hunchback
See the embryonic expression pattern of hb at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
Earliest zygotic hb expression is in the anterior 45% of the embryo. Maternal hb expression forms an anterior to posterior gradient that disappears by cellularization (Tautz, 1987).
HB is required for the formation and segmentation of two regions of the
Drosophila embryo: a broad anterior domain and a narrow posterior domain. Accumulation of HB
transcript in the posterior of the embryo occurs in two phases: an initial cap covering the terminal
15% of the embryo followed by a stripe at the anterior edge of this region. Zygotic transcripts are absent from the central domain of Krüppel expression (Tautz, 1987 and Margolis, 1995).
The tightly choreographed neuroblast (NB) expressions of Hb, Pdm-1 and Pdm-2, and Castor suggest temporally integrated processes participate in their formation (see Lateral views of Drosophila CNS). Clonal analysis of ventral cord NB lineages has revealed that many early delaminating NBs produce lineages that span most of the ventral cord's dorsal/ventral axis. For example the NB5-2, one of the first NBs to delaminate, generates a ventral-dorsal column of 17 to 26 cells. The dynamics of Hb, Pdm-1 and Cas expression in NBs indicates that many of the early S1 and S2 delaminating NBs may sequentially express all three and thereby produce lineages spanning all three compartments. Two such ventral cord candidates are the early NB5-2s and NB7-4s. Shortly after their delamination, during early stage 9, they activate Hb expression, while later, after several rounds of GMC divisions, they activate Cas expression. The fact that NBs co-expressing Hb/Pdm-1 or Pdm-1/Cas have been detected, but never Hb/Cas, further suggests that at least some of the early NBs make the Hb->Pdm->Cas transition. However, not all NBs undergo these transitions. This is particularly evident in NBs that enter the proliferative zone during later delamination waves. For example, the first ventral cord NBs to express Cas, the S3 NB6-1s, activate Cas shortly after delaminating from the ectoderm and do not express Hb (Kambadur, 1998).
During Drosophila embryonic CNS development, the sequential neuroblast (NB) expression of four proteins, Hunchback
(Hb), Pou-homeodomain proteins 1 and 2 (referred to collectively as Pdm), and Castor (Cas), identifies a transcription factor network regulating the temporal development of all ganglia. The Zn-finger proteins Hb and Cas, acting as repressors, confine
Pdm expression to a narrow intermediate temporal window; this results in the generation of three panneural domains whose cellular constituents are marked by expression of Hb, Pdm, or Cas. Seeking to identify the cellular mechanisms that generate these expression compartments, the lineage
development of isolated NBs in culture was studied. The Hb, Pdm, and Cas expression domains are generated by transitions in NB gene expression that are followed by gene product perdurance within sequentially produced sublineages.
These results also indicate that following Cas expression, many CNS NBs continue their asymmetric divisions and generate additional progeny, which can be identified by the expression of the bHLH transcription factor Grainyhead (Gh). Gh appears
to be a terminal embryonic CNS lineage marker. Taken together, these studies indicate that once NBs initiate lineage development, no additional signaling between NBs and the neuroectoderm and/or mesoderm is required to trigger the
temporal progression of Hb followed by Pdm and then Cas, and subsequently Gh expression during NB outgrowth (Brody, 2000).
Underpinning the formation of NB lineages are spatially and temporally regulated transcription factor networks that play pivotal roles in establishing
the unique cellular identities of NBs and their
progeny. Prior to NB delamination, during the initial specification of NBs,
two spatially regulated transcription factor networks subdivide
the ventral neuroectoderm along its anterior/posterior (A/P) x axis and dorsal/ventral (D/V) y axis. Later, during NB lineage
development, at least one additional network, acting over
several hours, gives rise to sequentially formed multilayered
basal (inner or dorsal) to apical (outer or ventral)
neuronal subpopulations. Along the basal/apical z axis, neuronal subpopulations in
all ganglia can be identified by their expression of the
transcription factors Hb, Pdm and Cas. Hb marks a deeper, basally distributed population of neurons that are born early, Cas marks a superficial, apically
distributed population of neurons that are born late,
and Pdm marks an intermediate population arrayed
between the Hb- and the Cas-expressing cells. Both genetic
and molecular analysis indicates that two Zn-finger proteins,
Hb and Cas, act as repressors to silence pdm
expression. By restricting pdm expression primarily to
intermediate-born neuronal precursors these structurally
different Zn-finger proteins help establish three pan-CNS
neural subpopulations whose cellular constituents are
marked by the expression of Hb, Pdm, or Cas (Brody, 2000).
To what extent are the z axis expression domains generated successively by invariant
gene expression programs, maintained in different
NBs, versus sequential gene expression programs within sublineages of single NBs?
To better understand the nature of the
temporal components regulating the CNS z axis network,
attempts were made to determine if the sequential expression of Hb,
Pdm-1, and Cas occurs during NB lineage development
in vitro. In order to analyze the capacity of individual NBs to
generate a full repertoire of Hb-, Pdm-, or Cas-expressing
sublineages, overnight cultures were simultaneously immuno-stained
for all three factors and the percentage of cells
within a clone that were positive for each factor was subsequently determined. Not all clones contained cells expressing each of the transcription factors.
The majority of clones containing Cas-expressing
cells also contain additional NB descendants marked by the
expression of Hb or Pdm-1. Triple-immunolabeling
studies have revealed that clones expressing only Cas are the
exception. Taken together the results indicate that many isolated S1 and S2
NBs, when maintained in culture, will generate neuronal
descendants that are marked by Hb, Pdm, or Cas expression.
