1
2
3
Morphogenesis is triggered by the action of upstream acting factors, the
morphogens.
In animals, the morphogenesis is accompanied by several changes in cells or
their groups, e.g. changes in adhesion, motility (or protrusion), shape and rate of
proliferation.
For those changes, the communication among the cells and their interaction with
the surrounding cellular environment is critical. In the following part, we will
discuss the major factors involved in those aspects of the cellular life.
4
In contrast to epithelial cells, the cells of mesenchyme are not in a tight contact
with each other. The intercellular spaces are filled with what is called
extracellular matrix.
The extracellular matrix consists of collagen, proteoglycans, glycoproteins and
signalling molecules and is highly hydrated.
The figure above demonstrates the differences between mesenchymal, epithelial
and plant cells. In plant morphogenesis (see later), the cell wall plays a role, too.
Some of the important proteins that are part of the extracellular matrix will be
described in the following slides.
5
6
Collagen is formed from the proline or hydroxyproline-rich chains of amino acids.
There are at least 18 types of collagen in vertebrates, all of them being composed
of tandem three-amino-acid repeats of the form (Gly-X-Y)n, in which Y is proline
or its hydroxylated form, the hydroxylproline.
There are several important posttranslational modifications of the collagen.
These are e.g. hydroxylation of some prolines to hydroxyprolines, glycosylations
and protease-mediated cleavage of peptides at both N- and C-terminal ends.
Further there are intramolecular disulfide bonds being formed at both N- and C-
termini.
Three of the protein chain subunits are joined together into triple helix via
intermolecular disulfide bonds that occur along the length of the whole subunit
chain.
Collagen synthesis, glycosylation, hydroxylation, helix formation and other
modifications occur still in a cell, while the final assembly takes place in the
extracellular space.
Importantly, the extracellular environment regulates the final assembly of
collagen. For example, salivary glands are composed of branched epithelial ducts
ending in the secretory acini. During their development, the epithelial cells
secrete the enzymes that is necessary for the final assembly of the collagen.
7
The different collagen types differ in the composition of individual procollagen
subunits and sometimes, the individual subunits are not identical in the final
triple helix (see the table above).
8
Laminins are another proteins that occur mostly in basal lamina between
mesenchyme and epithelial cells.
It is composed of three different protein chains that wound around each other at
their carboxyl ends. Different regions of the lamininin molecule include sites
recognized by other proteins.
B2 chain has a domain that interacts with type IV collagen, another important
component of the basal lamina; the chain A binds to a proteoglycan heparin.
9
Fibronectin is a heterodimer glycoprotein that serves as a bridge between
extracellular matrix and cellular integral proteins (see also slide #14).
Fibronectin contains cell binding motifs “RGD” and “RGDS”. These sequences are
important for the interaction of fibronectin with cell mebrane-associated
molecules called integrins.
The above described glycoproteins (collagen, laminin and fibronectin) form much
of the extracellular matrix.
10
A specific portion of the extracellular matrix macromolecules is formed by
proteoglycans.
Proteoglycans are formed of proteins and large amount of unusual
polysacharides called glycosaminoglycans (GAGs).
These macromolecules are composed of repeating disaccharide subunits
containing acidic sugars, usually glucuronic acid and the sugars usually have
sulfonated hydroxyl groups.
Long GAG polymers are very hydrophilic and bind huge amount of water, leading
to the huge volume relative to their mass. Recall the involvemnt of
proteoglycans-rich vacuoles in the water absorbance during notochord formation
(swelling of its cells, see Lesson 5).
Long GAG molecule, e.g. hyaluronic acid, which is connected via linker
glycoproteins to the small GAGs, e.g. chondroitin sulfate, forming thus typical
“brush” structure of glycoproteins that have important regulatory functions.
11
In contrast to mobile animal cells, the plant cells are engaged in the rigid cell
walls that precludes their movement during development. However, the plant
cells can expand during development in a process that is called elongation (or
elongation growth).
Directionality of the elongation is determined by the arrangement of cellulose
fibrils in the primary cell wall that are embedded in a matrix of hemicellulose
and pectins.
