Biology subjects

Behringer, R. R.

Publications and source records attributed to Behringer, R. R..

5 recordsLinked to original sources

bioRxiv2025

Poly(A) probe HCR RNA-FISH specifically marks pyriform nurse cells in the brown anole lizard ovary

Hybridization chain reaction RNA-fluorescent in situ hybridization (HCR RNA-FISH) is a powerful and increasingly used method for visualizing gene expression in cells and tissues. A probe set against polyadenylated RNA (poly(A)) is often used as a positive control for RNA integrity and staining quality. While optimizing this technique in the ovary of the brown anole lizard (Anolis sagrei), we found that the poly(A) probe produced a strikingly specific and intense signal in pyriform cells, a specialized lizard-specific nurse cell type. This staining pattern was found in both whole-mount samples and paraffin sections, suggesting that poly(A) signal intensity can serve as a robust molecular marker for this cell type. The specific and robust signal facilitated segmentation of volumetric data to create the first 3D models of pyriform cells and quantify their sphericity. We also observed unusually diffuse DAPI staining in pyriform cell nuclei, distinguishing them from surrounding granulosa cells, pointing to possible differences in chromatin structure or nuclear organization. Together, these findings highlight the potential of poly(A) probes used in HCR RNA-FISH not only as a technical control, but also as a tool to selectively label specific cell types with high transcriptional activity or storage of abundant poly(A) transcripts.

developmental biology↗

bioRxiv2019

CreLite: An Optogenetically Controlled Cre/loxP System Using Red Light

Precise manipulation of gene expression with temporal and spatial control is essential for functional studies and the determination of cell lineage relationships in complex biological systems. The Cre-loxP system is commonly used for gene manipulation at desired times and places. However, specificity is dependent on the availability of tissue- or cell-specific regulatory elements used in combination with Cre or CreER (tamoxifen-inducible). Here we present CreLite, an optogenetically-controlled Cre system using red light in developing zebrafish embryos. Cre activity is disabled by splitting Cre and fusing the inactive halves with the Arabidopsis thaliana red light-inducible binding partners, PhyB and PIF6. In addition, PhyB-PIF6 binding requires phycocyanobilin (PCB), providing an additional layer of control. Upon exposure to red light (660 nm) illumination, the PhyB-CreC and PIF6-CreN fusion proteins come together in the presence of PCB to restore Cre activity. Red-light exposure of transgenic zebrafish embryos harboring a Cre-dependent multi-color fluorescent protein reporter (ubi:zebrabow) injected with CreLite mRNAs and PCB, resulted in Cre activity as measured by the generation of multi-spectral cell labeling in various tissues, including heart, skeletal muscle and epithelium. We show that CreLite can be used for gene manipulations in whole embryos or small groups of cells at different stages of development. CreLite provides a novel optogenetic tool for precise temporal and spatial control of gene expression in zebrafish embryos that may also be useful in cell culture, ex vivo organ culture, and other animal models for developmental biology studies.

molecular biology↗

bioRxiv2019

Morphology and development of the Portuguese man of war, Physalia physalis

The Portuguese man of war, Physalia physalis, is a siphonophore that uses a gas-filled float as a sail to catch the wind. It is one of the most conspicuous, but poorly understood members of the pleuston, a community of organisms that occupy a habitat at the sea-air interface. The development, morphology, and colony organization of P. physalis is very different from all other siphonophores. Here, we propose a framework for homologizing the axes with other siphonophores, and also suggest that the tentacle bearing zooids should be called tentacular palpons. We also look at live and fixed larval and non-reproductively mature juvenile specimens, and use optical projection tomography to build on existing knowledge about the morphology and development of this species. Previous descriptions of P. physalis larvae, especially descriptions of budding order, were often framed with the mature colony in mind. However, we use the simpler organization of larvae and the juvenile specimens to inform our understanding of the morphology, budding order, and colony organization in the mature specimen. Finally, we review what is known about the ecology and lifecyle of P. physalis.