Given that Hb and Cas are repressors of pdm gene NB
expression, these observations also suggest that the overlapping
Hb/Pdm and Pdm/Cas expressions, both in vivo and in culture represent transition
states in NB gene expression. In other words, NBs undergo sequential transitions in gene expression, thus generating the multiple cell layers seen in vivo (Brody, 2000).
Triple-immunolabeling studies have revealed that many of
the overnight NB clones contain a subset of cells that do not
contain detectable levels of Hb, Pdm-1, or Cas. In many of
these in vitro lineages the putative NB is also unstained. The
bHLH transcription factor Gh is known to be expressed in CNS NBs but
only after stage 14. In view of the late
onset of Gh expression in NBs and the triple-staining
results identifying cells in o/n clones that do not express
Hb, Pdm-1, or Cas, it was hypothesized that these negative
cells may represent an additional late NB expression window
marked by Gh expression. To test this hypothesis, the
spatial/temporal expression dynamics of Gh were compared
to other members of the z axis network. Similar to its late
activation during in vivo development, Gh expression was
observed only in overnight cultures; when more than one
Gh-positive cell was detected in a clone they were consistently
found clustered together. Two-thirds of the Cas+
clones had at least one Gh+ cell and the average number of Gh+ cells
in all clones was 2.3. Approximately 2/3 of the Gh+
clones also contained Hb-immunopositive cells. While no
Hb-Gh coexpressing cells were observed, approximately
20% of the Gh+ cells also expressed Cas. Given
the late onset of Gh expression in both the embryo and the
cultured NB clones and the overlapping Cas and Gh expression,
it is likely that Gh marks a fourth temporal window
for NB transcription factor expression. In addition, because
there was an average of more than one cell in an o/n clone
that was immunopositive for Gh, it is likely that Gh is also expressed/maintained in a sublineage(s) born after the one marked by Cas expression (Brody, 2000).
The principle finding of this study is that built on top of the x and y axis
neural identity systems is an additional temporal network
that defines successive stages of lineage maturation in an
apical/basal z axis. This global CNS network, identified by
the temporal cascade of Hb followed by Pdm and subsequently Cas NB expression,
most likely ensures in part that each NB generates a
column of uniquely specified neuronal subtypes. The
shared transcription factor expression within a given temporal
layer also suggests that the cellular constituents of
these expression domains may also have similar patterns of
downstream target gene expression (Brody. 2000).
The following
model for the origin of the layer sublineages marked by
these transcription factors has been suggested. As each NB divides, generating a succession
of GMCs, it undergoes multiple transitions in
transcription factor expression. In succession, the NBs
express Hb, Pdm, Cas, and Gh. The first progeny generated
by the early S1 and S2 NBs express Hb, and the presence of
Hb protein persists in their neural progeny. These early S1
and S2 NBs go on to activate the expression of the Pdms
that, like Hb, persist in neural sublineages generated during
this temporal window. Subsequently Cas is activated in
NBs, represses Pdm transcription, and likewise persists in
neural sublineages. After Cas expression, a fourth neural
subpopulation, generated by dividing NBs, expresses Gh.
This Gh subpopulation most likely represents the terminal
sublineage of the embryonic NB. The data also reveal that
not all NBs generate cells that occupy all four layers, a
result that reflects the unique set of lineages, generated by
each NB. Most likely, each NB has a
preprogrammed time of delamination, but the timing of
transitions is synchronized in a global fashion. The model
further suggests that late delaminating NBs can be distinguished
from early NBs by their inability to activate Hb.
Although Hb is activated shortly after the S1s and S2s have
delaminated, Hb is never seen in the proliferative zone
during late delaminations (Brody, 2000).
What mechanism drives transitions in transcription
factor expression in NBs and in their GMC progeny?
It has been shown that Hb, Cas, and Pdm are involved
in a regulatory circuit in which Hb and Cas repress Pdm in
a cooperative, nonoverlapping fashion both early and late
within NB lineages. In addition,
Pdm is also required for the proper expression of Cas. It is
likely, therefore, that this Hb to Pdm followed by Cas network is
responsible for temporal transitions in transcription factors,
related to the generation of multiple cellular layers. This
conclusion must be tempered by the observation that less
than 50% of the cells in clones and, by implication, in the
CNS, are positive for even one of these transcription factors.
There must be other factors involved in sublineage
determination related to CNS layering. If the transitions
observed are not caused by the partitioning of mRNA and
proteins between NBs and their GMC, but by regulatory
interactions within the cells themselves, then there must
be additional mechanisms that are involved in the rapid
disappearance of these molecules. Expression of transcription
factors restricted to one or two generations of NB
development could be accomplished if these transcription
factors were autoregulatory, repressing their own expression
in NBs and in their progeny (Brody. 2000).
For more information on Drosophila neuroblast lineages, see Linking neuroblasts to their corresponding lineage, a site carried by Flybrain, an online atlas and database of the Drosophila nervous system.
