Howard Hughes Medical Institute, Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT 84112, USA
* Author for correspondence (e-mail: mario.capecchi{at}genetics.utah.edu)
Accepted 14 October 2003
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SUMMARY |
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Key words: Hox genes, Limb development, Limb defects, Chondrogenesis, Mouse
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Introduction |
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A conundrum in the field of limb development has been establishing where
Hox genes fit into the molecular-genetic program guiding formation and
patterning of the limb elements. Previous misexpression studies have shown
that Hoxc8, Hoxd11 and Hoxd13 can affect chondrocyte
proliferation or cartilage condensation size
(Goff and Tabin, 1997;
Yueh et al., 1998
). Clearly
Hox genes perform crucial roles in limb development as specific combinations
of Hox gene mutations result in severely deformed principal elements. For
example, in mice mutant for Hoxa10, Hoxc10 and Hoxd10, or
mutant for both Hoxa11 and Hoxd11, formation of the femur or
the radius and ulna are drastically affected, respectively
(Fig. 1)
(Davis et al., 1995
;
Wellik and Capecchi,
2003
).
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Finally, an analysis of mice with one functional allele of either Hoxa11 or Hoxd11 (i.e. Hoxa11+/- Hoxd11-/- or Hoxa11-/- Hoxd11+/- mice) reveals an additional role for Hoxa11 and Hoxd11 in postnatal growth of the forelimb zeugopod.
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Materials and methods |
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Embryos were fixed in Bouins or 4% paraformaldehyde/PBS overnight, rinsed in 5% sucrose phosphate and embedded in paraffin wax according to standard procedures. Some embryos were processed in an MVP tissue processor (Ventana Medical Systems). Sections of 10 µm were mounted on slides for standard Hematoxylin and Eosin (H & E) staining.
Newborn skeletons were fixed in 95% ethanol, stained in Alcian Blue (in 76% ethanol/20% acetic acid) at 37°C for 2 days. After rinsing in 95% ethanol, skeletons were then treated with 1% KOH for 4 to 5 hours, stained with Alizarin Red in 2% KOH for 1 hour, and cleared successively in 20% glycerol/1% KOH, 50% glycerol/1% KOH and 95% glycerol.
Cell proliferation and apoptosis assays
For cell proliferation assays, pregnant females were injected with a 10 mM
solution of BrdU in PBS (1 ml per 100 g weight), and embryos were collected 1
hour later. After fixation overnight in 4% paraformaldehyde in PBS at 4°C,
embryos were embedded in paraffin wax. Sections (5 µm) were stained with an
anti-BrdU antibody conjugated with alkaline phosphatase (Boehringer Mannheim).
Enzyme activity was detected using Fast Red (Boehringer Mannheim) or NBT/BCIP
as substrate. Sections developed with Fast Red were mounted in 90% glycerol
and NBT/BCIP sections were counterstained with nuclear Fast Red or Eosin and
mounted with Permount (Fisher Scientific).
For cell proliferation analysis of postnatal growth plates, 5 µm sections were incubated with the PCNA monoclonal antibody (Novocastra). Binding of the primary antibody was visualized using goat anti-mouse secondary and horse anti-goat tertiary antibodies, both conjugated with HRP. Enzyme reactions were carried out with DAB or Vector SG (Vector) as substrate, and sections were counterstained with nuclear Fast Red.
Apoptosis assays were performed on 10 µm paraffin wax sections of 4% paraformaldehyde fixed embryos using the in situ Cell Death Detection Kit, Fluorescein (Boehringer Mannheim) or the Apoptag kit (Oncor/Intergen).
Immunohistochemistry and in situ hybridization
An anti-type II collagen antibody (Developmental Studies Hybridoma Bank)
was used to stain 3% acetic acid/ethanol-fixed (E12.5) or Bouins-fixed
(newborn) paraffin wax-embedded sections. Goat anti-mouse secondary antibody
(Jackson Immunoresearch) conjugated with HRP and Vector SG (Vector) were used
to visualize primary antibody. The anti-type X collagen antibody was kindly
provided by R. Haronen and B. Olsen. After incubation with biotinylated goat
anti-rabbit secondary antibody and ABC reagent (Vector), enzyme reactions used
Vector SG. Newborn sections stained with these antibodies were pretreated with
hyaluronidase (1 mg/ml in PBS for 45 minutes at 37°C).
