University of Minnesota, Department of Genetics, Cell Biology and Development, 6-160 Jackson Hall, 321 Church St SE, Minneapolis, MN 55455, USA
e-mail: rougvie{at}cbs.umn.edu
SUMMARY
A fundamental challenge in biology is to understand the reproducibility of developmental programs between individuals of the same metazoan species. This developmental precision reflects the meticulous integration of temporal control mechanisms with those that specify other aspects of pattern formation, such as spatial and sexual information. The cues that guide these developmental events are largely intrinsic to the organism but can also include extrinsic inputs, such as nutrition or temperature. This review discusses the well-characterized developmental timing mechanism that patterns the C. elegans epidermis. Components of this pathway are conserved, and their links to developmental time control in other species are considered, including the temporal patterning of the fly nervous system. Particular attention is given to the roles of miRNAs in developmental timing and to the emerging mechanisms that link developmental programs to nutritional cues.
Introduction
How to synchronize developmental events among different tissues as an
organism progresses from a fertilized egg to its adult form is a problem that
all animals have solved. The molecular mechanism responsible for integration
of such events is emerging from studies in C. elegans, where a
forward genetics approach has identified components of a pathway that
temporally specifies cell identities. The original four members of this
`heterochronic' gene pathway were identified from screens for animals with
cell lineage or egg-laying defects (Ambros
and Horvitz, 1984; Chalfie et
al., 1981
), while additional members were revealed, in part,
through screens targeted to identify genes that regulate the timing of
developmental events. These screens included searches for mutants with
temporal alterations in reporter gene expression
(Abrahante et al., 1998
) or
stage-specific locomotion behavior
(Abrahante et al., 2003
), as
well as screens for suppressors of known heterochronic mutants
(Pepper et al., 2004
;
Reinhart et al., 2000
). In
addition to advancing our understanding of developmental timing mechanisms in
the worm, the subsequent analyses of the genes and proteins identified by
these studies has had two especially significant outcomes. First, phylogenetic
analyses have revealed that many of the key timing genes are conserved among
diverse organisms, and some of these homologs are also known or suspected to
be involved in the temporal patterning of development. Second, these studies
have spawned a new area of biology that has seen exponential growth in the
past few years: the biogenesis and function of microRNAs (for reviews, see
Bartel and Chen, 2004
;
He and Hannon, 2004
).
This review focuses on temporal control mechanisms employed in C. elegans and considers how components of this pathway intersect with biological processes in other organisms. Particular attention is devoted to the temporal specification of cell identities in the fly central nervous system (CNS), a process that employs a worm heterochronic gene homolog, thereby raising the possibility of functional conservation. Finally, interplay between the nutritional status of an organism and the execution of developmental timing mechanisms is also considered.
The C. elegans heterochronic gene pathway: keeping time with microRNAs
Diverse developmental events are under heterochronic gene control in the
worm, including the specification of cell lineage patterns in the epidermis
(often called hypodermis) and vulva, neuronal rewiring and formation of the
dauer larva (Box 1)
(Ambros and Horvitz, 1984;
Hallam and Jin, 1998
;
Liu and Ambros, 1989
).
Mutations in heterochronic genes alter the timing of such stage-specific
events relative to other unaffected events (e.g. gonad development or the
molting cycle). In general, these mutations do not act by appreciably
accelerating or retarding the life cycle or life span of the animal. Nor do
they alter cell fate per se. Rather, they change a the temporal identity of a
cell (or perhaps `temporal fate') to one normally expressed at a different
time within the same lineage, but usually restricted to a distinct life stage.
The observed temporal transformations in heterochronic mutants have been
likened to the homeotic mutants of flies, in which cell identities are
spatially, rather than temporally, transformed
(Ambros and Horvitz, 1987
;
Slack and Ruvkun, 1997
;
Thummel, 2001
).
The core members of the heterochronic gene pathway appear to act as developmental switches that program stage-specific cell identities. Thus, the activation or repression of a given heterochronic gene at a specific developmental time is often a crucial event, such that the corresponding change in activity level modulates the progression of a cell to its next temporal fate. Thus, mutations that increase or decrease heterochronic gene activity at inappropriate times often result in opposite temporal transformations. The ultimate readout of heterochronic gene activity in the worm epidermis in particular is the behavior of specialized `seam' cells, which are situated on the lateral midlines of the worm. These cells terminally differentiate during the final molt, the transition from the fourth larval stage to the adult, and they contribute to the synthesis of a morphologically distinct adult cuticle (Fig. 1). As described below, specific heterochronic genes alter the time of adult cuticle synthesis indirectly, by deleting or reiterating earlier cell identities, whereas other genes act more directly.