The Drosophila brain develops from the procephalic neurogenic region of the ectoderm. About 100 neural precursor cells (neuroblasts) delaminate from this region on either side in a reproducible spatiotemporal pattern. Neuroblast maps have been prepared from different stages of the early embryo (stages 9, 10 and 11, when the entire population of neuroblasts has formed), in which about 40 molecular markers representing the expression patterns of 34 different genes are linked to individual neuroblasts. In particular, a detailed description is presented of the spatiotemporal patterns of expression in the procephalic neuroectoderm and in the neuroblast layer of the gap genes empty spiracles, hunchback, huckebein, sloppy paired 1 and tailless; the homeotic gene labial; the early eye genes dachshund, eyeless and twin of eyeless; and several other marker genes (including castor, pdm1, fasciclin 2, klumpfuss, ladybird, runt and unplugged). Based on the combination of genes expressed, each brain neuroblast acquires a unique identity, and it is possible to follow the fate of individual neuroblasts through early neurogenesis. Furthermore, despite the highly derived patterns of expression in the procephalic segments, the co-expression of specific molecular markers discloses the existence of serially homologous neuroblasts in neuromeres of the ventral nerve cord and the brain. Taking into consideration that all brain neuroblasts are now assigned to particular neuromeres and individually identified by their unique gene expression, and that the genes found to be expressed are likely candidates for controlling the development of the respective neuroblasts, these data provide a basic framework for studying the mechanisms leading to pattern and cell diversity in the Drosophila brain, and for addressing those mechanisms that make the brain different from the truncal CNS (Urbach, 2003).
The cephalic gap genes are expressed in large domains of the procephalon and play a crucial role not only in the patterning of the peripheral ectoderm, but also in regionalizing the brain primordium. The segmental organization of the Drosophila brain is based on the expression pattern of segment polarity and DV patterning genes. To see whether the cephalic gap genes respect the neuromeric boundaries segment polarity and DV patterning genes, and to provide a basis for studying their potential role in the formation or specification of brain precursor cells, the expression was studied of orthodenticle, empty spiracles, sloppy paired 1, tailless, huckebein, and hunchback in the developing head ectoderm, as well as in the entire population of identified NBs during stages 9-11 (Urbach, 2003).
The head and thorax are missing in mutant embryos and abdominal segments 7 and 8 are fused (Tautz, 1987).
It is suggested that Hb and Castor act in a cooperative, non-overlapping manner to control POU gene expression during Drosophila CNS development. By silencing pdm expression in early and late NB sublineages, Hb and Cas establish three pan-CNS compartments whose cellular constituents are marked by the expression of either Hb, Pdm, or Cas. Embryos lacking Hb function suffer multiple defects. During CNS development, hb- embryos fail to develop labial and thoracic ganglia and gaps form between the subesophageal maxillary neuromeres and the abdominal ganglia. In addition, the seventh and eighth abdominal segments are fused due to the absence of parasegment 13. To further characterize the phenotypic consequences triggered by the loss of Hb and compare them with defects caused by loss of Cas, the axon fascicle organization was examined in hb null embryos. hb- embryos have severe axon guidance defects. Missing are the highly ordered ventral cord axon scaffolds made up of longitudinal connective and commissural fascicles. However, although axon fascicles do not form properly, significant numbers of neurons still generate axons, albeit misguided, in hb- embryos. Since many axon-guiding glia and pathfinding neurons are born from early NB sublineages, hb function may be essential for establishing correct axon guidance cues in these sublineages. Unlike the hb- phenotype and consistent with its late NB expression, loss of cas does not disrupt the formation of axon connective or commissure fascicles but it does reduce the number of late forming axons that participate in these fascicles (Kambadur, 1998).
The requirements for the multi sex combs (mxc) gene during development have been examined to gain
further insight into the mechanisms and developmental processes that depend on the important
trans-regulators forming the Polycomb group (PcG) in Drosophila. Although mxc has not yet been cloned, it is known to be allelic with the
tumor suppressor locus lethal (1) malignant blood neoplasm [l(1)mbn]. The mxc product
is dramatically needed in most tissues because its loss leads to cell death after a few divisions. mxc also has
a strong maternal effect. Hypomorphic mxc mutations are found to enhance other PcG gene
mutant phenotypes and cause ectopic expression of homeotic genes, confirming that PcG products are
cooperatively involved in repression of selector genes outside their normal expression domains. The mxc product is needed for imaginal head specification, through regulation of
the ANT-C gene Deformed. This analysis reveals that mxc is involved in the maternal control of early
zygotic gap gene expression known to involve some other PcG genes and suggests that the mechanism
of this early PcG function could be different from the PcG-mediated regulation of homeotic selector
genes later in development (Saget, 1998).
Induction of
uncontrolled growth and deregulation of Hox genes are linked in mammals, where Hox products can
induce leukemia. In Drosophila,
modification of homeotic gene expression causes homeosis, sometimes associated with increased
proliferation but not with uncontrolled tumorous growth, possibly because
the identity of each segment is specified by a combination of HOM products. Loss or gain of one
HOM gene will likely lead to a new combination that is found elsewhere in wild type, and cells
expressing this combination could be expected to follow the corresponding developmental pathway and give rise to homeotic
transformations. However, because each cellular identity apparently corresponds to a given
proliferation rate, loss or ambiguity of identity due to deregulation of several selector genes in a single
cell, such as mxc mutations apparently induce, could lead to loss of proliferation control. Identification
of mxc partners and targets, as well as of the molecular nature of the mxc product, may help throw
light on the genes and mechanisms involved in this process (Saget, 1998).