Elongation occurs in the young tissues where the cell walls are elastic, i.e. the
cellulose microfibrils are not cross-linked yet.
Cellulose is glucose homopolymer, while hemicellulose and pectin are
heteropolymeric molecules formed by branch-chained polysacharides. Cellulose,
hemicellulose and pectins are hold together via both covalent and non-covalent
bonds.
12
Integrins are specific molecules that allow interactions of cells with the individual
components of the extracellular matrix.
Extracellular portion of the heterodimer might interact with extracellular matrix
molecules. The intracellular portion might be connected to internal proteins and
transfers the signal to the cell via e.g. interaction with cytoskeleton (see the next
slide) or via regulation of the secondary messenger pathways.
Integrins are heterodimers consisting of two subunits called α and β. Each of the
subunits is encoded by the gene family, leading thus into many possible
combinations of formed heterodimers (see the table on the next slide).
13
Integrins might directly interact with cytoskeleton.
The interaction of integrins with cytoskeleton is complex and is mediated by
other proteins, e.g. talin, vinculin or α-actinin.
14
Different combinations of α and β subunits allow different binding specificity of
integrins.
However, the specificity is not complete, as could be seen e.g. in case of integrin
interactions with collagen or laminin. E.g. integrins consisting of β1 in
combination with α1 or α2 will bind to collagen and laminin, α4 or α5 combined
with β1 will bind to fibronectin while α3 β1 will bind to all three ligands.
Both collagen and fibronectin posses RGD binding motif, which mediates the
interaction with integrins.
The binding integrin specificity is reflected in the functional specificity, too. Some
integrins mediate cell adhesion while do not stimulate cell motility, the other
ones do the opposite.
Cell adhesion is critical for the cell movement (see later).
15
There are several types of intercellular connections in epithelial cells.
Tight junctions form a circumferential seal in the lateral plasma membranes. That results into functional
isolation of the epithelial surface (the lumen) from the lateral plasma membranes and the basal lamina.
Adherens junctions are often basal located to the tight junctions and besides connecting the cells to each
other, they connect the cell surface to the intracellular microfibrils, forming thus intracellular belt
circumventing the epithelial cell.
Desmosomes are specialized connections forming “spot welds”. The integral membrane proteins of
desmosomes connect to the intermediate filaments of the cytoplasm (e.g. keratins, see the figure). Both
desmosomes and adherens junctions utilize “gluer” proteins called cadherins (see later, slide # 25).
Recall the different cell junctions discussed in case of internal and external cell subpopulation and later
trophoblast cell connection during blastocyst formation in mammals (see Lesson 4).
Hemidesmosomes are similar to desmosomes except that they connect intermediate filaments to the
basal lamina, not to other cells.
Gap junctions consist of specific structures called connexons that are formed from membrane-spanning
proteins. When two connexons are connected together, that form an aqueous pore between the two
cells.
In plants, the term epidermis is being used instead of epithelial cells. In the plat cells, plasmodesmata
allow intercellular communications (see the slide #5).
The “welding” of the epithelial cells and thus formation of different junctions is a dynamic process. E.g.
during ectodermal organogenesis, the neural crest cell are derived from the originally tightly associated
cells of the neural epidermis. These cells re-associate again in the target destinations, e.g. in the adrenal
medulla.
16
There are several receptor types involved in the communication between
extracellular space and the cell environment.
These are
A. ion-channel-linked receptors,
B. enzyme-linked receptors and
C. G protein-linked receptors (see next slides).
17
Ad A.
The ion-channel-linked receptors changes its conformation in a response to the
ligand binding, leading to the open/close status of the ion channel.
18
Ad B.
Enzyme-linked receptors interact with ligand and dimerize upon its recognition.
That leads to the autophosphorylation of the intracellular domain. Dependent on
the substrate of the kinase, there are recognized serin/theronin or tyrosin kinase
receptors.
19
Detailed schematic representation of the series of events after activation of the
receptor tyrosin kinase (RTK).
