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Keywords:

  • calbindin;
  • catecholamine;
  • enteric nervous system;
  • serotonin;
  • transient phenotype;
  • tyrosine hydroxylase

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Funding
  11. Disclosures
  12. References

Background

During development of the enteric nervous system, a subpopulation of enteric neuron precursors transiently expresses catecholaminergic properties. The progeny of these transiently catecholaminergic (TC) cells have not been fully characterized.

Methods

We combined in vivo Cre-lox-based genetic fate-mapping with phenotypic analysis to fate-map enteric neuron subtypes arising from tyrosine hydroxylase (TH)-expressing cells.

Key Results

Less than 3% of the total (Hu+) neurons in the myenteric plexus of the small intestine of adult mice are generated from transiently TH-expressing cells. Around 50% of the neurons generated from transiently TH-expressing cells are calbindin neurons, but their progeny also include calretinin, neurofilament-M, and serotonin neurons. However, only 30% of the serotonin neurons and small subpopulations (<10%) of the calbindin, calretinin, and neurofilament-M neurons are generated from TH-expressing cells; only 0.2% of nitric oxide synthase neurons arise from TH-expressing cells.

Conclusions & Inferences

Transiently, catecholaminergic cells give rise to subpopulations of multiple enteric neuron subtypes, but the majority of each of the neuron subtypes arises from non-TC cells.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Funding
  11. Disclosures
  12. References

In the adult gut, there are many different functional types of neurons that differ in their shapes, projections, electrophysiological properties, and neurochemistry.[1-5] Enteric neurons arise predominantly from vagal neural crest cells that migrate into and along the gut.[6-9] Enteric neural crest-derived cells (ENCCs) then develop along neuronal or glial lineages at varying times after entering the gut.[8, 10, 11] Different enteric neuron subtypes vary in their birthdates,[12, 13] and in their time of appearance.[14] The transcription factors, Ascl1 (formerly known as Mash1)[15] and Hand2,[16] and signalling through Ret[17-20] and TrkC[21] receptors all appear to play roles in the development of some enteric neuron subtypes, but there is still no detailed understanding of the mechanisms controlling the development of different enteric neuron subtypes.[7, 8, 14, 22] Enteric neural crest-derived cells have been subdivided into developmental lineages, based on the expression of nestin,[23] or their requirement for Ascl1[15, 22, 24] or Hand2,[22] but the lineages have not been fully characterized.

During development, the first ENCCs to express pan-neuronal markers transiently express catecholaminergic properties including catecholamine storage, expression of the synthetic enzymes, tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DβH), and expression of the norepinephrine transporter (NET); these cells have consequently been named transiently catecholaminergic (TC) cells.[25-34] Although TC cells express pan-neuronal proteins and have a neuron-like morphology,[28, 33, 35] they proliferate and migrate[29, 36] and are therefore neuronal precursors rather than terminally differentiated neurons. In the mouse, TC cells are present between E10 and E13.[30] However, there is also a very small population of enteric dopaminergic neurons in the postnatal mouse intestine, which express TH but do not arise from TC cells.[37] Dopaminergic neurons develop perinatally and persist into adulthood.[37]

Deletion of Ascl1 in mice leads to a loss of TC cells and a marked reduction of enteric serotonin neurons, but no change in the numbers of calcitonin gene-related peptide (CGRP) or dopaminergic neurons.[15, 37] This has led to the idea that the Ascl1-dependent lineage gives rise to TC cells and serotonin neurons, but not to CGRP or dopaminergic neurons.[15, 22] However, knowledge of the fate of TC cells is still scant, and it is not clear whether TC cells give rise to enteric neuron subtypes other than serotonin neurons. To identify the enteric neuron subtypes that are the progeny of cells that express TH, we combined in vivo Cre-lox-based genetic fate-mapping, to permanently label TH-expressing cells, with phenotypic analysis. In concordance with previous studies[15] we show that a significant proportion of serotonin neurons in the adult gut arise from transiently TH cells. However, ~50% of the progeny of transiently TH cells are neurons that are immunoreactive for calbindin.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Funding
  11. Disclosures
  12. References

Mice

The TH-Cre mice[38] were obtained from the European Mutant Mouse Consortium, and were mated to Rosa26R-YFP reporter mice,[39] which were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). Mice were time plug-mated, and midday of the day the plug was found was designated as day E0.5. The TH-Cre;R26RYFP mice of the following ages, E10.5, E11.5, E13.5, P3 and adult (6 week old) were examined. Mice were killed by cervical dislocation. All mice used were housed and treated in accordance with the guidelines of the Anatomy & Cell Biology, Neuroscience, Pathology, Pharmacology and Physiology Animal Ethics Committee at the University of Melbourne.