It has been proposed that certain PcG genes are required for the
maintenance of the expression domains of knirps and giant, through a mechanism similar to the
regulation of homeotic genes. The regionalization of the Drosophila embryo depends on the maternally supplied products of bicoid
(bcd), hunchback (hb), and nanos (nos). Nos represses the translation of the maternal HB mRNA in
the posterior embryonic region. This permits the expression of the zygotic gap genes knirps (kni) and
giant (gt), which specify posterior identities. These genes would otherwise be repressed by Hb. Embryos from nos/nos mothers form no abdominal
segments, but this phenotype can be rescued by a total lack of hb in the maternal germline. It can also
be dominantly rescued by the mutation of maternally supplied regulator molecules that normally repress
kni and gt in the zygote. Pelegri and Lehmann (1994) have shown that certain mutant products
of the PcG genes E(z), Psc, and pleiohomeotic can partially rescue nos by such a maternal effect. To determine
if mutation of mxc also affects this regulation, the cuticles of embryos were examined from mxc/+;hb nos/nos mothers that were heterozygous for different mxc mutations. This
genetic background was used because a decrease in the amount of maternal hb product can partially rescue the
nos phenotype in F1 embryos. Such embryos can differentiate a few
abdominal denticle belts and form an adequate background to evaluate increased rescue of
nos. Thus loss-of-function PcG mutations should have a strong effect on rescue,
and the embryos from hb nos/nos mothers that have two PcG mutations in their genetic background
should permit increased rescue of the nos phenotype (Saget, 1998).
Any of three E(z)son (suppressor of nanos) alleles or a hypomorphic pleiohomeotic allele partially rescue the
phenotypes of hb nos/nos progeny by a maternal effect; deficiencies covering E(z) or the Psc/Su(z)2
complex also allow some maternal rescue of hb nos/nos progeny, yet the strongest effect is observed
with the gain-of-function E(z)son alleles. The
EMS-induced allele mxcG48 rescues the hb nos/nos progeny phenotype, whereas a deficiency of mxc
does not. Some rescue with the
Psc/Su(z)2 complex deletion Df(2)vgB is also observed and
strong rescue (consistently >50%) is observed with an EMS-induced pleiohomeotic allele phob, described as
amorphic. This suggests that phob and mxcG48 are probably not
amorphic alleles, and that maternal rescue of hb nos/nos progeny by a PcG gene is most efficient with
a non-null mutation (Saget, 1998).
Segmentation of embryos from transheterozygous mothers was also examined. Because neither a reduction of wild-type PcG product nor two PcG mutations in trans in the hb
nos/nos mothers increases nos rescue, these data strongly suggest that, whatever the mechanism of gap
gene regulation by these PcG mutations may be, it does not function like the PcG-mediated
maintenance of homeotic gene expression in embryos and in imaginal discs. The strong rescue provided by
several non-null EMS-induced mutations, which may produce mutant proteins, leads to a proposal that
modified PcG proteins are poisoning a normal process. How this process depends on wild-type
regulation by PcG products has yet to be established (Saget, 1998).
Neural precursors often generate distinct cell types in a specific order, but the intrinsic or extrinsic cues regulating the timing of cell fate specification are poorly understood. Drosophila neuroblasts sequentially express the transcription factors Hunchback->Krüppel->Pdm->Castor, with differentiated progeny maintaining the transcription factor profile present at their birth. Hunchback is necessary and sufficient for first-born cell fates, whereas Krüppel is necessary and sufficient for second-born cell fates: this is observed in multiple lineages and is independent of the cell type involved. It is proposed that Hunchback and Krüppel control early-born temporal identity in neuroblast cell lineages (Isshiki, 2001).
To begin investigating birth order dependent cell fate specification in the Drosophila CNS, the morphological and molecular features that distinguish early- versus late-born neurons were investigated. The axon projections and cell positions of first-born neurons were examined from datasets of DiI-labeled neuroblast clones, and find that first-born neurons typically occupy the deepest (most internal) position in the clone and have the longest axon projections of any cell in the clone (see also Brody, 2000). Conversely, later-born neurons lie in more superficial positions (nearest the ventral epithelium) and have relatively short projections (Isshiki, 2001).
What genes might regulate these birth order-specific neuronal properties? It is known that deep layer neurons are Hb+, middle layer neurons are Pdm+, while superficial layer neurons are Cas+ (Kambadur, 1998). These findings have been confirmed in this study and Kr is identified as a new deep layer transcription factor. Kr is weakly detected in Hb+ neurons, and strongly Kr+ neurons define a deep layer that lies between the Hb+ and Pdm+ layers. Some neurons showing coexpression of Kr/Pdm and Pdm/Cas are also detected. Tests were made to see whether Hb, Kr, Pdm, and Cas are expressed in a temporal order within neuroblasts at the time each layer of neurons is being generated. Indeed, sequential, transient expression of Hb -> Kr -> Pdm -> Cas in neuroblasts was observed that is 'stabilized' in progeny born during each window of gene expression (Kambadur, 1998; Brody, 2000; Isshiki, 2001). In addition, Pdm is transiently expressed in a subset of newborn Hb+ neuroblasts and their first-born GMCs, probably due to persistence of Pdm from the neuroectoderm, but it is usually not maintained in their Hb+ neuronal progeny (Kambadur, 1998; Isshiki, 2001).
Interestingly, the temporal expression pattern of Hb, Kr, Pdm, and Cas within neuroblasts parallels the spatial pattern of these genes during segmentation. Hb, Kr, Pdm, and Cas are detected in progressively more posterior domains at cellular blastoderm, respectively. Thus, the spatial order of these genes during segmentation is the same as their temporal order in neuroblasts, raising the possibility of a conserved gene cassette used in both segmentation and neurogenesis (Isshiki, 2001).
