Biology subjects

Kawabe, Y.

Publications and source records attributed to Kawabe, Y..

5 recordsLinked to original sources

bioRxiv2025

Retinoic acid-responsive hox genes in hoxba and hoxbb clusters direct pharyngeal pouch formation in zebrafish

The segmented pharyngeal apparatus is crucial for organ development specific to vertebrates, and its formation relies on the proper development of pharyngeal pouches. While retinoic acid (RA) is known to influence pouch formation, the downstream genes involved have been unclear. In this study, we demonstrate that zebrafish mutants lacking both the hoxba and hoxbb clusters--teleost-specific duplicates of the ancestral HoxB cluster--exhibit a significant loss of posterior pharyngeal pouches and related skeletal elements. This phenotype resembles that observed in raldh2 and pax1a;pax1b mutants. We identify hoxb1a and hoxb1b as RA-dependent genes expressed in the pharyngeal region that are essential for pouch formation. Morpholino-mediated knockdown of these genes replicated the pouch defects and decreased pax1a expression, indicating a regulatory pathway linking RA, Hox, and pax1. Our findings uncover a previously unrecognized role of Hox genes in early pouch segmentation and suggest that RA-responsive HoxB clusters were co-opted during vertebrate evolution to initiate pharyngeal regionalization.

developmental biology↗

bioRxiv2025

Database Chemistry for Genomics-Based Safety and Quality Evaluation of Biologics

Genomics-based safety and quality evaluation studies are advancing the bioindustry by enhancing various aspects, including viral safety, host cell protein (HCP) control, product heterogeneity control, cellular heterogeneity control, and process reproducibility. High-throughput instruments and genome-scale databases are essential in genomics, with the reference genome sequence being the most critical database. The completeness and accuracy of these genome sequences depend on DNA quality, sequencing instruments, read coverage, and assembly strategies. Significant efforts are being made to perfect genome assembly and continuously improve it. However, the quantitative impact of reference genome sequence accuracy on the safety of biologics is not yet fully understood. In this study, we compared and benchmarked six Chinese hamster genomes, including four newly sequenced genomes derived from Chinese hamster cell lines, from an industrial perspective. We also developed database assembly techniques to enhance the safety of biologics. We recommend using two or more independent reference genomes for viral safety studies. For HCP control, we suggest using protein sequences in which trypsin degradation peptides that overlap with high-risk proteins should be masked and unified. Additionally, we can predict microenvironments using single-cell transcriptome data. In bioengineering processes, any nucleotide samples have potential commercial benefits.

bioengineering↗

bioRxiv2025

Genetic evidence: zebrafish hoxba and hoxbb clusters are essential for the anterior-posterior positioning of pectoral fins

Vertebrate paired appendages, such as the pectoral fins in fish and the forelimbs in tetrapods, arise at specific regions along the anterior-posterior axis of the body. Hox genes have long been considered prime candidates for determining the anteroposterior positioning of these paired appendages during development. Evidence from various model organisms, including mouse and chick, supports a role for Hox genes in limb positioning. However, despite extensive phenotypic analyses of numerous single and compound Hox knockout mice, clear genetic evidence for substantial defects in limb positioning has been limited, leaving questions unresolved. In a previous study, we generated seven distinct hox cluster-deficient mutants in zebrafish. Here, we provide genetic evidence that zebrafish hoxba;hoxbb cluster-deleted mutants specifically exhibit a complete lack of pectoral fins, accompanied by the absence of tbx5a expression in pectoral fin buds. In these mutants, tbx5a expression in the pectoral fin field of the lateral plate mesoderm fails to be induced at an early stage, suggesting a loss of pectoral fin precursor cells. Furthermore, the competence to respond to retinoic acid is lost in hoxba;hoxbb cluster mutants, indicating that tbx5a expression cannot be induced in the pectoral fin buds. We further identify hoxb4a, hoxb5a, and hoxb5b as pivotal genes underlying this process. Although the frameshift mutations in these hox genes do not recapitulate the absence of pectoral fins, we demonstrate that deletion mutants at these genomic loci show the absence of pectoral fins with low penetrance. Our results suggest that, by establishing the expression domains along the anteroposterior axis, hoxb4a, hoxb5a, and hoxb5b within hoxba and hoxbb clusters cooperatively determine the positioning of zebrafish pectoral fins through the induction of tbx5a expression in the restricted pectoral fin field. Our findings also provide insights into the evolutionary origin of paired appendages in vertebrates.

developmental biology↗

bioRxiv2022

Molecular engineering of emicizumab to improve hemophilia A treatment

Emicizumab, a factor (F)VIIIa-function mimetic bispecific antibody (BsAb) to FIXa and FX, has become an indispensable treatment for people with hemophilia A (PwHA). Although emicizumab is very potent, long-term outcomes from the clinical studies suggest that a small proportion of PwHA still experiences bleeds. Additionally, non-clinical studies indicate that the maximum cofactor activity of emicizumab is lower than international standard activity (100 IU/dL of FVIII). An increased cofactor activity BsAb would benefit such patients. Here, we report NXT007, a BsAb binding FIXa and FX developed through further engineering of emicizumab. Emicizumab has a common light chain, but through advances in antibody engineering, we were able to create a more potent BsAb with two new non-common light chains. After extensive optimization of the heavy and light chains, the resulting BsAb, NXT007, exerted in vitro thrombin generation (TG) activity in hemophilia A plasma equivalent to 100 IU/dL of FVIII when triggered by tissue factor. NXT007 demonstrated potent hemostatic activity in an acquired hemophilia A model in non-human primates at a much lower dosage than emicizumab, consistent with an around 30-fold dose shift in the in vitro TG activity between NXT007 and emicizumab. Moreover, together with Fc engineering that enhanced FcRn binding and reduced in vivo clearance, we demonstrate that NXT007 could be effective at a much lower dosage with a longer dosing interval compared to emicizumab. These non-clinical results suggest that NXT007 could maintain a non-hemophilic range of coagulation potential in PwHA and provides a rationale for its clinical testing.

bioengineering↗

bioRxiv2022

FUS regulates RAN translation through modulating the G-quadruplex structure of GGGGCC repeat RNA in C9orf72-linked ALS/FTD

Abnormal expansions of GGGGCC repeat sequence in the noncoding region of the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). The expanded repeat sequence is translated into dipeptide repeat proteins (DPRs) by noncanonical repeat-associated non-AUG (RAN) translation. Since DPRs play central roles in the pathogenesis of C9-ALS/FTD, we here investigate the regulatory mechanisms of RAN translation, focusing on the effects of RNA-binding proteins (RBPs) targeting GGGGCC repeat RNAs. Using C9-ALS/FTD model flies, we demonstrated that the ALS/FTD-linked RBP FUS suppresses RAN translation and neurodegeneration in an RNA-binding activity-dependent manner. Moreover, we found that FUS directly binds to and modulates the G-quadruplex structure of GGGGCC repeat RNA as an RNA chaperone, resulting in the suppression of RAN translation in vitro. These results reveal a previously unrecognized regulatory mechanism of RAN translation by G-quadruplex-targeting RBPs, providing therapeutic insights for C9-ALS/FTD and other repeat expansion diseases.

neuroscience↗