Embryos for in situ hybridization were fixed in 4% paraformaldehyde in
PBS/0.1% Tween 20 at 4°C for 1-3 days. Whole-mount in situ hybridization
was carried out as described previously
(Boulet and Capecchi, 1996).
Radioactive in situ hybridizations were performed on 6 µm paraffin wax
sections using 33P-labeled RNA probes. Sections were pretreated
with proteinase K, re-fixed, dehydrated and hybridized overnight at
55-56°C. Sections were washed, RNAse-treated and dipped in emulsion
according to standard protocols.
The Sox9 and Ihh probes were generated by PCR
amplification of genomic mouse DNA. The Sox9 probe contains 388
nucleotides from exon 3 (Wright et al.,
1995) and the Ihh probe contains 631 nucleotides from
exon 3 (GenBank X76291). The Pthlh probe was obtained from K. Lee,
the Pthr probe was a gift from B. St-Jacques and K. Lee, the
Bmp2 probe was obtained from B. Hogan, the Ptch probe was a
gift from M. Scott, and Se-Jin Lee kindly provided the Gdf5 probe.
The Fgf10 probe was obtained from D. Ornitz and C. Deng. The
Fgf8 probe was transcribed from a 280 bp Pst to Sac
fragment from exon 5 and the 3' UTR.
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Results |
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The TUNEL assay was used to visualize apoptotic cells in forelimbs of E11.5, E12.5 and E13.5 embryos. The majority of apoptotic cells at E11.5 are observed in the `opaque patch' located between the developing radius and ulna mesenchymal condensations. The number of apoptotic cells in this region was comparable in double mutant embryos and wild-type littermate controls (data not shown). By contrast, when embryos between E12.5 and E13.5 (stage 8-9 limbs) were examined, double mutant embryos displayed an increase in the total number of apoptotic cells relative to control littermates. Excessive TUNEL-positive cells were located in regions showing significant, but lower, levels of apoptosis in normal embryos (Fig. 2I-L).
Chondrogenesis appears to initiate normally in Hoxa11/d11 double mutants
The reduced zeugopod condensation size in double mutant embryos may also
reflect a failure of discrete populations of mesenchymal cells to activate
genes required to turn on the chondrogenic program in the absence of
Hoxa11 and Hoxd11 function. The expression patterns of
several genes involved in the establishment of prechondrogenic condensations
were examined.
The Sox9 gene product is a transcription factor essential for
chondrocyte differentiation, and is expressed in all chondroprogenitors
(Ng et al., 1997;
Zhao et al., 1997
). Cells
lacking Sox9 are unable to switch on expression of chondrocyte-specific genes
or participate in the process of mesenchymal condensation
(Bi et al., 1999
). The
Sox9 expression pattern in
Hoxa11-/-Hoxd11-/- sections appears
comparable with the controls, although the pattern reflects the reduced
zeugopod condensation size characteristic of E12.5 mutant embryos
(Fig. 3A-D and data not
shown).
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We also examined the expression patterns of Bmp2 and Gdf5, but were unable to detect any differences between control and double mutant embryos (data not shown).
Delayed forelimb bud outgrowth is associated with reduction of Fgf8 and Fgf10 expression
The forelimb buds of E9.5 and E10.5 double mutant embryos (limb stage 2-3)
appear identical to those of control littermates (data not shown). At E11.5
(limb stage 5-6), forelimb bud appearance of some double mutants was
distinguishable from control littermates of the same crown-rump length
(Fig. 4A,B). By 12.5 days of
gestation, there was an obvious delay in forelimb bud growth relative to
littermate control embryos, particularly with respect to development of the
handplate (Fig. 4C,D). By
E13.5, double mutant forelimbs were readily distinguishable from normal
littermates (Fig. 4E,F). The
forearms were noticeably shorter and indentations between digits were less
prominent.
|
|
Chondrocyte maturation is severely delayed in Hoxa11/d11 double mutant radius and ulna
After perichondrium formation, chondrocytes in the center of each cartilage
anlage begin to undergo the process of maturation. Prehypertrophic cells
differentiate at the center of the condensation and subsequently mature to
form hypertrophic cells. Growth plates that form at the ends of long bone
cartilage precursors consist of ordered arrays of cells (i.e. in reserve,
proliferating, prehypertrophic and hypertrophic zones)
(Poole, 1991). As shown
previously, condensed mesenchyme that gives rise to the radius and ulna of
Hoxa11/d11 double mutant embryos makes the initial transition to a
type II collagen-expressing chondrocyte fate. However, histological
examination of the double mutants demonstrated that chondrocytes in the radius
and ulna fail to mature in a normal manner. In the double mutant at E16.5,
chondrocytes in radius and ulna condensations showed a uniform appearance with
no evidence of hypertrophic cells, whereas chondrocytes in the humerus and
digits progress normally through their maturation pathway
(Fig. 6A,B and data not
shown).
