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Curr Mol Med. 2018 Mar 9;17(7):509-517. doi: 10.2174/1566524018666180212145714.

The bHLH Protein Nulp1 is Essential for Femur Development Via Acting as a Cofactor in Wnt Signaling in Drosophila.

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The Center for Heart Development, State Key Laboratory of Development Biology, Key Laboratory of MOE for Development Biology and Protein Chemistry, College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China.
Cardiovascular Surgery, Guangdong Cardiovascular Institute, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510100, China.
Department of Pediatrics, the Third Xiangya Hospital, Central South University, Changsha, 410013, China.
Development, Aging and Regeneration Program, Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, CA 92037, United States.



The basic helix-loop-helix (bHLH) protein families are a large class of transcription factors, which are associated with cell proliferation, tissue differentiation, and other important development processes. We reported that the Nuclear localized protein-1 (Nulp1) might act as a novel bHLH transcriptional factor to mediate cellular functions. However, its role in development in vivo remains unknown.


Nulp1 (dNulp1) mutants are generated by CRISPR/Cas9 targeting the Domain of Unknown Function (DUF654) in its C terminal. Expression of Wg target genes are analyzed by qRT-PCR. We use the Top-Flash luciferase reporter assay to response to Wg signaling.


Here we show that Drosophila Nulp1 (dNulp1) mutants, generated by CRISPR/Cas9 targeting the Domain of Unknown Function (DUF654) in its C terminal, are partially homozygous lethal and the rare escapers have bent femurs, which are similar to the major manifestation of congenital bent-bone dysplasia in human Stuve- Weidemann syndrome. The fly phenotype can be rescued by dNulp1 over-expression, indicating that dNulp1 is essential for fly femur development and survival. Moreover, dNulp1 overexpression suppresses the notch wing phenotype caused by the overexpression of sgg/GSK3β, an inhibitor of the canonical Wnt cascade. Furthermore, qRT-PCR analyses show that seven target genes positively regulated by Wg signaling pathway are down-regulated in response to dNulp1 knockout, while two negatively regulated Wg targets are up-regulated in dNulp1 mutants. Finally, dNulp1 overexpression significantly activates the Top-Flash Wnt signaling reporter.


We conclude that bHLH protein dNulp1 is essential for femur development and survival in Drosophila by acting as a positive cofactor in Wnt/Wingless signaling.


CRISPR/Cas9; Drosophila.; Wnt/Wg signaling; bHLH; dNulp1; femur

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