The interaction with ligand leads to the cross-phosphorylation of the dimer that
allows interaction with proteins possessing domains (called SH2 or SH3) that
specifically interact with the phosphorylated tyrosine.
20
Ad C.
Interaction of the G protein-linked receptor activates G protein that binds GTP
(its α subunit). That results into dissociation of the β and γ subunits, which
further activate other downstream targets, e.g. phospholipase C (recall the
phospholipase C-mediated signaling leading to Ca2+ release after fertilization
discussed in lesson 1).
The different receptor types could be combined in frame of one signalling
pathway, e.g. G protein-coupled receptors are combined with ion-channel-linked
receptors in case of phospholipase C-mediated signalling regulating the egg
response after fertilization (see Lesson 1).
21
22
Classical experiments by the Johannes Holtfreter showing the specificity of cell
aggregations.
Cells of the amphibian gastrula were dissected using pipette in the solution
lacking Ca2+. The cells were then cultivated in the medium with Ca2+, which led
to the cell re-aggregation.
Interestingly, when cells derived from different germ layers were mixed together,
the cells were able to sort-out according to their origin, i.e. ectodermal cells
associated with ectodermal, endodermal with endodermal and so on.
Further, the cells were oriented to each other in specific arrangements. E.g. when
the prospective epidermis and mesoderm were mixed together, the epidermal
cells were always oriented on the surface and mesodermal cell in the inner
space.
This suggests that there is a certain specificity of cell-to-cell aggregations.
23
Malcolm Steinberg at the Princeton University find out that the relative strength
of the adhesion leads to the specific cellular pattern. The more adhering cells
tend to locate to the more central positions.
Here, the re-association of cartilage, heart and liver progenitors is shown with
cartilage cells having the strongest adhesion and locating in the centre.
24
There are plenty of cell surface molecules involved in the specific cell adhesion. Two classes of these
molecules are shown in the figure above.
First class is called cell adhesion molecules (CAMs).
The extracellular portion of CAMs consists of 5 looped domains (in the figure above, only three of them
are shown for simplicity) that bear similarity with immunoglobulins; therefore, they are considered as Iglike
molecules.
There are different types of CAMs that are either free or associated with the membrane via glycolipid
anchor or have a hydrophobic domain that passes through the membrane.
Usually, the interactions allowing specific cell adhesion require Ca2+ and are homophilic, i.e. the cells
expressing the same CAM type adhere. However, there are also cell interactions that are heterophilic
and/or do not require Ca2+.
The other class are cadherins.
Cadherins consist of extracellular portion that contains several Ca2+ domains and typically have
hydrophobic transmembrane domain. Cadherins always require Ca2+ and the mediated interactions are
homophilic.
Different tissues are characterized by different cadherin expression. E.g. in the placenta there are Pcadherins,
in the epithelial tissue are E-cadherins and N-cadherins are typical for nervous tissues.
Cadherins are also part of the adherens and desmosome junctions, where cadherins interact at the
cytoplasmic portion of these junctions via linker proteins called catenins with actin or intermediate
filaments.
25
The table shows examples of cell adhesion molecules and types of their
interactions.
26
Scheme of the experiment performed by Masatoshi Takeichi in Kyoto.
Takeichi and his colleagues expressed P- or E-cadherin in mouse L cells that do
not adhere at all. The transformed cells acquired the ability to adhere and when
mixed, the cells expressing the same type of cadherins aggregated (homophilic
type of interaction).
Furthermore, the level of P-cadherins was distinctive for the sorting-out of the
cells and their relative position. I.e. the cells expressing higher amount
aggregated together and located centrally in comparison to cells expressing lower
amounts of P-cadherin.
27
28
There are several of cellular movements or what is called “morphogenic
maneuvers” taking place during morphogenesis. The individual processes listed
here are usually rather complex and it is not easy to discriminate among them
during embryogenesis.
However, the basic classification is useful.
A. Epiboly, i.e. stretching of cells in one direction leads to the elongation of
the cell layer in one direction and thinning of the cells. The resulting
epithelium is sometimes called squamous. The epiboly occurs during
gastrulation in amniotes (discussed in the Lesson 3) in the region of the
prospective dorsal posterior portion of the embryo.