Immunohistochemistry

Immunohistochemical analysis was performed either on whole embryonic gut or on whole mount preparations of myenteric or submucosal plexus of P3 and adult gut. Sympathetic ganglia were also examined as controls. The tissue was fixed in 4% paraformaldehyde in 0.1% mol L−1 phosphate-buffered saline overnight at 4 °C and processed as described previously.[40] Primary antisera used are listed in Table 1; the anti-GFP antisera also bind to YFP. The secondary antisera used were as follows: donkey anti-rabbit Alexa 594 (1 : 400; Molecular Probes, Mulgrave, Vic., Australia), donkey anti-sheep FITC (1 : 100; Jackson Immunobiologicals, West Grove, PA, USA), donkey anti-sheep Alexa 594 (1 : 100; Molecular Probes), donkey anti-human Texas red (1 : 100; Jackson), donkey anti-human Alexa 647 (1 : 200, Jackson), donkey anti-rabbit FITC (1 : 200; Jackson), donkey anti-chicken FITC (1 : 100; Jackson) and donkey anti-rabbit Alexa 647 (1 : 400; Molecular Probes). Neurons were counted using a Zeiss Axio Imager.M1 microscope (North Ryde, NSW, Australia) and a ×40 objective lens. Quantitative analyses of the overlap between YFP expression and immunoreactivity for a variety of myenteric neuron subtype markers were performed on the jejunum (middle third of the small intestine) from three adult TH-Cre;R26R-YFP mice. The range in the numbers of YFP+ neurons examined in each mouse for each neurochemical marker were as follows: Hu: 46–89 YFP+ neurons; neuronal nitric oxide synthase (nNOS): 84–107; TH: 154–174; calbindin: 118–163; calretinin: 77–128; serotonin: 110–142; NF-M: 72–99. Preparations were imaged using a Zeiss Pascal confocal laser scanning microscope. Data are presented as mean ± standard error of the mean. The proportions of Hu-immunostained myenteric neurons in the jejunum of two TH-Cre;R26R-YFP mice that were nNOS-IR or calretinin-IR were determined from single optical sections taken using a ×20 lens on a Zeiss Pascal confocal microscope.

Table 1. Primary antisera used
 HostSource and referenceConcentration
SerotoninRabbitImmunostar1 : 2000
CalbindinRabbitSwant1 : 1600
CalretininGoatSwant1 : 100
GFPRabbitMolecular Probes1 : 500
GFPGoatRockland1 : 400
GFPChickenAbcam1 : 2000
Hu c/dHumanGift from Vanda Lennon[58]1 : 5000
Neurofilament MRabbitChemicon1 : 2000
nNOSSheepGift from Piers Emson[46, 59]1 : 2000
THSheepChemicon1 : 2000
THRabbitChemicon1 : 500

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Funding
  11. Disclosures
  12. References

The TH-Cre mice were mated to R26R-YFP Cre reporter mice; on Cre-mediated recombination, YFP is permanently expressed in cells that express TH and in their differentiated progeny. Thus, cells that expressed TH only transiently will be YFP+, but not TH-immunoreactive (IR). In this study, we refer to YFP-expressing, non-TH cells in the gut of TH-Cre;R26R-YFP mice as ‘transiently TH cells’, but use the term ‘TC cells’ when discussing earlier literature, as TH was not the only marker of a catecholaminergic phenotype used in previous studies.

Expression of the YFP reporter gene by TH-IR neurons in the adult jejunum, and embryonic and adult sympathetic ganglia, of TH-Cre;R26R-YFP mice

As a control, we first examined whether TH-IR neurons in the jejunum and sympathetic ganglia of adult mice were YFP+. In the small intestine of adult mice, TH-IR neurons comprise only a very small subpopulation of myenteric neurons,[3, 37, 41] but make up 22% of submucosal neurons.[42] In agreement with a previous study on wild-type mice on a C57BL/6 background,[42] we found that TH-IR neurons comprised 23% of Hu+ submucosal neurons in TH-Cre;R26R-YFP mice (= 509 Hu+ submucosal neurons examined from two mice). In the jejunum of adult TH-Cre;R26R-YFP mice, 100% of TH-IR neurons (n = 31 neurons from three mice) in the myenteric plexus, and 95.5% of TH-IR neurons (n = 112 neurons from two mice) in the submucosal plexus (Fig. 1A–C) were YFP+, which indicates almost complete Cre-mediated recombination. Furthermore, in the rostral sympathetic ganglia of E11.5 TH-Cre;R26R-YFP mice (Fig. 1D–F), and in the superior cervical ganglion of adult TH-Cre;R26R-YFP mice (Fig. 1G–I), almost all TH-IR neurons were also YFP+. Thus, TH-Cre;R26R-YFP mice provide a robust lineage-tracing tool to identify the progeny of the transiently TH cells in the embryonic and adult gut.

