DOI: 10.1126/science.1070343
, 1858 (2002);296Science
et al.Geeta Bharathan,
Expression During Development
GeneKNOXIHomologies in Leaf Form Inferred from
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Homologies in Leaf Form
Inferred from KNOXI Gene
Expression During Development
Geeta Bharathan,1
* Thomas E. Goliber,2
* Christopher Moore,2
†
Sharon Kessler,2
Thinh Pham,2
‡ Neelima R. Sinha2
§
KNOTTEDI-like homeobox (KNOXI) genes regulate development of the leaf from
the shoot apical meristem (SAM) and may regulate leaf form. We examined KNOXI
expression in SAMs of various vascular plants and found that KNOXI expression
correlated with complex leaf primordia. However, complex primordia may
mature into simple leaves. Therefore, not all simple leaves develop similarly, and
final leaf morphology may not be an adequate predictor of homology.
In simple-leaved species (maize, rice, Arabidopsis,
tobacco, snapdragon) KNOXI genes
are expressed in the SAM and unexpanded
axis and are down-regulated after leaf initiation,
suggesting fundamental differences between
the indeterminate shoot and determinate
leaves (1–5). Overexpression of KNOXI
in simple-leaved plants results in distorted
leaves and ectopic shoots (6–8). By contrast,
in tomato, a complex-leaved plant, KNOXI
genes are up-regulated in leaf primordia and
down-regulated in the mature leaf (9–11).
Overexpression of KNOX1 in tomato results
in increased leaf complexity (9–11). To determine
how general these patterns of expression
are, we studied KNOXI expression in
developing simple- and complex-leaved
shoots in different species of a genus, Lepidium,
and within a single species, Neobeckia
aquatica (both in Brassicaceae, eurosid II).
All species showed down-regulation of
KNOXI protein at sites of initiation of leaf
primordia (P0), but differed in whether or not
KNOX1 was expressed later in leaf development.
The simple-leaved Lepidium africanum
had high KNOXI protein expression in the
SAM, but not later (Fig. 1, A and B), whereas
in the complex-leaved L. perfoliatum and L.
hyssopifolium, KNOX1 expression appeared
later in leaf primordia (Fig. 1, C to H). L.
oleraceum produces simple leaves (Fig. 1I)
and unexpectedly shows KNOXI expression
in its leaf primordia (Fig. 1K) [see supplementary
material (12)]. Scanning electron
microscopy revealed that the simple leaves of
L. oleraceum had primordia that, in early
development (primary morphogenesis), produced
marginal outgrowths (Fig. 1J) typical
of early complex leaf development (e.g., L.
hyssopifolium: Fig. 1G) [see supplementary
material (12)]. This early complex form of L.
oleraceum was later subsumed by inner blade
growth [by a process of secondary morphogenesis
(13, 14)], and the early marginal
outgrowths were apparent only as coarse
teeth in the simple mature leaf (Fig. 1I). That
KNOXI expression correlates with early leaf
development but not necessarily with final
leaf form implies that other regulatory changes
influence leaf blade expansion.
The species of Lepidium studied here share
an ancestor with complex leaves, as inferred
from parsimony reconstructions of mature leaf
form (Fig. 1L) on a phylogenetic hypothesis for
the genus (15). Within this lineage simple leaves
arose in a group that included L. africanum and
L. oleraceum, and later, there was a reversal
back to complex leaves in L. hyssopifolium (Fig.
1L). We infer that the complex-leaved common
ancestor had complex primordia with early
KNOXI expression and that simple leaves
evolved by either turning off KNOXI in the
primordia (L. africanum) or modifying secondary
morphogenesis (L. oleraceum) (16).
This correlation between KNOXI expression
and primordium form was also observed
within individuals of a single species, N.
aquatica (Brassicaceae). This aquatic species
has two kinds of leaves: simple or complex
aerial leaves and submerged complex leaves.
Emergent leaf form varies with light intensity:
Simple leaves are produced under high
light, and complex leaves under low light.
