Shikaya, Y. | Search bioRxiv

bioRxiv2022

Newly raised anti-c-Kit antibody detects interstitial cells of Cajal in the gut of chicken embryos

The gut peristaltic movement, a wave-like propagation of a local contraction, is important for the transportation and digestion of ingested materials. Among three types of cells, the enteric nervous system (ENS), smooth muscle cells, and interstitial cells of Cajal (ICCs), the ICCs have been thought to act as a pacemaker, and therefore it is important to decipher the cellular functions of ICCs for the understanding of gut peristalsis. c-Kit, a tyrosine kinase receptor, has widely been used as a marker for ICCs. Most studies with ICCs have been conducted in mammals using commercially available anti-c-Kit antibody. Recently, the chicken embryonic gut has emerged as a powerful model to study the gut peristalsis. However, since the anti-c-Kit antibody for mammals does not work for chickens, cellular mechanisms by which ICCs are regulated have largely been unexplored. Here, we report a newly raised polyclonal antibody against the chicken c-Kit protein. The specificity of the antibody was validated by both Western blotting analyses and immunocytochemistry. Co-immunostaining with the new antibody and anti- smooth muscle actin (SMA) antibody successfully visualized ICCs in the chicken developing hindgut in the circular muscle- and longitudinal muscle layers: as previously shown in mice, common progenitors of ICCs and smooth muscle cells at early stages were double positive for SMA and c-Kit, and at later stages, differentiated ICCs and smooth muscle cells exhibited only c-Kit and SMA, respectively. A novel ICC population was also found that radially extended from the submucosal layer to circular muscle layer. Furthermore, the new antibody delineated individual ICCs in a cleared hindgut. The antibody newly developed in this study will facilitate the study of peristaltic movement in chicken embryos.

developmental biology↗

bioRxiv2021

Distribution map of peristaltic waves in the chicken embryonic gut reveals importance of ENS and inter-region cross talks along the gut axis

Gut peristaltic movements recognized as the wave-like propagation of a local contraction are crucial for effective transportation and digestion/absorption of ingested materials. Although the physiology of gut peristalsis has been well studied in adults, it remains largely unexplored how the cellular functions underlying these coordinated tissue movements are established along the rostral-caudal gut axis during development. The chicken embryonic gut serves as an excellent experimental model for elucidating the endogenous potential and regulation of these cells since peristalsis occurs even though no ingested material is present in the moving gut. By combining video-recordings and kymography, we provide a spatial map of peristaltic movements along the entire gut posterior to the duodenum: midgut (jejunum and ileum), hindgut, caecum, and cloaca. Since the majority of waves propagate bidirectionally at least until embryonic day 12 (E12), the sites of origin of peristaltic waves (OPWs) can unambiguously be detected in the kymograph. The spatial distribution map of OPWs has revealed that OPWs become progressively confined to specific regions/zones along the gut axis during development by E12, and that such specific zones are largely conserved between different individuals implying genetic regulation for OPW determination. We have also found that the enteric nervous system (ENS) is essential for the OPW patterning since an ablation of ENS or blocking neural activity by tetrodotoxin disrupts the confined pattern of OPWs, resulting in a failure of transportation of inter-luminally injected ink. Finally, we have discovered a functional coupling of the endpoint of hindgut with the cloaca. When surgically separated, the cloaca ceases its acute contractions that would normally occur concomitantly with the peristaltic rhythm of the hindgut. Our findings shed light on the intrinsic regulations of gut peristalsis, including unprecedented ENS contribution and inter-region cross talk along the gut axis. Contribution to the field statementIt has been well accepted that the gut peristalsis is important in adults, where a luminal content (ingested material) mechanically influences peristalsis. However, the endogenously regulated cellular mechanisms that initiate and sustain peristalsis have poorly been explored, and this might be a reason why few therapeutic treatments have been available for gut diseases associated with peristaltic dysfunction. Recent studies have shown that peristaltic movements occur in the embryonic gut, suggestive of genetic involvement. However, how the peristaltic movement is coordinately patterned along the gut axis remains unknown, because most studies have used isolated fragments of the gut. In our study, we examined the entire gut posterior to the duodenum of chicken embryos to produce a spatial map of peristaltic movements during gut development. Using the map of origins of peristaltic waves (OPWs), we found previously unappreciated roles of enteric nervous system for the OPW patterning and for the physiological functions of embryonic gut tissues. Furthermore, we propose that peristaltic movements might mediate inter-region crosstalk along the gut axis. Our findings provide novel insights into the mechanisms by which the gut peristalsis is established during development at the cellular basis.

developmental biology↗