Immunohistochemical and ultrastructural analysis of the maturing larval zebrafish enteric nervous system reveals the formation of a neuropil pattern

Abstract

The gastrointestinal tract is constructed with an intrinsic series of interconnected ganglia that span its entire length, called the enteric nervous system (ENS). The ENS exerts critical local reflex control over many essential gut functions; including peristalsis, water balance, hormone secretions and intestinal barrier homeostasis. ENS ganglia exist as a collection of neurons and glia that are arranged in a series of plexuses throughout the gut: the myenteric plexus and submucosal plexus. While it is known that enteric ganglia are derived from a stem cell population called the neural crest, mechanisms that dictate final neuropil plexus organization remain obscure. Recently, the vertebrate animal, zebrafish, has emerged as a useful model to understand ENS development, however knowledge of its developing myenteric plexus architecture was unknown. Here, we examine myenteric plexus of the maturing zebrafish larval fish histologically over time and find that it consists of a series of tight axon layers and long glial cell processes that wrap the circumference of the gut tube to completely encapsulate it, along all levels of the gut.By late larval stages, complexity of the myenteric plexus increases such that a layer of axons is juxtaposed to concentric layers of glial cells. Ultrastructurally, glial cells contain glial filaments and make intimate contacts with one another in long, thread-like projections. Conserved indicators of vesicular axon profiles are readily abundant throughout the larval plexus neuropil. Together, these data extend our understanding of myenteric plexus architecture in maturing zebrafish, thereby enabling functional studies of its formation in the future.

Enteric glia as a source of neural progenitors in adult zebrafish

“…Immunostaining signal for GFAP has previously reported in the ENS (Baker et al, 2019;Kelsh and Eisen, 2000), however in our experiments the expression is not apparently within the NC-derived lineages. Consistent with the immunostaining, expression of the Tg(gfap:GFP) transgene (Bernardos and Raymond, 2006) was also undetectable in the gut of 7dpf larvae (Fig.…”

“…Immunostaining signal detected with two antibodies specific for zebrafish GFAP ( Baker et al, 2019 ; Trevarrow et al, 1990 ) was likely to represent cross-reactivity with non-neuroectodermal gut tissues, as it persisted in ret mutant larvae, which lack enteric neuroglia networks ( Figure 1—figure supplement 1G–J ; Heanue et al, 2016a ). Immunostaining signal for GFAP has previously been reported in the ENS ( Baker et al, 2019 ; Kelsh and Eisen, 2000 ), however in our experiments the expression is not apparently within the NC-derived lineages. Consistent with the immunostaining, expression of the Tg(gfap:GFP) transgene ( Bernardos and Raymond, 2006 ) was also undetectable in the gut of 7 dpf larvae ( Figure 1E ).…”

“…1D and Figure 1-figure supplement 1E-F). Immunostaining signal detected with two antibodies specific for zebrafish GFAP (Baker et al, 2019;Trevarrow et al, 1990) was likely to represent crossreactivity with non-neuroectodermal gut tissues as it persisted in ret mutant larvae, which lack enteric neuroglia networks ( Figure 1-figure supplement 1G-J) (Heanue et al, 2016a).…”

Unbiased intestinal single cell transcriptomics reveals previously uncharacterized enteric nervous system populations in larval zebrafish

“…Three papers reported contradicting findings regarding expression of canonical glial genes such as gfap, the traditional marker for enteric glia in human and mouse. Baker et al showed Gfap+ enteric glia in the outer layer of the intestine of 7 and 18 dpf fish, encapsulated by a layer of enteric neurons (10). Transmission electron microscopy showed the presence of granular vesicles and filiform processes wrapping the muscularis and caveolae, which are typical characteristic of glia (10).…”

“…showed Gfap+ enteric glia in the outer layer of the intestine of 7 and 18 dpf fish, encapsulated by a layer of enteric neurons (10). Transmission electron microscopy showed the presence of granular vesicles and filiform processes wrapping the muscularis and caveolae, which are typical characteristic of glia (10). McCallum et al .…”