KNOXI expression was absent in simple leaf
primordia of emergent shoots under high
light, but present in complex primordia made
under low-light conditions (17). Thus,
KNOXI expression can be modulated by light
conditions, perhaps through hormonal changes
that often accompany alterations in light
quality and quantity (18).
Phylogenetic analyses of leaf evolution (Fig.
2) reveal that the ancestral angiosperm had simple
leaves (19, 20), and that complex leaves
repeatedly arose from these simple-leaved ancestors
(on average 29 “gains”) and reverted (on
average six “losses”) to the ancestral simple
form [see supplemental material (12)]. This indicates
that neither all simple nor all complex
leaves are homologous [similar owing to common
ancestry (21)]. Complex leaves are generally
assumed to be nonhomologous (22), but
simple leaves are generally assumed to be homologous
and, therefore, developmentally similar.
Our observations in Lepidium suggest that
the latter assumption may not always be correct.
1
Department of Ecology and Evolution, State University
of New York, Stony Brook, NY 11794–5245, USA.
2
Section of Plant Biology, University of California–
Davis, Davis, CA 95616, USA.
*These authors contributed equally to this work.
†Present address: Department of Genetics, Stanford
University, Stanford, CA 94305, USA.
‡Present address: Department of Pathology, Genentech,
South San Francisco, CA 94080, USA.
§To whom correspondence should be addressed. Email:
nrsinha@ucdavis.edu
Fig. 1. Consistent correlation
between leaf
form and KNOX expression
in Brassicaceae.
(A and B) L.
africanum; (C to E) L.
perfoliatum; (F to H)
L. hyssopifolium; (I to
K) L. oleraceum. The
final leaf form is
shown in (A), (C), (F),
and (I). Protein expression
is present in (B),
(D), (H), and (K) in the
SAM (*); absent in P0
and P1 (O); and
present in developing
leaflets of complex
leaf (ٙ). The same
symbols are used in
Figs. 3 and 4. (E, inset)
Whole-mount reverse
transcription–polymerase chain reaction in situ hybridization shows expression of the KNOX gene
STM1 in developing leaflets. Scanning electron micrographs of L. hyssopifolium (G) and L. oleraceum
(J) developing leaves. (L) Phylogenetic patterns of leaf evolution in Lepidium (Brassicaceae,
eurosid I). Colors indicate ancestors reconstructed with simple (blue) or complex (red) leaves. Bars:
(A, C, F, I) 1 cm, (G and J) 50 ␮m, (B, D, H, K) 100 ␮m. KNOX expression patterns: complex with
no secondary simplification (asterisk), complex with secondary simplification (circled asterisk), or
simple (filled yellow circle).
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Does the correlation between KNOXI expression
and primordium form hold for a broad
range of taxa? We surveyed simple and complex
leaves across vascular plants and found that (i)
modification of complex primordia through secondary
morphogenesis is common in simple
leaves across eudicots; (ii) the presence of
KNOXI expression is associated with complex
primordia and its absence with simple primordia;
and (iii) KNOXI protein expression is
down-regulated at the site of leaf initiation (P0),
except in ferns. Taxa were drawn from cycads,
ferns, and various angiosperms that represent
independent instances of origins of complex
leaves or reversals to simple leaves.
KNOXI protein expression reflective of simple
leaves was seen in the simple primordia of
Amborella trichopoda (Fig. 3, A to C), a putative
basal extant angiosperm (23–25), and in the
simple primordia of grasses (26, 27) [fig. S1
(12)]. Because primordia in basal angiosperms
are simple (28), this expression pattern is inferred
to represent the ancestral state in the
angiosperms. This inference is reinforced by our
observation of the “simple” pattern in the simple-leaved
gymnosperm, Welwitschia [fig. S1
(12)]. This is the pattern in most simple-leaved
species studied to date (1–5) and in this study (L.
