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. 2024 Aug 14;40(1):225.
doi: 10.1007/s00383-024-05809-7.

A lmod1a mutation causes megacystis microcolon intestinal hypoperistalsis in a CRISPR/Cas9-modified zebrafish model

Alvin Santoso Kalim  1 , Kouji Nagata  2 , Yukihiro Toriigahara  1 , Takeshi Shirai  1 , Kosuke Kirino  1 , Zhang Xiu-Ying  1 , Takuya Kondo  1 , Naonori Kawakubo  1 , Junko Miyata  1 , Toshiharu Matsuura  1 , Tatsuro Tajiri  1
Affiliations

A lmod1a mutation causes megacystis microcolon intestinal hypoperistalsis in a CRISPR/Cas9-modified zebrafish model

Alvin Santoso Kalim et al. Pediatr Surg Int. .

Abstract

Purpose: Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is defined as a congenital visceral myopathy with genetic mutations. However, the etiology and pathophysiology are not fully understood. We aimed to generate a gene leiomodin-1a (lmod1a) modification technique to establish a zebrafish model of MMIHS.

Methods: We targeted lmod1a in zebrafish using CRISPR/Cas9. After confirming the genotype, we measured the expression levels of the target gene and protein associated with MMIHS. A gut transit assay and spatiotemporal mapping were conducted to analyze the intestinal function.

Results: Genetic confirmation showed a 5-base-pair deletion in exon 1 of lmod1a, which caused a premature stop codon. We observed significant mRNA downregulation of lmod1a, myh11, myod1, and acta2 and the protein expression of Lmod1 and Acta2 in the mutant group. A functional analysis of the lmod1a mutant zebrafish showed that its intestinal peristalsis was fewer, slower, and shorter in comparison to the wild type.

Conclusion: This study showed that targeted deletion of lmod1a in zebrafish resulted in depletion of MMIHS-related genes and proteins, resulting in intestinal hypoperistalsis. This model may have the potential to be utilized in future therapeutic approaches, such as drug discovery screening and gene repair therapy for MMIHS.

Keywords: Animal model; CRISPR/Cas9; Lmod1a; MMIHS; Zebrafish.

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