Example grant courtesy Dr. Shubha Tole, TIFR

Format for submission of projects

Project Title
Role of Lhx2 in Olfactory Bulb formation
Project summary (Not to exceed one page. Please use separate sheet) 
The Olfactory bulb is a specialized structure that receives sensory information from the nasal epithelium.  It has a distinctive morphology and cytoarchitecture that is crucial to its function in olfactory processing.  We are interested in the mechanisms that regulate the formation of this structure, and also the regulation of axon guidance to and from the olfactory bulb.  In particular, we will focus on a transcription factor of the LIM-homeodomain family, Lhx2, that appears to be important in the formation of the olfactory bulb, since we find in the Lhx2 mutant embryo, the olfactory bulb appears to be morphologically disrupted.  We propose to examine the role of Lhx2 in the specification of the olfactory bulb. We will also examine axon guidance in the Lhx2 mutant olfactory system.  In additon, will use RNAi and gene transfection in utero to selectively perturb the system.

Technical details

Introduction (under the following heads)
1. Origin of the proposal
    The broad question underlying this proposal is one of pattern formation.  How does a sheet of neuroepithelium, apparently uniform in morphology and cell composition, become “patterned” i.e. regionally specialized, generating neurons with distinct molecular and morphological features as well as specialized innervation patterns?  The Olfactory Bulb (OB) is a specialization of the most rostral part of the forebrain.  How this structure and its unique cell types are formed is the focus of this proposal.
    The central nervous system arises from the neural tube, formed by rolling up a sheet of embryonic neuroepithelium.  By embryonic day (E) 9.5 in the mouse, neural tube formation is complete, with morphologically visible divisions such as forebrain, midbrain, hindbrain, and spinal cord.  The forebrain gives rise to the OB at its most rostral point, first observed at E12.5 in the mouse.  By E14.5, a distinct morphological protuberance is seen, which forms the OB.  Sensory innervation from the nasal epithelium provides input to the OB.  Projection neurons from the OB, the mitral cells, innervate the olfactory cortex and amygdala.   The genetic mechanisms that control the formation of the OB and its afferent and efferent innervation are not well understood.
2. Rationale of the study supported by cited literature
In this proposal, we will explore these questions in the light of a new observation in a mouse mutant for the gene Lhx2, which at least morphologically, appears to be lacking the olfactory bulb.
Lhx2 is a member of the LIM-HD (LIM-Homeodomain) family, which encodes a group of transcription factors that are involved in cell fate specification in diverse invertebrate as well as vertebrate systems (reviewed in Subramanian et al., 2003).  Multiple LIM-HD genes act combinatorially to specify neuronal identity in the Drosophila ventral nerve cord, and in the vertebrate spinal cord (Tsuchida et al., 1994; Appel et al., 1995; Thor at el., 1999).  Several members of this family have been implicated in regulating specific aspects of neuronal identity, differentiation, and axon pathfinding (Hobert and Westphal, 2000). 

LIM-HD protein function is modulated by regulatory interactions via the LIM domains.  Lmo proteins have only LIM domains, but no homeodomain, and these molecules can interfere with the function of LIM-HD proteins by competing for essential co-factors encoded by Clim genes (Milan and Cohen, 1999).  LIM-HD protein function may thus be modulated by the presence of Lmo molecules.    
The functions of the LIM-HD group of genes have been examined to some extent in the brain.  For example, Lhx5 knockout mice have a disrupted hippocampal morphology, while Lmx1a knockout mice have neuronal migration defects (Zhao et al., 1999; Costa et al., 2001).  However, the OB was not specifically examined in these mice.  Our own previous work has shown that Lhx2 is crucial for an early event in the formation of the cortex (Bulchand et al., 2001).  In addition, we have preliminary evidence that the formation of the olfactory bulb is abnormal in the Lhx2 mutant.  This serves as a basis for setting up the hypotheses we propose to test (detailed in “objectives”).

