Lunatic fringe, which encodes a glycosyltransferase that modulates Notch signaling, is cyclically expressed in the chick and mouse PSM, although it is not expressed cyclically in zebrafish. Instead, deltaC, which encodes a Notch ligand, is expressed in a cyclical manner in zebrafish, although its related chick and mouse genes, delta1 and delta3, are expressed uniformly throughout the PSM. In mouse, mutations for Lfng result in random and incomplete somite segmentation. Lfng is thus functionally important for the coordinated somite segmentation. Constitutive expression of Lfng also disturbs somite segmentation in chick and mouse. Dynamic expression of Lfng is not a result of posttranscriptional regulation such as cyclic changes in mRNA stability but rather a result of periodic activation and repression of the Lfng promoter. Region 2 also known as region A in the promoter of Lfng contains a binding site for CBF1/ RBP-J, a mediator of Notch signaling. Notch signaling induces oscillatory expression of Lfng through the CBF1/RBP-J site. Lfng also establishes a negative feedback loop in chick. In addition to Lfng mRNA, Lfng protein exhibits oscillatory expression in the PSM. Activation of Notch signaling induces Lfng transcription but Lfng protein inhibits Notch signaling and thereby represses its own transcription. Thus, Lfng also periodically represses its own expression. Axin2 in the Wnt/b-catenin signaling is cyclic in mouse PSM and induces cyclic transcription. Wnt3a mRNA is expressed in the mouse tailbud and it has been proposed that Wnt3a protein forms a posterior-anterior gradient in the PSM, because Axin2 is downstream of Wnt signaling and oscillates with a higher amplitude in posterior PSM than in anterior PSM. Axin2 is related to Axin, a critical component of the Wnt signaling pathway that acts as a scaffold for the b-catenin destruction complex. Wnt signaling drives transcription of Axin2 mRNA, and Axin2 protein inhibits Wnt signaling. Snail1 is a cyclic gene of the Fgf clock and oscillates largely in synchrony with the NOTCH cyclic genes, but its expression is independent of NOTCH signaling and relies upon WNT3A signaling. Misexpression of Snail1 in the chick PSM blocks Lfng and Meso1 expression. Periodic expression of Snail1 is independent of NOTCH, but is downstream of the WNT Pathway.
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A negative feedback loop is a mechanism by which the expression of a gene is repressed by its own protein product. It has been suggested by several authors that the mechanism that drives the oscillations of the segmentation genes relies indeed on feedback inhibition. It is possible to consider the existence of three types of negative feedback loops by incorporating data from zebrafish, chick and mouse, although the possible interactions between these loops are still not understood: 1) a direct feedback loop that generates the cyclic expression of Hairy/ Enhancer-of-Split family of bHLH repressors (Her1 and Her7 in zebrafish and Hes1 and Hes7 in the mouse); 2) an indirect feedback loop that establishes periodic activation of Notch signalling (DeltaC in zebrafish and Lfng in the chick); 3) another indirect feedback loop that promotes periodic activation of Wnt signalling (Axin2 in the mouse)
The wavefront position seems to be regulated by Fgf and Wnt signalling, by retinoic acid (RA) signalling and possibly by an unknown pathway involving the T-box gene, tbx24. In chick, either inhibiting or overexpressing Fgf8 at the level of the determination front alters the position of somitic boundaries, inducing the formation of larger or smaller somites, respectively. Fgf8 maintains posterior PSM cells in an immature state, thus negatively regulating the wavefront of differentiation. Fgf/mitogen-activated protein kinase (MAPK) signaling is functioning in the posterior PSM and it maintains these cells in an immature state. Fgf signalling determines the position of segment border formation in Zebrafish. In the mouse, wnt3a seems to play a similar role to the one attributed to Fgf signalling in both chick and zebrafish PSM. wnt3a is strongly expressed in the tail bud and it acts upstream of fgf8 in the regulation of the wavefront position. Since there is evidence that Fgf signalling may enhance Wnt/β-catenin signalling, Fgf8 might act as a relay enhancer of Wnt signalling in the PSM of mouse embryos.
