Backtransplantation of chick cardiac neural crest cells cultured in LIF rescues heart development

Abstract

The cardiac neural crest is essential for normal development of the cardiovascular system. Cardiac neural crest cells are derived from the neural folds located between the mid-otic placodes and the caudal limit of somite 3. These crest cells can differentiate into a variety of mesenchymal cell types that support cardiovascular development, in addition to neurogenic cells. When cultured, many express a-smooth muscle actin or neurofilaments and lose their undifferentiated neural crest phenotype as shown by a decrease in HNK-1 reactivity. We wanted to determine whether cultured cardiac neural crest cells maintained the potency to support normal heart development when backtransplanted into embryos lacking their native cardiac neural crest. Under usual circumstances removal of the cardiac neural crest results in 80-100% incidence of persistent truncus arteriosus. The present study reports a system in which cardiac neural folds are cultured for 3 days and the cells backtransplanted into chick embryos after laser-induced ablation of the intrinsic cardiac neural folds, Rescue of heart development was improved 50% when cultured cells were backtransplanted and almost 200% when the backtransplanted cells had been cultured in leukemia inhibitory factor (LIF). To determine whether the cultured cells are capable of following normal migratory routes, cultured homospecific cardiac neural crest cells were tagged with DiI. Initially, fluorescent cells were found concentrated around the neural tube. By the second day following backtransplantation, the cells had migrated to the circumpharyngeal crest, populated the pharyngeal arches and aortic arch arteries, and were in the region of the cardiac outflow tract. By the third day, the labeled cells had dispersed, but could be found around the neural tube, esophagus, cardiac outflow tract, and within the dorsal root ganglia. Interestingly, a cranial migration to the periphery of the eyes was also noted. With the exception of the cranial migration to the eyes, cultured and backtransplanted cardiac neural crest cells followed normal migratory pathways to the cardiac outflow tract. LIF is used for the in vitro maintenance of the pluripotential phenotype of embryonic stem cells. In an effort to understand why LIF improves the ability of cul-tured neural crest cells to support normal heart development, we have examined the relationship of neural crest expression of HNK-1 antigen, a-smooth muscle actin, and neurofilament protein in neural crest cells cultured in LIF. LIF treatment resulted in an expanded period of expression of HNK-1 antigen, associated with a decrease in expression of a-smooth muscle actin. The expanded period of HNK-1 expression is associated with proliferation of the HNK-1 positive cell population. Expression of the neurofilament was the same in cells cultured with or without LIF.0 1993 Wiley-Liss, Inc.

Cardiac Chamber Formation: Development, Genes, and Evolution

“…As we have seen, pacemaking activity is already present in the hearts of the chordate ancestor in which neural crest cells have not yet developed (114). An ECG (232,277,313), indicating coordinated depolarization of the distinct cardiac compartments, can be recorded concomitantly with chamber formation at a stage in which neural crest cells (106,107,158,159) as well as cells derived from the epicardium (103,191) have not arrived in the heart. Although a role for these cells in the maturation of the conduction system can be envisaged, the essential electrical configuration of the heart has already been laid down before these cells arrive at the heart and before the different components of the conduction system can be morphologically identified.…”

Induction of Purkinje fiber differentiation by coronary arterialization

“…We tested this hypothesis in vivo by probing for Purkinje fiber differentiation in the embryonic chicken heart after either inhibition or activation of coronary artery development. In the first set of experiments, coronary arterial branching was inhibited by ablating cardiac NC at E2 prior to its emigration from the dorsal neural tube (12,15). NC-ablated embryos developed to E15, at which stage Purkinje fibers can be discriminated by expression of distinct marker genes (4).…”

Shortened Outflow Tract Leads to Altered Cardiac Looping After Neural Crest Ablation

“…Neural crest-ablated, sham-operated, and control embryos were prepared as described previously. 12 The number of embryos used in each analysis is shown in the Table. For shell-less culture, the eggshell contents were transferred to a hexagonal polystyrene weigh boat (Fisher Scientific) at stage 12. The weigh boat was placed in a Petri dish with 0.5-cm distilled water and incubated at 37°C.…”