3. Hypothesis 
Several fundamental questions arise with respect to the development of the olfactory bulb.  The precise genetic cascade of events required to specify the unique cell types in the olfactory bulb are largely unknown (reviewed in López-Mascaraque and de Castro, 2002).  There is only a little information on the genes that might regulate the positioning of the olfactory bulb at the rostral end of the telencephalon, and the related issue of its morphological structure.  Are the issues of specification, i.e. creating the correct cell types, distinct from the morphological “out-pouching” of tissue to form the bulb?  Can an olfactory bulb be created at incorrect locations in the forebrain, and is a mislocated olfactory bulb able to receive innervation from and send projections to the correct areas?  We propose to address these questions via the specific objectives b:

4.  Key questions
a)  To test the role of LIM-HD gene Lhx2 in the specification of the OB by a detailed analysis of the Lhx2 mutant brain, and by perturbing Lhx2 function using in utero electroporation. 
b)  To examine the regulation of axon pathfinding from the nasal epithelium to the olfactory bulb in control and Lhx2 mutant mice.
5. Current status of research and development in the subject (International and National)
International status
This is a frontier area of Neuroscience.  The olfactory bulb is a structure about which much is known in terms of how it receives its input, but little is known about the mechanisms that control its formation or that regulate the exit of information to higher areas (reviewed in López-Mascaraque and de Castro, 2002).  To date, a handful of molecules have been identified that participate in this function (Frantz, 1994; Yoshida et al., 1997; Bulfone et al., 1998; Jimenez et al., 2000), but the inter-relationships between them, and identification of a well-defined pathway with “steps” or hierarchy of action is as yet far from reach.  Partly, this is because all the players in this process have yet to be identified; in particular, the role of the LIM-HD family in this process has not been previously addressed.  This proposal seeks to expand our knowledge of genetic mechanisms in the olfactory bulb.
National status
The development of the brain in the embryo is being examined in very few laboratories in India, and to our knowledge none focus on the vertebrate olfactory bulb as the structure of interest.  There is, however, a growing number of laboratories that work on vertebrate development focusing on other structures:  Dr. Maneesha Inamdar’s laboratory (JNCASR) studies angiogenesis; Dr. Jyotsna Dhawan’s group (CCMB) focuses on myogenesis; Prof. Mitradas Panickar (NCBS) studies early embryonic development; Dr. Shyamala Mani (NBRC) examines embryonic stem cell differentiation as well as neural development.  These systems share common molecules and mechanisms with those that operate in the developing brain.  Furthermore, Prof. Veronica Rodrigues’ laboratory at TIFR has a long-standing interest and expertise in the field of Drosophila olfactory lobe development.  There are many parallels in the fly and mouse olfactory lobe organization, and this presents an exciting avenue to explore in the context of conservation of LIM gene function.
6 Relevance and Expected outcome of the proposed study
This work is unique for two reasons:
1)  We have identified a strong candidate gene for the specification of the olfactory bulb.  Our results will have evolutionary implications, since olfactory structures are present in non-mammalian vertebrates, and the mechanisms that govern their development may be conserved also.  Furthermore, by the end of this proposed research, we will be in a position to benefit from the expertise in the Drosophila olfactory system that has been developed in Prof. Veronica Rodrigues’ lab, by beginning an exploration of conserved mechanisms of LIM-HD gene function in the mouse and fly olfactory systems.  Such conservation of function of LIM-HD genes has been demonstrated in the wing/limb of the two species, (Rodrigues-Esteban  et al., 1998) so it will be significant to extend such a parallel to the brain structures.
Elucidating the mechanisms of formation of embryonic brain structures is crucial to understanding developmental deficits that underlie disorders of brain function.  Our work in the mouse cortex will set up a foundation for analysis of defective human development underlying behavioural deficits of anosmia. 

2)  This proposal uses an elegant and powerful technology for perturbing gene expression in mammalian embryos.  In utero electroporation selectively perturbs a group of cells in a normal background.  Since the perturbation can be done after early embryonic development is complete, and because electroporation is only performed in the forebrain, the rest of the embryo is unaffected. Only a few laboratories in the world have successfully used this cutting-edge technology.   We have recently succeeded in obtaining preliminary data with in utero electroporation, demonstrating that we are very close to establishing this technique in our lab.  This will open up new avenues of exploration for any developmental question, and will be shared with other researchers who want to learn it.  Our laboratory will take in students from other Institutes for training, and since this technique does not require large equipment support, it will speed the progress of research in mammalian development in India

7. Preliminary work done so far
We have previously shown that one member of the LIM-HD family, Lhx2, plays a major role in regulating a crucial boundary in the early cortical neuroepithelium.  In the absence of Lhx2, the cortex is greatly shrunken and specific structures like the hippocampal formation are undetectable.  These findings indicate a crucial role for Lhx2 at an early step in telencephalic patterning, that of specifying the boundary between presumptive cortex and the cortical hem (Bulchand et al., 2001).  In addition, there appears to be a morphologically observable defect in the olfactory bulb in this mutant, suggesting that Lhx2 may also play an important role in the development of this structure.