The somatic mesoderm promotes maturation events, which are correlated with the activation of RA signalling in rostral PSM and somites, as indicated by the expression of the RA-synthesizing enzyme raldh2. Furthermore, RA downregulates the expression of fgf8 in the PSM. Conversely, Fgf8 soaked beads placed in the chick PSM represses the expression of raldh2, which indicates that Fgf signalling regulates the onset of RA synthesis in presomitic tissue. Thus Fgf and RA signalling pathways are mutually inhibitory and point to an important role of RA in inducing PSM cells maturation, in opposition to Fgf8. tbx24 is expressed in anterior and intermediate PSM, its function seems to be restricted to the rostral PSM. tbx24 plays a role in the maturation process of anterior PSM cells and it might be independent of the molecular clock.
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Fgf and Wnt signalling pathways seem to control the positioning of the wavefront of differentiation. Since the formation of somitic boundaries is such a finely tuned process it is conceivable that a signal in the anterior PSM controls the precise site of the determination front.
Somite formation begins as paraxial mesoderm cells become organized into whorls of cells called somitomeres. The somitomeres become compacted and bound together by an epithelium, and eventually separate from the presomitic paraxial mesoderm to form individual somites. Somite formation correlates with the expression of the cyclic genes of Notch, Wnt and Fgf. Initially the expression of the cyclic genes is seen in the caudal half of a newly formed somite, as well as in the posterior portion of the presomitic mesoderm and in a thin band that will form the caudal half of the next somite. It is the anterior boundary of FGF8/Wnt3a that determines where the somites form. The wavefront regresses posteriorly and its anterior boundary correlates with the Determination boundary. Determination front is the point where there is least expression of both RA (expressing anteriorly) and WNT/FGF8 (expressing posteriorly) in a gradient fashion. The clock oscillations stop and there is expression of new sets of gene called as Meso1. Then a caudal fissure begins to separate the somite from the presomitic mesoderm. The posterior region of cyclic gene expression extends anteriorly The newly formed somite retains the expression of in its caudal half, as the posterior domain of gene expression moves farther anteriorly and shortens. Once the formation of somite is completed, the anterior region of what had been the posterior cyclic genes expression pattern is now the anterior expression pattern. The molecular relationship between the clock and wavefront is described while describing the wavefront itself.
Answer 2: Part B
The cell generation time is almost fourfold slower in snake than in chicken. Remarkably, the calculated number of cell generations required to generate the more than 300 somites in the snake (~21 generations) is only slightly greater than for the 65 somites in the mouse (~17 generations) or 55 somites in the chicken (~13 generations), although much larger than for the 31 somites in the zebrafish (~2.8 generations). Therefore, the exceptionally large number of somites in the snake, compared with that in other amniotes, is not primarily the result of a large number of generations of Presomitic Mesoderm growth, but reflects a clock rate that is rapid in relation to the cell-cycle rate in the elongating axis. Thus, the reason there are more somites in the snake is not that there are significantly more cell divisions that make significantly more cells; rather, in a single cell generation in the snake the clock ticks more times than in a single cell generation in the chicken or mouse. By ticking of clock more number of times I mean that there is more rapid oscillation of cyclic genes as compared to other species in snake. (Gomez, C., -zbudak, E.M., Wunderlich, J., Baumann, D., Lewis, J., Pourquié, O. (2008). Control of segment number in vertebrate embryos. Nature DOI: 10.1038/nature07020)
The morphological criteria the authors used to distinguish between the digits are:
Loss of associated carpal or tarsal: I think it is a good way to identify digits because for digits 2,3 and 4, the shape and size of articulating carpals/tarsals are distinctive. But since it is not distinctive between 4 and 5, there may be a confusion as to which digit is actually lost, if one of the digit is still there.
Digit attenuation: Observed as intermediate stage or partially developed stage. For anticipation of next digit to be lost. I believe this is a good criteria as long as the digit attenuation is not confused with smaller digits. Always a wild type control would be required while using this criteria.
Metapodial shape at the proximal articular end. This is not a good way to identify because the shape at the articular end may not be very distinct especially within the digits 3,4 and 5, and there may be confusion.
Element length and timing of ossification: Used mainly for secondary confirmation. It is good only for secondary confirmation because the it is variable even within the wild type embryos.
