Actin filament-organized local cortical endoplasmic reticulum aggregations in developing stomatal complexes of grasses

Endoplasmic reticulum (ER) immunolabeling in developing stomatal complexes and in the intervening cells of the stomatal rows (ICSRs) of Zea mays revealed that the cortical-ER forms distinct aggregations lining locally expanding wall regions. The polarized subsidiary cell mother cells (SMCs), displayed a cortical-ER-patch lining the wall region shared with the inducing guard cell mother cell (GMC), which disorganized during mitosis. In dividing SMCs, ER persisted in the preprophase band region and was unequally distributed in the mitotic spindle poles. The subsidiary cells (SCs) formed initially an ER-patch lining the common wall with the GMC or the young guard cells and afterwards an ER-ring in the junction of the SC wall with the neighboring ones. Distinct ER aggregations lined the ICSR wall regions shared with the SCs. The cortical-ER aggregations in stomatal cells of Z. mays were co-localized with actin filament (AF) arrays but both were absent from the respective cells of Triticum turgidum, which follow a different morphogenetic pattern. Experimental evidence showed that the interphase ER aggregations are organized by the respective AF arrays, while the mitotic ER aggregations by microtubules. These results revealed that AF and ER demarcated “cortical cytoplasmic domains” are activated below the locally expanding stomatal cell wall regions, probably via a mechanosensing mechanism triggered by the locally stressed plasmalemma/cell wall continuum. The probable role(s) of the local ER aggregations are discussed.     

Continuous culture of the postimplantation rat conceptus

A procedure for continuous culture of rat conceptuses during organogenesis with a number of advantages over existing methods has been established. In this method, rat conceptuses of pregnancy Day 10 (embryonic age 9.5 days; Witschi Stage 13) with embryos at pre- or early somite neurula stage were cultured for 96 h in roller bottles fitted with New Brunswick swivel caps. These caps have 5 inlets which permit continuous gassing of culture bottles and withdrawal of samples or supply of growth medium. The culture medium used in this study was immediately centrifuged, heat-inactivated fresh male rat serum. Continuous gassing of roller bottles with humidified gas mixtures of 5% CO2 and increasing O2 concentrations (5, 20, 40 and 95%), and balanced N2 provided optimal progressive conceptus development and differentiation. The average pO2 of the medium rose from 73.4 to 427.3 mm Hg, while the pCO2 and pH remained relatively stable. During the 96-h culture period, growth and differentiation of conceptuses were considerable, reaching Witschi Stage 27/28. Cultured embryos developed 48-52 somites with extensive differentiation of various organs: brain and sensory organs, heart and circulatory system, limb bud and hepatic prominence, and numerous internal visceral organs. Embryonic DNA and protein contents increased 100- to 200-fold from the initial values. Therefore, this improved procedure with periodic progressive increases in pO2 and stable low pCO2 and physiologic pH in the medium permits growth and differentiation of rat conceptuses in vitro over a prolonged period of time.