Biology subjects

Iben, J.

Publications and source records attributed to Iben, J..

8 recordsLinked to original sources

bioRxiv2025

Developmental regulation of intestinal best4+ cells

best4+/CFTR-high expressing cells are a recently described intestinal epithelial cell type potentially altered in inflammatory bowel disease and colorectal cancer. However, their developmental origin, developmental regulation, and functions remain undefined. This study identifies their conserved transcriptional program and uses zebrafish to dissect their developmental regulation in vivo. Lineage tracing identified that best4+ cells arise from atoh1b+ secretory progenitors. We identify that Notch signaling, mediated by dll4, specifies best4+ cells at the expense of enterochromaffin cells. Downstream of Notch, meis1b confers best4+ cell identity. best4+ cells then exhibit regionalized gene expression, regulated by pbx3a. Additionally, this study demonstrates a system where best4+ cells can be manipulated, observed, and removed in an organismal context. Live imaging and electron microscopy of best4+ cells identified dynamic cellular projections, suggesting a sensory or communicative function. Removal of best4+ cells in vivo eliminated previously proposed functions: they are not required to restore intestinal pH following acidic challenge and do not absorb nutrients. However, we identify region-specific intracellular pH differences that suggest potential functional heterogeneity. Altogether, this study presents a comprehensive description of best4+ cell development from birth to spatial regulation that will be instrumental to understand how best4+ cells change in disease or might be therapeutically manipulated and presents the tools to dissect their function in vivo.

developmental biology↗

bioRxiv2025

Specialized gas-exchange endothelium of the zebrafish gill

The pulmonary vasculature plays a critical role in gas exchange and in lung pathologies, but it is challenging to observe and experimentally manipulate deep within the lungs of living mammals. Unlike mammalian lungs, externally located zebrafish gills are readily accessible for high-resolution optical imaging and experimental manipulation, suggesting zebrafish might provide an excellent comparative vertebrate model for studying the development and function of gas-exchange organs and the gas-exchange blood vasculature. To characterize their resident cell populations, we performed single-cell RNA sequencing (scRNAseq) on adult zebrafish gills, revealing numerous cell types with transcriptional similarities to those found in mammalian lungs. We uncovered and characterized several different endothelial cell populations, including distinct clusters of arterial endothelial cells and lymphatic endothelial cells. The largest endothelial cell cluster closely resembles Cap2 or "Aerocyte" endothelial cells, a recently discovered unusual mammalian endothelial cell type found exclusively in lung alveoli. Zebrafish aerocytes localize to the analogous gas-exchange structures in fish, the highly vascularized gill lamellae. We use confocal and super-resolution imaging of transgenic and hybridization chain reaction-probed zebrafish, array tomography, and focused ion beam scanning electron microscopy to carry out a detailed and comprehensive characterization of gill aerocytes including 3-D ultrastructural reconstruction of one of these cells, showing that as in mammals these cells are closely associated with gas-exchange epithelia and that they possess unique properties that may help facilitate their gas-exchange function. Together, our findings help establish a new, experimentally accessible comparative vertebrate model for studying the gas-exchange blood vasculature.

developmental biology↗

bioRxiv2024

The axillary lymphoid organ - an external, experimentally accessible immune organ in the zebrafish

Lymph nodes and other secondary lymphoid organs play critical roles in immune surveillance and immune activation in mammals, but the deep internal locations of these organs make it challenging to image and study them in living animals. Here, we describe a previously uncharacterized external immune organ in the zebrafish ideally suited for studying immune cell dynamics in vivo, the axillary lymphoid organ (ALO). This small, translucent organ has an outer cortex teeming with immune cells, an inner medulla with a mesh-like network of fibroblastic reticular cells along which immune cells migrate, and a network of lymphatic vessels draining to a large adjacent lymph sac. Noninvasive high-resolution imaging of transgenically marked immune cells can be carried out in the lobes of living animals, and the ALO is readily accessible to external treatment. This newly discovered tissue provides a superb model for dynamic live imaging of immune cells and their interaction with pathogens and surrounding tissues, including blood and lymphatic vessels. TeaserA newly characterized external zebrafish lymphoid organ provides a powerful model for live imaging of immune cell dynamics

developmental biology↗

bioRxiv2024

Yeast poly(A)-binding protein (Pab1) controls translation initiation in vivo primarily by blocking mRNA decapping and decay

Poly(A)-binding protein (Pab1 in yeast) is involved in mRNA decay and translation initiation, but its molecular functions are incompletely understood. We found that auxin-induced degradation of Pab1 reduced bulk mRNA and polysome abundance in a manner suppressed by deleting the catalytic subunit of decapping enzyme (dcp2{Delta}), demonstrating that enhanced decapping/degradation is the major driver of reduced mRNA abundance and protein synthesis at limiting Pab1 levels. An increased median poly(A) tail length conferred by Pab1 depletion was also nullified by dcp2{Delta}, suggesting that mRNA isoforms with shorter tails are preferentially decapped/degraded at limiting Pab1. In contrast to findings on mammalian cells, the translational efficiencies (TEs) of many mRNAs were altered by Pab1 depletion; however, these changes were broadly diminished by dcp2{Delta}, suggesting that reduced mRNA abundance is a major driver of translational reprogramming at limiting Pab1. Thus, assembly of the closed-loop mRNP via PABP-eIF4G interaction appears to be dispensable for normal translation of most yeast mRNAs in vivo. Interestingly, histone mRNAs and proteins are preferentially diminished on Pab1 depletion dependent on Dcp2, accompanied by activation of internal cryptic promoters in the manner expected for reduced nucleosome occupancies, revealing a new layer of post-transcriptional control of histone gene expression.