Identified neuroblast lineages were assayed to test the hypothesis that transient Hb -> Kr -> Pdm -> Cas expression in neuroblasts is stably maintained in neuronal progeny born during each window of gene expression. Three model neuroblast lineages were characterized, an early forming neuroblast (7-1) and two late-forming neuroblasts (7-3 and 2-4), where specific neuronal progeny were tracked from birth to differentiation. Although early- and late-forming neuroblasts begin their cell lineages hours apart, all show the same sequential, transient Hb -> Kr -> Pdm -> Cas pattern of expression (Isshiki, 2001).
Neuroblast 2-4 sequentially expresses Hb/Kr, Kr, Kr/Pdm, Pdm, Pdm/Cas, and Cas. GMCs and neurons with most of these expression patterns can be detected in deep to superficial layers of the CNS, respectively, except Kr+/Pdm+ or Pdm+ GMCs were rarely observed. In addition, GMC-1 is transiently Pdm+. To track gene expression patterns at the level of identified neuronal/glial progeny, gene expression was examined in the neuroblast 7-3 and 7-1 lineages (Isshiki, 2001).
Neuroblast 7-3 produces only three GMCs: GMC-1 generates the EW1 interneuron and the GW motoneuron, GMC-2 produces the EW2 interneuron and an EW2 sibling that rapidly undergoes programmed cell death, and GMC-3 differentiates directly into the EW3 interneuron. These neurons are referred to here by the abbreviated names of 1/1G, 2, 3 (respectively) to reflect their birth order. Neuroblast 7-3 sequentially expresses Hb, Hb/Kr, Kr, Kr/Pdm, Pdm; it is never Cas+. In addition, the newborn neuroblasts 7-3 and GMC-1 are transiently Pdm+. GMC-1 is Hb+ Kr+ and generates the Hb+ Kr+ 1/1G neurons; GMC-2 is Kr+ and generates the Kr+ interneuron 2, and GMC-3 is Pdm+ and generates the transiently Pdm+ interneuron 3 (Isshiki, 2001).
Neuroblast 7-1 generates over 20 GMCs, but only the first five GMCs express the nuclear marker Even-skipped (Eve). Each of the five Eve+ GMCs produces one Eve+ motoneuron (named U1, U2, U3, U4, U5, based on birth order) and one Eve- sibling neuron which cannot be tracked. Neuroblast 7-1 sequentially expresses Hb/Kr, Kr, Kr/Pdm, Pdm, and Pdm/Cas. The Hb+ Kr+ neuroblast produces two Hb+ Kr+ GMCs which develop into Hb+ Kr+ U1 and U2 motoneurons; the Kr+ neuroblast generates a Kr+ GMC-3 which produces the Kr+ U3 motoneuron; and GMC-4 and GMC-5 are born during the Pdm+ and Pdm+ Cas+ phases of gene expression, respectively, and produce the Pdm+ U4 motoneuron and the Pdm+ Cas+ U5 motoneuron (Isshiki, 2001).
Three conclusions are drawn from this detailed gene expression analysis: (1) nearly all of the 30 known neuroblasts go through an invariant temporal pattern of Hb -> Kr -> Pdm -> Cas gene expression, including early-forming and late-forming neuroblasts; (2) Hb -> Kr -> Pdm -> Cas gene expression is transient in neuroblasts, but is maintained in differentiated neuronal progeny; (3) Hb -> Kr -> Pdm -> Cas gene expression is correlated with birth order and not a particular cell type. For example, Hb+ progeny are all early-born, but can differentiate as interneurons, motoneurons, or glia depending on their parental neuroblast (Isshiki, 2001).
In the wild-type neuroblast 7-3 lineage, the first-born GMC and its 1/1G neuronal progeny are Hb+ Kr+; later-born interneurons 2 and 3 do not express Hb. In addition, the molecular markers Zfh-1, Zn finger homeodomain 2 (Zfh-2), and the neurotransmitter corazonin label different subsets of these neurons. In embryos specifically lacking Hb in the CNS (hb CNS mutants), either a duplication of interneuron 2 at the expense of the first-born 1/1G neurons is observed (11%), consistent with a duplication of GMC-2 fate, or a specific loss of the first-born 1/1G neurons (89%) is observed (Isshiki, 2001).
It cannot be distinguish whether loss of first-born 1/1G neurons is due to cell death or due to 'skipping' of the GMC-1 fate (i.e., the lineage begins with GMC-2). In contrast, when neuroblast 7-3 is forced to continuously express Hb ('UAS-hb'), there are additional neurons in the lineage (as many as 11 cells -- average of 6.3 cells), and all exhibit first-born 1/1G fates based on molecular markers, neurotransmitter expression, and characteristic axon projections. It is concluded that Hb is necessary for normal GMC-1 development, but not later-born cell fates; that continuous Hb can transform all progeny toward a GMC-1 fate, and that continuous Hb expression leads to an extension of the neuroblast cell lineage (Isshiki, 2001).
In the wild-type neuroblast 7-1 lineage, the first two GMCs and their two Eve+ U1, U2 motoneuron progeny are Hb+; later progeny do not express Hb. In hb CNS mutants, Eve+ GMC-1/GMC-2 or their Eve+ U1, U2 motoneuron progeny were rarely detected; however, later-born GMCs and neurons develop normally based on molecular marker expression. Whether loss of the Eve+ U1, U2 motoneurons is due to cell death or due to skipping of the first two GMCs fates cannot be distinguised. In contrast, when neuroblast 7-1 is forced to continuously express Hb, there are extra Eve+ cells (as many as 19 cells; average of 14.4 cells), and all differentiate as early-born U1/U2 motoneurons based on molecular marker expression. Thus, Hb is necessary for normal GMC-1 and GMC-2 development without affecting later-born cell fates, and continuous Hb can transform many or all progeny toward a GMC-1/GMC-2 fate (Isshiki, 2001).
