Expression of an engrailed -like gene during development of the early embryonic chick nervous system

The engrailed gene has been identified in Drosophila as an important developmental gene involved in the control of segmentation. Here we describe the embryonic expression of a chicken gene, ChickEn (Darnell et al.: J Cell Biol 103(5):311a, 1986), which contains homology to the Drosophila engrailed gene. Northern blots of early chick embryo tissue poly(A) + RNA resulted in hybridization to at least three bands expressed predominantly in the brain/head region when probed with ChickEn genomic fragments. Eight cDNA clones generated from embryonic day 6 (stage 29–30) chick brain poly(A) + RNA are identical in their nucleotide sequence with the ChickEn genomic clone. In situ hybridization to sections of 4-day (stage 24) embryos indicated that ChickEn transcripts were concentrated in the posterior mesencephalon and anterior metencephalon. In cultures of chick cranial neural crest cells (eight to nine somites; stage 9) ChickEn transcripts were localized in a subset (approx. 8%) of cells examined after 2 days in culture. A mouse monoclonal antibody, inv-4D9D4, made by Coleman and Kornberg recognizes the engrailed -like homeo domain of the engrailed and invected proteins (Martin-Blanco, Coleman, and Kornberg, personal communication). Patel, Coleman, Kornberg and Goodman (unpublished) have shown that this antibody binds to the hindbrain of 2-day-old chick embryos. We have confirmed these results and shown that this antibody binds to the same region of 4-day (stage 24) chick brains that in situ hybridization showed contained ChickEn transcripts. This antibody also recognizes a homeo domain-containing ChickEn peptide expressed as a beta-galactosidase fusion protein in Drosophila cell culture. We have not detected ChickEn protein in any tissue prior to eight to nine somites (stage 9). These results delineate the major expression pattern of the ChickEn gene during early (prior to stage 30) embryonic development in the chick.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50222/1/490210234_ftp.pd

Robo2-Slit1 dependent cell-cell interactions mediate assembly of the trigeminal ganglion

Vertebrate cranial sensory ganglia, responsible for sensation of touch, taste and pain in the face and viscera, are composed of both ectodermal placode and neural crest cells. The cellular and molecular interactions allowing generation of complex ganglia remain unknown. Here, we show that proper formation of the trigeminal ganglion, the largest of the cranial ganglia, relies on reciprocal interactions between placode and neural crest cells in chick, as removal of either population resulted in severe defects. We demonstrate that ingressing placode cells express the Robo2 receptor and early migrating cranial neural crest cells express its cognate ligand Slit1. Perturbation of this receptor-ligand interaction by blocking Robo2 function or depleting either Robo2 or Slit1 using RNA interference disrupted proper ganglion formation. The resultant disorganization mimics the effects of neural crest ablation. Thus, our data reveal a novel and essential role for Robo2-Slit1 signaling in mediating neural crest–placode interactions during trigeminal gangliogenesis

Antigen recognized by monoclonal antibodies to mesencephalic neural crest and to ciliary ganglion neurons is involved in the high affinity choline uptake mechanism in these cells

High-affinity choline uptake mechanisms are among the characteristics of cholinergic neurons such as the ciliary and choroid subpopulations in the ciliary ganglion (Barald and Berg, 1979). We have produced three monoclonal antibodies (Mabs), two of which were made to 8-day embryonic chick ciliary ganglion (CG) neurons (CG-1, CG-4) (Barald, 1982) and one of which was made to cultured mesencephalic neural crest (NC) cells (CG-14) removed from the embryo 31 hr after incubation. We have shown that all three Mabs label a common 75 kD antigen present on the cell surface of both CG neurons and NC cells (Barald, 1988). Here we report that the CG-1 and CG-4 antibodies, used in the same ratios in which they are synergistically cytotoxic for both the CG and NC cells (Barald, 1988), and Mab CG-14 alone, have specific effects on the high-affinity choline uptake mechanism (HACU) of CG neurons and isolated antigen-positive NC cells in the absence of complement. CG-1 and CG-4 in ratios of 8/1 (the same ratios that are used to kill the CG and the NC subpopulation), but neither singly, inhibit the HACU of CG neurons by 40% and that of isolated antigen-positive NC cells by 75%. However, CG-14 alone, at 1 Μg/ml, inhibits the HACU of both CG neurons and isolated NC cells by 95%. None of the antibodies had an effect on numbers of ouabain binding sites (a measure of the Na + /K + ATPase) or cell surface acetylcholinesterase (AChE) of CG neurons or NC cells isolated by “no-flow” fluorescence cytometry with a Meridian Instruments ACAS470 cytometer. CG or NC cells grown in the presence of the antibodies without complement grow and remain healthy for many weeks. They exhibit no difference in morphology, protein content, lactate dehydrogenase activity (LDH), or division time from untreated sister cultures. Therefore, the antigen recognized by all three Mabs may be involved in a high-affinity choline uptake mechanism, a common characteristic of cholinergic neurons. The Mabs themselves may possibly label some element of the high-affinity transporter or a proximal membrane component. This implies that such a high-affinity uptake mechanism is present in the subpopulation of NC cells at early times in development. If these cells in fact are destined to contribute to the avian CG, these characteristics are present in the subpopulation before the NC cells take on a neuronal morphology.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50220/1/490210205_ftp.pd

Ornithine decarboxylase activity during development of the mouse inner ear in vivo and in vitro

Ornithine decarboxylase activity was determined during the development of the peripheral auditory system in the murine otocyst with the goal of understanding the role of this enzyme in the morphological and functional maturation of the inner ear. At gestational days 11 and 12 enzyme activity was more than 10-fold higher than adult levels. A sharp decline occured between day 12 and 13 after which activity rose to a peak around day 15. Activity then dropped continuously until near-adult levels were reached at birth. A lower specific activity of ODC but a similar time-course was seen in otocysts explanted at gestational day 13 and subsequently cultured for 6 days. For two stages of development, enzyme activity and binding of 3 H-α-difluoromethylornithine were compared. The four-fold difference in enzymatic activity on gestational days 15 and 17 was paralleled by a similar difference in binding. Ornithine decarboxylase activity during inner ear development therefore seems primarily regulated at the level of protein synthesis. Ornithine decarboxylase activity correlates with major inductive events in the morphogenesis of the cartilagenous otic capsule that serves as a template for the formation of the bony labyrinth. The pattern of activity may reflect the changes in the head mesenchyme that is recruited by the otocyst to aggregate and form its protective otic capsule.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47689/1/441_2004_Article_BF00340878.pd