A. NK-2 Homeobox Gene. The NK-2 homeobox gene is expressed in nuclei in the ventral half of the ventrolateral neurogenic anlage
very early in the development of part of the CNS of Drosophila. The distribution of NK-2 RNA in various mutants was determined
to identify genes that regulate NK-2 expression. Four genes were found that encode DNA binding proteins that regulate NK-2
gene expression. In addition, the 5-flanking region of the NK-2 gene was shown to contain many binding sites for NK-2 protein,
which suggests that NK-2 protein may be required to maintain NK-2 gene expression. These results suggest that the NK-2 gene
receives and integrates information from the ventral-dorsal and anterior-posterior gradients of gene regulators to generate
a pattern of clusters of neuroectodermal cells that synthesize NK-2 RNA and are precursors of different types of neuroblasts.
The NK-2 homeodomain was shown by NMR to have a novel secondary structure.
B. Gene Expression in the Developing Nervous System. A Drosophila gene was found that encodes a novel zinc finger protein
that is restricted to the CNS. Homozygous P-element insertions are lethal and are accompanied by massive morphological defects
in the ventral nerve cord. Another cDNA clone was identified as a DNA binding protein which is widely distributed during early
embryonic development, but is expressed exclusively in the nervous system during later embryonic development. Another cDNA
clone was found that corresponds to a Drosophila gene that encodes a novel member of the kinesin heavy chain gene family.
Other cDNAs were found that correspond to genes that encode novel proteins that are specifically expressed in the developing
Cell Recognition And Synapse Formation
NK-2 Homeobox Gene. The first known step in the zygotic development of a considerable portion of the Drosophila CNS is the
expression of the NK-2 gene. During the past year, proteins that regulate the expression of the NK-2 gene were identified
by determining the expression of the NK-2 gene in embryos with mutations in various genes. The results show that the NK-2
gene is activated in the ventral half of the embryo, presumably by dorsal protein, which is distributed in nuclei in a ventral-dorsal
concentration gradient. The NK-2 gene is activated but not expressed in the most ventral horizontal stripe of nuclei, the
mesodermal anlage, due to repression by snail, a zinc finger protein, or in the adjacent horizontal stripe of nuclei, the
mesectodermal anlage, due to repression by single-minded and Enhancer of split m8, which are basic, helix-loop-helix proteins.
However, the NK-2 gene is expressed by nuclei in the ventral half of the ventrolateral neurogenic anlage early in Drosophila
embryonic development as the nuclei undergo commitment to the neuroblast pathway of differentiation, or soon thereafter. Initially,
the NK-2 gene is expressed fairly uniformly in a horizontal stripe of nuclei about 7 nuclei in width on each side that extends
over 90% of the length of the embryo. During gastrulation, the horizontal stripe of cells expressing NK-2 is converted to
12 vertical stripes by repression of the NK-2 gene in some cells. Later, 26 clusters of cells that express the NK-2 gene are
formed on each side, presumably by repression of the NK-2 gene in additional cells. Therefore, 2 clusters of neuroectodermal
cells that synthesize NK-2 RNA are formed per hemisegment that are the precursors of many neuroblasts in the ventral nerve
Twenty high-affinity and 13 low-affinity NK-2 binding sites were found in 2.2 kb of DNA from the 5'-upstream region of
the NK-2 gene, which suggests that NK-2 protein may be required to maintain the expression of the NK-2 gene. Other putative
sites for proteins that overlap or are adjacent to the NK-2 protein binding sites were found. The conversion of neuroectodermal
cells to neuroblasts is accompanied by activation of the snail gene in the neuroblasts, thereby repressing activation of the
NK-2 gene by dorsal protein. The results suggest that the NK-2 gene receives and integrates information from the ventral-dorsal
and anterior-posterior gradients of gene regulators, which is needed to generate a pattern of clusters of neuroectodermal
cells that synthesize NK-2 RNA that are precursors of different types of neuroblasts.
One of the major goals in neurobiology is to understand how the nervous system is assembled. Studies on the NK-2 homeobox
gene led to some novel ideas and to a hypothesis which predicts the overall strategy of the gene program (that is the rules)
for the early development of part of the CNS of Drosophila. Every aspect of the hypothesis can be tested experimentally using
the NK-2 gene. With a slight modification the hypothesis also applies to the assembly of part of the mammalian CNS.
Circular dichroism measurements and 1D NMR spectra showed that the tm for denaturation of the NK-2 homeodomain, NK-2H, is
approximately 25 C at pH 4.4 and that denaturation is fully reversible. NK-2H was shown to have relatively little [alpha]-helical
content. No dramatic change in the CD spectra was observed on addition of an oligodeoxynucleotide with a high-affinity NK-2
binding site. The results show that NK-2H has an unusual homeodomain secondary structure.
Genes Expressed In The Developing Nervous System. Transposition of a P-element that contains the [beta]-galactosidase gene
from 1 site in the Drosophila genome to another yielded many transgenic fly lines that express [beta]-galactosidase only in
the nervous system during embryonic development. The developmental time and location of [beta]-galactosidase expression then
is determined by regulatory signals of the genes that contain the inserted P-element DNA. DNA flanking the P-element insertion
sites were cloned from 15 of the most interesting transgenic fly lines and corresponding cDNA clones were obtained and were
sequenced partially. Clone 393C-2 was shown to encode Drosophila high-mobility-group protein D (HMG-D), a DNA binding protein.
A homologous mammalian protein, HMG-1, recognizes DNA conformation rather than nucleotide sequence; HMG-1 binds to cruciform
DNA and to DNA with axial distortion due to cisplatin. The functions of HMG-1 and HMG-D proteins have not been identified;
however, the proteins are thought to play a role in chromatin structure. Also the HMG domain has been found in many DNA binding
proteins that regulate transcription. We find that the HMG-D gene is expressed ubiquitously during early embryonic development
but later in development is expressed exclusively in the nervous system. The homozygous P-element insertion is a lethal mutation
and is accompanied by striking morphologic defects in the central nervous system.
Clone 367C-3 DNA corresponds to a novel gene that encodes a zinc finger protein that is expressed in the CNS and anterior
sensory organs. The homozygous P-element insertion is a lethal mutation that results in extraordinary morphologic defects
in the ventral nerve cord of developing embryos. Clone 7D3C-1 corresponds to a novel Drosophila gene that encodes a member
of the kinesin heavy chain gene family. Kinesin functions as a molecular motor for axonal fast transport of organelles or
cell membranes on microtubule tracks from soma of neurons towards axon tips. Clone 314-4C-2 encodes a protein that is similar
to the human QM protein, an apparent suppressor of Wilm's tumor, a pediatric nephroblastoma. Sequence analysis of other
cDNA clones suggest that the cDNAs correspond to novel genes that are expressed in the nervous system.