Is Hb required for the specification of all first-born neurons? To broaden this analysis, the first-born progeny were assayed from the well-characterized 1-1 and 4-2 lineages. Both lineages produce an Eve+ GMC-1; in the 1-1 lineage, the NB generates the Eve+ aCC motoneuron/pCC interneuron siblings, whereas in the 4-2 lineage, the NB produces the Eve+ RP2 motoneuron and its Eve- sibling. In hb CNS mutants, the first-born Eve+ neurons typically survive but abnormally express Zfh-2, a marker for later-born neurons, and the aCC and RP2 motoneurons fail to project to their proper dorsal muscle target; both phenotypes are consistent with a transformation of GMC-1 to a later-born GMC fate. In contrast, when all neuroblasts are forced to continuously express Hb, there are duplications of the first-born aCC/pCC (17%), duplications of the first-born RP2 (6%), or triplications of RP2 (4%). These results show that hb regulates first-born motoneuron and interneuron cell fates in the neuroblast 1-1 and 4-2 lineages (Isshiki, 2001).
If hb controls first-born identity in all neuroblast lineages, then severe motoneuron axon projection defects should be observed because many motoneurons derive from Hb+ first-born GMCs. Wild-type embryos have about 35 motoneurons with a stereotyped projection pattern to ventral and dorsal bodywall muscles, including the Hb+ aCC, RP2, U1, and U2 motoneurons that innervate dorsal muscles. hb CNS mutants show a reduction in the number of motoneuron projections, particularly to the dorsal muscles. Embryos where hb is misexpressed in all neuroblasts show the opposite phenotype: a striking increase in the number of motoneurons, particularly to the dorsal muscles. These results suggest that hb regulates first-born cell fate and/or axon projection patterns in most or all of the many neuroblast lineages that produce first-born motoneurons (Isshiki, 2001).
These results suggest that hb is required for specifying first-born GMC identity in lineages where first-born GMCs produce motoneurons or interneurons. To determine whether hb specifies first-born cell fate in lineages that produce glia, the thoracic 6-4 (6-4T) neuroblast lineage, in which the first-born GMC produces glia, and the 7-4 lineage, in which only later-born GMCs generate glia, were examined. The 6-4T lineage produces a Hb+ GMC-1, which produces the two Hb+ MM-CB glia that migrate to the midline, whereas neuroblast 7-4 first generates Hb+ interneurons, and then produces many Hb- glia, including two dorsoventral channel glia located at the midline. hb CNS mutants lack the first-born MM-CB glia at the midline, but have no change in the number of later-born channel glia at the midline. In contrast, forced misexpression of hb in all neuroblasts results in extra MM-CB glia at the midline and a decrease in the number of midline channel glia. These results, together with the neuronal phenotypes, strongly suggest that Hb regulates first-born temporal identity, rather than cell type identity, within multiple neuroblast lineages (Isshiki, 2001).
In the wild-type neuroblast 7-3 lineage, the first-born GMC-1 and its 1/1G neuronal progeny are Hb+ Kr+, while the second-born GMC-2 and interneuron 2 are Hb- Kr+. In embryos lacking Kr CNS expression (Kr CNS mutants), the first-born 1/1G sibling neurons are variably affected: both can be missing (17%), 1G can be missing (73%), or both can be normal (10%); however, the second-born interneuron 2 is always missing (100%) and the third-born interneuron 3 is almost always normal (90%). Absence of interneuron 2 could be due to cell death or due to skipping of the GMC-2 fate. In contrast, when neuroblast 7-3 is forced to continuously express Kr ('UAS-Kr,'), there are extra cells in the lineage (4-8; average of 5.5), and all but two cells differentiate as GMC-2 derived interneuron 2; the two unaffected cells are the GMC-1 derived 1/1G neurons. It is concluded that Kr contributes to GMC-1 development (where it is expressed with Hb) and is essential for GMC-2 development (where it is expressed without Hb); that continuous Kr can transform all progeny except GMC-1 toward a GMC-2 fate, and that continuous Kr leads to an extension of the neuroblast cell lineage (Isshiki, 2001).
In the wild-type neuroblast 7-1 lineage, the first two GMCs and their U1/U2 motoneuron progeny are Hb+ Kr+, GMC-3 and its U3 motoneuron progeny are Kr+, and subsequent GMCs do not express Kr. In Kr CNS mutants, one of the U3/U4 motoneurons is frequently missing (73%), although all earlier- and later-born neurons develop normally. It is suspected that the missing neuron is the normally Kr+ U3, based on cell position, but markers to distinguish U3/U4 in Kr mutant embryos are not available. As in the 7-3 lineage, the Kr phenotype may arise through cell death or a skipping of the GMC-3 fate. In contrast, continuous expression of Kr in neuroblast 7-1 results in extra Eve+ neurons (8-14; average of 10.0) with all differentiating as U3 or U4 motoneurons, except the normal pair of early-born U1/U2 motoneurons. It is suspected all neurons have the normally Kr+ U3 fate, but markers are not available to distinguish U3/U4 fates. It is concluded that Kr is necessary for U3 motoneuron development, and that continuous Kr can transform most or all 7-1 progeny, except the first-born U1/U2 neurons, toward a U3 neuron fate (Isshiki, 2001).
