Deep Mutational Scanning to Predict Escape from Bebtelovimab in SARS-CoV-2 Omicron Subvariants.

The major concern with COVID-19 therapeutic monoclonal antibodies is the loss of efficacy against continuously emerging variants of SARS-CoV-2. To predict antibody efficacy against future Omicron subvariants, we conducted deep mutational scanning (DMS) encompassing all single mutations of the receptor-binding domain of the BA.2 strain utilizing an inverted infection assay with an ACE2-harboring virus and library spike-expressing cells. In the case of bebtelovimab, which preserves neutralization activity against BA.2 and BA.5, a broad range of amino acid substitutions at K444, V445, and G446, and some substitutions at P499 and T500, were indicated to achieve the antibody escape. Among subvariants with current rises in case numbers, BA2.75 with G446S partially evaded neutralization by bebtelovimab, while complete evasion was observed in XBB with V445P and BQ.1 with K444T. This is consistent with the DMS results against BA.2, highlighting the potential of DMS as a predictive tool for antibody escape.

Androgen-regulated MafB drives cell migration via MMP11-dependent extracellular matrix remodeling in mice.

While androgen is considered a pivotal regulator of sexually dimorphic development, it remains unclear how it orchestrates the differentiation of reproductive organs. Using external genitalia development as a model, we showed that androgen, through the transcription factor MafB, induced cell migration by remodeling the local extracellular matrix (ECM), leading to increased cell contractility and focal adhesion assembly. Furthermore, we identified the matrix metalloproteinase Mmp11 as a MafB target gene under androgen signaling. MMP11 remodels the local ECM environment by degrading Collagen VI (ColVI). The reduction of ColVI led to the fibrillar deposition of fibronectin in the MafB-expressing bilateral mesenchyme both in vivo and ex vivo. The ECM remodeling and development of migratory cell characteristics were lost in the MafB loss-of-function mice. These results demonstrate the requirement of mesenchymal-derived androgen signaling on ECM-dependent cell migration, providing insights into the regulatory cellular mechanisms underlying androgen-driven sexual differentiation.

Exposure to the Organophosphate Pesticide Fenitrothion Directly Induced Defects in Mouse Embryonic External Genitalia.

Many industrial chemicals have been reported as antiandrogenic substances. Exposure to these substances represents a potential risk to human health, particularly to the development of reproductive organs such as embryonic external genitalia (eExG). Currently, there is a need for more assay systems that can elucidate the toxicological actions and mechanisms of endocrine-disrupting chemicals. In this study, we show that the eExG slice culture assay is useful for the evaluation of the differing modes of action of endocrine-disrupting chemicals on urethra formation. We assessed the possible endocrine-disrupting activity of 3 chemicals with reported antiandrogenic function, diazinon, dibutyl phthalate, and fenitrothion (FNT) on eExG slices. Exposure to FNT, but not diazinon and dibutyl phthalate, induced defects of androgen-induced urethral masculinization and reduced expression of the androgen-target gene Mafb. Live imaging analyses showed that FNT treatment inhibited androgen-dependent MAFB induction within 12 h. Furthermore, FNT-treated tissue slices showed reduced expression of the androgen receptor. These results indicate that FNT disrupts androgen signaling by reduction of androgen receptor expression during androgen-induced eExG masculinization. This study thus highlights the importance of animal models, which allow for the effective assessment of tissue-specific endocrine-disrupting activity to further reveal the etiology of chemical-induced congenital anomalies.

Stage-dependent function of Wnt5a during male external genitalia development.

External genitalia development in mice involves multiple developmental processes under the regulation of various signaling pathways. Wnt5a, one of the major Wnt ligands, is a crucial developmental regulator of outgrowing organs such as the limb, the mandible, and the external genitalia. Defects in Wnt5a signaling have been linked to Robinow syndrome, a genetic disorder in which male patients manifest a micropenis and defective urethral tube formation. Whereas Wnt5a is required for cell proliferation during embryonic external genitalia outgrowth, its role for urethral tube formation has yet to be understood. Here, we show that Wnt5a contributes to urethral tube formation as well as external genitalia outgrowth. Wnt5a is expressed in the embryonic external genitalia mesenchyme, and mesenchymal-specific conditional Wnt5a knockout mice resulted in hypospadias-like urethral defects. Early deletion of Wnt5a at E10.5 showed severe defects in both external genitalia outgrowth and urethral tube formation, along with reduced cell proliferation. The severe urethral tube defect persisted during later timing deletion of Wnt5a (E13.5). Further analyses revealed that loss of Wnt5a disrupted cell polarity and led to a reduction of the phosphorylated myosin light chain and the focal adhesion protein, vinculin. Altogether, these results suggest that Wnt5a coordinates cell proliferation and directed cell migration in a stage-dependent manner during male external genitalia development. Furthermore, Wnt5a may regulate cell polarity, focal adhesion formation, and cell contractility, leading to directed cell migration during male-type urethral formation in a manner that has not been reported in other organ fusion events.

Mesenchymal actomyosin contractility is required for androgen-driven urethral masculinization in mice.

The morphogenesis of mammalian embryonic external genitalia (eExG) shows dynamic differences between males and females. In genotypic males, eExG are masculinized in response to androgen signaling. Disruption of this process can give rise to multiple male reproductive organ defects. Currently, mechanisms of androgen-driven sexually dimorphic organogenesis are still unclear. We show here that mesenchymal-derived actomyosin contractility, by MYH10, is essential for the masculinization of mouse eExG. MYH10 is expressed prominently in the bilateral mesenchyme of male eExG. Androgen induces MYH10 protein expression and actomyosin contractility in the bilateral mesenchyme. Inhibition of actomyosin contractility through blebbistatin treatment and mesenchymal genetic deletion induced defective urethral masculinization with reduced mesenchymal condensation. We also suggest that actomyosin contractility regulates androgen-dependent mesenchymal directional cell migration to form the condensation in the bilateral mesenchyme leading to changes in urethral plate shape to accomplish urethral masculinization. Thus, mesenchymal-derived actomyosin contractility is indispensable for androgen-driven urethral masculinization.

Developmental mutant mouse models for external genitalia formation.

Development of external genitalia and perineum is the subject of developmental biology as well as toxicology and teratology researches. Cloaca forms in the lower (caudal) end of endoderm. Such endodermal epithelia and surrounding mesenchyme interact with various signals to form the external genitalia. External genitalia (the anlage termed as genital tubercle: GT) formation shows prominent sexually dimorphic morphogenesis in late embryonic stages, which is an unexplored developmental research field because of many reasons. External genitalia develop adjacent to the cloaca which develops urethra and corporal bodies. Developmental regulators including growth factor signals are necessary for epithelia-mesenchyme interaction (EMI) in posterior embryos including the cloaca and urethra in the genitalia. In the case of male type urethra, formation of tubular urethra proceeds from the lower (ventral) side of external genitalia as a masculinization process in contrast to the case of female urethra. Mechanisms for its development are not elucidated yet due to the lack of suitable mutant mouse models. Because of the recent progresses of Cre (recombinase)-mediated conditional target gene modification analyses, many developmental regulatory genes become increasingly analyzed. Conditional gene knockout mouse approaches and tissue lineage approaches are expected to offer vital information for such sexually dimorphic developmental processes. This review aims to offer recent updates on the progresses of these emerging developmental processes for the research field of congenital anomalies.