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. 2010 Oct 15;346(2):215-23.
doi: 10.1016/j.ydbio.2010.07.026. Epub 2010 Aug 4.

Mouse atonal homolog 1 directs intestinal progenitors to secretory cell rather than absorptive cell fate

Kelli L VanDussen  1 , Linda C Samuelson
Affiliations

Mouse atonal homolog 1 directs intestinal progenitors to secretory cell rather than absorptive cell fate

Kelli L VanDussen et al. Dev Biol. .

Abstract

The Notch-regulated transcription factor mouse atonal homolog 1 (Math1) is required for the development of intestinal secretory cells, as demonstrated by the loss of goblet, endocrine and Paneth cell types in null mice. However, it was unknown whether Math1 is sufficient to induce the program of secretory cell differentiation. To examine the function of Math1 in the differentiation of intestinal epithelial cells, intestinal morphology and epithelial and mesenchymal cell fate were examined by histological staining and marker gene expression in transgenic mice expressing a villin-regulated Math1 transgene. Late prenatal transgenic founders exhibited a gross cellular transformation into a secretory epithelium. The expansion of secretory cells coupled with the almost complete loss of absorptive enterocytes suggested reprogramming of a bipotential progenitor cell. Moreover, Math1 expression inhibited epithelial cell proliferation, as demonstrated by a marked reduction in Ki67 positive cells and blunted villi. Unexpectedly, the transgenic mesenchyme was greatly expanded with increased proliferation. Several mesenchymal cell types were amplified, including smooth muscle and neurons, with maintenance of basic radial patterning. Since transgenic Math1 expression was restricted to the epithelium, these findings suggest that epithelial-mesenchymal signaling is altered by the cellular changes induced by Math1. Thus, Math1 is a key effector directing multipotential precursors to adopt secretory and not absorptive cell fate.

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Conflict of interest statement

DISCLOSURES: The authors have no conflicts to disclose.