LIN-14 and LIN-28 are core components of the mechanism that programs early
epidermal cell fate transitions, which can be recognized by specific cell
division patterns (Fig. 1A) as
development proceeds through the first three larval stages
(L1L2
L3) (Ambros and Horvitz,
1984
; Ambros and Horvitz,
1987
; Moss et al.,
1997
). LIN-14 and LIN-28 protein levels are high at hatching and
subsequently decay; LIN-14 disappears from the epidermis by the end of the L1
stage and LIN-28 by the end of the L2 stage
(Fig. 1B)
(Ruvkun and Giusto, 1989
;
Seggerson et al., 2002
).
LIN-14 is a nuclear protein, whereas LIN-28 is cytoplasmic and contains
hallmark RNA-binding domains (Moss et al.,
1997
; Ruvkun and Giusto,
1989
). Although the precise functions of these proteins and the
identities of their potential targets remain unknown, molecular genetic
studies have provided insights into their roles in developmental timing.
lin-14 is required for the execution of wild-type L1-stage cell
division patterns; in its absence, L2 patterns occur instead, and subsequent
patterns are each advanced by one stage, leading to a precocious phenotype and
to the synthesis of an adult-type cuticle one stage too early
(Fig. 1A,E)
(Ambros and Horvitz, 1984).
lin-28 mutants skip the proliferative double division at the start of
the L2 stage, and substitute the L3 pattern in its place, again leading to a
precocious adult cuticle phenotype, albeit with a somewhat different
underlying basis. The lin-28 phenotype suggests that LIN-28 has a
role in promoting the L2 stage proliferative division. However, this appears
not to be the case; mutations in lin-46, a lin-28
suppressor, allow the L2 stage fate to be expressed in the complete absence of
lin-28 activity (Pepper et al.,
2004
). Thus, in wild-type animals, the activity of lin-28
postpones L3 fates, allowing for the expression of the L2 stage division
pattern. The temporal decay of LIN-14 and LIN-28 levels is therefore a key
factor for seam cell identity to progress through the early larval fates.
Indeed, the continued expression of these proteins at inappropriately late
times produces phenotypes that are essentially opposite to their
loss-of-function (lf) phenotypes: development is retarded because of
the reiteration of L1 (lin-14) or L2 (lin-28) stage
patterns, and subsequent patterns are delayed
(Ambros and Horvitz, 1984
;
Moss et al., 1997
). A crucial
question to answer in terms of selecting temporal fates during early larval
stages then logically moves upstream is how is the temporal decay of LIN-14
and LIN-28 managed? The answer is found in lin-4.
|
The regulation of the early larval stage timer is more complex than the
simple downregulation of lin-14 and lin-28 by the
lin-4 miRNA. Additional levels of control are built into the temporal
regulation of these early cell lineages. A complex feedback mechanism acts to
further fine-tune lin-14 and lin-28 levels; each is required
for optimal expression of the other (Arasu
et al., 1991; Moss et al.,
1997
). Moreover, elegant genetics experiments have revealed that
lin-28 is also controlled by a lin-4-independent mechanism
(Moss et al., 1997
).
Downregulation of LIN-28 occurs in the absence of lin-4 when
lin-14 levels are reduced. Here too, the regulation occurs through
the lin-28 3'UTR (Seggerson
et al., 2002
), raising the possibility that additional miRNAs act
to modulate lin-28 expression. Various experiments indicate that
other heterochronic genes (including lin-42, hbl-1 and
daf-12, which are discussed in more detail later) also act in this
early time window to ensure the proper temporal progression of development in
the epidermis.
Given the hundreds of miRNAs that are now known, it is amazing that the
second miRNA to be discovered, let-7, was also identified in worms as
a key temporal regulator of seam cell identity
(Reinhart et al., 2000).
let-7 miRNA is detected during the L3 and later stages, and is
deployed to downregulate targets stage specifically in the epidermis,
temporally guiding development to the adult stage. A major target of the
let-7 miRNA in the epidermis is lin-41, which encodes a
cytoplasmic protein that has RNA-binding motifs
(Slack et al., 2000
). Loss of
lin-41 function causes a precocious phenotype in which seam cell
lineage patterns appear to be wild type until the L3 molt, when terminal
differentiation occurs one stage too early. The disappearance of LIN-41 from
the hypodermis during the L4 stage depends upon the presence of its
3'UTR and the let-7 miRNA
(Slack et al., 2000
).