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Overall I think since the authors used all the above mentioned criteria, it can be said that their interpretation on digit identity would be correct.
Shh specifies A-P digit identity: When Shh-soaked bead was implanted at the anterior portion of the limb bud of a WT embryo before the development of digits (before 9.5pdc), mirror image duplication of normal development was observed from the graft (after 14.5dpc when entire digits are formed). Also the level of duplication was observed in a concentration dependent manner. When beads with high levels of Shh (16mg/ml) were placed, complete duplication was observed. However on placing bead with lower concentration (0.75mg/ml) of Shh, only anterior duplications were observed.
Shh controls digit growth: Shh -/- mutant mice donot have 2,3,4 and 5 digits. These digits are entirely lost and only digit 1 is seen in such mice.
Answer 3F : Author's Interpretation of their data in terms of
The role of Shh in specifying A-P digit identity: They say that Shh does specify the A-P digit identity, only during the initial phases when the Shh is expressed. Although the way is specifies digits are digit 1 is independent of Shh expression. The more posterior digits 2,3,4 and 5 develops in a way that digit 3 is most Shh dependent digit followed by digit 5, digit 2 and digit 4.
The role of Shh in controlling digit growth: They say that the removal of Shh activity alters both cell survival and proliferation in limb mesenchyme. Both of these effects may contribute to the resulting decreased number of cells in Shh mutant limbs. Shh removal reduce the total number of cells proportionate to the duration of its absence.
The authors have a biphasic model. They say that Shh acts early and transiently to specify digits properly, and thereafter is required mainly for the regulation of cell numbers. They say that the activity of Shh in specifying digit identity and conferring A-P polarity is only confined to a narrow window when the Shh is first observed. Harfe et al., 2004 however showed that d3 partly and d4-d5 completely derive from cells that previously expressed Shh, showing that Shh patterning occurs by signal transduction over time.
I do believe the author's interpretation of their data and their model. The way they used to identify the different digits is the most appropriately used method till date. I think their idea that A-P polarity is conferred during early expression of Shh is weakly supported. To prove this to be true there can be performed an experiment when the Shh is allowed to express early and then should be shut off using tamoxifen induced cre in Shh flox/null mice. Then growth factors can be added to the embryo (either by harvesting the mice and growing the embryo in vitro) or by some means to the in vivo system. I think it would be difficult to grow the embryos in vivo by adding growth factors for allowing the phalanges to develop. So the mice can be harvested after tamoxifen induced cre is active and no more Shh is expressed. Then in vitro we can add growth factors specifically to the limb buds for it to stimulate the growth of limb.
Results and Interpretation: If the embryo shows development of tarsals, metatarsals and phalanges, similar to the normal digit growth, then the model is validated.
Answer 4 A
The Mutant 1 phenotype (similar to lin 28 mutnat) shows precocious heterochronic phenotype because the formation of adult stage occurs earlier than usual. The lineage pattern as compared to WT shows that Second larval stage is skipped and a division pattern seen at later stage is observed. Because when cells go from L1 to L2 at moulting, from 1 L1 seam cell, four cells are formed. However, in mutant 1, only 2 cells are formed from one cell at the time of moulting.
Answer 4 B
Mutant 2 (similar to Lin-29 mutant) shows a retarded heterochronic phenotype because the formation of adult stage is delayed and the fates are reiterated. The lineage pattern reveals that L4 stage is repeated because the division is normal until L4 and then it instead of forming adult alae, it goes on repeating the L4 stage. At the time point indicated with an arrow, the cuticle will look like a larval cuticle instead of an adult cuticle. The seam cells will not fuse to form the adult lateral seam and no secretion of specialized cuticle structure alae will be there.
When the authors used RNA interference, using miRNA and shRNA, under the control of avian retroviral vector, RCASBP, all the embryos that were genetically male (ZZ) showed feminization. Histologically, the left gonad showed female like histology, the testis cords were disorganized. When looked at the expression levels of sex related markers, the marker for maleness showed lower expression in DMRT1 knockdown ZZ embryos. However marker for femaleness was shown to be ectopically expressed in such embryos. Sox9 was used as the marker for maleness, whereas Aromatase was used as a marker for femaleness.
