Mechanism of Lmx1b-regulated Limb Dorsalization

Lmx1b, a LIM homeodomain transcription factor is necessary for limb, kidney, eye, and central nervous system development. Lmx1b knockout (KO) mice display; abnormal limb dorsalization with distal ventral-ventral limbs, nephropathy, ocular malformations, loss of dopaminergic/serotonergic neurons, and reduced cerebellum and tectum formation. The developmental mechanisms utilized by Lmx1b remain largely unknown; to investigate downstream targets, we have used the limb as a model because of its accessibility and striking Lmx1b KO phenotype. To identify candidate genes, we compared gene arrays from Lmx1b KO and wildtype mouse limbs during limb dorsalization, i.e., 11.5, 12.5, and 13.5 days post coitum. We identified 54 target genes differentially expressed in all three stages. An abundance of extracellular matrix-related targets were found to be regulated by Lmx1b, including collagens and proteoglycans. Three Lmx1b-regulated proteoglycans are clustered together at one genomic locus, Keratocan, Lumican, and Decorin (KLD). To determine whether Lmx1b's regulation of these targets is direct, we used in silico comparative genomic analysis and identified a potential regulatory region with a known Lmx1b binding site (Peak 3). We hypothesized that Lmx1b directly regulates KLD via Peak 3. We isolated Peak 3 and linked it to a green fluorescent protein (GFP) reporter. This construct was electroporated into chick embryos at the primitive streak stage (Hamburger-Hamilton (HH) 4) and during early neural tube formation (HH10). GFP expression was detected in neural tube and surrounding the developing midbrain and cerebellum. Expression of Lmx1b and KLD mRNA overlaps the activity of this regulatory region. Our data suggests Lmx1b regulates morphogenesis by altering the extracellular matrix composition such as KLD proteoglycans. Furthermore, the activity of KLD-associated Peak 3 localized to Lmx1b-dependant CNS structures during development supports a direct role for Lmx1b regulation.

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Basic Sciences

Year

2012

Author dc:creator

• Feenstra, Jennifer

Contributors dc:contributor

• Oberg, Kerby C.
• Fallon, John: Payne, Kimberly
• Subburaman, Mohan
• Wall, Nathan

• Medical Anatomy
• Central Nervous System -- Growth and Development; Embryonic Development; Kidneys -- Growth and Development; Eye -- Growth and Development; Extremities -- Growth and Development
• Limb Dorsalization
• Central Nervous System Development
• LIM Homeodomain Transcription Factor
• Kidney Development
• Eye Development

Statement dc:rights

• This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Language dc:language

English

Repository record dc:identifier

https://scholarsrepository.llu.edu/etd/80

OAI identifier oai:identifier

oai:scholarsrepository.llu.edu:etd-1079