Moreover, the lin-41 3'UTR can direct temporal downregulation
of a heterologous reporter gene, and this control is abrogated in a
let-7 mutant background (Reinhart
et al., 2000
). The temporal decay of LIN-41 activity presages the
accumulation of LIN-29, the most downstream member of the pathway
(Ambros, 1989
;
Slack et al., 2000
). LIN-29 is
a zinc-finger transcription factor that triggers the switch to the adult fate,
in part through the stage specific control of collagen gene expression
(Fig. 1B)
(Rougvie and Ambros,
1995
).
In summary, the lin-4 and let-7 miRNAs are key to guiding
the gene expression transitions that temporally pattern the worm epidermis.
Although many of the core components of the lin-4-directed early
timer and the let-7-directed late timer have been identified and
characterized, our knowledge of how these genes collectively convey temporal
information during development is still incomplete. The identification of the
precise functions, possible interaction partners and direct regulatory targets
of LIN-14, LIN-28 and LIN-41 are future challenges. Direct regulators of
lin-29 activity also await identification, as does the mechanism that
links the early and late timers. Whether molecularly related developmental
timing mechanisms operate in other organisms also remains unknown. As
lin-4 and let-7 miRNAs, and some of their targets, are
conserved in other species (Lagos-Quintana
et al., 2002; Pasquinelli et
al., 2000
), some temporal control mechanisms might share a common
origin.
Keeping time with microRNAs in other organisms
We now know that miRNAs are neither restricted to C. elegans nor
are they unique to developmental timing pathways. miRNAs function in diverse
processes, including the specification of neuronal asymmetry
(Chang et al., 2004;
Johnston and Hobert, 2003
),
insulin secretion (Poy et al.,
2004
) and programmed cell death
(Brennecke et al., 2003
;
Xu et al., 2003
), and their
mis-regulation has been linked to cancer
(Calin et al., 2002
;
Calin et al., 2004
;
Takamizawa et al., 2004
;
Johnson et al., 2005
). The
study of these small non-coding RNAs has become one of the fastest-paced
fields of research in both animal systems and plants (for reviews, see
Ambros, 2004
;
Bartel and Chen, 2004
;
He and Hannon, 2004
;
Kidner and Martienssen,
2005
).
let-7 led the march for the expansion of miRNA biology outside of
C. elegans, when Ruvkun and colleagues discovered that it is
conserved broadly in bilaterian animals, including humans
(Pasquinelli et al., 2000). In
fact, let-7 is extremely highly conserved: the mature let-7
miRNA sequence is identical between worms and humans (it is encoded by
multiple genes in humans) (Fig.
2C). Moreover, let-7 is a member of a larger gene family
in both species; additional members contain imperfect sequence identity
(Lagos-Quintana et al., 2001
;
Lagos-Quintana et al., 2002
;
Lau et al., 2001
;
Lim et al., 2003
). miRNA
cloning efforts have now also identified lin-4-related genes in
worms, flies and vertebrates (Fig.
2D) (Ambros et al.,
2003
; Lagos-Quintana et al.,
2002
; Lim et al.,
2003
).
Whether lin-4 and let-7-related miRNAs have conserved
roles in developmental timing in other organisms has not been established at
the level of mutational analysis; however, there is considerable evidence in
favor of such a role. For example, in flies and zebrafish, let-7
expression is activated late during development
(Pasquinelli et al., 2000;
Wienholds et al., 2005
),
indicating that its function in promoting late-stage cell fate decisions, such
as the induction of terminal differentiation programs, may be a general
feature of the gene. In flies, let-7 miRNA is first detected as pupal
formation begins and remains high in adults
(Pasquinelli et al., 2000
;
Sempere et al., 2002
). The
timing of this activation suggests that it is regulated by ecdysone, a steroid
hormone that triggers stage transitions during fly development, including
pupal formation and metamorphosis
(Riddiford, 1993
). However,
depletion of the ecdysone receptor by RNAi has little, if any, effect on
let-7 miRNA levels, yet it effectively eliminates expression of known
ecdysone early response genes (Bashirullah
et al., 2003
). Moreover, these early response genes are rapidly
induced in cultured cells in response to ecdysone treatment, whereas
let-7 miRNA is not. These experiments suggest that let-7
induction occurs by a mechanism distinct from that of ecdysone early
responders and is independent of the ecdysone receptor. Nevertheless, the
temporal let-7 miRNA accumulation profile, together with its
widespread expression pattern (Sempere et
al., 2002
), suggests that its expression is under systemic
hormonal control possibly via a distinct receptor. Curiously, the best match
to the lin-4 miRNA in flies, miR-125
(Fig. 2D) (Lagos-Quintana et al., 2002
),
is co-transcribed with let-7
(Bashirullah et al., 2003
).
