Effect of orthodenticle expression on anterior regionalization

Anterior regionalization in A. tepidariorum:

In other higher level organisms and Drosophila, it is a well documented fact that the bicoid gene creates a gradient for anterior posterior formation. It is used extensively in pattern formation in Drosophila with its morphogen gradient mechanism (Royet 1995). However, cellular membranes in arthropods prevent gradient formation and it was previously unknown how regionalization occurred. It is now known that anterior regionalization begins before cellular membranes form, ie before the blastoderm forms and is crucially affected by the orthodenticle gene. This paper seeks to explore how the orthodenticle gene affects anterior regionalization.

What is orthodenticle (otd)?

Orthodenticle (otd) is a homeobox gene that is involved in diversified mechanisms in many steps of development. It is a highly conserved gene in many  insects and arthropods including Drosophila and other species of spider. The gene encodes DNA binding proteins that then express in stages of development, often regulating the development of the head and specifically the eyes (Simmonet et al, 2006).

In A. tepidariorumthere are two types of otd  expressed at different moments of regionalization, and only At-otd-1 is involved in early development.  Atd-otd-1 is integral

A. tepidariorum

for the establishment of positional information. In development, it is first found in the germ disk at stage 5 and eventually moves to the head domain that corresponds to the ocular region. Studies have shown that Atd-otd-1 is integral in the formation of all the structures in the pedipalpal region of the insect. It also has another function in determining the positional information for the corresponding dynamic hairy and hedgehog genes (At-h and At-hh). While it does not determine the functionality nor the onset of the two genes, it does determine the dynamic repositioning of the hairy gene (At-h) and  the hedgehog gene (At-hh).


Experimental procedure:

In order to first determine what elements were involved in regionalization, three segmentation genes were analyzed: Hairy(At-h), hedgehog(At-hh), and orthodenticle(At-otd-1). After finding that At-otd-1 has the  most crucial place among the three, the exact function of otd was explored by using pRNAi embryos.


Results:

When gene expression was examined in A. tepidariorum, it was found that At-otd-1 was a primary gene that was expressed and it affected the At-h and At-hh genes in the following ways.

Expression of At-odt-1, At-h, At-hh at different stages of embryo formation (Pechmann 2009)

Stage 5: At-otd-1 is expressed in a ring of the germ disk; at-h is expressed in a ring at the rim and in the center of the ring; at-hh is also in ring on germ disk

Stage 6: At-otd-1 ring breaks and begins to form the anterior of the organism; at-h clears out leaving a stripe that corresponds to other development stuff; at-hh appears as ring at rim of germ disk

Stage 7: At-otd-1 moves towards posterior side of organism; at-h more posterior and splits into 2 stripes

Stage 8: At-otd-1 moves toward head domain

Based on the gene expressions, it can be seen that dynamic gene expression is a critical mechanism by anterior regionalization is achieved. In this case, there is a single wave of expression of each gene and out of these waves, based on RNAi tests, otd is the most crucial for anterior regionalization because without it, the other two do not function.

At-otd-1 pRNAi results in loss of anterior structures. including in the pedipalpal region (Pechmann 2009) showing that otd is necessary for anterior regionalization because it pinpoints hairy and hedgehog genes expression.

In experiments done on orthodenticle pRNAi embryos it was found that otd is the crucial gene in anterior regionalization. In embryos whose mothers had been injected with At-otd-1 dsRNA, anterior expression was greatly reduced. They also showed head defects (showing that otd’s other functions are located in the head region), and no development in the regions anterior to the pedipalpal region (showing that otd is also crucial for pedipalpal formation). The lack of At-Pax6 and At-six3 also confirms the crucial role otd plays  in pedipalpal formation because those are the two crucial genes . It was determined that otd expression directs hairy and hedgehog to form stripes in the anterior of the spider embryo which in turn define the pedipalpal region.

Future Research/Paper critique

It is unclear what signaling pathways are involved, but there the Wnt pathway is the most likely candidate. Insects and spiders develop similarly, but insects use the Bicoid gene for pattern formation.

The paper ends by saying that many of the other pathways could be conserved in Drosophila and mice however it is unclear which pathways are being discussed and how they suggest researching them.

In general, the paper was very clear and concise however many of the results were not given in quantitative form but were rather discussed in a very general manner. It would be beneficial to see more precise explanation of the results. The paper would also benefit from a further study into the mechanisms of the dynamic gene expression and the signalling mechanism used for the dyanamic gene expression.

References:

Pechmann, M., A. P. McGregor, et al. (2009). “Dynamic gene expression is required for anterior regionalization in a spider.” Proc Natl Acad Sci U S A 106(5): 1468-1472.

Simonnet, Franck, Marie-Louise Célérier, and Eric Quéinnec. “Orthodenticle and Empty Spiracles Genes Are Expressed in a Segmental Pattern in Chelicerates.” Development Genes and Evolution 216.7-8 (2006): 467-80. Print.

Kotkamp, K., M. Klingler, and M. Schoppmeier. “Apparent Role of Tribolium Orthodenticle in Anteroposterior Blastoderm Patterning Largely Reflects Novel Functions in Dorsoventral Axis Formation and Cell Survival.” Development 137.11 (2010): 1853-862. Print.

Royet, Julien, and Robert Finkletstein. “Pattern Formation in Drosophila Head Development: The Role of the Orthodenticle Homeobox Gene.” Development 121 (1995): 3561-572. Print

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