Hu neuronal proteins are expressed in proliferating neurogenic cells

Abstract

We have utilized immunochemical techniques to investigate the developmental expression of the Hu proteins, a neuron-specific family of RNA binding proteins in vertebrates. Previous work suggests that these proteins may play an important role in neuronal development and maintenance. For the present study, we developed a monoclonal antibody (MAb 16A11) that binds specifically to an epitope present in gene products of all known Hu genes, including HuD, HuC, and Hel-N1. Using brief pulses (1-2 h) of the DNA precursor analog bromodeoxyuridine (BrdU) in conjunction with MAb 16A11, we observed Hu+/BrdU+ cells in nascent sensory and sympathetic ganglia in vivo, and in populations of cultured neural crest cells. In addition, a few Hu+ cells were ambiguously BrdU+ in the neural tube. We conclude that Hu+ cells first appear in avian neurogenic populations immediately before neuronal birthdays in the peripheral nervous system, and at the time of withdrawal from the mitotic cycle in the central nervous system. Consistent with these conclusions, we have also observed neural crest-derived cells that are both Hu+ and in metaphase of the cell cycle. We suggest that Hu proteins function early in neurogenic differentiation.

Appearance of neurons and glia with respect to the wavefront during colonization of the avian gut by neural crest cells

“…The subsequent formation of ganglia at these foci might occur by the movement of additional Huϩ cells to these aggregation centers or by local recruitment of neuroblasts from nearby neural crest cells. Experiments in the developing avian dorsal root ganglion (Marusich et al, 1994) indicate that Hu-IR appears either at or just after the time of cell cycle exit. It is not known whether a similar process occurs in the gut, whether the terminal division is symmetrical (both daughters are Huϩ) or whether the Hu-labeled cells continue to divide or to advance.…”

Cytoplasmic localization is required for the mammalian ELAV‐like protein HuD to induce neuronal differentiation

“…To examine the endogenous levels of Hu proteins in PC12 cells, we ®rst performed an immuno¯uorescence analysis using monoclonal antibody 16A11, which speci®cally recognizes the mammalian Hu proteins, except for HuR (Marusich et al 1994) (Fig. 1A).…”

“…For indirect immuno¯uorescence analysis, cells were ®xed with 4% formaldehyde, and incubated with the appropriate mouse monoclonal antibodies as follows: 16A11 antibody (Marusich et al 1994), anti-NF-H antibody (Sigma), anti-NF-M antibody (Sigma), anti-T7 epitope antibody (Novagen), anti-FLAG epitope antibody (Eastman Kodak), or anti-myc epitope 9E10 antibody (Michael et al 1997). Subsequently, cells were incubated with FITC-labelled anti-mouse IgG secondary antibody (DACO) or Cy3-labelled anti-mouse IgG antibody (Amersham) and analysed with the Photometrics SenSys 0400 or Zeiss confocal microscopy systems.…”

Manipulation of Neural Precursors in situ Induction of Neurogenesis in the Neocortex of Adult Mice

“…25 , NO . 6 First, subsets of BrdU ϩ precursors expressed Doublecortin, a protein expressed exclusively in immature, typically migrating neurons (Gleeson et al 1999;Francis et al 1999), and Hu, an early neuronal marker (Marusich et al 1994;Barami et al 1995). We observed no new neurons in control, intact cortex.…”

“…The continued differentiation of these newborn neurons was examined using antibodies to Hu, an RNAbinding protein that begins to be expressed in neuronal nuclei and somata soon after differentiation (Marusich et al 1994;Barami et al 1995). BrdUϩ/Huϩ neurons with large, ovoid nuclei were found in the cortex of experimental mice two weeks after induction of apoptosis ( Figure 5), whereas no BrdUϩ/Huϩ cells were found in control mice.…”