|
Expression of Ihh is perturbed in Hoxa11/d11 double mutant embryos
To determine the nature of the block in chondrocyte development, we assayed
the expression of a number of genes that are transcribed during the course of
chondrocyte maturation. Type X collagen is specifically expressed in
chondrocytes that have undergone hypertrophy
(Schmid and Linsenmayer,
1985). At E16.5, no type X collagen could be detected in the
radius and ulna of the double mutants (Fig.
6D). Strong staining for type X collagen was visible in the
zeugopod of control embryos at this stage and in the humerus and digits of
both controls and double mutants (Fig.
6C,D and data not shown).
Indian hedgehog plays crucial roles in the regulation of
chondrocyte proliferation and maturation
(Karp et al., 2000;
St-Jacques et al., 1999
).
Initially, Ihh is expressed in the central region of each long bone
cartilage element. At E12.5, transcripts of both Ihh and patched
(Ptch), the Ihh receptor, were detected in the radius and
ulna condensations of Hoxa11/Hoxd11 double mutant embryos, but the
levels of expression of Ihh and Ptch were significantly
reduced relative to littermate controls
(Fig. 7A,B and data not shown).
Subsequently, Ihh expression is progressively restricted to
postmitotic prehypertrophic chondrocytes in growth plates at the proximal and
distal ends of long bones (Bitgood and
McMahon, 1995
; Iwasaki et al.,
1997
; Vortkamp et al.,
1996
). At E15.5 (stage 12 of forelimb development) and E16.5,
Ihh transcripts could not be detected in double mutant radius and
ulna (Fig. 7C,D and data not
shown).
|
Finally, Pthr, the receptor for Pthlh, is normally expressed in the
prehypertrophic cells of the growth plate
(Lee et al., 1996).
Transcripts for Pthr can be detected in the radius and ulna of
wild-type embryos as early as E13.5 (stage 10,
Fig. 7G). However, in
Hoxa11/d11 double mutants at the same limb stage, no Pthr
signal could be seen in the zeugopod (data not shown). Pthr mRNA was
still undetectable in the double mutant radius and ulna at E16.5, although
Pthr expression was readily detectable in the digits of double
mutants at this stage (Fig.
7H). Therefore as late as 16.5 days of gestation, the double
mutant radius and ulna show no detectable histological or molecular signs of
chondrocyte maturation.
To facilitate interpretation of the observed defects in chondrocyte maturation, we examined the expression patterns of Hoxa11 and Hoxd11 in mid-gestation mouse embryos. During the early stages of chondrocyte maturation in the radius and ulna, Hoxa11 and Hoxd11 transcripts are detected in cells surrounding the radius and ulna condensations (Fig. 7I,J). Expression in chondrocytes was not detectable above background.
Although no molecular evidence of chondrocyte maturation was evident at E16.5, a few cells that resemble hypertrophic chondrocytes were discernible in the center of the radius and ulna of newborn Hoxa11/d11 double mutant specimens (Fig. 8A,B,E,F). Small areas of mineralization in the centers of both skeletal elements were revealed by Alizarin Red staining (Fig. 1). Furthermore, Ihh and Pthr transcripts, and type X collagen, were also readily detected in these aberrant maturation centers (Fig. 8C,D,G,H and data not shown).
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Expression of Ihh (Fig. 9G,I) and Pthr (Fig. 9H,J) in the Hoxa11-/-Hoxd11+/- newborn radius and ulna were similar to that observed in controls.
The expression patterns of Hoxa11 and Hoxd11 at late embryonic stages and in newborn forelimb have not previously been reported. In situ hybridization detected Hoxa11-expressing cells surrounding the distal ends of the zeugopod cartilages up to E16.5, while Hoxd11 transcripts could still be distinguished in a similar pattern at E16.5 (Fig. 9K) and E17.5. However, expression of neither Hoxa11 nor Hoxd11 could be detected above background in newborn forelimbs (data not shown).