zoology↗

bioRxiv2018

A three-dimensional staging system of mouse endometrial gland morphogenesis

Endometrial or uterine glands secrete substances essential for uterine receptivity to the embryo, implantation, conceptus survival, development, and growth. Adenogenesis is the process of gland formation within the stroma of the uterus that occurs after birth. In the mouse, uterine gland formation initiates at postnatal day (P) 5. Subsequently, the developing uterine glands invade into the adjacent stroma. Mouse uterine gland morphology is poorly understood because it is based on two-dimensional (2D) histological observations. To more fully describe uterine gland morphogenesis, we generated three-dimensional (3D) models of postnatal uterine glands from P0 to P21, using light sheet microscopy. At birth (P0), there were no glands. At P8, we found bud- and teardrop-shaped epithelial invaginations. By P11, the forming glands were elongated epithelial tubes. By P21, the elongated tubes had a sinuous morphology. These morphologies are homogeneously distributed along the anterior-posterior axis of the uterus. To facilitate uterine gland analyses, we propose a novel 3D staging system of uterine gland morphology during postnatal development in the mouse. We define 6 stages: Stage 0: Aglandular, Stage 1: Bud, Stage 2: Teardrop, Stage 3: Elongated, Stage 4: Sinuous, and Stage 5: Primary Branches. This staging system provides a standardized key to assess and quantify uterine gland morphology that can be used for studies of uterine gland development and pathology. In addition, our studies suggest that gland formation initiation occurs during P8 and P11. However, between P11 and P21 gland formation initiation stops and all glands elongate and become sinuous.

developmental biology↗

bioRxiv2017

Osterix functions downstream of anti-Mullerian hormone signaling to regulate Mullerian duct regression

In mammals, the developing reproductive tract primordium of male and female fetuses consists of the Wolffian duct and the Mullerian duct (MD), two epithelial tube pairs surrounded by mesenchyme. During male development, mesenchyme-epithelia interactions mediate MD regression to prevent its development into a uterus, oviduct and upper vagina. It is well established that transforming growth factor-beta family member anti-Mullerian hormone (AMH) secreted from the fetal testis and its type 1 and 2 receptors expressed in MD mesenchyme regulate MD regression. However, little is known about the molecular network regulating downstream actions of AMH signaling. To identify potential AMH-induced genes and regulatory networks controlling MD regression in a global non-biased manner, we examined transcriptome differences in MD mesenchyme between males (AMH signaling on) and females (AMH signaling off) by RNA-Seq analysis of purified fetal MD mesenchymal cells. This analysis found 82 genes up-regulated in males during MD regression and identified Osterix (Osx)/Sp7, a key transcriptional regulator of osteoblast differentiation and bone formation, as a novel downstream effector of AMH signaling during MD regression. Osx/OSX was expressed in a male-specific pattern in MD mesenchyme during MD regression. OSX expression was lost in mutant males without AMH signaling. In addition, transgenic mice ectopically expressing human AMH in females induced a male pattern of Osx expression. Together these results indicate that AMH signaling is necessary and sufficient for Osx expression in the MD mesenchyme. In addition, MD regression was delayed in Osx null males, identifying Osx as a new factor that regulates MD regression.\n\nSignificanceIn mammals, each embryo forms both male and female reproductive tract organ progenitor tissues. Anti-Mullerian hormone (AMH) secreted by fetal testes acts on mesenchyme cells adjacent to the Mullerian duct (MD) epithelium, the progenitor tissue of the female reproductive tract, to induce MD regression. While AMH and early AMH signaling components are elucidated, downstream gene networks directing this process are largely unknown. A global non-biased approach using whole transcriptome sequencing of fetal MD mesenchymal cells identified 82 factors as potential target genes of AMH including Osterix (Osx). Our findings provide in vivo evidence Osx is an AMH-induced gene that regulates MD regression. Identification of Osx may provide key insights into gene regulatory networks underlying MD regression and male sex differentiation.

developmental biology↗