An atypical basement membrane forms a midline barrier during left-right asymmetric gut development in the chicken embryo.

Correct intestinal morphogenesis depends on the early embryonic process of gut rotation, an evolutionarily conserved program in which a straight gut tube elongates and forms into its first loops. However, the gut tube requires guidance to loop in a reproducible manner. The dorsal mesentery (DM) connects the gut tube to the body and directs the lengthening gut into stereotypical loops via left-right (LR) asymmetric cellular and extracellular behavior. The LR asymmetry of the DM also governs blood and lymphatic vessel formation for the digestive tract, which is essential for prenatal organ development and postnatal vital functions including nutrient absorption. Although the genetic LR asymmetry of the DM has been extensively studied, a divider between the left and right DM has yet to be identified. Setting up LR asymmetry for the entire body requires a + midline barrier to separate the two sides of the embryo, without it, embryos have lethal or congenital LR patterning defects. Individual organs including the brain, heart, and gut also have LR asymmetry, and while the consequences of left and right signals mixing are severe or even lethal, organ-specific mechanisms for separating these signals are poorly understood. Here, we uncover a midline structure composed of a transient double basement membrane, which separates the left and right halves of the embryonic chick DM during the establishment of intestinal and vascular asymmetries. Unlike other basement membranes of the DM, the midline is resistant to disruption by intercalation of Netrin4 (Ntn4). We propose that this atypical midline forms the boundary between left and right sides and functions as a barrier necessary to establish and protect organ asymmetry.