Selected Grantee Publications
Rbbp4 Loss Disrupts Neural Progenitor Cell Cycle Regulation Independent of Rb and Leads to Tp53 Acetylation and Apoptosis
Schultz-Rogers et al., Developmental Dynamics. 2022.
https://www.doi.org/10.1002/dvdy.467
Retinoblastoma binding protein 4 (Rbbp4) is a component of transcription regulatory complexes that control cell cycle gene expression by cooperating with the Rb tumor suppressor to block cell cycle entry. The authors used genetic analysis to examine the interactions of Rbbp4, Rb, and Tp53 in zebrafish neural progenitor cell cycle regulation and survival. Rbbp4 is upregulated across the spectrum of human embryonal and glial brain cancers, and it is essential for zebrafish neurogenesis. Rbbp4 loss leads to apoptosis and γ-H2AX in the developing brain that is suppressed by tp53 knockdown or maternal zygotic deletion. Mutant retinal neural precursors accumulate in M phase and fail to initiate G0 gene expression. Rbbp4; Rb1 double mutants show an additive effect on the number of M phase cells. The study demonstrates that Rbbp4 is necessary for neural progenitor cell cycle progression and initiation of G0, independent of Rb, and suggests that Rbbp4 is required for cell cycle exit and contributes to neural progenitor survival. Supported by ORIP (R24OD020166) and NIGMS.
HDAC Inhibitor Titration of Transcription and Axolotl Tail Regeneration
Voss et al., Frontiers in Cell and Development Biology. 2021.
https://pubmed.ncbi.nlm.nih.gov/35036404/
New patterns of gene expression are enacted and regulated during tissue regeneration. Romidepsin, an FDA-approved HDAC inhibitor, potently blocks axolotl embryo tail regeneration by altering initial transcriptional responses to injury. Regeneration inhibitory concentrations of romidepsin increased and decreased the expression of key genes. Single-nuclei RNA sequencing at 6 HPA illustrated that key genes were altered by romidepsin in the same direction across multiple cell types. These results implicate HDAC activity as a transcriptional mechanism that operates across cell types to regulate the alternative expression of genes that associate with regenerative success versus failure outcomes. Supported by ORIP (P40OD019794, R24OD010435, R24OD021479), NICHD, and NIGMS.
MIC-Drop: A Platform for Large-scale In Vivo CRISPR Screens
Parvez et al., Science. 2021.
https://pubmed.ncbi.nlm.nih.gov/34413171/
CRISPR screens in animals are challenging because generating, validating, and keeping track of large numbers of mutant animals is prohibitive. These authors introduce Multiplexed Intermixed CRISPR Droplets (MIC-Drop), a platform combining droplet microfluidics, single-needle en masse CRISPR ribonucleoprotein injections, and DNA barcoding to enable large-scale functional genetic screens in zebrafish. In one application, they showed that MIC-Drop could identify small-molecule targets. Furthermore, in a MIC-Drop screen of 188 poorly characterized genes, they discovered several genes important for cardiac development and function. With the potential to scale to thousands of genes, MIC-Drop enables genome-scale reverse genetic screens in model organisms. Supported by ORIP (R24OD017870), NIGMS, and NHLBI.
Loss of Gap Junction Delta-2 (GJD2) Gene Orthologs Leads to Refractive Error in Zebrafish
Quint et al., Communications Biology. 2021.
https://pubmed.ncbi.nlm.nih.gov/34083742/
Myopia is the most common developmental disorder of juvenile eyes. Although little is known about the functional role of GJD2 in refractive error development, the authors find that depletion of gjd2a (Cx35.5) or gjd2b (Cx35.1) orthologs in zebrafish cause changes in eye biometry and refractive status. Their immunohistological and scRNA sequencing studies show that Cx35.5 (gjd2a) is a retinal connexin; its depletion leads to hyperopia and electrophysiological retina changes. They found a lenticular role; lack of Cx35.1 (gjd2b) led to a nuclear cataract that triggered axial elongation. The results provide functional evidence of a link between gjd2 and refractive error. Supported by ORIP (R24OD026591), NIGMS, and NINDS.
Cell-Specific Transcriptional Control of Mitochondrial Metabolism by TIF1γ Drives Erythropoiesis
Rossmann et al., Science. 2021.
https://pubmed.ncbi.nlm.nih.gov/33986176/
Transcription and metabolism both influence cell function but dedicated transcriptional control of metabolic pathways that regulate cell fate has rarely been defined. The authors discovered that inhibition of the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) rescues erythroid differentiation in bloodless zebrafish moonshine (mon) mutant embryos defective for transcriptional intermediary factor 1 gamma (tif1γ). Upon tif1γ loss, CoQ levels are reduced, and a high succinate/α-ketoglutarate ratio leads to increased histone methylation. A CoQ analog rescues mon's bloodless phenotype. These results demonstrate that mitochondrial metabolism is a key output of a lineage transcription factor that drives cell fate decisions in the early blood lineage. Supported by ORIP (R24OD017870), NIGMS, NHLBI, and NCI.