B. Intercalation is reduction of the number of cell layers via introgression of
the two adjacent cell layers. Radial and mediolateral intercalation was also
discussed in case of the amniotes’ gastrulation (Lesson 3).
C. Convergent extension is directional movement of the cells in one direction
leading to the reduction of the cell layer width and its directed elongation.
D. Invagination is movement of the whole portion of the cell layer into the
internal cavity, as observed e.g. in the blastopore formation in amniotes or
ventral furrow formation in Drosophila.
29
E. Involution is the movement of cellular layers to each other, leading into
formation of internal and external cell layers. That could be observed e.g.
during gastrulation in amniotes during involution of the blastopore lip (see
Lesson 3).
F. Migration is the change of the cell motility and adhesion that leads to the
change of its position. This process take place e.g. during neural crest cells
migration (see Lesson 4).
G. Ingression is detaching of the individual cells from the cellular layer and
their migration in the internal cavity. That occurs e.g. in the formation of
neuroblasts from ectoderm during gastrulation of Drosphila (see Lesson 2).
H. Proliferation is change of the shape and position of cellular layers via cell
division that could be targeted (mostly in plants) or not.
30
There are several examples of the above mentioned processes, taking place in
different organisms and in different stages of development.
E.g. invagination could be observed in the neural furrow formation during
gastrulation of Drosophila. The process is driven by expression of two genes,
SNAIL and TWIST. The figure above shows individual stages of the neural furrow
formation and staining of cells that express TWIST using antibodies against TWIST
protein (see figure on the left-hand side, arrows).
Regulation of morphogenesis could be also demonstrated by the regulation of
proliferation in the limb formation in mouse embryo. The apical ectodermal ridge
(AER, discussed in Lesson 5) regulates the proliferation of the adjacent
mesoderm via growth factor production. If the AER is removed and the embryo is
cultured in the medium with fibroblast growth factor FGF4, that leads to the
deregulation of the proliferation and ectopic formation of additional tissues (see
the figure above).
31
32
Changes in motile behavior is involved the regulation of the targeted migration of the
primordial germ cells (PGCs).
In the chicken embryo, PGCs migrate from the extraembryonic endoderm via blood
circulation to the genital ridge. In mammals, the PGCs migrate from the yolk sac
endoderm to the germ ridge via “overland” route through the mesenchyme. In
mammals, it is not clear if the migration occurs also through the circulation and if yes, to
which extent.
The interaction of the RTK cKit, encoded by the W gene with its ligand Mgf, encoded by
the STEEL gene is involved in the complex process of the PGCs migration.
The migrating PGCs express the cKit and the presence of Mgf is congruent with the
migration pathway (see the figure above). The cKit/Mgf interaction ensures that the
PBCs remain attached in the germinal ridge and do not travel outside.
Mutations in genes for either the receptor or the ligand lead to the defects in the
directed migrations of PGCs, as well as defective migration of melanoblasts arising from
the neural crest and of hematopoiesis.
Accordingly, Mgf /cKit colocalization is present in the migrating population of other cell
types, e.g. melanoblasts of the neural crest (see the dorsolateral localization of Mgf
expression in the figure above).
33
Another example of the specificity in the motile behavior is directed migration of neural
crest cells (for the basics of the neural crest migration see Lesson 4).
Neural crest forms from the dorsal portion of the neural tube. The necessary preposition
of the induction of neural crest formation is that the edge of the neural crest must be
adjacent to the prospective epidermis. Production of BMP4 by the epidermal cells was
shown to stimulate neural crest formation.
There are two major routes of the neural crest cells, the earlier route, leading ventrally
along the lateral portion of the neural tube (2 in the figure above) and the later, leading
more dorsal (3).
In the dorsal route, the extracellular matrix contains chondroitin sulfate proteoglycans
that probably hinder the migration during early stages. Later, the inhibitory activity of
the matrix lessens in the dorsolateral route, which allows neural crest migration through
that path during later stages.