africanum, Neobeckia, Amborella) and contrasts
with the pattern seen in complex leaves of tomato
[fig. S1 (12)]. However, as in L. oleraceum,
development of simple leaves from complex
primordia through secondary morphogenesis
was also observed in various eudicot lineages
[euasterid II: Apiales, Pimpinella (Fig. 3,
D to F); euasterid I: Gentianales, Coffea; rosids:
Vitales, Vitis sp. (fig. S2) (12)]. This
molecular developmental dissimilarity may
Fig. 3. Comparison of mature
leaf form, leaf primordia, and
KNOX1 immunolocalization pattern
in angiosperms with simple
leaves. “Simple” expression: (A
to C) A. trichopoda. “Complex”
expression: (D and E) P. anisum
(Apiaceae). Scanning electron
micrographs (B and E) show early
development. KNOXI protein expression
can be seen in shoot
apices in (C) and (F). Symbols as
in Fig. 1. Bars: (A and D) 1 cm, (B
and E) 50 ␮m, (C and F) 100 ␮m.
Fig. 4. Vascular plants with complex pattern and complex leaves. (A and B) D. carota (Apiaceae).
(C to E) C. congestum ( Vitaceae). (F and G) Anogramma chaeophylla. (H to J) Zamia floridans.
Symbols as in Fig. 1. Bars: (A, C, F, H) 1 cm, (B, E, G, I), 100 ␮m, (D) 50 ␮m.
Fig. 2. Evolution of leaf form. Summary of
phylogenetic patterns inferred from parsimony
reconstructions of ancestral states with data on
leaf form in 557 genera of angiosperms (24,
39). The ancestral angiosperm (0), eudicot (1),
rosid (2), and asterid (3) had simple leaves.
Some groups at the tips (in red) are equally
likely to have had ancestors with simple or
complex leaves; within all other taxa in color
there are multiple origins (“gains”) and reversals
(“losses”) of complex leaves; taxa in black
have only simple leaves. If reconstructions were
done with terminals coded according to the
state in the family (to include polymorphisms),
then ancestors of taxa in green were reconstructed
as having complex leaves. If polymorphic
families were coded as having complex
leaves, then the ancestral eudicot was equally
likely to have had simple or complex leaves (1,
closed red circle); if the ancestral eudicot is
assumed to have had complex leaves, then a
“loss” (4, open red circle) to simple leaves was
followed by a new “gain” of complex leaves in
the rosids (5, closed red circle). Under this
scheme alone, it is possible that complex leaves
of rosids (excluding Saxifragales) are homologous.
KNOX expression patterns: as in Fig. 1.
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reflect the nonhomology of simple leaves in
eudicots. KNOXI expression of the “complex”
pattern was seen in the complex primordia
of different eudicot lineages [euasterid II:
Apiaceae–Daucus carota (Fig. 4, A and B);
rosids: Vitaceae–Cissus (Fig. 4, C to E)].
Sampling suggests that the molecular hypothesis
regarding an association of KNOXI activity
with complexity of the leaf primordium
is supported across most eudicots [fig. S3
(12)]. One exception is a group of legumes,
including peas, which have complex leaves
but no KNOXI expression in leaf primordia
(29, 30). We believe that, in this group,
KNOXI genes ceased to be part of the genetic
cascade leading to the complex leaf form and
that a different gene, PEAFLO (29, 30), became
part of the cascade (31). The unusual
KNOXI expression pattern in this group of
legumes is striking, given our observation that
the correlation of “complex” KNOXI protein
expression with primordium complexity was
present in ferns and gymnosperms (Fig. 4, F
to J), representing stages early in the evolution
of vascular plants.
Regardless of final leaf form, KNOXI expression
is down-regulated at sites of leaf initiation
(P0) in flowering plants and gymnosperms
[Figs. 1, 3, and 4; figs. S1 to S3 (12)]. This
suggests a mechanism that denotes “determinacy”
during initiation of the leaf. Unlike in
seed plants, KNOXI is not down-regulated in
the P0 of ferns (Fig. 4G) (32). This result is
consistent with current understanding that
leaves of ferns and seed plants evolved independently
(33) and may have different developmental
characteristics (34).