Accurate temporal regulation of Hb, Kr, and Cas is critical for proper CNS development, so it is important to determine the mechanisms that regulate sequential gene expression in neuroblasts. Regulatory interactions between Hb, Kr, Pdm, and Cas can be detected using misexpression assays: overexpression of Hb activates Kr and represses Pdm and Cas; overexpression of Kr activates Pdm, represses Cas, but has no effect on Hb expression; and Pdm positively regulates Cas expression (Brody, 2000), leading to the model that each gene can activate the next gene in the pathway and repress the 'next plus one' gene. These interactions are not necessary for driving sequential gene expression, however, as it is observed that hb, Kr, or cas mutations have only subtle alterations in the remaining gene expression profiles, and loss of hb or Kr does not appear to affect the fate of cells born later in the lineage based on existing markers. Thus, an independent pathway must also drive the sequential expression of Hb -> Kr -> Pdm -> Cas in neuroblasts. This mechanism involves cell cycle progression, directly or indirectly, because newly formed neuroblasts remain Hb+ Kr+ if they are cell cycle-arrested before their first division. They rarely if ever make the transition to Kr+ Hb-, Pdm+, or Cas+ (Weigmann, 1995 and Cui, 1995). It is concluded that a cell cycle-dependent 'clock' is required to drive the transitions in Hb -> Kr -> Pdm -> Cas gene expression (Isshiki, 2001).
The data do not support a model in which global temporal cues trigger gene expression transitions simultaneously in all neuroblasts. Most of the 30 neuroblasts, including the earliest neuroblasts to form (e.g., 7-1 and 7-4) and some of the latest neuroblasts to form (e.g., 2-4 and 7-3), go through the same Hb -> Kr -> Pdm -> Cas gene expression cascade. Exceptions are neuroblasts 2-1, 3-3, 5-1, and 6-1 (which start with Kr, Pdm, or Cas). Thus, early-forming neuroblasts can generate Cas+ progeny at the same time that late-forming neuroblasts produce Hb+ progeny (Isshiki, 2001).
A model is favored in which the timing of Hb -> Kr -> Pdm -> Cas expression is regulated primarily by a cell cycle-dependent clock but also by regulation within the Hb -> Kr -> Pdm -> Cas pathway (Kambadur, 1998). Evidence for the latter mechanism is that misexpression studies show that Hb, Kr, Pdm, and Cas typically activate the next gene in the pathway and repress the 'next plus one' gene in the pathway; and that loss of function mutations can result in premature expression of later genes in the pathway and the skipping of GMC fates. Evidence for the cell cycle-dependent clock mechanism is that hb and Kr mutants have relatively subtle changes in hb, Kr, pdm, or cas expression or in later-born GMC fates; that cell cycle arrested neuroblasts remain Hb+ Kr+ and fail to make a transition to Hb- Kr+, Pdm+, or Cas+; and that when neuroblast 1-1 is cell cycle arrested for several hours prior to its first cell division and then triggered to divide, it will produce an Eve+ GMC-1 instead of a later-born Eve- GMC (Weigmann, 1995), highlighting the importance of the cell cycle progression rather than developmental time in regulating GMC identity (Isshiki, 2001).
All early developing neuroblasts, such as 7-1, produce two Hb+ GMCs before downregulating Hb expression, whereas many late developing neuroblasts, such as 7-3, produce just one Hb+ GMC. How do neuroblasts regulate the number of Hb+ GMCs produced? All neuroblasts could express Hb for the same length of time but have different cell cycle rates, or all neuroblasts may have the same cell cycle rate but vary the length of Hb expression (Isshiki, 2001).
Hb, Kr, and Cas are transiently expressed in neuroblasts but maintained in GMC and neural progeny (Pdm can be transient or stable in neuroblast progeny, depending on the lineage) (Kambadur, 1998; Brody, 2000; Isshiki, 2001). By inheriting and maintaining the gene expression profile of their parental neuroblasts, GMCs can 'memorize' their birth order. This seems a powerful and efficient way for stem cells to make a variety of fate-restricted progeny in invariant sequence. A similar mechanism may be used during vertebrate cortical and retinal development, where precursors transiently express genes that are maintained in a subset of differentiated progeny. It is currently unknown what distinguishes transient neuroblast expression from persistent GMC/neuronal expression (Isshiki, 2001).
Loss of Hb or Kr from early-born GMCs results in loss or transformation of neurons normally derived from these GMCs, but later-born neurons develop normally based on nuclear markers, neurotransmitter expression, and axon projections. Neuronal loss in hb and Kr mutants is likely due to multiple mechanisms: (1) Cell death. Necrotic Eve+ or Eg+ neurons can be detected in the 7-1 or 7-3 lineages of Kr CNS mutants, supporting a cell death model. Although necrotic Eve+ or Eg+ neurons in hb mutants have not been detected, more early 7-3 lineages are seen in which three Prospero+ GMCs are born than mature 7-3 lineages with progeny from all three GMCs, suggesting that cell death also occurs in hb mutants. (2) GMC skipping (i.e., the neuroblast skips a GMC fate without duplicating a later-born fate). In Kr mutants, neuroblast 7-3 typically produces two Prospero+ GMCs at the time GMC-1 and GMC-2 are normally born, consistent with a skip of GMC-2 fate. Similarly, hb mutants often produce only two Prospero+ GMCs in the 7-3 lineage, consistent with a skip of the GMC-1 fate. It is not possible to assay for GMC skipping in the 7-1 lineage. (3) GMC transformation. hb mutants clearly show transformation of GMC-1 to GMC-2 fate in the 7-3 lineage, based on the observed duplication of interneuron 2 at the expense of the 1/1G sibling neurons. This phenotype may arise if the endogenous Kr in GMC-1 is sufficient to induce GMC-2 fate in the absence of Hb. In Kr mutants, a similar transformation of GMC-2 into the GMC-3 fate is never observed, perhaps because the Kr- GMC-2 does not prematurely express Pdm. Thus, there is evidence that hb mutants show all three phenotypes, GMC death, skipping, and transformation, while Kr mutants show only GMC death and skipping phenotypes (Isshiki, 2001).
