Inhibition of cell death results in hyperganglionosis: implications for enteric nervous system development

Abstract

The enteric nervous system (ENS) is derived from vagal and sacral neural crest cells (NCC) that delaminate from the neural tube and undergo extensive migration and proliferation in order to colonize the entire length of the gut and differentiate into many millions of neurons and glial cells. Although apoptotic programmed cell death is an essential physiological process during development of the majority of the vertebrate nervous system, apoptosis within early ENS development has not been comprehensively investigated. The aim of this study was to determine the presence and extent of apoptosis within the vagal NCC population that gives rise to most of the ENS in the chick embryo. We demonstrated that apoptotic cells, as shown by terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling and active caspase-3 immunoreactivity, are present within an electroporated green fluorescent protein (GFP) and human natural killer-1 (HNK-1) immunopositive NCC population migrating from the vagal region of the neural tube to the developing foregut. Inhibition of caspase activity in vagal NCC, by electroporation with a dominant-negative form of caspase-9, increased the number of vagal NCC available for ENS formation, as shown by 3-dimensional reconstruction of serial GFP or HNK-1 labelled sections, and resulted in hyperganglionosis within the proximal foregut, as shown by NADPH-diaphorase whole gut staining. These findings suggest that apoptotic cell death may be a normal process within the precursor pool of pre-enteric NCC that migrates to the gut, and as such it may play a role in the control of ENS formation.

Isolation and live imaging of enteric progenitors based on Sox10‐Histone2BVenus transgene expression

“…This behavior strongly suggests that cell elimination does occur among migrating ENPs populating the intestine. Given the role of programmed cell death in neurons and proliferating neural precursors (Enomoto, 2009) and a recent report that that inhibition of cell death leads to hyperganglionosis of the foregut (Wallace et al ., 2009), this elimination process likely plays a critical role in normal development of the ENS.…”

“…Cells exhibiting nuclear fragmentation were observed at a relatively low frequency, 0.7-3% of total Sox10 -H2BVenus+ cells in each viewing field exhibited fragmenting nuclei in images taken every 15-20 minutes. Multiple efforts have been made previously to identify apoptosis of ENPs in the developing intestine using static methods (Gianino et al ., 2003; Kruger et al ., 2003; Maka et al ., 2005; Wallace et al ., 2009; Wallace et al ., 2010). Only a single study has previously identified apoptotic cells within the walls of the fetal gut using static methods and established that approximately 0.2% of all Sox10+ cells were caspase3+ throughout the length of the fetal gut at 11.5dpc (Wallace et al ., 2010).…”

“…The ability to visualize cell elimination during population of the ENS is tantalizing and raises multiple questions about the signaling pathways that mediate this process. It has been established that cell death is an important process in the ENS using dominant negative forms of caspase-9 in the chick (Wallace et al ., 2009). However, other efforts to identify apoptotic cells in gut cross-sections from normal fetal mice did not find evidence of caspase-3 mediated programmed cell death, nor were enteric neuron numbers reduced in Bax−/− or Bid−/− mutants (Gianino et al ., 2003).…”

Enteric neural progenitors are more efficient than brain-derived progenitors at generating neurons in the colon

“…It has been previously shown that both cell death and proliferation play important roles in the normal development of the ENS (20,56,68,69). Proliferation is essential for the migration of ENS progenitors (63), and some of the genes associated with Hirschsprung disease play a role in proliferation (46).…”

Phactr4 regulates directional migration of enteric neural crest through PP1, integrin signaling, and cofilin activity

“…Sacral NCCs make a small contribution of neurons and glial cells by colonizing the hindgut at E15.5 (Druckenbrod and Epstein 2005). Different cellular processes such as neural crest specification, proliferation, differentiation, and migration are important for complete innervation of the gut (Asai et al 2006;Simpson et al 2007;Okamura and Saga 2008;Wallace et al 2009). The study of enteric NCC (ENCC) migration has revealed complex cellular behaviors at the migratory wave front.…”