Our results suggest that at least two different
modes of development have evolved to generate
simple leaves (e.g., L. africanum with simple
pattern and Pimpinella anisum with complex
pattern). By contrast, the same, complex,
pattern of KNOXI expression characterizes
independently evolved complex leaves
(e.g., Cissus congestum and Daucus carota;
N. aquatica, L. perfoliatum, and L. hyssopifolium).
Complex leaves may thus be partially
indeterminate. Several studies on vascular
development in leaves suggest that
leaf shape and venation patterns parallel
each other. However, it is unclear whether
one directs the other and, if so, which one.
Analysis of venation patterns in developing
leaves with secondary morphogenesis may
provide some information on this aspect of
leaf development. Our results are similar to
those for Crustacea, a group of animals, in
which Hox expression is correlated with the
specialization of limbs into feeding appendages
(35). As in that case, these results
highlight the value of comparative studies
in augmenting and/or refuting hypotheses
that emerge from experimental studies, and
in suggesting new hypotheses that may be
tested experimentally.
References and Notes
1. E. Vollbrecht, B. Veit, N. Sinha, S. Hake, Nature 350,
241 (1990).
2. C. Lincoln, J. Long, J. Yamaguchi, K. Serikawa, S. Hake,
Plant Cell 6, 1859 (1994).
3. A. Nishimura, M. Tamaoki, M. Matsuoka, Plant Cell
Physiol. 39, S60 (1998).
4. A. Nishimura, M. Tamaoki, Y. Sato, M. Matsuoka,
Plant J. 18, 337 (1999).
5. R. Waites, H. R. N. Selvadurai, I. R. Oliver, A. Hudson,
Cell 93, 779 (1998).
6. N. Sinha, R. E. Williams, S. Hake, Genes Dev. 7, 787
(1993).
7. G. Chuck, C. Lincoln, S. Hake, Plant Cell 8, 1277
(1996).
8. R. Schneeberger, M. Tsiantis, M. Freeling, J. A. Langdale,
Development 125, 2857 (1998).
9. D. Hareven, T. Gutfinger, A. Parnis, Y. Eshed, E. Lifcshitz,
Cell 84, 735 (1996).
10. J.-J. Chen, B.-J. Janssen, A. Williams, N. Sinha, Plant
Cell 9, 1289 (1997).
11. B.-J. Janssen, L. Lund, N. Sinha, Plant Physiol. 117, 771
(1998).
12. Supplementary figures and details of experimental
procedures are available on Science Online at www.
sciencemag.org/cgi/content/full/296/5574/1858/
DC1.
13. W. Hagemann, S. Gleissberg, Plant Syst. Evol. 199,
121 (1996).
14. N. G. Dengler, H. Tsukaya, Int. J. Plant Sci. 162, 459
(2001).
15. J. L. Bowman, H. Bruggemann, J.-Y. Lee, K. Mummenhoff,
Int. J. Plant Sci. 160, 917 (1999).
16. A recent phylogenetic hypothesis of relationships
among many more Lepidium species on the basis of
chloroplast sequences (36) suggests that these two
shifts to simple leaves occurred independently. However,
extensive hybridization may underlie this difference
in phylogeny (37), and it is not possible to
comment further on the independence or otherwise
of this morphological shift.
17. N. R. Sinha et al., unpublished data.
18. J. Chory, J. Li, Plant Cell Environ. 20, 801 (1997).
19. D. W. Taylor, L. J. Hickey, Flowering Plant Origin,
Evolution and Phylogeny (Chapman & Hall, New York,
1996).
20. J. A. Doyle, P. K. Endress, Int. J. Plant Sci. 161, S121
(2000).
21. Here we use the phylogenetic definition of homology.
The present usage is prevalent in the field of
evolution of development (38). Other uses of the
term are not implied in this work.
22. S. Gleissberg, J. W. Kadereit, Int. J. Plant Sci. 160, 787
(1999).
