Our lab currently investigates the roles of two genes known to be involved in the regulation of area patterning: Emx1 and Emx2.  Both genes are expressed in developing neural precursors in a gradient where the highest level of expression is located posterio-medially (i.e. back and midline of the cortex).

 Previous research demonstrated that Emx2 plays an important role regulating area fate utilizing constitutive knock-out and over-expression approaches.  This research demonstrated that loss of Emx2 causes a posterio-medial shift of the location of all primary sensory areas.  This posterior shift results in an effective reduction in the size of sensory areas that reside along the posterior edge; more specifically the primary visual area is reduced in size.  This posterior shift also results in an effective increase in the size of sensory areas that reside along the anterior edge; more specifically the frontal-motor area is increased in size.  Conversely gain of Emx2 function causes an anterio-lateral shift of areas.  Increasing Emx2 expression thus results in changes opposite of deletion of Emx2 with respect to sensory area size.

Figure caption: Flattened horizontal (tangential) sections of cortical hemispheres that have been immunostained for serotonin (5HT).  5HT staining elucidates thalamic terminations in layer IV of primary sensory areas thus providing a useful marker of the primary sensory areas.  Constitutive heterozygous deletion of Emx2 (Emx2 +/-) causes a posterior shift of sensory areas and reduction in primary visual area (V1) size.  Over-expression of Emx2, either heterozygous (Ne-Emx2 het) or homozygous (Ne-Emx2 homo) gain of function, causes a proportional anterior shift of sensory areas and increase in V1 size.

F/M denotes mixed frontal-motor area; S1 denotes primary somatosensory areas; A1 denotes primary auditory areas; V1 denotes primary visual areas; asterisk denotes the location of the C3 barrel within the posteromedial barrel subfield of S1; dotted line denotes anterior edge of V1 in wild type (control) animals; percentages denote the amount of Emx2 gene expression as determined by qPCR.

This figure is taken from the O'Leary et al., 2012 review chapter.

The findings yielded by the various deletion approaches were confirmed by our recent published data utilizing a conditional deletion approach (Zembrzycki et al., 2015).  This recent publication further demonstrates that changes in the level of Emx2 expression in the developing neocortex not only result in alterations in primary visual area size but also the higher order visual sensory areas (responsible for additional processing of visual information acquired from the eyes and relayed through the thalamus to the neocortex).

Figure caption: Whole mount in situ hybridization stains for the recently described markers of higher order areas: Cadherin 8 (Cad8) and Lmo4.  As described in the figure above, increasing or decreasing Emx2 level results in a corresponding change in V1 size.  These newly described markers allowed for the first analysis to be performed on changes in higher order area size following genetic changes to area patterning genes.  The higher order visual areas (VHO) also increase or decrease corresponding to Emx2 levels, and the changes very closely mirror the changes observed in V1 (i.e. the different visual areas, VHO and V1, scale linearly or exhibit the same proportional change).

V1 denotes primary visual areas; VHO denotes the higher order visual areas which are also outlined by the white dotted lines; Aud denotes primary auditory area in the schematic; S1 denotes primary somatosensory area in the schematic; scale bar = 1 mm.

This figure is modified from Zembrzycki et al., 2015.

Previous research failed to discover a link between Emx1 and area patterning, but the field has actively developed many useful tools and procedures that greatly enhance our ability to identify primary sensory areas.  Utilizing these new markers and approaches, we were able to demonstrate that Emx1 also regulates area patterning (Stocker & O’Leary, 2016).  The changes in primary sensory area location following deletion of Emx1 mirror what has been observed following the cortex-specific deletion of Emx2.  Thus both Emx genes contribute to area patterning in a similar fashion.

Figure caption: Flattened horizontal (tangential) sections of cortical hemispheres that have been processed for in situ hybridization against the ROR-beta probe.  ROR-beta expression is present in layer IV of primary sensory areas providing another useful marker of the primary sensory areas.  Deletion of Emx1 causes a posterior shift of the primary sensory areas and a reduction in primary visual area size (V1).  Two different Emx1 deletion approaches were employed to ensure that the changes observed were due to the deletion of the gene and not strain or background specific alterations.

F/M denotes mixed frontal-motor area; PMBSF denotes the posteromedial barrel subfield (which is the most readily identifiable portion of the primary somatosensory area); V1 denotes primary visual areas; arrow denotes the location of the C3 barrel within the PMBSF; borders of the aforementioned regions as well as for the entire neocortex (cortex) are outlined; scale bar = 1 mm.

This figure is modified from Stocker & O'Leary, 2016.

Future research will further explore the roles the Emx genes play in area patterning regulation.  Conditional and constitutive genetic deletion approaches of either Emx gene will be employed to pursue these questions.  Students who join the team to help investigate these questions will learn a number of different experimental approaches. Students will learn how to tag and genotype mice.  Students will learn how to perform transcardial perfusions and remove intact murine brains via microdissection.  Students will also learn how to prepare tissue for and embed into paraffin wax, then subsequently how to section tissue utilizing a microtome.  Finally, students will learn how to detect the presence of specific proteins or messenger RNA in tissue (i.e. immunohistochemistry or in situ hybridization).  While the diversity and complexity of the approaches employed by our laboratory can seem daunting, we progress through these at a pace that ensures people slowly become proficient in each task.  Science is not always an easy undertaking, but few worthy pursuits are easy to master and this is no different.  If you are interested, visit the get involved page to learn how to join the team.

Web design by A. Stocker

Minnesota State University Moorhead, Biosciences Dept.