Mirk/Dyrk1B controls ventral spinal cord development via Shh pathway.

Cross-talk between Mirk/Dyrk1B kinase and Sonic hedgehog (Shh)/Gli pathway affects physiology and pathology. Here, we reveal a novel role for Dyrk1B in regulating ventral progenitor and neuron subtypes in the embryonic chick spinal cord (SC) via the Shh pathway. Using in ovo gain-and-loss-of-function approaches at E2, we report that Dyrk1B affects the proliferation and differentiation of neuronal progenitors at E4 and impacts on apoptosis specifically in the motor neuron (MN) domain. Especially, Dyrk1B overexpression decreases the numbers of ventral progenitors, MNs, and V2a interneurons, while the pharmacological inhibition of endogenous Dyrk1B kinase activity by AZ191 administration increases the numbers of ventral progenitors and MNs. Mechanistically, Dyrk1B overexpression suppresses Shh, Gli2 and Gli3 mRNA levels, while conversely, Shh, Gli2 and Gli3 transcription is increased in the presence of Dyrk1B inhibitor AZ191 or Smoothened agonist SAG. Most importantly, in phenotype rescue experiments, SAG restores the Dyrk1B-mediated dysregulation of ventral progenitors. Further at E6, Dyrk1B affects selectively the medial lateral motor neuron column (LMCm), consistent with the expression of Shh in this region. Collectively, these observations reveal a novel regulatory function of Dyrk1B kinase in suppressing the Shh/Gli pathway and thus affecting ventral subtypes in the developing spinal cord. These data render Dyrk1B a possible therapeutic target for motor neuron diseases.

Cloning, expression and localization of human BM88 shows that it maps to chromosome 11p15.5, a region implicated in Beckwith-Wiedemann syndrome and tumorigenesis.

Porcine BM88 is a neuron-specific protein that enhances neuroblastoma cell differentiation in vitro and may be involved in neuronal differentiation in vivo. Here we report the identification, by Western blotting, of homologous proteins in human and mouse brain and the isolation of their respective cDNAs. Several human and mouse clones were identified in the EST database using porcine BM88 cDNA as a query. A human and a mouse EST clone were chosen for sequencing and were found both to predict a protein of 149 amino acids, with 79.9% reciprocal identity, and 76.4% and 70.7% identities to the porcine protein, respectively. This indicated that the clones corresponded to the human and mouse BM88 homologues. In vitro expression in a cell-free system as well as transient expression in COS7 cells yielded polypeptide products that were recognized by anti-BM88 antibodies and were identical in size to the native BM88 protein. Northern-blot analysis showed a wide distribution of the gene in human brain whereas immunohistochemistry on human brain sections demonstrated that the expression of BM88 is confined to neurons. The initial mapping assignment of human BM88 to chromosome 11p15.5, a region implicated in Beckwith-Wiedemann syndrome and tumorigenesis, was retrieved from the UniGene database maintained at the National Centre for Biotechnology Information (NCBI, Bethesda, MD, U.S.A.). We confirmed this localization by performing fluorescence in situ hybridization on BM88-positive cosmid clones isolated from a human genomic library. These results suggest that BM88 may be a candidate gene for genetic disorders associated with alterations at 11p15.5.

Purification and cDNA cloning of mouse BM89 antigen shows that it is identical with the synaptic vesicle protein synaptophysin.

The BM89 antigen, first identified in porcine brain by means of a monoclonal antibody, is a neuron-specific molecule widely distributed in the mammalian central and peripheral nervous system (Merkouri and Matsas: Neuroscience 50:53-68, 1992). Here we describe the purification of BM89 antigen from porcine and mouse brain by immunoaffinity chromatography using, respectively, the previously described BM89 monoclonal antibody which belongs to the IgM class and a specific polyclonal antibody generated in the present study. This antibody was also used for the cDNA cloning of the BM89 antigen from mouse brain. cDNA sequencing revealed that the mouse BM89 antigen is identical with the synaptic vesicle protein synaptophysin which is implicated in the control of regulated exocytosis and neurotransmitter release. Mouse BM89 antigen/synaptophysin exhibits, except for one extra amino acid, 100% identity with rat synaptophysin and substantial sequence identity with bovine (92.5% identity) and human (94.8% identity) synaptophysin, but only 59.8% identity with Torpedo synaptophysin. Northern and Western blot analyses confirmed that the mouse BM89 antigen/synaptophysin is expressed only in neural tissues.