Recently, mutations in ZFHX1B, the gene that encodes Smad-interacting protein-1 (SIP1), were found to be implicated in the etiology of a dominant form of Hirschsprung disease-mental retardation syndrome in humans. To clarify the molecular mechanisms underlying the clinical features of SIP1 deficiency, we generated mice that bear a mutation comparable to those found in several human patients. Here, we show that Zfhx1b-knockout mice do not develop postotic vagal neural crest cells, the precursors of the enteric nervous system that is affected in patients with Hirschsprung disease, and they display a delamination arrest of cranial neural crest cells, which form the skeletomuscular elements of the vertebrate head. This suggests that Sip1 is essential for the development of vagal neural crest precursors and the migratory behavior of cranial neural crest in the mouse. Furthermore, we show that Sip1 is involved in the specification of neuroepithelium.
One of the most notable features of the vertebrate body plan organization is its bilateral symmetry, evident at the level of vertebrae and skeletal muscles. Here we show that a mutation in Rere (also known as atrophin2) leads to the formation of asymmetrical somites in mouse embryos, similar to embryos deprived of retinoic acid. Furthermore, we also demonstrate that Rere controls retinoic acid signalling, which is required to maintain somite symmetry by interacting with Fgf8 in the left-right signalling pathway. Rere forms a complex with Nr2f2, p300 (also known as Ep300) and a retinoic acid receptor, which is recruited to the retinoic acid regulatory element of retinoic acid targets, such as the Rarb promoter. Furthermore, the knockdown of Nr2f2 and/or Rere decreases retinoic acid signalling, suggesting that this complex is required to promote transcriptional activation of retinoic acid targets. The asymmetrical expression of Nr2f2 in the presomitic mesoderm overlaps with the asymmetry of the retinoic acid signalling response, supporting its implication in the control of somitic symmetry. Misregulation of this mechanism could be involved in symmetry defects of the human spine, such as those observed in patients with scoliosis.
In mouse embryos, the Zfhx1 transcription factor genes, Sip1 and ␦EF1, are expressed in complementary domains in many tissues. Their possible synergism in embryogenesis was investigated by comparing the phenotype of Sip1؊/؊;␦EF1؊/؊ double homozygotes with single homozygous embryos. Unexpectedly, in Sip1؊/؊ embryos ␦EF1 was ectopically activated, suggesting a negative regulation of ␦EF1 expression by Sip1. Sip1؊/؊;␦EF1؊/؊ embryos were similar to Sip1؊/؊ embryos in short somite production and developmental arrest around E8.5, but showed more severe defects in dorsal neural tube morphogenesis accompanied by a larger reduction of Sox2 expression, ascribable to the loss of the ectopic ␦EF1 expression. Sip1؉/؊;␦EF1؊/؊ embryos develop various morphological defects after E10 that were absent in ␦EF1؊/؊ embryos even in tissues without significant overlap of Sip1 and ␦EF1 expression, and arrested during mid gestation earlier than ␦EF1؊/؊ embryos. These findings indicate that complex synergistic interactions occur between Zfhx1 transcription factor genes during mouse embryogenesis. Developmental Dynamics 235: 1941-1952, 2006.
Periodical production of somites provides an excellent model system for understanding genesis of metameric structures underlying embryonic development. This study reports production of somites with roughly half rostro-caudal length in homozygous Sip1 (Smad-interacting protein 1) knockout mouse embryos. This altered periodicity of somitogenesis is caused by the rostral expansion of the expression domain of genes involved in the maintenance of unsegmented state of paraxial mesoderm, e.g., Fgf8, Wnt3a, Dll3, and Tbx6. This is accompanied by the rostral extension of oscillatory gene expression such as L-fng, Hes7, and Dll1, and the rostrally shifted termination of Raldh2 expression that continues from the anterior embryonic side. The phenotype of Sip1؊/؊ embryo introduces a new molecular component SIP1 in positioning of somite boundaries, and provides support for the current "clock and wavefront" model. Developmental Dynamics 234:332-338, 2005.
The nuclear RNA export factor (NXF) family proteins have been implicated in various aspects of post-transcriptional gene expression. This study shows that mouse NXF7 exhibits heterologous localization, i.e. NXF7 associates with translating ribosomes, stress granules (SGs) and processing bodies (P-bodies), the latter two of which are believed to be cytoplasmic sites of storage, degradation and/or sorting of mRNAs. By yeast two-hybrid screening, a series of heterogeneous nuclear ribonucleoproteins (hnRNPs) were identified as possible binding partners for NXF7. Among them, hnRNP A3, which is believed to be involved in translational control and/or cytoplasmic localization of certain mRNAs, formed a stable complex with NXF7 in vitro. Although hnRNP A3 was not associated with translating ribosomes, it was co-localized with NXF7 in P-bodies. After exposing to oxidative stress, NXF7 trans-localized to SGs, whereas hnRNP A3 did not. In differentiated neuroblastoma Neuro2a cells, NXF7 was co-localized with hnRNP A3 in cell body and neurites. The amino terminal half of NXF7, which was required for stable complex formation with hnRNP A3, coincided with the region required for localization in both P-bodies and neuronal RNA granules. These findings suggest that NXF7 plays a role in sorting, transport and/or storage of mRNAs through interactions with hnRNP A3.
Bilateral symmetry is a striking feature of the vertebrate body plan organization. Vertebral precursors, called somites, provide one of the best illustrations of embryonic symmetry. Maintenance of somitogenesis symmetry requires retinoic acid (RA) and its coactivator Rere/Atrophin2. Here, using a proteomic approach we identify a protein complex, containing Wdr5, Hdac1, Hdac2 and Rere (named WHHERE), which regulates RA signaling and controls embryonic symmetry. We demonstrate that Wdr5, Hdac1, and Hdac2 are required for RA signaling in vitro and in vivo. Mouse mutants for Wdr5 and Hdac1 exhibit asymmetrical somite formation characteristic of RA-deficiency. We also identify the Rere-binding histone methyltransferase Ehmt2/G9a, as a RA coactivator controlling somite symmetry. Upon RA treatment, WHHERE and Ehmt2 become enriched at RA target genes to promote RNA polymerase II recruitment. Our work identifies a protein complex linking key epigenetic regulators acting in the molecular control of embryonic bilateral symmetry.
The vertebrate Zfhx1 transcription factor family comprises dEF1 and Sip1, which bind to CACCT-containing sequences and act as transcriptional repressors. It has been a longstanding question whether these transcription factors share the same regulatory functions in vivo. It has been shown that neural crest (NC) delamination depends on the Sip1 activity at the cranial level in mouse and chicken embryos, and it remained unclear how NC delamination is regulated at the trunk level. We observed that the expression of dEF1 and Sip1 overlaps in many tissues in chicken embryos, including NC cells at the trunk level. To clarify the above questions, we separately knocked down dEF1 and Sip1 or in combination in NC cells by electroporation of vectors expressing short hairpin RNAs (shRNAs) against respective mRNAs on the dorsal side of neural tubes that generate NC cells. In all cases, the migrating NC cell population was significantly reduced, paralleled by the decreased expression of dEF1 or Sip1 targeted by shRNAs. Expression of Sox10, the major transcription factor that regulates NC development, was also decreased by the shRNAs against dEF1 or Sip1. We conclude that the trunk NC delamination is regulated by both dEF1 and Sip1 in an analogous manner, and that these transcription factors can share equivalent regulatory functions in embryonic tissues.
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