Unraveling whether these genes are required for specifying late-stage cell
identities in the fly is thus likely to require targeted mutation of the
let-7/miR-125 locus coupled to transgenic expression of each single
miRNA.
|
let-7 family member binding sites are also conserved in the
lin-41 3'UTRs of flies and fish
(Fig. 2B)
(Pasquinelli et al., 2000). A
reporter gene bearing a lin-41 3'UTR can be
post-transcriptionally downregulated if artificially co-expressed with
let-7 in early fish embryos
(Kloosterman et al., 2004
),
but additional experiments are required to test whether the
lin-41::let-7 partnership has been maintained in vivo and whether
control of temporal identity is an aspect of its function. These vertebrate
studies are confounded by the presence of multiple lin-4 and
let-7 family members that are identical in their 5' `seed'
sequence (nucleotides 2-8; Fig.
2C,D) (Doench and Sharp,
2004
). Basepairing between this region and a 3'UTR-binding
site is a key factor in target recognition (see
Fig. 2A,B), and, consequently,
these miRNA family members have the ability to regulate common targets if
co-expressed. This property complicates the functional analysis of these
miRNAs through gene knockout experiments and makes it difficult to assess the
miRNA dependence of target gene expression patterns.
Additional players in the C. elegans heterochronic gene pathway
Two central players in the C. elegans pathway do not fit neatly
into either the early lin-4-directed timer or the late
let-7-directed timer: lin-42 and hbl-1 (also known
as lin-57). Mutations in these genes cause strong precocious
phenotypes, with seam cell terminal differentiation occurring during the L3
molt (Abrahante et al., 2003;
Jeon et al., 1999
;
Lin et al., 2003
), but
additional analyses suggest that they act at multiple points in the
pathway.
LIN-42 is a worm homolog of the Period (Per) family of proteins, originally
identified in insects and mammals (Jeon et
al., 1999). The Per proteins function in a second biological
timing mechanism, the control of circadian rhythms, raising the possibility
that this protein family has a conserved role in timing events. Per proteins
contain a hallmark protein interaction domain, the PAS domain, and the fly and
mammalian homologs have been shown to function, at least in part, by
interfering with transcriptional activator proteins (for a review, see
Glossop and Hardin, 2002
).
Another feature of Per genes is their extremely dynamic expression pattern:
their message and protein levels oscillate with a 24-hour period. A truly
striking aspect of lin-42 conservation is that, similar to
per, its expression levels cycle, although with a shorter period that
is coupled to molting cycles rather than to day length. This reiterative
expression pattern (Fig. 1B),
with high mRNA levels during each intermolt [a pattern that is reflected at
the protein level (J. Tennessen and A.R., unpublished)] sets lin-42
apart from the other members of the pathway and is suggestive of multiple or
repeated roles for lin-42 during postembryonic development. Genetic
interactions with a weak lin-14 allele suggest that lin-42
has an early role in controlling the proliferative L2 division (Z. Liu, PhD
Thesis, Harvard University, 1990). In this sensitized background, the
proliferative division is omitted, indicating an early, albeit redundant, role
for lin-42. By contrast, inactivation of lin-42 alone does
not appear to alter the early lineages, suggesting that it may have a later
role in the pathway, a position supported by genetic studies that place
lin-42 in parallel to, or downstream from, lin-46 and
let-7 (Pepper et al.,
2004
; Reinhart et al.,
2000
).
Although there have been reports of circadian behaviors in worms
(Kippert et al., 2002;
Saigusa et al., 2002
), they
have yet to be associated with lin-42 function. However, worm genes
with sequence relatedness to other circadian rhythm proteins have also been
identified in the genome (Banerjee et al.,
2005
; Clayton et al.,
2001
; Jeon et al.,
1999
). Postembryonic RNAi-based depletion experiments for two of
these, kin-20 [a fly doubletime (dbt; dco
FlyBase) homolog] and, to a lesser extent, tim-1 [a
timeless (tim)/timeout homolog], reveal phenotypes
and genetic interactions that suggest that these genes act in the timing
pathway; their loss of function causes some aspects of the terminal
differentiation program (e.g. cell fusion) to be activated precociously
(Banerjee et al., 2005
).