We have made some progress towards setting up the experimental design for this proposed research.  Specifically, the following steps have been completed:

a)Construct making has begun:  We have generated a full length, sequence-verified construct encoding Lmo3, a potential competitior of Lhx2 function.  This construct is GFP tagged to allow us to detect cells that express it.  Other constructs, encoding Lhx2, DN-Lhx2, and RNAi constructs, remain to be made and will be part of the proposed work.

b)In vitro electroporation works:  We have successfully tested that EGFP fusion constructs express in explant cultures of embryonic cortex.   The following figure shows GFP fluoresence from one such experiment, where after the GFP image was recorded, the explant was processed for in situ hybridization for the gene of interest.  There is a good match between the GFP and in situ signal, illustrating that the flourescent cells are indeed expressing mRNA of the gene of interest that was transfected by the method of electroporation.

In vitro electroportion of construct: 

GFP image In situ hybridization image
c)In utero electroporation works:  We have successfully transfected control GFP vectors into embryos by in utero electroporation.  When harvested 2-3 days later, sections from these brains reveal fluoresence in restricted regions, as shown below. We are now poised to initiate experiments with misexpression constructs.

In utero electroporation: 

Expression in the hippocampus and in the striatum
e)  A panel of markers that identifies the OB has been established:
 Lmo4 (E14.5)   Steel (E15.5)  
AP2 (E12.5) c-kit (E15.5)   

Shown here are images of different preparations processed for in situ hybridization:  embryonic heads (AP2); intact forebrain (Lmo4); single hemispheres (Steel and c-kit).   AP2, Steel, and c-kit are markers that label the olfactory bulb (black arrowhead) at different embryonic ages.  The optimal age for each marker is shown. 

Specific objectives

a)  To test the role of LIM-HD gene Lhx2 in the specification of the OB by a detailed analysis of the Lhx2 mutant brain, and by perturbing Lhx2 function using in utero electroporation. 

b)  To examine the regulation of axon pathfinding from the nasal epithelium to the olfactory bulb in control and Lhx2 mutant mice.
Experimental methodology will make use of RNAi constructs and expression of competitive inhibitors to downregulate gene function; misexpression and overexpression to upregulate gene function. 

Work Plan:
    In situ hybridization will be performed using non-radioactive (digoxigenin) labeled probes on tissue sections and whole forebrain of control and mutant embryos.  We have considerable expertise in this technique (preliminary data in this proposal; Bulchand et al., 2001; 2003; Vyas et al., 2003).
    Pathfinding experiments will be done by placing minute crystals of DiI in the nasal epithelium or in the olfactory bulb of fixed preparations of embryonic brain taken from different stages.  These will be examined after 2-3 week incubation by sectioning the tissue and using confocal microscopy to examine the axon trajectories.
In utero electroporation will be performed using procedures that have been approved by the Institute Animal Ethics Committee.  Pregnant mice will be anaesthesised using a standardized dosage of freshly prepared Avertin.  After testing for absence of reflexes, the uterus will be exposed by laprotomy.  The embryos will be illuminated using a cold light source to enable visualization of the telencephalic vesicles.  DNA will be injected using a pulled glass micropipette.  Square wave electric pulses of 25-40V will be applied in a sequence of 5 pulses, 50 ms pulse duration, 1 sec. gap between pulses.  This is the sequence of pulses that have given good expression in our hands (see preliminary data). After this procedure is performed on 4-6 embryos, sterile saline will be used to restore any lost fluid during the operation.  The animal will be sutured and allowed to recover in a warm chamber.  Recovery will be monitored closely for the first 2-4 hours, and then periodically for the first 24 hours.  The embryos will be collected 4-8 days after the surgery, fixed, and sectioned to examine the effects of the perturbations.  Molecular markers will reveal whether OB specification has been disrupted, and DiI placements will be used to assess axon pathfinding.
These experiments will be performed using RNAi constructs, as well as full length misexpression constructs.

Objective 1:  Examining the Lhx2 mutant OB phenotype
We will examine Lhx2 mutant embryos with a panel of molecular markers of the olfactory bulb.  We have markers that identity the OB primordium from E12.5.  Early markers will reveal if there is any defect in the morphogenesis at early stages.  Later markers will reveal if the development of the OB is merely slowed, or affected permanently.  Since the Lhx2 mutant dies at E15.5, this will be the oldest stage to be examined.
Interpretation:  if we find no expression of OB markers, we will conclude that this structure is not specified in the mutant.  If we find expression of the markers, we will assess if the bulb is disorganized, or mislocalized, or some combination of these defects.  This analysis is necessary to study questions of axon pathfinding.