image

Figure 1. Overlap between YFP expression and TH immunoreactivity in TH-Cre;R26R-YFP mice. (A–C) Wholemount preparation of submucosal plexus of jejunum from a 6 week old TH-Cre;R26R-YFP mouse. Almost all (95.5%) of TH-IR neurons expressed YFP (filled arrows). Around 40% of YFP+ neurons (open arrows) did not show TH immunoreactivity, indicating that some submucosal neurons arise from cells that only transiently express TH. (D–I) Wholemount preparation of rostral sympathetic chain from an E11.5 TH-Cre;R26R-YFP mouse (D–F) and cryosection through the superior cervical ganglion (G–I) of an adult TH-Cre;R26R-YFP mouse. There is complete overlap between YFP expression and TH immunoreactivity, indicating highly efficient Cre-mediated expression and recombination. Scale bars: A–C and G–I = 100 μm; D–F = 50 μm.

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YFP expression in the developing gut of TH-Cre;R26R-YFP mice in relation to TH, calbindin and nNOS immunoreactivities

At E10.5, the migratory wavefront of ENCCs is midway along the small intestine.[9] Only a small number of YFP+ cells was present in the small intestine of E10.5 TH-Cre;R26R-YFP mice; all were TH-IR, but they comprised only a small proportion of the TH-IR cells (Fig. 2A–C). By E11.5, ENCCs have colonized the entire small intestine. YFP+ cells were abundant throughout the small intestine, and nearly 90% of the TH-IR cells were YFP+ (n = 33 cells in the small intestine from four preparations) (Fig. 2D–F). At E11.5, around 20% of YFP+ cells did not show detectable TH immunoreactivity (= 37 cells from four preparations), but by E12.5, TH-IR cells were very sparse and so YFP+ cells rarely showed TH-IR. These dynamic changes in TH immunoreactivity and YFP expression probably reflect the time taken for reporter gene activation, and then the downregulation of TH from E11.5, as reported previously.[35]

image

Figure 2. Wholemount preparations of small intestine from E10.5 and E11.5 TH-Cre;R26R-YFP mice. (A–F) Overlap between YFP expression and TH immunoreactivity. (A–C) At E10.5, multiple TH-IR neurons are present in the rostral small intestine, some of which have long processes projecting caudally. Only a small proportion of TH-IR cells is YFP+ (arrow), while most of the TH-IR cells do not yet express YFP. (D–F) The proportion of TH-IR that show YFP expression increases after E10.5, and almost complete overlap of YFP expression and TH immunoreactivity is observed at E11.5 throughout the small intestine. (G–I) Overlap between calbindin immunoreactivity and YFP expression in the E11.5 rostral small intestine. A subpopulation of YFP+ cells is calbindin-IR (arrows). Scale bars = 50 μm.

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The first enteric neuron subtype markers to be expressed in the mouse gut are calbindin and nNOS.[12, 33, 43, 44] Neuronal nitric oxide synthase-IR and calbindin-IR neurons can first be detected at E11.5,[44] when TC cells are also present. We therefore examined whether YFP+ cells in the small intestine of E11.5 TH-Cre;R26R-YFP mice were calbindin-IR or nNOS-IR: Around 40% of YFP+ cells (= 64 YFP+ cells from four preparations) were calbindin-IR, and 66% of calbindin-IR cells (= 38 calbindin-IR cells from four preparations) were YFP+ (Fig. 2G–I). In contrast, YFP+ cells that showed nNOS-IR were rare. For example, in the E13.5 gut, only 2.3% of YFP+ cells in the small intestine were nNOS-IR (= 264 YFP+ cells from five preparations).