Neural crest cells must migrate through long distances and arrive at spatiotemporally
precisely defined locations. That behavior requires complex regulations of the cell
adhesion, i.e. the adhesion is first reduced and later acquired again.
Migrating neural crest cells show reduced levels of N- and E-cadherins and later reduced
levels of N-CAM that were discussed before. That is consistent with their temporary
reduced cell adhesivity.
34
As discussed previously (see Lesson 4), migrating neural crest cells prefer
transition through the antherior (nostral) portion of somites. Interaction of
Ephrin B3 receptor (RTK) with its ligand, Ephrin B1, is probably involved in that
type of specific cell migration.
Neural crest cells as well as anterior portion of somites express Eph3 receptor,
while the posterior somites portion express Ephrin B1 that is tightly bound to
them. In the experiments, where the migrating crest cells were flooded with
soluble Eph B1, the neural cells migrated through both posterior and anterior
portions of somites.
That suggests that if the migrating cell tries top migrate through the posterior
portion, it become “trapped” by the substrate-bound Eph B1. That is supported
by the findings that the neural crest cells can migrate to the area expressing
substrate-bound Eph B1, but their leading protrusions collapse soon.
35
As an example of the complex tissue interactions affecting the morphogenesis in
animals, the kidney formation is shown here.
A. Kidney forms from the mesenchyme that is invaginated by the Wolfian duct. The
mesenchyme produces glials derived neurotrophic factor (GDNF) that induces
formation of ureteric bud (UB), growing from the Wolfian duct into the
metanephrogenic mesenchyme (MM).
B. Ureteric bud evaginates and mesenchyme cells response by the production of
proteoglycans; the bud produces signalling molecule from the Wnt family, Wnt11
(see also next slide).
C. The interaction of growing bud then induces formation of epithelial cells from the
surrounding mesenchyme, leading to mesenchymal cell agregations (A) and
epithelium formation of the tubular kidney system. Remaining mesenchymal cells
form the stroma (S).
D. The ureteric bud further growths and branches that is associated with further
aggregation and mesenchyme production of what is called renal vesicles.
E. Finally, the ureteric bud forms ureter (U) and collecting ducts (CD), which connect
to the nephrons forming from the various kinds of epithelial aggregates in the
mesenchyme. G, glomerulus.
36
The molecular interactions during induction of kidney tubules.
A. As just discussed (see the previous slide), metanephrogenic mesenchyme
induces nephrogenic bud outgrowth via production of GDNF. That signal is
received via heterodimeric RTK cRet. That leads to the expression of Wnt11
and production of proteoglycans (PG). This stimulates its own growth and
also signals the mesenchyme. BMP7 from the duct further stimulates the
surrounding mesenchyme.
B. The ureteric bud induces formation of tubules in the surrounding
mesenchyme. This is done via expression of emX2 expression that further
stimulates production of other signalling molecules, e.g. Wnt11, BMP7 and
various FGFs. These signals help pattern of the formation of tubules and
stroma in the metanephrogenic mesenchyme. Signals are produced by
both, ureteric bud and the mesenchyme (e.g. Wnt4 or BF2 that is produced
by the stroma cells of the developing kidney). These signals further
stimulate differentiation of the tubules in the mesenchyme and in the bud.
37
38
Leaves are formed by the shoot apical meristem (SAM) as previously discussed in
the lesson on the plant embryogenesis (see Lesson 7).
39
Here, we will discuss the mechanisms of leaf primordium development, i.e. how
does a leaf primordium become a leaf.
40
During the leaf formation, the primordium dramatically increases in size. There is
approximately 2500 fold increase in the primordium size during leaf
development.
41
The increase in size is achieved via both cell division (i.e. cell proliferation, thus
similarly as in animals, see above) and cell expansion.
The cell division, however, must be precisely controlled. The unregulated cell
division in plants leads to the formation of undifferentiated and disorganized
mass of plant tissue called callus.
42
In contrast to animals, there is no cell movement during plant development and
organogenesis.
The morphogenetic processes relay mostly on the tight spatiotemporal
regulation of cell division and cell differentiation.
43
44
Plants can be identified by the shape of their leaves. Field guides contain
classification keys based on leaf shape.
