Smo-AW4: A novel hypomorphic allele Open Access

Ben-Ami, Johanna Rae (2016)

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Vertebrate hedgehog signaling is essential for patterning and cell proliferation during development. Overactivation of the Sonic Hedgehog (Shh) signaling pathway is the cause of some cancers, and both increased and decreased signaling can cause intellectual disability. Within this pathway Smoothened (Smo) is the obligate transducer, thus necessary for activation of downstream signal targets. We have isolated a novel mutant, SmoAW4, which survives longer than the null allele. Our mutant is able to survive until birth, but dies thereafter. We identified an asparagine to lysine change at residue 223 in SmoAW4, which I hypothesize acts as a hypomorphic allele. The mutant presents with craniofacial, limb, and neural tube abnormalities, consistent with Smo's importance in Shh signaling transduction. During development, protein expression is essential in patterning the neural tube, which becomes the organism's future spinal cord. Shh signaling plays a vital role in specification of progenitor cells via Smo. I analyzed neural patterning using fluorescent antibody staining at e9.5, e10.5, and e11.5. I found the patterning to be abnormal, with ventral expansion of the motor neuron progenitors indicating lower Shh activity, consistent with SmoAW4 being a hypomorphic allele. Next, I examined fibroblasts derived from the mutants by quantitative RT-PCR looking at downstream pathway targets Gli1 and Patched1, which I hypothesized would be reduced from the wild type but still present. I found Patched1 RNA expression to be significantly reduced in the mutants when Shh treated, and Gli1 RNA expression was also reduced, with a strong trend toward significance. Primary cilia are involved in detecting sensory information for the cell, and are where Shh signaling occurs. Although there are clear links between primary cilia and Shh signaling, the mechanism is not fully understood. During vertebrate Shh signaling, Smo is enriched within the cilium. I looked for enrichment of Smo within the cilium, and preliminary results suggest that the mutant allele does not have this enrichment when Shh treated. Taken together, the results indicate that SmoAW4 is a hypomorphic allele and suggest that ciliary enrichment is not absolutely required for pathway activation.

Table of Contents

Introduction 1

Materials and Methods 6

Mouse Information 6

Immortalization of MEFs 6

Cell Culture 7

Immunofluorescent Antibody Staining 8

Quantitative Reverse Transcription-PCR 10

Statistical Analysis 11

Results 11

Discussion 16

References 20

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