However, the observed phenotypes are weaker and less penetrant than that
produced by of lin-42 inactivation, suggesting that these genes may
play less central roles in the timing mechanism. In flies, Tim binds Per,
contributing to its ability to interfere with transcriptional activators and
the generation of its oscillatory expression pattern
(Darlington et al., 1998
;
Lee et al., 1999
). Dbt is a
casein kinase that phosphorylates Per, thereby possibly potentiating its
repressor activity (Nawathean and Rosbash,
2004
). Future studies will reveal whether the worm homologs of
these proteins act to modulate LIN-42 activity in ways similar to their
control of Per. C. elegans tim-1 is also a component of the cohesin
complex (Chan et al., 2003
),
but how and whether this function relates to the reported postembryonic RNAi
phenotype has not been addressed.
Similar to lin-42, hbl-1, which encodes the worm homolog of the
Drosophila Hunchback (Hb) transcription factor, may
influence seam cell temporal identity at multiple points. Omission of the L2
proliferative division occurs when hbl-1 activity is depleted by RNAi
(Abrahante et al., 2003),
suggesting it has an early timing role. hbl-1 might also have a later
function, as indicated by the presence of many putative let-7-binding
sites in its 3'UTR, and by the observation that, genetically,
hbl-1 appears to be partially redundant with the let-7
target lin-41 (Abrahante et al.,
2003
; Lin et al.,
2003
). Curiously, hbl-1 mis-expression has not been
detected in the epidermis of let-7 mutants, a finding that may be
explained by the existence of the three let-7-related miRNA genes:
mir-48, mir-84 and mir-241
(Fig. 2C) (Lim et al., 2003
). The
presence of multiple let-7 family members in worms, as in
vertebrates, raises the possibility of functional redundancy and implicates
these additional family members in developmental time control. Because these
four miRNAs share perfect identity in the 5' seed, they are likely to
act through similar or overlapping sets of binding sites. All four family
members show temporally restricted expression patterns
(Lau et al., 2001
;
Lim et al., 2003
), and if also
expressed in the epidermis, these other family members might regulate
hbl-1 and perhaps other members of the pathway. Indeed,
overexpression of mir-84 results in precocious seam cell phenotypes,
suggesting that it may time the terminal differentiation of this tissue in
wild-type animals (Johnson et al.,
2005
). The ultimate test of whether hbl-1 is regulated by
let-7 family member(s) in the epidermis awaits the generation of
C. elegans strains that are null for all these miRNA genes, a project
that is now under way (V. Ambros, personal communication).
HBL-1 and fly Hb share strongest sequence identity in their central four Cys2-His2 zinc fingers, which bind DNA in the fly, indicating that this function might be conserved. More impressive than this simple sequence conservation are recent experiments that suggest that these proteins share a common biological function control of developmental time.
Hunchback homologs in worms and flies time developmental events
The conservation of Hb sequence between flies and worms provides a
molecular link between developmental timing mechanisms in these organisms.
Although perhaps best known for its role in spatial patterning
(Lehmann and Nusslein-Volhard,
1987; Tautz et al.,
1987
), hb is a key regulator of temporal identity in the
Drosophila CNS (Isshiki et al.,
2001
). The fly CNS also provides an exquisite system for examining
temporal control mechanisms in animals. Similar to the blast cells of the
C. elegans epidermis, fly neuroblasts (NBs) divide in invariant stem
cell-like lineage patterns (Fig.
3), and cell identities can be determined by their position and
the expression of molecular markers (for a review, see
Skeath and Thor, 2003
). The
NBs divide asymmetrically, giving rise to a smaller ganglion mother cell
(GMC), which divides to produce post-mitotic neurons, and a NB, which retains
stem cell character and repeats the cycle. The sequential expression of
several transcription factors [Hb
Krüppel (Kr)
Pdm1
Castor
(Cas)] in the NB, and maintained in the GMC progeny, specifies temporal
identity. Hb determines the identity of the first born progeny, Kr the second,
and so on. In a manner remarkably reminiscent of heterochronic genes in worms,
Hb loss- and gain-of-function situations cause opposite temporal
transformations in cell fate: Hb loss of function causes the first born fate
to be omitted and subsequent identities to be expressed too early, whereas Hb
gain of function (i.e. continued Hb expression in NBs at inappropriately late
developmental times) results in the reiteration of the first-born fate
(Isshiki et al., 2001
).
|
Intriguingly, the temporal transitions downstream of the HbKr switch
do not require cell division
(Grosskortenhaus et al.,
2005
); when the cell cycle is blocked in the absence of Hb, the
Kr
Pdm
Cas transcription factor progression is correctly specified.