We can check the DMRT1 expression in males and females at day 6, when sex is determined or at later stages. For this purpose, in-siu hybridizations (ISH) or qPCR can be done. Insitus or even Immnohistochemistry (IHC) can be done to identify where the DMRT1 is expressed, whereas qPCR will quantify the amount of DMRT1 expression and will make it easy to compare male and female.
The possible outcome:
ISH and IHC: Can find expression area of DMRT1 gene in males. However, in females there might not be any area where DMRT1 expression could be seen. If DMRT1 is expressed in the internal organs and could not be observed, then at first sectioning could be done and then the expression could be checked by using ISH or IHC.
The entire embryo could be used to isolate RNA. qPCR should show higher expression of DMRT1 in males as compared to females.
ISH and IHC: DMRT1 is expressed only in males and thus show that it might have a role in the determination of male sex. There is no role of DMRT1 in the females. This shows that DMART1 is not required for development of any other morphology other than the gonads.
qPCR results further validate the results obtained in the above set of experiments.
Another approach which is not feasible could be to make transgenic chicken for DMRT1 under a cre and then can express cre (using tamoxifen) at day 6. All the embryos irrespective of their genotype would show feminization if DMRT1 is required for maleness.
DMRT1 protein is expressed in gonads of male embryos in the nuclei of developing sertoli and germ cells within testis cords. It is not expressed in the female gonad. I believe that DMRT1 functions to get expressed in the primordial germ cells (PGC) in male sex (ZZ genotype embryos) to control sexual fate. DMRT1 starts getting expressed in the PGC at the time when cleavage and blastulation occurs and the PGC is derived. In males the DMRT1 gets expressed in these PGC to finally develop testis and sertoli cells.
Experiment: Can be done at the blastocyst stage or at the stage when the fertilized egg is laid by hen. Since the segregation of cells into groups of special function (tissues) occurs before laying and then the embryo goes into stage of inactive embryonic life, day 1 after egg is laid could be the best stage for our experiment. We can look at the expression of DMRT1 in this stage. Genotypically male embryos will show the expression of DMRT1. But the presence of expression would show that DMRT1 gets expressed in ZZ genotype embryos to finally become males.
Two copies of Z chromosome is required for the induction of expression of DMRT1 for sex determination. However, only one copy of Y chromosome is required for SRY.
DMRT1 gets expressed in both male and female cells due to its presence on Z chromosome, however, SRY is only expressed in males due to its presence on Y chromosome.
The components of germplasm are in cytoplasm, called pole plasm but are in form of non membrane bound cytoplasmic structures called nuage or germplasm granules (called as P granules in C.elegans), also referred as polar granules. Germplasm granules are ribonucleoprotein particles implicated in translational control. In the developing germ line P granules are associated with nuclear membrane and detach from it in the maturing oocyte. In the early embryo P granules co-segregate into the germline progenitor cells. The germplasm also harbors a large number of RNAs that likely play specific roles in germ cell specification, migration, and germ cell fate. Among these, nanos (nos protein is required for germ cell fate and migration), germ cell less (gcl protein is required for germ cell formation), and polar granule component (pgc protein is required for transcriptional silencing in germ cells) RNAs become enriched in the germplasm during oogenesis.
Because the cells remain connected by cytoplasmic bridges, there is movement of cytoplasm from one cells to other. This helps as even after Meiosis is done still every cell can get both X and Y chomosome expressed proteins. There is synchronization of cell division which leads to formation of gametes with similar age and thus helpful in fertilization. The cells donot get clustered with each other and each cell gets equal opportunity to grow.
Advantages: A ) Spatialy better. Occupies less space and better anchor for cells. B) Better for sperms, as they are produced in clusters and thus easy for movement. C) All sperm and ova from one lineage mature at the same time. This is helpful for efficient fertilization.
Problem could be that if one cell gets affected in cell division, there would be change in the protein expression in those cells. So the adjacent cells will also be affected by getting more/less protein depending on the problem occurred during cell division. Infection of one cell by any pathogen will affect all the cells sharing the same syncitial environment.