Gene Development and Inheritance - Experiment 5
Fig 1 shows the microchaete pattern of a wild type Drosophila, as you can see the pattern consists of rows and columns of hairs that are evenly spaced and cover the top portion of the notum of the Drosophila completely. Fig 2 shows a very different pattern, most noticeably is the presence of two long inverted triangular stripes running side by side down the back of the fly, these triangles also have a more dense packing of hairs around the edge and a lighter patch of hairs in the middle. Flanking the two inverted triangles is another set of hair patches, two on each side that grow towards each other. Fig 3 has significantly less hair than the other two, there are two small stripes of hair growing between the macrochaete on the middle of the notum and a few individual hairs at the top of the notum but generally that is it.
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The components that make up a bristle begin as undifferentiated cells, these cells can either differentiate into sensory mother cells (which are early neural precursors) or epidermal precursors. The sensory mother cells (SMC's) are produced 8-12hr after puparium formation (APF) which coincides with the heat shock period (6-12hr APF) of the flies in Fig 2. The effect of the heat shock caused more bristles to develop in certain areas of the notum as described in question one, this can be interpreted as an overproduction of SMC's during puparium development. The mutant in Fig 2 has a temperature sensitive Notch allele which when heated at 30ºC renders the gene inactive, so during the heat shock period there is no Notch protein and the production of SMC's increased to produce the phenotype seen in Fig 2. We can therefore conclude that Notch plays a role in preventing the production of SMC's and/or promotes the formation of epidermal precursors. This is illustrated in the diagrams below, Fig 4 shows that when Notch is active the majority of the cells become epidermal precursors, however when Notch is inactivated during the heat shock period as shown in Fig 5 the net result is an increased production in SMC's. It is also important to note that there is not the same complete coverage of the notum in the Fig 2 fly as seen in the Fig 1 wild type fly, this suggests that even though there are more SMC's to develop into bristles there has not been enough time after the heat pulse for the development of the external bristle structures to take place, hence why you see the striped pattern and not complete coverage.
After the production of the SMC's and epidermal precursors (12hrs APF), the SMC,s undergo two phases of mitosis. During the first round of mitosis the SMC's can divide into either A cells or B cells, the A cells are precursors for external components of the bristles and the B cells are precursors for the internal components of the bristles. The external and internal components are formed after the second phase of mitosis, this is illustrated in Fig 6 below. During the development of the pupa before the 14-20hr heat shock period we can assume that Notch has already caused the formation of SMC's. When Notch was heat inactivated during 14-20hr only a few bristles were produced and the majority of the fly looked hairless as described in question 1, this suggests that Notch has another function. Because almost no external structures were produced Notch must be preventing the formation of the A cells so all the SMC's then divide to produce B cells which form internal components and cannot be seen, this is illustrated below in Fig 7. There is a two hour window between the development of the SMC's and the heat shock period, this window is long enough for the very first SMC's that begin to develop to divide into A cells, this explains why there was a few bristles near the macrochaete and the fly was not completely bare.
The experiment that produced the bristle pattern in Fig 2 was replicated throughout the whole class and almost the exact same bristle pattern was found in each case. This evidence suggests that the bristle pattern is conserved and follows a fixed pattern from pre determined points. It appears that the development of bristles begins at the bottom of the notum near the macrochaete because of the evidence found in Fig 3, however this only an assumption and other heat shock time frames would be needed to confirm this hypothesis. The change in pattern from Fig 3 to Fig 2 indicates that there could be a morphogen gradient such as a Notch gradient that spreads up towards the head of the Drosophila and then outwards towards the sides/wings during development as time elapses, which is why there is an inverted triangle shape/striped appearance. This type of mechanism is not unusual during development of Drosophila and many other morphological features develop using protein and chemical gradients.
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From the observations made in the previous questions we can conclude that Notch is an essential gene in determining cell fate during the development of the notum epidermis. Firstly it is involved in preventing cells from becoming SMC's and/or promotes their development into epidermal cells, secondly it is involved in promoting the mitosis of the SMC's into bristle precursor A cells.
The bristle pattern of the wild type fly in Fig 1 is even with no area of the notum containing bristles which have formed right next to each other in groups, however when you look at the triangular pattern in the absence of Notch in Fig 2 the dark concentrated patches of bristles show growth of bristles proximal to each other. Therefore in order for Notch to to produce an even pattern with spacing in between its mechanism of action must be on neighbouring cells and not directly on the SMC's , this is described in Fig 8 below.
Fig 8 - This diagram shows a proposed model for the mechanism of action of Notch, the red squares represent SMC's, the white squares are the neighbouring cells. The repression arrows that protrude out of the SMC's represent the inhibition of SMC development via the secretion of Notch. This mechanism would produce an even, regularly spaced pattern just like in the wild type flies. The removal of Notch in this mechanism would allow bristles to grow proximal to each other which can be seen in Fig 2.
There are two explanations for why the macrochaete's are not affected during after the heat shocks. Firstly, their cell fate could have already been determined before puparium formation by the action of other genes aswell as Notch. Secondly, the alternative is that their cell fate is also determined APF but before the first heat shock period i.e. before 6hrs, if this is the case then no discrepancies in the phenotype would be seen in either of the two mutants because the heat cycles are post macrochaete development..