Misexpression of Hb or Kr can transform all GMCs toward a first-born or second-born fate, respectively. This is likely to be a transformation of GMC identity, rather than an increase in the proliferation of early-born GMCs, because the extra early-born neurons are produced at the expense of the later-born neurons in the lineage, and the extra cells are not reduced in size (extra embryonic divisions lead to reduced cell size). Interestingly, Kr misexpression fails to transform early-born Hb+ GMCs into the later-born Kr+ fate. Kr is induced early enough to affect GMC-1 in the 7-3 lineage because GMC transformations are seen in other lineages (e.g., 7-1) prior to the time GMC-1 is born in the 7-3 lineage, yet it has no effect. The model that first-born Hb+ fates are dominant over second-born Kr+ fates is favored. In the future, cell type-specific Hb misexpression studies could be used to determine precisely when birth order-specific cell fates become fixed: in neuroblasts, GMCs, or neurons? It would also be interesting to determine if a pulse of Hb expression midway through a neuroblast lineage is sufficient to induce first-born cell fates, and if so, does the temporal program resume or reset to the beginning of the lineage after the Hb pulse ends (Isshiki, 2001)?
hb, Kr, pdm, and cas are not the only genes controlling temporal identity in neuroblast lineages. Some markers for first-born fate occur normally in hb mutants, such as Eve expression in the 1-1 and 4-2 lineage. Moreover, Hb misexpression may not fully transform every cell in the 7-3 lineage to a first-born fate: a full transformation would produce equal numbers of interneuron 1/motoneuron 1G siblings, but typically only 2-3 motoneurons and 5-6 interneurons are seen, suggesting that only the first 2-3 GMCs are fully transformed to a first-born fate. Finally, in most lineages, there are GMCs produced after Cas expression ends; additional genes such as grainyhead (Brody, 2000) may specify the temporal identity of these GMCs (Isshiki, 2001).
Hb is expressed in virtually all first-born GMCs, and these can differentiate into motoneurons, interneurons, or glia, depending on the neuroblast lineage. Similarly, high level Kr is detected in virtually all second-born GMCs (i.e., the GMCs following the Hb+ GMCs), and they can differentiate into motoneurons, interneurons, or glia. Not only are Hb and Kr expressed in multiple cell types, but they are necessary and sufficient for the proper cell fate specification of motoneurons, interneurons, or glia, depending on the neuroblast lineage (Isshiki, 2001).
An extremely interesting question is how GMC 'temporal identity' (regulated by Hb and Kr) is coordinated with individual 'neuroblast identity' to achieve the proper sequence of cell types that characterizes each neuroblast lineage. The Hb protein, its putative mammalian ortholog Ikaros, and the mammalian Kr-related EKLF protein all associate with chromatin remodeling proteins, and both Hb and Kr are thought to regulate chromatin-mediated heritable expression of homeotic genes. Thus, Hb and Kr may modulate chromatin structure such that different subsets of genes are accessible for transcription in first-born versus second-born GMCs, with the palette of genes expressed by a first-born or second-born GMC, dependent on the neuroblast-specific transcription factors they inherit. In this manner, neuroblast identity might be integrated with GMC temporal identity to create the unique cell types characterizing each neuroblast lineage. This is conceptually similar to homeotic genes and tissue-specific genes working together to uniquely specify distinct cell types in each tissue at different anterior-posterior levels of the body axis (Isshiki, 2001).
When neuroblast 7-3 is forced to continuously express Hb or Kr, it generates an extended lineage of up to ten neurons instead of four neurons and one programmed cell death. The increase does not appear to be due to an extra round of cell division by the normally postmitotic neurons because the extra cells are produced at the expense of later-born cell types and because smaller cells are not seen (as expected, since extra cell divisions in the embryo lead to smaller cell size. It is proposed that Hb or Kr misexpression results in production of extra GMCs, and it is suggested that each neuroblast has an intrinsic mechanism for triggering quiescence that is related to successful transition from Hb and Kr expression to later genes in the hierarchy. These data also show that Hb and Kr can regulate features of neuroblast cell biology (cell cycle control) in addition to regulating GMC temporal identity (Isshiki, 2001).
The temporal gene expression in neuroblasts (early to late: Hb -> Kr -> Pdm -> Cas) mimics the major domains of gene expression at cellular blastoderm (anterior to posterior: Hb -> Kr -> Pdm -> Cas). Additional studies will be needed to discern common and distinct regulatory features between Hb, Kr, Pdm, and Cas expression during segmentation and neurogenesis. The expression of all genes at both stages of development raises the question of which function is ancestral. Hb is detected in the CNS of various arthropod, leech, and C. elegans embryos. In mammals, Hb-related genes of the Ikaros family are best known for regulating immune development, but some also show CNS expression. A mammalian Pdm homolog, SCIP/Oct-6, is expressed in specific cortical layers of the brain, and a mammalian Cas ortholog exists but has not been characterized. It will be interesting to determine whether genes regulating temporal identity in Drosophila neuroblasts have similar functions in the mammalian CNS or immune system (Isshiki, 2001).
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