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Discussion |
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However, the most informative finding is that formation of growth plates at each end of the radius and ulna condensations is extremely delayed. The enormous growth extension of these long bones along the proximodistal axis is crucially dependent on the presence of growth plates at both ends of the condensation. Normally, chondrocyte proliferation and maturation is restricted to the growth plates and longitudinal growth relies on the appropriate balance of several signaling molecules and their receptors within the growth plate. Ihh expression is initially weak in the double mutants and disappears completely before normal localization to separate domains at each end of the condensations occurs. Although the percentage of cells incorporating BrdU in Hoxa11/Hoxd11 double mutants at E16.5 was similar to that observed in control embryos, the spatial distribution of dividing cells was very different. BrdU-positive nuclei were randomly distributed within the radius and ulna condensations, whereas with control embryos, dividing cells were largely restricted to the ends (i.e. within the growth plates). In the absence of growth plates resulting from the enormous delay in chondrocyte maturation, chondrocytes continue to proliferate throughout the condensation in a disorganized manner, and grossly misshapen and shortened bony elements are produced instead of the normal radius and ulna. Extremely delayed formation of abnormal growth plates at the ends of the zeugopod elements in the double mutants suggests that an important function of these Hox genes is to refine the pattern of the zeugopod cartilage elements to ensure appropriate growth of the bones along the proximodistal axis. Further experiments would be required to determine unequivocally whether defective growth plate formation is directly due to the absence of Hoxa11 and Hoxd11 or a secondary consequence of reduced condensation size.
Finally, abnormal growth plate architecture is evident in mice containing
only a single functional allele of Hoxa11 and Hoxd11 (i.e.
Hoxa11+/-Hoxd11-/- or
Hoxa11-/-Hoxd11+/- mice). The radius
and ulna of adult mice of these genotypes are approximately half as long as in
wild-type littermates (Davis et al.,
1995). However, the discrepancy in the length of these long bones
relative to controls becomes evident primarily post-birth, suggesting the
importance of establishing the correct growth plate cellular architecture in
allowing normal postnatal growth of these bones. The observed reduction in
cell density is in contrast to the effect of mutations in the pRB related
genes, p130 and p107 (Cobrinik et al.,
1996
), which are required for the cell cycle withdrawal that
accompanies chondrogenic differentiation, and could indicate that cells are
exiting the cell cycle or differentiating prematurely.
Previous studies on the functions of Hox genes in limb development fall
into two categories: loss-of-function and gain-of-function. By combining
loss-of-function alleles for two or three Hox genes, we now know that the most
3' members of the posterior Hox gene (Abd-B) group (e.g. Hox9
and Hox10 paralogs) are crucial for proper development of the most
proximal limb skeletal element, the stylopod, while Hox11 paralogous
genes function mainly in patterning the zeugopod, and the 5' genes, the
Hox13 paralogs, function predominantly in specification of the most
distal structures, the elements of the autopod
(Davis et al., 1995;
Fromental-Ramain et al.,
1996a
; Fromental-Ramain et
al., 1996b
; Wellik and
Capecchi, 2003
). However, little could be learned regarding
mechanisms of Hox gene function by simple examination of the mutant
phenotypes. More thorough studies have hinted at effects on multiple
processes. For example, while heterochrony or a change in the rate and timing
of cartilage formation was seen in Hoxd13 mutant limbs
(Dollé et al., 1993
), a
loss of cell adhesion and chondrogenic capacity was discovered in
Hoxa13 mutant limb bud cells
(Stadler et al., 2001
).
Gain-of-function studies point to potential roles for Hox genes in control of
proliferation of undifferentiated limb bud mesenchymal cells, as well as cells
in the proliferative zone of developing cartilage
(Goff and Tabin, 1997
;
Morgan and Tabin, 1994
). The
result of controlled overexpression of Hoxc8 suggests that Hox genes
have the capacity to regulate chondrocyte differentiation
(Yueh et al., 1998
). Our
analysis of Hoxa11/Hoxd11 double mutant limbs confirms
multiple roles for Hox genes in patterning of the limb skeleton. Not only did
we find that Hox genes are required for the proper level of cell
proliferation, as evidenced by delayed limb bud outgrowth and reduced BrdU
incorporation, but, in addition, we have obtained further evidence supporting
a role for Hox genes in the control of chondrocyte differentiation, in
particular in the formation of normal growth plates at the ends of the radius
and ulna cartilage elements.