In the gut of E13.5 and P3 TH-Cre;R26R-YFP mice, a regular, but low density, network of YFP+ cells was present; most myenteric ganglia along the small intestine of P3 mice contained 0–3 YFP+ neurons (Fig. 3).

image

Figure 3. (A) Intact, wholemount preparation of the duodenum of an E13.5 TH-Cre;R26R-YFP mouse showing the network of YFP+ cells and fibers. (B) Wholemount preparation of myenteric plexus and external muscle from the jejunum of a P3 TH-Cre;R26R-YFP mouse. The intestine had been opened down the mesenteric border and pinned prior to fixation, and then the mucosa removed after fixation. There are around 1–2 YFP+ cells per myenteric ganglion. Scale bars = 100 μm.

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Neurochemical phenotype of YFP+ cells in the myenteric ganglia of the jejunum of adult TH-Cre;R26R-YFP mice

The neurochemical phenotype of myenteric neurons that arise from cells that transiently express TH (YFP+/TH-negative cells) was investigated by immunohistochemical analysis of YFP+ neurons in the jejunum of adult TH-Cre;R26R-YFP mice using antibodies to the pan-neuronal marker, Hu, and the neuron subtype markers serotonin, calbindin, neurofilament-M, calretinin, and nNOS.[3] The data are summarized in Figs 4 and 5. Only around 5% of YFP+ myenteric neurons were TH-IR (Fig. 5A), and thus 95% of myenteric YFP+ neurons expressed TH only transiently. For technical reasons, we were unable to examine the overlap between TH-immunoreactivity and the other neuron subtype markers in this study. However, previous studies have shown that the TH neurons in the mouse small intestine are a different subpopulation from the serotonin neurons,[37] and that the TH neurons appear to be a rare, but separate, subpopulation from other characterized neuron subtypes.[3]

image

Figure 4. Wholemount preparations of jejunal myenteric plexus/longitudinal muscle of 6 week old TH-Cre;R26R-YFP mice. (A) All YFP+ cells were neurons as demonstrated by co-staining with antibodies to the pan-neuronal maker Hu (A–C; arrows). YFP+ neurons comprise only around 3% of the Hu-IR neurons in the myenteric plexus. (D–F) About 50% of YFP+ cells were calbindin-IR (arrows), but YFP+ neurons comprised only 8% of the calbindin-IR neurons. (G–I) A small proportion of calretinin-IR neurons expressed YFP (arrow). (J–O) Serotonin neurons comprised about 6% of YFP+ cells (arrow in J–K), but not all serotonin-IR neurons expressed YFP (yellow arrow in M–O). Scale bars = 50 μm.

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image

Figure 5. Phenotypic characterization of YFP expressing cells in the myenteric plexus of the jejunum of 6 week old TH-Cre;R26R-YFP mice. (A) Percentage of YFP+ cells that show immunoreactivity to a variety of neuron subtype markers. Note, that all YFP+ cells showed Hu immunoreactivity, and around 50% of the YFP+ cells was calbindin-IR. (B) Percentage of neuron subtypes that express YFP. Data are shown as mean ±SEM;= 3.

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All YFP+ cells in the myenteric plexus of the jejunum were Hu-IR, and thus TH-expressing cells give rise only to neurons and not glial cells. YFP+ neurons comprised only 2.7 ± 0.8% of the total (Hu-IR) neurons in the myenteric plexus (= 7038 Hu-IR neurons from three preparations) (Figs 4A–C; 5), and hence only a small proportion of myenteric neurons in the adult jejunum are the progeny of transiently TH-expressing cells. Nearly 30% of serotonin-IR neurons (= 82 serotonin-IR neurons from three mice) in the myenteric plexus was YFP+, but only 6.3 ± 1.2% of YFP+ cells (= 376 YFP+ neurons from three mice) were serotonin-IR (Figs 4J–O; 5), indicating that serotonin neurons are not the main derivative of the TC cells. Calbindin-IR neurons represented about 50% of the YFP+ neurons, while neurofilament-M-IR and calretinin-IR neurons comprised around 20% and 10%, respectively, of the YFP+ neurons (Figs 4D–I; 5). Only 2% of YFP+ neurons were nNOS-IR (Fig. 5). Although, calbindin-IR, neurofilament-M-IR and calretinin-IR show the largest overlap with YFP expression, YFP+ neurons comprise only 8.3 ± 1.0%, 7.1 ± 2.5%, and 0.9 ± 0.1% of these neuron subtypes, respectively (Fig. 5). To determine if there are any major differences in the proportions of neuron subtypes in the myenteric plexus of TH-Cre;R26R-YFP mice, we quantified the proportions of total (Hu-IR) neurons that exhibit nNOS- or calretinin-immunoreactivity; nNOS-IR and calretinin-IR neurons are two of the best characterized myenteric neuronal subtypes in the mouse ENS.[3, 4, 12, 19, 23, 31, 41, 45, 46] The proportions of Hu-IR myenteric neurons in the jejunum of two TH-Cre;R26R-YFP mice that were nNOS-IR (33% and 31%, = 832 and 692 Hu-IR neurons, respectively) or calretinin-IR (44% and 47%, = 615 and 630 Hu-IR neurons) were similar to previous reports of wild-type mice.[3, 4, 19, 46]