45
Plants can be identified by the shape of their leaves. Field guides contain
classification keys based on leaf shape, margins.
46
Plants can be identified by the shape of their leaves. Field guides contain
classification keys based on leaf shape, margins, and complexity.
47
48
49
As mentioned previously, the size and the shape of the leaf is determined by the
differential cell division and cell expansion.
50
51
The AINTEGUMENTA (ANT) gene controls organ size by promoting
expression of cyclin D3. Higher levels of ANT mean more cell cycles and
larger leaves.
ANT is required for control of cell proliferation and encodes a putative
transcriptional regulator similar to AP2. Loss of function alleles have reduced
fertility, abnormal ovules and abnormal lateral organs. Expressed specifically in
the chalaza and in floral organ primordia.
52
The imporatance of the cell divison patterns might be demonstrated on the
differences between the shape of monocotyledonous and dicotyledonous plant
leaves.
53
In long leaves of monocotyledonous plants, the cell division and expansion
occurs mostly in unidirectional orientation.
Initially the primordium expands isodiametrically (in all
directions).
At later stages, the leaf grows by uni- or bi-directional cell
division and expansion.
An animated video of this slide (leaf development over time) is available at:
http://www.vcbio.science.ru.nl/en/virtuallessons/leaf/formation/.
54
In contrast to that, in leaves of dicotyledonous plants, the cell division patterns
are more complex and cell divisions contribute to the width of the blade. Leaf
shapes are formed by persistent growth in isolated regions of the
developing blade.
E.g. In case of what are called serrated or lobbed leaves, the cell divisions occur
in specific positions at the leaf margin. Factors regulating that process (e.g. auxin
maxima formation) will be discussed later.
An animated video of this slide (leaf development over time) is available at:
http://www.vcbio.science.ru.nl/en/virtuallessons/leaf/formation/
55
The spatiotemporal distribution of cell division might be estimated using
transgenic lines carrying transcriptional fusion of CYCLIN gene promoter with
reporter gene, e.g. GUS.
In those plants, the blue staining corresponds to dividing cells. Darker blue
indicates higher levels of gene expression.
56
57
Factors driving the coordinated cell divisions across the leaf blade are mostly not
known.
However, mutations in TCP genes encoding for TF are affected in the coordinated
cell divisions.
In wt of Antirrhinum, growth arrests occurs in a concave wave from
tip to base, resulting in a flat blade (see the inset on the righthand
side).
However, in the mutant in the gene from the TCP family,
CINCINNATA (CIN), the cell cycle progression is affected, with
growth arrest occurring in a convex wave. Too much cell
proliferation at margins causes leaves leads to formation of extra
tissue at the leaf margins that causes the leaf to curl.
58
TCP gene family is named for identified members of the
family:TEOSINTE BRANCHED1 (TB1) (from maize),
CYCLOIDEA (CYC) (from Antirrhinum ), and PROLIFERATING
CELL FACTOR (PCF) (from rice).
TCP genes encode basic-helix-loop-helix transcription factors,
some of which can regulate cell proliferation.
59
Overexpression of miR-JAW lessens the amount of transcription factor made,
resulting in a phenotype that is similar to the cincinnata loss of function mutant
phenotype (for details on miRNAs production and function see the Lesson 10).
60
Arabidopsis leaves are serrated. Cell divisions in sinuses arrest before
those in teeth.
61
The boundary gene CUP-SHAPED COTYLEDONS2 (CUC2)
contributes to the formation of serrations.
As shown in the transgenic lines carrying transcriptional fusion of
the CUC2 promoter with GUS, CUC2 is expressed in leaf
sinuses.
CUC2 is part of the large NAM/CUC3 family of plant-specific
transcription factors, some of which specify organ boundaries.
62
Loss-of CUC2 function causes smooth margins. CUC2 is
necessary for division arrest in sinuses.
In contrast to that, the ectopic expression of CUC2 due to mutation of the
negative regulator of CUC2 expresion, miR164, results into more serrated leaves.