The temporal transitions in this late timer are thus triggered in a
mechanistically distinct manner from the Hb
Kr transition. Moreover, Hb
activity must play a key role in the inhibition of the cell cycle-independent
late timer. But how? Grosskortenhaus et al. distinguish between two models. In
one, Hb would block the initiation of the late timer. Think of a power-cut at
3 AM the clock stalls until power returns at, say, 7 AM, at which time
it resumes and marks the intervening hours 4, 5, etc. In the other model, Hb
would simply inhibit the expression of the transcription factors, while the
timer itself continues to progress, as in a power-cut to a computer
the timer progresses and when power returns its clock resumes from 7 AM, with
the omission of the intervening times (or transcription factor profiles). Hb
function fits the former model. Continued Hb expression at inappropriately
late times causes the reiteration of first-born fates, and when it is
subsequently removed, the temporal program resumes sequential transcription
factor expression (Kr
Pdm
Cas) without the deletion of intermediate
expression patterns. Hb is required to specify the first-born fate and to
postpone the subsequent fates until the appropriate time. Thus, Hb negatively
regulates the late timer, and its level must decline to allow the temporal
progression of neuronal differentiation in the wild-type CNS. One can consider
the downregulation of Hb expression as playing an important role in the
diversification of neurons in the fly CNS; Hb decay allows the temporal
progression of NB identity and the subsequent expansion of neuronal cell
types.
Hb activity is thus a key component of the temporal identity timer that
specifies neuronal cell fates. However, important questions remain. How does
Hb activity specify the first-born fate? How does it inhibit the late timer?
How is Hb expression spatially and temporally restricted within the NB lineage
and downregulated to allow the transition to later fates? Answers to the first
two questions await the identification of the direct Hb targets in the CNS and
determination of its mode of action. Answers to the last question are
beginning to emerge from studies of Hb expression. The regulation of Hb in the
CNS appears to be largely transcriptional
(Grosskortenhaus et al.,
2005), although additional post-transcriptional modulation has not
been ruled out. Such a post-transcriptional system could reinforce a primarily
transcriptional mechanism, contributing to the observed rapid decay in Hb
levels. Translational control of Hb plays a major role in the spatial
patterning of the early fly embryo. Interestingly, this mechanism acts through
the 3'UTR and employs Brat, a protein with sequence relatedness to
LIN-41 (Sonoda and Wharton,
2001
), which, similar to HBL-1, also acts downstream of
let-7 in the worm heterochronic gene pathway. Although
let-7-binding sites are not present in the fly hb
3'UTR, other miRNAs could potentially play a secondary role in
fine-tuning Hb levels (Abrahante et al.,
2003
; Lin et al.,
2003
).
Hb is expressed in the NB and first GMC daughter, and this expression is
maintained in the GMC and its post-mitotic neuronal descendents. Although
activators of Hb in the NB lineage have not been identified, some models of Hb
regulation have been ruled out. In contrast to the situation in the fly
epidermis, where Hb maintains its own expression, neuronal expression of Hb in
the GMC and its progeny is not maintained by an auto-feedback loop and must
rely on heterologous activators. The loss of Hb from the NB, a key to late
fate transitions, is not simply due to its asymmetric localization to the GMC
when the NB divides (Grosskortenhaus et
al., 2005). Rather, recent studies indicate that Hb expression is
transcriptionally downregulated in the NB
(Kanai et al., 2005
).
One key to the puzzle of Hb regulation is found in the orphan nuclear
receptor Seven-up (Svp) (Kanai et al.,
2005). Alterations in svp expression cause temporal cell
fate transformations that are essentially opposite to those caused by varying
hb expression patterns. svp(lf) leads to reiteration of the
early-born fate, similar to that observed with prolonged hb
expression, whereas forced precocious expression of svp causes loss
of the early-born fate, similar to hb(lf). These observations suggest
that svp might negatively regulate hb expression in early
stage NBs. Indeed, svp is activated in the NB after the first GMC
daughter is born, at the time of the transition from Hb
Kr expression.
Moreover, prolonged expression of hb is observed in svp(lf)
mutants and proper downregulation of a hb promoter:lacZ
fusion is dependent on svp activity, raising the possibility that
this regulation may be direct. These studies demonstrate nicely that the
Hb
Kr transition is mediated by Svp, thereby allowing the transition to
the late timer. In addition to identifying a new component of the NB identity
timer, these studies also highlight the issue of how developmental time is
controlled in the NB lineage. How does svp expression become
activated in the early NB in order to orchestrate the subsequent temporal
transitions? Is cytokinesis required? If so, why and how? Is there a signal
from the newly born GMC? Only time will tell.