23. S. Mathews, M. Donoghue, Science 286, 947 (1999).
24. Y.-L. Qiu et al., Nature 402, 404. (1999).
25. D. E. Soltis et al., Bot. J. Linn. Soc. 133, 381 (2000).
26. L. G. Smith, D. Jackson, S. Hake, Dev. Genet. 16, 344
(1995).
27. Y. Sato et al., Proc. Natl. Acad. Sci. U.S.A. 93, 8117
(1996).
28. W. Troll, Vergleichende Morphologie der hoheren
Pflanzen Bd I. Vegetationsorgane (Gebrueder Borntraeger,
Berlin, 1939).
29. J. Hofer, L. Turner, R. Hellens, M. Ambrose, P. Matthews,
Curr. Biol. 7, 581. (1997).
30. C. W. Gourlay, J. M. I. Hofer, T. H. N. Ellis, Plant Cell
12, 1279 (2000).
31. N. R. Sinha et al., unpublished data.
32. Similar results were observed in Ceratopteris richardii
with a cloned KNOX1 gene (39).
33. P. Kenrick, P. R. Crane, The Origin and Early Diversification
of Land Plants: A Cladistic Study (Smithsonian
Institution Press, Washington, DC, 1997), pp. xiii–
441.
34. Y. L. Ma, T. A. Steeves, Ann. Bot. 70, 277 (1992).
35. M. Averof, N. H. Patel, Nature 388, 682 (1997).
36. K. Mummenhoff, H. Bruggemann, J. L. Bowman, Am. J.
Bot. 88, 2051 (2001).
37. K. Mummenhoff, personal communication.
38. Abouheif et al., Trends Genet. 13, 432 (1997).
39. J.-A. Banks, personal communication.
39. W. P. Maddison, D. R. Maddison, MacClade: Interactive
Analysis of Phylogeny and Character Evolution
(Sinauer Associates, Sunderland, MA, 1992).
40. We thank J. Harada, A. Doust, P. Stevens, and B.
Grabowski for critical comments; T. Kellogg, C. Kuhlemeier,
and members of the Sinha lab for helpful discussions;
J. Jernstedt for discussions, help with dissections
of the fern, cycad samples and, along with B. Hall, for
the Amborella samples; T. Metcalf and E. Sandoval
(Section of Plant Biology Conservatory) for plant materials;
and S. Hake and D. Jackson for providing the
antibodies to KNOTTED1. Supported by NSF
IBN-9983063 and IBN-0092599 (N.R.S.) and SHARP
(HHMI) undergraduate fellowships (C.M. and T.P.).
29 January 2002; accepted 15 April 2002
Amacrine-Signaled Loss of
Intrinsic Axon Growth Ability
by Retinal Ganglion Cells
Jeffrey L. Goldberg,* Matthew P. Klassen, Ying Hua, Ben A. Barres
The central nervous system (CNS) loses the ability to regenerate early during
development, but it is not known why. The retina has long served as a simple model
system for study of CNS regeneration. Here we show that amacrine cells signal
neonatal rat retinal ganglion cells (RGCs) to undergo a profound and apparently
irreversible loss of intrinsic axon growth ability. Concurrently, retinal maturation
triggers RGCs to greatly increase their dendritic growth ability. These
results suggest that adult CNS neurons fail to regenerate not only because of
CNS glial inhibition but also because of a loss of intrinsic axon growth ability.
Neurons in the CNS lose the ability to regenerate
their axons early in development, but it
is not known why. A currently prevailing
view is that a strongly inhibitory glial environment
causes regenerative failure in the
adult CNS (1, 2), as CNS glial cells, both
astrocytes and oligodendrocytes, inhibit regenerating
axons after injury (3–6). A crucial
question is whether overcoming these inhibitory
cues will be sufficient to promote rapid
regeneration or whether adult CNS neurons
have undergone a developmental loss of intrinsic
regenerative ability (7–11). For example,
CNS neurons in slices are less able to
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