In the submucosal plexus of the jejunum, 35% of Hu+ neurons were YFP+, and TH-IR/YFP+ neurons comprised 62% of the YFP+ submucosal neurons (= 172 YFP+ submucosal neurons examined from two mice). Therefore, around 13% of submucosal neurons (YFP+/TH-negative neurons) arise from cells that transiently express TH.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Funding
  11. Disclosures
  12. References

Cells in the embryonic gut that express a transiently catecholaminergic phenotype (TC cells) were first identified over 30 years ago.[25, 27, 32, 34] We performed a genetic fate-mapping study of TH-expressing cells to characterize the progeny of the cells that express TH. We show that cells that express TH transiently give rise to <3% of myenteric neurons in the small intestine of adult mice, and the progeny include subpopulations of calbindin, serotonin, calretinin, and neurofilament-M-expressing neurons. Transiently TH cells give rise to around 30% of serotonin neurons, and <10% of the calbindin, neurofilament-M and calretinin neurons in the myenteric plexus.

Serotonin neurons

In the small intestine of adult mice, serotonin neurons comprise around 1% of myenteric neurons, and are descending interneurons.[3] During development of the enteric nervous system, serotonin neurons also play a key role as spontaneous release of serotonin from neurons regulates the survival and/or differentiation of the precursors of some enteric neuron subtypes.[41] In the gut of fetal Ascl1 null mutant mice, TH-expressing cells are absent, and there is also a reduction in the number of serotonin neurons.[15] It was therefore concluded that TC cells give rise to serotonin neurons.[15, 22, 24] Our data largely confirm this idea, although only a minority (~30%) of serotonin neurons appear to arise from TH-expressing cells. Serotonin neurons are one of the first enteric neuron subtypes to exit the cell cycle, and are born between E8 and E14.[13] It is possible that the serotonin neurons that do not arise from TH-expressing cells are born prior to (E8 or E9), or after (E14), the transient expression of TH by enteric neuron precursors.

Calbindin neurons

This study showed that over 50% of the progeny of TH-expressing cells in the myenteric plexus are calbindin neurons. In the guinea-pig small intestine, calbindin is a selective marker of intrinsic sensory neurons.[1, 47-49] In the mouse small intestine, the expression of calbindin by different enteric neuron subtypes has not been fully characterized, but it appears to be expressed by multiple enteric neuron subtypes.[3, 4] We found that only 8% of calbindin neurons arise from TH-expressing cells. Cells expressing calbindin appear early during development of the enteric nervous system,[44] and this study showed that many of the early calbindin cells in TH-Cre;R26R-YFP mice were YFP+. Calbindin neurons exit the cell cycle over a wide period of time (E12.5-E17.5, although E12.5 was the youngest age examined),[12] and it is likely that the calbindin neurons that arise from TC cells are those that exit the cell cycle early.

Neurofilament-M neurons

Based on shape and projections, it has been suggested that intrinsic sensory neurons in the mouse small intestine can be identified by the expression of NF-M.[3] This study showed that around 20% of the progeny of TH-expressing cells are NF-M neurons, but only 7% of NF-M neurons arise from TH-expressing cells. The birth dates of NF-M neurons in the mouse small intestine have not yet been examined.

nNOS neurons

Neuronal nitric oxide synthase neurons in the gut appear early, prior to the downregulation of TH expression,[44] and there is a small amount of overlap between TH and nNOS immunoreactivities at E12.5.[35] Although TH expression is down-regulated by enteric neuron precursors in vivo, TH expression is maintained in explants of embryonic gut grown in vitro[30]; following growth of E11.5 gut explants in vitro for 4–5 days, we reported previously that 80–90% of the TH+ cells are also nNOS+. We therefore proposed that nNOS neurons are one of the main derivatives of the TC cells.[35] However, this earlier conclusion is incorrect, as in this study we found that only 2% of the progeny of TH-expressing cells were nNOS neurons, and that ~0.2% of nNOS neurons arise from TH-expressing cells. The high degree of overlap between TH and nNOS in cultured gut explants suggests that there are significant differences in the phenotype of enteric neurons in cultured explants compared to in vivo. Thus, although some nNOS neurons develop early during development, and although TH-expressing cells give rise to enteric neurons of a variety of phenotypes, very few TH-expressing cells give rise to nNOS neurons in the adult gut.