Nutritional inputs into developmental timing
The temporal control of neuronal differentiation has also been revealed
through genetic studies of fly eye development, but with a unique twist: these
studies have unexpectedly linked temporal control mechanisms to nutritional
inputs and growth control. Target of rapamycin (Tor) and insulin receptor
(InR) signaling pathways monitor nutrient status and mediate cell growth in
animals (Fig. 4A) (for reviews,
see Long et al., 2004;
Neufeld, 2004
). An intriguing
new study reveals that increased signaling through the Tor/InR pathways causes
precocious neuronal differentiation, whereas reduced activity delays
differentiation (Bateman and McNeill,
2004
).
The fly eye consists of hundreds of ommatidia, clusters of eight
photoreceptors (R1-R8) that are arrayed in a stereotypic pattern. The
differentiation of these receptors occurs in the larval eye imaginal disc,
which is initially an undifferentiated epithelium. As the visually distinct
morphogenetic furrow passes through the epithelium from posterior to anterior,
the differentiation of photoreceptors is induced by an epidermal growth factor
(Egf)/Ras/Mapk pathway (Wolff,
2003).
Bateman and McNeill (Bateman and
McNeill, 2004) monitored the temporal profile of photoreceptor
differentiation by assaying neuronal markers in clones of mutant cells that
spanned the morphogenetic furrow. Clones of cells bearing mutations that
increased Tor/InR signaling differentiated before their wild-type neighbors
and expressed neuronal markers too early
(Fig. 4B-D). By contrast,
decreased Tor/InR signaling delayed differentiation. Moreover, the expression
of components of the Egf/Ras/Mapk pathway appeared normal in mutant clones,
suggesting that the Tor/InR pathways exert temporal control on the
differentiation program at a downstream step in this signal cascade or through
a parallel pathway. Importantly, the observed disruption of temporal control
was not simply a result of altered cell size caused by the disruption of
Tor/InR signaling. Increased cell mass caused by other means (such as
increased activity of cyclin D) failed to induce precocious
differentiation.
|
Although this example of temporal transformation is not on the same scale
as the life-stage temporal transformations seen in C. elegans, it
nevertheless significantly expands our repertoire for studies of developmental
time control in animals. Once more, a common theme arises timing
molecules need to be precisely controlled to establish developmental synchrony
between tissues; too much or too little signaling activity causes opposite
temporal transformations. In addition, the possibility that timing molecule
activity is modulated by miRNAs again looms on the horizon because a miRNA has
been implicated in the control of insulin secretion in mammals
(Poy et al., 2004). Future
work will need to determine whether there is an intersection between this
nutrient-sensitive mechanism in flies and the heterochronic gene pathway, and
to test, for example, whether Tor or InR signaling pathways, parts of which
are conserved in worms (Hara et al.,
2002
; Jia et al.,
2004
; Long et al.,
2002
), influence developmental time control in the nematode
relative to nutritional status.
It has been established that nutritional status influences multiple steps
of the heterochronic gene pathway. If worms hatch in the absence of food, the
L1 hatchlings arrest development and can survive several weeks. In this state
of `L1 diapause', postembryonic cell divisions do not occur and the
heterochronic gene pathway is not initiated. LIN-14 levels remain elevated
(Arasu et al., 1991) and
lin-4 is not activated. The heterochronic gene timer is thus
ultimately dependent upon external food signal(s), although how directly this
timing mechanism is linked to nutritional status is unknown. One approach to
investigating this problem would be to work backwards from lin-4
activation at present lin-4 activation, which occurs
12
hours after L1 larvae are place on food
(Feinbaum and Ambros, 1999
),
is the most upstream step in the heterochronic gene pathway. The
identification of the transcriptional regulator(s) of lin-4 and the
testing of whether their activity is altered by nutritional status, will be
important next steps.
Nutritional cues also affect the heterochronic gene pathway with respect to
the developmental choice between proceeding directly through the L3 stage or
instead forming a dauer larva, an alternative third larval stage specialized
for stress-resistance and dispersal
(Cassada and Russell, 1975).
Adverse conditions such as food shortage and high population density trigger
dauer larva formation. A key player in this decision is daf-12, which
encodes a nuclear hormone receptor that also acts in the heterochronic pathway
(Antebi et al., 1998
;
Antebi et al., 2000
). The
daf-12 locus is complex. Null mutations are dauer defective, whereas
specific alleles have highly penetrant heterochronic phenotypes, including
reiteration of the proliferative L2 division during subsequent stages, a
retarded heterochronic phenotype. In an interesting twist to the heterochronic
gene pathway, daf-12 has recently been identified as a let-7
target in the hypodermis, and its mis-regulation at late larval stages is
likely to contribute to the let-7 mutant phenotype
(Großhans et al.,
2005
).
