle, as opposed to the nerves ("neuropathies" or "neurogenic" disorders) or elsewhere (e.g., the brain etc.). Muscle cramps, stiffness, and spasm can also be associated with myopathy Centronuclear myopathy (or myotubular myopathy) (in which the nuclei are abnormally found in the center of the muscle fibers) is a rare muscle wasting disorder that occurs in three forms: * The most severe form is present at birth, inherited as an X-linked genetic trait, and can cause severe respiratory muscle weakness. This is the form of centronuclear myopathy currently referred to as myotubular myopathy. * A less severe form of centronuclear myopathy that may present itself at birth or in early childhood progresses slowly and is inherited as an autosomal recessive genetic trait. * The least severe of the three forms of centronuclear myopathy first appears during the second and third decades of life and is slowly progressive; it is inherited as an autosomal dominant genetic trait. Becker's muscular dystrophy (also known as Benign pseudohypertrophic muscular dystrophy) is an X-linked recessive inherited disorder characterized by slowly progressive muscle weakness of the legs and pelvis. It is a type of dystrophinopathy, which includes a spectrum of muscle diseases in which there is insufficient dystrophin produced in the muscle cells, resulting in instability in the structure of muscle cell membrane. This is caused by mutations in the dystrophin gene, which encodes the protein dystrophin. Becker's muscular dystrophy is related to Duchenne muscular dystrophy in that both result from a mutation in the dystrophin gene, but in Duchenne muscular dystrophy no functional dystrophin is produced making DMD much more severe than BMD. Both Duchenne and Becker's muscular dystrophy have traditionally been called "X-linked" recessive diseases, but in view of modern molecular biology and identification of the dystrophin gene, it might be more appropriate to say they are X-chromosome recessive diseases. MyoD- The inactivation of MyoD in mice results in delayed muscle development in limbs, an increased number of satellite cells, and a deficient regenerative processes. The inactivation of both MyoD & Myf-5 causes complete absence of myoblasts. Myostatin – In humans, myostatin mutation causes muscle hypertrophy. High levels of myostatin could cause HIV infection with muscle wasting, while low levels in muscle could cause muscular dystrophy. In animals inactivation of myostatin causes an increase in muscle mass due to hyperplasia & hypertrophy.