Figures

Figure 1
Figure 1
Abnormal intestinal morphology in Vil-Math1 E18.5 transgenic mice. (A) The Vil-Math1 transgene contained mouse villin sequences, including 5′ flanking sequence, the first untranslated exon (UTR) and intron, to regulate expression of the mouse Math1 cDNA, and SV40 sequences (hatched) to provide a polyA site. (B) Transgenic (Tg) intestines were fluid-filled and translucent (observed in M12, M41, and M48) compared to Nontransgenic (Ntg) controls. Bars: 2 mm. (C) Total Math1 mRNA (transgene plus endogenous) was measured by quantitative reverse transcriptase-PCR (qRT-PCR) analysis of proximal intestine, distal intestine, and colon RNA. Six Vil-Math1 founders exhibited significantly higher Math1 mRNA levels in at least one region. Values were normalized to Gapdh expression and reported as fold-change relative to the corresponding region of Ntg littermate controls (***P<0.001). n.d., not determined. (D) Immunostaining for Math1 in proximal intestine of Ntg and two representative transgenic founders. Math1 is normally expressed in nuclei of secretory progenitor cells and mature secretory cells (arrowheads); transgenics had widespread expression of Math1 throughout the epithelium. Hematoxylin nuclear stain. Bars: 100 μm.
Figure 2
Figure 2
Math1 promotes the development of goblet cells throughout the intestine. Histological analysis of Ntg (A, D) and Tg (B, C, E, F) intestines from E18.5 founders. Transgenics M3 and M48 are shown to demonstrate the range in phenotypes. (A–C) H&E staining of proximal intestine showed increased numbers of goblet-like cells in Tg mice, apparent in the higher magnification insets (arrowheads), as well as mesenchymal tissue expansion. (D–F) PAS/Alcian blue staining confirmed increased goblet cells in the proximal intestine of Tg mice. Bars: 100 μm.
Figure 3
Figure 3
Math1 stimulates the endocrine cell differentiation program. (A–C) Immunostaining for chromogranin A (CgA; green) showed increased endocrine cell numbers (arrowheads) in proximal Tg intestines, which was associated with loss of lateral inhibition (C, inset shows confocal image of 2 cell doublets with one doublet outlined). DAPI (blue/red) nuclear stain. Bars: 100 μm; 10 μm (inset). (D) Quantification of endocrine cells from CgA-stained sections. Values were normalized to epithelial area (μm2) and statistical significance was determined by comparison to Ntg (**P<0.01; ***P<0.001). (E) qRT-PCR analysis of CCK mRNA abundance in proximal intestine. Values were normalized to Gapdh expression and reported as fold-change relative to Ntg (**P<0.01; ***P<0.001). (F–H) Immunostaining proximal intestines for Neurogenin 3 (Neurog3; green nuclear stain) demonstrated increased numbers of Neurog3-positive cells (arrowheads) in Vil-Math1 transgenics, which were often mislocated outside of the intervillus zone. DAPI (pseudocolored red) nuclear stain. Bars: 100 μm. (I) Quantification of endocrine progenitor cells from Ngn3-stained sections. Values were normalized to epithelial area (μm2) and statistical significance was determined by comparison to Ntg (**P<0.01; ***P<0.001). (J) qRT-PCR analysis of Neurog3 mRNA abundance in distal intestine. Values were normalized to Gapdh expression and reported as fold-change relative to Ntg (*P<0.05; **P<0.01; ***P<0.001).
Figure 4
Figure 4
Emergence of a Paneth-like cell in the intervillus zones and colons of Vil-Math1 transgenic intestine. (A–E) Immunostaining for lysozyme (green) showed faintly-staining cells scattered in Ntg distal intestine (A) and no positive-staining in the Ntg colon (D). Tg founders had increased numbers of lysozyme-positive cells predominantly in the intervillus zone of the distal intestine. Arrowheads denote some lysozyme-positive cells (B, C). Lysozyme-positive cells were also apparent in the Tg colon (E). DAPI (blue) nuclear stain. Bars: 100 μm. (F, G) qRT-PCR analysis of lysozyme mRNA abundance in distal intestine (F) and cryptdin (G) mRNA abundance in distal intestine and colon. Values were normalized to Gapdh expression and reported as fold-change relative to Ntg (**P<0.01; ***P<0.001).
Figure 5
Figure 5
Loss of the absorptive lineage in E18.5 Vil-Math1 transgenics. Ntg (A, D) and Tg (B, C, E, F) proximal (A–C) and distal (D–F) intestine sections were stained for the enterocyte brush border enzyme alkaline phosphatase (red) and counterstained with hematoxylin (blue). Bars: 100 μm. (D) Lactase mRNA abundance was measured by qRT-PCR analysis of distal intestine. Values were normalized to Gapdh expression and reported as fold-change relative to Ntg (***P<0.001).
Figure 6
Figure 6
Altered epithelial and mesenchymal cell proliferation in E18.5 Vil-Math1 intestine. (A–D) Proliferation was assessed in Ntg (A) and Tg (B–D) proximal intestine by Ki67 immunostaining (red) co-immunostained with desmin (green) to mark mesenchymal cells and DAPI (blue) nuclear stain. Arrowheads identify some proliferating cells in the mesenchyme. Bars: 50 μm. (E) Quantification of epithelial cell proliferation by morphometric analysis of Ki67 immunostaining of proximal intestine. Data are presented as Ki67-positive nuclei per epithelial area (μm2) (*P<0.05 compared to Ntg). (F) qRT-PCR analysis of the stem cell markers Lgr5 and Prom1 in distal small intestine. Values were normalized to Gapdh expression and reported as fold-change relative to Ntg (*P<0.05; **P<0.01; ***P<0.001).
Figure 7
Figure 7
Remodeled mesenchyme in E18.5 Vil-Math1 intestine. (A, B) qRT-PCR analysis of vimentin (A) and SM22α (B) mRNA abundance was performed with distal small intestine RNA. Values were normalized to Gapdh expression and reported as fold-change relative to Ntg (*P<0.05; **P<0.01; ***P<0.001). (C–E) Co-immunostaining of proximal intestine sections with α-smooth muscle actin (red) and desmin (green) with DAPI (blue) nuclear stain. Differentiated smooth muscle is yellow due to co-expression of these two markers. (F–G) Immunostaining for the neuronal marker neurofilament (brown), with hematoxylin counterstain (blue) shows expansion of the enteric nervous system in Vil-Math1 transgenics. Bars: 100 μm.
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