Interestingly, the daf-12 alleles that produce strongly retarded
phenotypes contain mutations in the ligand-binding domain of the protein
(Antebi et al., 2000),
suggesting that hormonal inputs are key to wild-type DAF-12 function and
prevention of these phenotypes. The hormonal control of daf-12
through its ligand binding domain is also supported by the finding that
daf-9, which acts just upstream of daf-12, encodes a
cytochrome P450, a class of enzyme required for steroid hormone biosynthesis
(Gerisch et al., 2001
;
Jia et al., 2002
). Binding of
DAF-12 by hormone has been proposed to promote reproductive development at the
L2 molt, while the unbound form triggers dauer formation
(Gerisch and Antebi, 2004
;
Mak and Ruvkun, 2004
).
Wild-type levels of daf-9 expression, and possibly downstream hormone
levels, depend on a variety of factors, including activity of the worm insulin
receptor (DAF-2) and feedback regulation by daf-12. Such nutritional
and hormonal inputs to the heterochronic gene pathway, mediated through
DAF-12, could provide a means for integrating nutritional signals and
coordinating the progression of temporal cell fates throughout the animal.
Conclusions and future directions
Temporal control is an important facet of the developmental mechanisms
employed to produce the complex body plans of multicellular organisms.
Considerable progress has been made in elucidating the molecular components
deployed to time developmental events, particularly within the C.
elegans epidermis. However, several key issues remain to be resolved.
Aside from a few miRNA:target interactions, direct connections between
heterochronic gene pathway members are essentially unknown. This problem is
exemplified by the dynamic expression pattern of lin-42, which must
reflect short, and possibly regulated, half-lives of both its protein and
mRNA. How this pattern is established and what its functional significance is
are unknown, as is the precise mechanism by which LIN-42 acts and the identity
of its interaction partners. This deficit of functional information is true of
most pathway members, including the miRNA components to some extent, and its
remedy is key to deciphering the timing pathway in the epidermis. And yet,
elucidation of the molecular mechanism that times stage identity in the
epidermis is one piece of a much larger puzzle: how are developmental events
throughout the animal synchronized? Answering this question will require
delineating the timing pathway components used in other tissues and the
molecules that coordinate these pathways. This coordination is likely to
employ cues that act systemically, and highlights the importance of searching
for hormonal inputs, such as those predicted to modulate of DAF-12 activity
(Antebi et al., 2000).
Recent advances in understanding temporal control have also been made in
Drosophila, including the important finding that alterations in
signaling through the insulin/Tor pathway can alter the time of cell
differentiation (Bateman and McNeill,
2004). This finding brings environmental cues and the issue of the
nutritional status of an organism into the equation of developmental time
control, thereby suggesting additional avenues to explore in worms for the
signals that mediate food inputs to lin-4 activation. Understanding
the mechanism that links the Tor/InR pathway to fly neuronal differentiation
will require the identification of its downstream components, perhaps
involving translational control mechanisms, as used by these pathways in
growth control.
Still unanswered is the extent to which conserved components of the worm heterochronic gene pathway time developmental events in other organisms. At present, the only clear example is the timing of NB identity by Drosophila hb, but it is still not clear whether there are other molecules shared by these two timing pathways. Tests of other conserved genes in flies and vertebrates (including lin-28, lin-41, and the lin-4 and let-7 miRNAs) will require inactivation studies, preferably by mutational analysis, to determine whether they act in temporal control mechanisms. However, the possibility remains that timing mechanisms in other organisms might be largely distinct from that of nematodes. Thus, further insights into developmental time control in other species, vertebrates in particular, might also require forward genetics, e.g. the design of zebrafish screens around reporter gene temporal mis-expression strategies.
Importantly, in each of the systems discussed in this review, the mutations analyzed alter temporal cell identities independently of cell fate. Thus, such studies are greatly expanding our understanding of developmental time control, thereby closing the knowledge gap between our understanding of spatial and temporal control mechanisms. An additional challenge for the future will be to decipher the mechanisms that integrate spatial and temporal information, together with cues that specify sexual cell identity, as an organism develops.
ACKNOWLEDGMENTS
I thank J. Tennessen, M. Fukuyama and M. O'Connor for helpful discussions and critical reading of the manuscript, and J. Tennessen, A. Daul and H. McNeill for contributions to figure preparation. The Rougvie laboratory is supported by the National Institutes of Health and the National Science Foundation.
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