Effects on the expression patterns of Fgf8 and Fgf10,
molecules known to be crucial for limb outgrowth, were identified in double
mutant forelimb buds. Fgf10 mutant mice completely lack limbs
(Min et al., 1998;
Sekine et al., 1999
). In the
absence of Fgf8 expression, limb bud outgrowth is delayed and some
skeletal elements are reduced in size or completely absent
(Dollé et al., 1989
;
Lewandoski et al., 2000
;
Moon and Capecchi, 2000
).
Prior to E10.75, the expression patterns of Hoxa11 and
Hoxd11 overlap in the progress zone
(Davis and Capecchi, 1994
;
Dollé et al., 1989
;
Haack and Gruss, 1993
;
Small and Potter, 1993
).
Because Hoxa11 and Hoxd11 are expressed in the mesenchyme,
but not in the ectoderm of the limb bud, we presume that decreased
Fgf10 expression is more likely to be the primary effect. The reduced
expression of Fgf8 in the anterior AER would then be a secondary
consequence of reduced Fgf10 expression in the anterior distal
mesenchyme (Min et al., 1998
;
Ohuchi et al., 1997
;
Sekine et al., 1999
). The
effects on the anterior regions of both expression patterns do not correlate
obviously with any aspect of the skeletal phenotype of
Hoxa11/Hoxd11 double mutants. However, reduced Fgf
expression levels may translate into a more wide-ranging effect on limb
outgrowth or may reflect a yet to be determined effect on the function of
another crucial limb bud pathway. Previous results on the effects of
mis-expression of Hoxd11 (Goff
and Tabin, 1997
; Morgan and
Tabin, 1994
) and Hoxd12
(Knezevic et al., 1997
) might
be partially explained in terms of an increase in Fgf10 expression in
the anterior limb bud which could, in turn, lead to enhanced growth and
additional condensations (as observed after elevated levels of bFgf
are induced in the chick wing (Riley et
al., 1993
). However, no effect on Shh expression was
detected in Hoxa11/Hoxd11 double mutants at E9.5 or E10.5
(data not shown).
Analysis of Ihh mutants demonstrated that, in addition to a
Pthlh-dependent role in the control of chondrocyte maturation,
Ihh is also essential for normal chondrocyte proliferation
(Karp et al., 2000;
St-Jacques et al., 1999
). In
Ihh mutants, a 50% reduction in cell division in the humerus was seen
between E12.5 and E14.5 (St-Jacques et
al., 1999
). Therefore, it seemed reasonable to postulate that the
failure to maintain Ihh expression might contribute to the shorter
radius and ulna of double mutants. However, the percentage of cells
incorporating BrdU in the 16.5-day Hoxa11-/-
Hoxd11-/- radius and ulna was not reduced relative to
wild-type growth plates. Therefore, although Ihh transcripts had been
below detectable levels for at least 1 day, the mitotic activity of
chondrocytes was not significantly altered. As Pthlh transcripts are
undetectable at the articular surfaces of skeletal elements in
Ihh-/- animals
(St-Jacques et al., 1999
), the
reduction in Pthlh expression seen in double mutants could be a
secondary effect of the absence of Ihh. However, Pthlh
transcripts are present in Wnt5a mutants in the absence of detectable
levels of Ihh (Yang et al.,
2003
).
Development of secondary ossification centers at the distal ends of the
radius and ulna is severely affected in mice with three mutant alleles.
Ossification of the epiphyses is thought to occur by a distinct
Ihh-independent pathway (Vu et
al., 1998). Hoxa11 and Hoxd11 may play a
specific role in the allocation of the population of cells destined to
contribute to the secondary ossification centers or in the maturation of these
cells, again resulting in the refinement of bone pattern.
In summary, it appears that Hoxa11 and Hoxd11 intersect the developmental pathway leading to forelimb zeugopod formation at multiple steps, from affecting the size and shape of the early condensations, to controlling the rate of chondrocyte maturation, and to ensuring that growth plates are properly positioned at the ends of the condensations. Mice with three mutant alleles further revealed a role of these genes in formation of the secondary ossification centers at the ends of these bones. Future experiments will be directed at determining the precise targets for Hox gene regulation in these pathways.
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ACKNOWLEDGMENTS |
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