Objective 2:  Axon pathfinding in the Lhx2 mutant
If the OB primordium is not present in the Lhx2 mutant, we will ask if nasal epithelium axons nevertheless project into the telencephalon, and if so, what area they innervate?  This will be done by placing DiI crystals in the nasal epithelium. 
If the OB primordium is present in the mutant, we will ask a) if nasal epithelium axons innervate it normally by placing DiI in the nasal epithelium   b) if OB axons grow normally to form a lateral olfactory tract that innervates the olfactory cortex.  This will be done by placing DiI crystals in the site of the mutant OB.

Objective 3:  Misexpression of Lhx2
We will use in utero electroporation to overexpress/misexpress Lhx2.  This will reveal if Lhx2 is sufficient to specify aspects of the OB identity in adjacent regions of the telencephalon, and create an enlarged OB.  This experiment is complementary to the Lhx2 mutant, since examining the mutant asks if Lhx2 is necessary for aspects of OB formation.  Misexpressing Lhx2 will ask if it is sufficient for these functions.

Objective 4:  Spatially restricted perturbation of Lhx2: 
Since the Lhx2 mutant dies at E15.5, the questions that can be asked are limited.  Spatially restricted perturbation of Lhx2 will be achieved by electroporation by injecting the following perturbation constructs into the forebrain and applying the electrodes selectively in the rostral brain region.  Electroporation is ideal for this since it will result in a small population of perturbed cells in a normal background.  Furthermore, we will allow the electorporated pups to develop to term, and will be able to examine the patterning of a partially defective bulb through postnatal or adult ages.
4a)  An Lmo3-EGFP construct will be electroporated in the rostral portion of the E12.5 telencephalon.  The embryos will be harvested at E15.5.  We expect to recapitulate some or all of the Lhx2 mutant phenotype, since Lmo3 is expected to act as a competitor for function of all LIM-HD genes.
4b)  Electroporation of a DN-Lhx2 construct will be expected to specifically interfere with Lhx2 function, giving a phenotype that may be more selective than Lmo3 electroporation. 
4c)  RNAi for Lhx2 will also be expected to give a similar phenotype to DN-Lhx2.  These phenotypes will be examined in terms of cell-autonomous or non-autonomous effects. 

Alternate strategies (if the proposed experimental design or method does not work what is the alternate strategy)

Since we have preliminary data on all the techniques we propose to use, including in utero electroporation and in situ hybridization, we do not expect that the proposed experimental design will not work. 
DiI placements pose a technical challenge due to the small size of embryonic brains.  However we have published studies that have data from embryonic DiI placements (Lakhina et al., 2007), demonstrating our competence with this technique.
Potential pitfalls:  If Lhx2 RNAi or DN-Lhx2 does not achieve adequate knockdown of Lhx2, we will use a conditional knockout of Lhx2 that we are in the process of obtaining from a collaborator.  Electroporation of Cre-GFP into this conditional knockout will achieve spatial and temporal control on Lhx2 loss of function.  These experiments are proposed for the 3rd year of this project which will give us the time to import the conditional knockout and expand the colony.


Year1:  a)  Analyse the Lhx2 mutant with OB markers using in situ hybridization.
b) Begin axon pathfinding studies on the mutant nasal epithelium; also on the mutant OB if it is specified.
c) Begin in utero electroporation experiments using the previously engineered Lmo3-EGFP construct.  Perform in situ hybridization for panel of OB markers, and also do DiI placements.  Analyse data.
Year 2:  a) Make constructs for misexpression of Lhx2; DN-Lhx2; also design RNAi constructs to knockdown Lhx2 function.   Test expression of these constructs in vitro by electroporating explants in tissue culture.
b)  Begin in utero electroporation experiments using misexpression constructs for Lhx2.  Perform in situ hybridization for panel of OB markers, and also do DiI placements.
Year 3:  a)  Complete electroporation experiments of Lhx2 and data analysis
b)  Begin in utero electroporation experiments using DN-Lhx2 and RNAi constructs for Lhx2 knockdown.  Perform in situ hybridization for panel of OB markers, and also do DiI placements.  Analyze data.
Analyze Lhx2 mutant phenotype                                     
Analyze pathfinding in Lhx2 mutant                                                        
Begin in utero electroporation for Lmo3 misexpression; analyze data                                     
Make constructs for Lhx2, DN-LHX2 and also RNAi constructs; test expression in vitro                                      
Begin in utero  electroporation for Lhx2 misexpression                                      
Complete Lhx2 misexpression experiments and data analysis                                     
Begin  in utero electroporation of DN-Lhx2 and RNAi expreriments and analyze data                                      
   Year1  Year2  Year3



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