63
64
NPA is a specific inhibitor of polar auxin transport; PIN1 is an auxin efflux carrier
(discussed previously, see Lesson 7 and 8).
65
Summary of the complex regulations affecting the cell division
pattern at the leaf margins in Arabidopsis.
At the sinus, cell cycle arrest by CUC genes is controlled by TCP
and MIR164 genes.
Outgrowth of the tip of the serration is specified by polar auxin
transport, i.e, by the formation of auxin concentration maxima.
As a result, the cell proliferation controls leaf shape. Genes that
promote or restrict division are precisely regulated through
interactions with each other, miRNAs and auxin.
66
67
Compound leaves in the Cardamine hirsuta occurs via induction of
what is called leaflets during early stages of the leaf
development.
White arrowheads in panel g indicate leaflets initiating on leaf primordia 3 and 4.
68
Polar auxin transport mediated by the efflux carriers from the
PIN family is necessary for the leaflet induction and thus the
compound leaf formation.
In the loss-of-function pin1 mutant of Cardamine hirsuta, or in
leaves treated with an inhibitor of polar auxin transport (NPA),
leaflet formation is suppressed.
Arrowhead in (a) indicates initiating leaflet which is absent in a leaf of similar age
in the pin1 mutant (b).
69
PIN1 gene is expressed and PIN1 protein polar localized in the developing leaf in
the position of the prospective leaflet formation. The polar localization of PIN1
results into auxin maxima formation that could be visualized via DR5 activity (see
the right-hand panel).
Up to now it is not completely clear whether auxin accumulation or enhanced
auxin transport is triggering novel organ formation.
The arrowheads point to initiating leaflets. Arrows point to larger leaflets in
which PIN1 is oriented towards the tip of the leaflet. Auxin accumulation is
determined by expression of the auxin-responsive DR5 promoter fused to YFP
(right-hand figure).
70
Auxin maxima precede leaf initiation (yellow arrow), leaflet
initiation (white) and lobe initiation (red).
Auxin flow is indicated by red arrows.
71
72
Besides the role of auxin in the leaflet initiation, members of the what is called
KNOX genes, encoding for KNOTTED1-like homeobox TF in Arabidopsis and
Cardamine hirsuta, is important regulator of leaflet growth.
Downregulation of the SHOOTMERSTEMLESS (STM) gene lead to absence of 4
parallel leaflets typical for WT Cardamine and to the formation of single leaves
(left-handed figure).
In contrast, the ectopic overexpression of KNOTTED1 (KN1), another member of
the homeobox genes, resulted into increased number of leaflet formation
suggesting positive role of KNOX genes in the leaflet formation.
73
In most of the plants with simple leaves, the KNOX genes are
expressed in shoot apical meristem (SAM) but not in incipient
leaf primordia.
74
However, in plants with compound leaves, the KNOX1 is usually induced in the
leaf primordia, leading to the leaflets formation.
Some plants with KNOX expression in leaves make simple leaves due to
secondary morphogenesis – see Bharathan et al., (2002).
75
In plants with compound leaves, over-expression of KNOX1 genes can
make leaves ultra-compound.
76
KNOX1 genes are repressed at the site of leaf primordium
initiation.
Simple leaves (e.g. Arabidopsis) KNOX1 genes stay off .
Compound leaves (e.g. tomato) KNOX1 genes turn on again in
developing leaf primordia, conferring prolonged organogenic
activity on the leaf edges.
77
Boundary genes coordinate auxin gradients and gene
expression patterns during leaf and leaflet initiation and leaf
serrations.
The tomato GOBLET (GOB) gene is related to CUC2 and sets
up boundaries throughout tomato leaf development. GOBLET
expression is shown in red.
In early P3 primordia, GOB is expressed at the leaf margin prior to leaflet
initiation and inhibits maturation in the adjacent area, enabling future leaflet
initiation.
During primary leaflet formation, restricted GOB expression in space and time
allows proper leaflet separation.
In the terminal leaflet, GOB expression enables the development of lobes and
serrations. In plastochron 5 (P5), stripes of GOB expression in the primary leaflet
flanks enable initiation and separation of secondary leaflets.
78
79