Minor contribution of transiently TH cells to the adult myenteric neuron population

The first ENCC to express pan-neuronal markers during ENS development also express TH.[30, 36] Our data show that, overall, transiently TH cells give rise to <3% of myenteric neurons. Thus, the vast majority of myenteric neurogenesis appears to occur after the transient expression of TH, which occurs between E10 and E13.[30] This is perhaps not surprising as the total number of neurons present in the gut of E10-E13 mice is probably only a very small percentage of the number of myenteric neurons in the adult gut. Although transiently TH cells can proliferate,[29] our data suggest that they do not undergo extensive proliferation.

Some submucosal neurons arise from transiently TH cells

Enteric neural crest-derived cells first settle in the outer mesenchyme in the site of the future myenteric plexus, and the submucosal plexus develops several days later from a secondary migration of cells from the myenteric region.[13, 50-57] We and others have found that around 22% of submucosal neurons in the adult mouse express TH[42]; however, we found that around 35% of YFP+ neurons in submucosal ganglia did not show TH immunoreactivity in TH-Cre;R26R-YFP mice, showing that around 13% of submucosal neurons arise from cells that transiently express TH. The phenotypes of the YFP+/TH-negative neurons in the submucosal plexus were not examined in this study.

Technical considerations

We examined the proportion of TH-IR neurons in myenteric, submucosal, and sympathetic ganglia that express YFP in TH-Cre;R26R-YFP mice, and our data suggest efficient Cre expression and recombination. Thus, the expression of YFP in only a subpopulation of particular enteric neuron subtypes is unlikely to be due to failure of Cre expression or inefficient recombination. The maximum proportion of any neuron class to express YFP was 30% of serotonin neurons. Some of the phenotypic classes of neurons examined, for example the calbindin, calretinin, and nNOS neurons, comprise multiple functional subtypes, and so we cannot rule out the possibility that 100% of some small functional enteric neuron subtypes do arise from TC cells. However, serotonin and NF-M neurons appear to be single functional classes of neurons, and not all serotonin or NF-M neurons arise from TC cells.

In previous studies, fate-mapping of TC cells was mainly based on the assumption that TC cells retain expression of some catecholaminergic genes, such as DβH and NET.[30, 31] Immunoreactivity of DβH was reported to coincide with expression of neuropeptide Y, substance P and with uptake of 5-HT by enteric neurons in adult rats.[30] In addition, all serotonin and nNOS neurons were shown to also express NET in the gut of adult mice.[31] Our data suggest that NET expression may not be restricted to the progeny of TH-expressing cells.

Although the first cells in the ENS to express pan-neuronal markers transiently express a catecholaminergic phenotype,[25-34] we cannot rule out the possibility that some of the YFP+/TH-negative neurons we observed in the adult gut transiently expressed TH at later ages, perhaps even postnatally. To examine whether there are transiently TH cells at a variety of prenatal and postnatal ages, it will be necessary to use mice expressing an inducible form of Cre recombinase under the control of TH.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Funding
  11. Disclosures
  12. References

Transient expression of a catecholaminergic phenotype does not appear to identify specific enteric neuron subtype lineages. Our finding that TC cells give rise to significant subpopulations of serotonin and calbindin neurons is consistent with the hypothesis that TC cells mainly generate neurons that emerge early in development.[15] However, not all neurons that develop early are the progeny of TC cells as very few nNOS neurons, which are also detectable during early ENS development[43, 44] seem to originate from TC cells.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Funding
  11. Disclosures
  12. References

We thank Annette Bergner and Jan Morgan for excellent technical assistance, and Andrew Allen and Toby Merson for the provision of mice.

Author Contributions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Funding
  11. Disclosures
  12. References

HY designed the research study; FO, LS, CA and HY performed research; FO and LS analyzed the data; FO and HY wrote the paper.

Funding

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Funding
  11. Disclosures
  12. References

This work was supported by NHMRC Project Grants #628349 and 1047953. FO is supported by a Research Fellowship from the German Research Foundation (DFG, OB 381/1-1).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. Author Contributions
  10. Funding
  11. Disclosures
  12. References