Biology subjects

Norton, P.

Publications and source records attributed to Norton, P..

5 recordsLinked to original sources

bioRxiv2024

An improved isochronous pulse after lentiviral knock-down of STEP in Area X of juvenile zebra finches

The zebra finch is one of the most commonly used animal models for studying the genetic mechanisms underlying vocal learning. To investigate the genetic basis of vocal learning, various genes have been knocked down in Area X--a brain region involved in birdsong acquisition--during the critical learning period. All genes that affect speech when mutated in humans similarly impair song learning in zebra finches. To date, no study has demonstrated that not all genes downregulated in Area X result in decreased song learning. Therefore, we sought a candidate gene to knock down in Area X that could either have no effect or potentially improve song learning. STEP is a protein that dephosphorylates many targets in the brain, and its knockout in mice has resulted in enhanced learning. In this study, a lentiviral knockdown of STEP in Area X during song learning resulted in birds producing normal song in almost all parameters and levels of song analysis. This stands in contrast to the knockdown of other genes like all FoxP subfamily members, which resulted in diminished song learning in previous studies. The only parameter positively affected by STEP knockdown was song rhythmicity, as evidenced by a lower deviation of song element onsets from an isochronous pulse than even their tutors. These results demonstrate for the first time that not all knockdowns in Area X lead to deterioration of song learning and validate the specificity of the method and previous findings. Significance StatementThis is the first coding gene knockdown in Area X without negatively affecting song learning.

animal behavior and cognition↗

bioRxiv2024

Performance of low-threshold, population replacement gene drives in cage populations of the yellow fever mosquito, Aedes aegypti

Aedes aegypti is the predominant vector for arboviruses including dengue, Zika, and chikungunya viruses, which infect over 100 million people annually. Mosquito population replacement strategies in which pathogen-susceptible mosquitoes in the field are replaced by laboratory-engineered pathogen-resistant strains are novel genetic control measures to inhibit the spread of malaria or arboviral diseases in endemic regions. To suppress arbovirus transmission following this approach, a mosquito strain needs to be transgenically modified to express an antiviral effector molecule which is linked to a gene drive (GD) system to both inhibit viral replication in the mosquito and drive the engineered resistance trait throughout a wild-type population. As a proof-of-concept, we tested the performance of two single locus CRISPR-Cas9 based GD for Ae. aegypti population replacement in small cage populations over 12 generations. Starting from a low release threshold of 1:9 GD bearing males, we observed two GD constructs in which Cas9 was expressed from different promoters increase in frequency in all discrete, non-overlapping cage populations. By generation 12, 56-79% of mosquitoes in six cage populations had at least one GD copy. The allele frequencies of the GD increased from <5% at release to >50% by G7 post-release for the nanos-driven Cas9 GD and by G10 in populations harboring the zpg-driven Cas9 GD. Insertion and deletion mutation (indel) frequency was measured for each discrete generation in pooled samples from the six populations harboring GD. We found that populations with Cas9 expression under control of the nanos-promoter accumulated gene drive blocking indels (GDBI) at more than twice the rate of populations harboring the zpg-promoter driven GD. Both GD produced de novo mutations at similar rates, with a difference in selection being the primary cause of greater indel accrual in the nanos-driven GD populations. Our results demonstrate that two single-locus, CRISPR-Cas9-based homing GD located at an intergenic locus exhibit continuous super-Mendelian inheritance in populations of Ae. aegypti. We further analyze the effects of fitness cost on the stability of low-threshold CRISPR/Cas9 based GD in populations of Ae. aegypti. This study demonstrates the feasibility of low-threshold, single-locus Cas9 gene drives for Ae. aegypti population replacement.

genetics↗

bioRxiv2024

Expression and regulation of SETBP1 in the song system of male zebra finches (Taeniopygia guttata) during singing

Rare de novo heterozygous loss-of-function SETBP1 variants lead to a neurodevelopmental disorder characterized by speech deficits, indicating a potential involvement of SETBP1 in human speech. However, the expression pattern of SETBP1 in brain regions associated with language remains poorly understood, along with the underlying molecular mechanisms linking it to speech. In this study, we examined SETBP1 expression in the brain of male zebra finches, a well-established model for studying vocal production learning. We demonstrated that zebra finch SETBP1 exhibits a greater number of exons and isoforms compared to its human counterpart. We characterized a SETBP1 antibody and showed that SETBP1 colocalized with FoxP1, FoxP2, and Parvalbumin in key song nuclei. Moreover, SETBP1 expression in neurons in Area X is significantly higher in zebra finches singing alone, than those singing courtship song to a female, or non-singers. Importantly, we found a distinctive neuronal protein expression of SETBP1 and FoxP2 in Area X only in zebra finches singing alone, but not in the other conditions. We demonstrated SETBP1s regulatory role on FoxP2 promoter activity in vitro. Taken together, these findings provide compelling evidence for SETBP1 expression in brain regions to be crucial for vocal learning and its modulation by singing behavior.

neuroscience↗

bioRxiv2023

An Automated Scientist to Design and Optimize Microbial Strains for the Industrial Production of Small Molecules

Engineering microbes to synthesize molecules of societal value has historically been a time consuming and artisanal process, with the synthesis of each new non-native molecule typically warranting its own separate publication. Because most microbial strain engineering efforts leverage a finite number of common metabolic engineering design tactics, we reasoned that automating these design steps would help create a pipeline that can quickly, cheaply, and reliably generate so-called microbial factories. In this work we describe the design and implementation of a computational system, an Automated Scientist we call Lila, which handles all metabolic engineering design and optimization through the design-build-test-learn (DBTL) paradigm. Lila generates metabolic routes, identifies relevant genetic elements for perturbation, and specifies the design and re-design of microbial strains in a matter of seconds to minutes. Strains specified by Lila are then built and subsequently phenotyped as part of a largely automated in-house pipeline. Humans remain in-the-loop to curate choices made by the system, helping for example to refine the metabolic model or suggest custom protein modifications. Lila attempted to build strains that could produce 454 biochemically diverse molecules with precursors located broadly throughout the metabolism of two microbial hosts, Saccharomyces cerevisiae and Escherichia coli. Notably, we observed the highest published titers for the molecule naringenin, the metabolic precursor to flavonoids. In total we created hundreds of thousands of microbial strains capable of overproducing 242 molecules, of which 180 are not native to S. cerevisiae or E. coli.

synthetic biology↗

bioRxiv2021

Feed-forward inhibition fine-tunes response timing in auditory-vocal interactions

The ability to regulate vocal timing is a fundamental aspect of communicative interactions for many species, including conversational speech among humans, yet little is known about the neural circuitry that regulates the input-dependent timing of vocal replies. Exploring this topic in the zebra finch premotor area HVC, we identify feed-forward inhibition as a key regulator of vocal response timing. Based on a spiking network model informed by behavioral and electrophysiological data from communicating zebra finches, we predicted that two different patterns of inhibition regulate vocal-motor responses. In one scenario, the strength of production-related premotor inhibition translates into plasticity in vocal response delays. In the other scenario, fast transient interneuron activity in response to auditory input results in the suppression of call production while a call is heard, thereby reducing acoustic overlap between callers. Extracellular recordings in HVC during the listening phase confirm the presence of auditory-evoked response patterns in putative inhibitory interneurons, along with corresponding signatures of auditory-evoked activity suppression. The proposed model provides a parsimonious framework to explain how auditory-vocal transformations can give rise to vocal turn-taking and highlights multiple roles of local inhibition for behavioral modulation at different time scales.

neuroscience↗