Characterization of Axolotl Lampbrush Chromosomes by Fluorescence In Situ Hybridization and Immunostaining
Keinath et al., Experimental Cell Research. 2021.
https://pubmed.ncbi.nlm.nih.gov/33675804/
The lampbrush chromosomes (LBCs) in oocytes of the Mexican axolotl (Ambystoma mexicanum) were identified by their relative lengths and predicted centromeres; they have never been associated completely with the mitotic karyotype, linkage maps, or genome assembly. The authors identified 9 of the axolotl LBCs using RNA sequencing to identify actively transcribed genes and 13 bacterial artificial clone probes containing pieces of active genes. This study presents a simple and reliable way to identify each axolotl LBC cytologically and to anchor chromosome-length sequences to the LBCs by immunostaining and fluorescence in situ hybridization. This data will facilitate a more detailed analysis of LBC loops. Supported by ORIP (P40OD019794, R24OD010435) and NIGMS.
The SARS-CoV-2 Receptor and Other Key Components of the Renin-Angiotensin-Aldosterone System Related to COVID-19 are Expressed in Enterocytes in Larval Zebrafish
Postlethwait et al., Biology Open. 2021.
https://bio.biologists.org/content/10/3/bio058172.article-info
Hypertension and respiratory inflammation are exacerbated by the Renin-Angiotensin-Aldosterone System (RAAS), which normally protects from dropping blood pressure via Angiotensin II (Ang II) produced by the enzyme Ace. The Ace paralog Ace2 degrades Ang II and serves as the SARS-CoV-2 receptor. To exploit zebrafish to understand the relationship of RAAS to COVID-19, the group conducted genomic and phylogenetic analyses. Results identified a type of enterocyte as the expression site of zebrafish orthologs of key RAAS components, including the SARS-CoV-2 co-receptor. Results identified vascular cell subtypes expressing Ang II receptors and identified cell types to exploit zebrafish as a model for understanding COVID-19 mechanisms. Supported by ORIP (R24OD026591, R01OD011116), NIGMS, NICHD.
A Chromosome-Level Genome of Astyanax mexicanus Surface Fish for Comparing Population-Specific Genetic Differences Contributing to Trait Evolution
Warren et al., Nature Communications. 2021.
https://pubmed.ncbi.nlm.nih.gov/33664263/
Identifying the genetic factors that underlie complex traits is central to understanding the mechanistic underpinnings of evolution. Cave-dwelling Astyanax mexicanus populations are well adapted to subterranean life and many populations appear to have evolved troglomorphic (morphological adaptation of an animal to living in the constant darkness of caves) traits independently, while the surface-dwelling populations can be used as a proxy for the ancestral form. Warren et al. present a high-resolution, chromosome-level surface fish genome, enabling the first genome-wide comparison between surface fish and cavefish populations. Using this resource, they performed quantitative trait locus (QTL) mapping analyses and found new candidate genes for eye loss (dusp26). They also generated the first genome-wide evaluation of deletion variability across cavefish populations to gain insight into this potential source of cave adaptation. The surface fish genome reference now provides a more complete resource for comparative, functional and genetic studies of drastic trait differences within a species. Supported by ORIP (R24OD011198), NIA, NICHD, NIGMS, amd NIDCR.
Deploying MMEJ using MENdel in Precision Gene Editing Applications for Gene Therapy and Functional Genomics
Martínez-Gálvez et al., Nucleic Acids Research. 2021.
https://academic.oup.com/nar/article/49/1/67/6030233
Gene-editing experiments commonly elicit the error-prone non-homologous end joining for DNA double-strand break (DSB) repair. Martinez-Galvez et al. compared three DSB repair prediction algorithms - MENTHU, inDelphi, and Lindel. MENTHU correctly identified 46% of all PreMAs available, a ∼2- and ∼60-fold sensitivity increase compared to inDelphi and Lindel, respectively. The investigators report the new algorithm MENdel, a combination of MENTHU and Lindel, that achieves the most predictive coverage of homogeneous out-of-frame mutations. They suggest that the use of MENdel helps researchers use MMEJ at scale for reverse genetics screenings to be viable for nearly all loss-of-function based gene editing therapeutic applications. Supported by ORIP (R24OD020166) and NIGMS.
Sequence Diversity Analyses of an Improved Rhesus Macaque Genome Enhance its Biomedical Utility
Warren et al., Science. 2020.
https://science.sciencemag.org/content/370/6523/eabc6617
Investigators sequenced and assembled an Indian-origin female rhesus macaque (RM) genome using a multiplatform genomics approach that included long-read sequencing, extensive manual curation, and experimental validation to generate a new comprehensive annotated reference genome. As a result, 99.7% of the gaps in the earlier draft genome are now closed, and more than 99% of the genes are represented. Whole-genome sequencing of 853 RMs of both sexes identified 85.7 million single-nucleotide variants and 10.5 million indel variants, including potentially damaging variants in genes associated with human autism and developmental delay. The improved assembly of segmental duplications, new lineage-specific genes and expanded gene families provide a framework for developing noninvasive NHP models for human disease, as well as studies of genetic variation and phenotypic consequences. Supported by ORIP (P51OD011106, P51OD011107, P51OD011132, P51OD011104, U42OD024282, U42OD010568, R24OD011173, R24OD021324, R24OD010962), NHGRI, NIMH, NHLBI, and NIGMS.