genomics↗

bioRxiv2023

Dkk2 interacts with Pax9 in palate mesenchyme to pattern and tune osteogenesis

Multiple genetic and environmental etiologies contribute to the pathogenesis of cleft palate, which constitutes the most common among the inherited disorders of the craniofacial complex. Insights into the molecular mechanisms regulating osteogenic differentiation and patterning in the palate during embryogenesis are limited and needed for the development of innovative diagnostics and cures. This study utilized the Pax9-/- mouse model with a consistent phenotype of cleft secondary palate to investigate the role of Pax9 in the process of palatal osteogenesis. While prior research had identified upregulation of Wnt pathway modulators Dkk1 and Dkk2 in Pax9-/- palate mesenchyme, limitations of spatial resolution and technology restricted a more robust analysis. Here, data from single-nucleus transcriptomics and chromatin accessibility assays validated by in situ highly multiplex targeted single-cell spatial profiling technology suggest a distinct relationship between Pax9+ and osteogenic populations. Loss of Pax9 results in spatially restricted osteogenic domains bounded by Dkk2, which normally interfaces with Pax9 in the mesenchyme. These results suggest that Pax9-dependent Wnt signaling modulators influence osteogenic programming during palate formation, potentially contributing to the observed cleft palate phenotype.

developmental biology↗

bioRxiv2021

Detection of precisely edited CRISPR/Cas9 alleles through cointroduced restriction-fragment length polymorphisms

CRISPR/Cas9 is a powerful tool for producing genomic insertions and deletions (indels) to interrogate gene function. Modified CRISPR/Cas9 protocols can produce targeted genetic changes that are more precise than indels, but founder recovery is less efficient. Focusing on producing missense mutations in zebrafish using single-stranded oligo deoxynucleotide (ssODN) donor templates, we pioneered a strategy of adding synonymous changes to create novel restriction-enzyme (RE) sites, allowing detection of rare precise edits in a modified fluorescent-PCR fragment assay. We have named this process TIARS (test for incorporation of added recognition sites). Aided by TIARS, we induced two distinct amino-acid substitutions (T979I and P1387S) in the atp7a gene among somatic tissues of CRISPR-Cas9-treated F0 zebrafish. One of these F0s transmitted the allele to atp7aT979I/+ F1 progeny, and trans-heterozygosity of this allele against a null atp7a allele causes hypopigmentation, consistent with more severe pigment deficits in zebrafish or humans carrying only null mutations in atp7a/ATP7A. Design of ssODNs with novel RE recognition sites is labor-intensive, so we developed an in silico tool, TIARS Designer, and performed bioinformatic validation indicating that TIARS should be generalizable to other genes and experimental systems that employ donor template DNA.

genetics↗

bioRxiv2019

Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway

Nuclear pore complexes (NPCs) are important for many processes beyond nucleocytoplasmic trafficking, including protein modification, chromatin remodeling, transcription, mRNA processing and mRNA export. The multi-faceted nature of NPCs and the slow turnover of their components has made it difficult to understand the role of basket nucleoporins (Nup153, Nup50 and Tpr) in these diverse processes. To address this question, we used an Auxin-Induced Degron (AID) system to distinguish roles of basket nucleoporins: Loss of individual nucleoporins caused distinct alteration in patterns of nucleocytoplasmic trafficking and gene expression. Importantly, Tpr elimination caused rapid and pronounced changes in transcriptomic profiles within two hours of auxin addition. These changes were dissimilar to shifts observed after loss of Nup153 or Nup50, but closely related to changes after depletion of mRNA export receptor NXF1 or the GANP subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex. Moreover, GANP association to NPCs was specifically disrupted upon TPR depletion. Together, our findings demonstrate a unique and pivotal role of Tpr in regulating gene expression through GANP- and/or NXF1-dependent mRNA nuclear export.

cell biology↗

bioRxiv2017

An epigenetic mechanism for cavefish eye degeneration

Coding and non-coding mutations in DNA contribute significantly to phenotypic variability during evolution. However, less is known about the role of epigenetics in this process. Although previous studies have identified eye development genes associated with the loss of eyes phenotype in the Pachon blind cave morph of the Mexican tetra Astyanax mexicanus1-6, no inactivating mutations have been found in any of these genes2,3,7-10. Here we show that excess DNA methylation-based epigenetic silencing promotes eye degeneration in blind cave Astyanax mexicanus. By performing parallel analyses in Astyanax mexicanus cave and surface morphs and in the zebrafish Danio rerio, we have discovered that DNA methylation mediates eye-specific gene repression and globally regulates early eye development. The most significantly hypermethylated and down-regulated genes in the cave morph are also linked to human eye disorders, suggesting the function of these genes is conserved across the vertebrates. Our results show that changes in DNA methylation-based gene repression can serve as an important molecular mechanism generating phenotypic diversity during development and evolution.

evolutionary biology↗