Hoxd13 expression in the developing limbs of the short-tailed fruit bat,

Carollia perspicillata

Chih-Hsin Chen,a,b Chris J. Cretekos,a John J. Rasweiler IV,c and Richard R. Behringera,b,�

aDepartment of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USAbGraduate Program in Genes and Development, University of Texas Graduate School of Biomedical Sciences at Houston,

Houston, TX 77030, USAcDepartment of Obstetrics and Gynecology, SUNY Downstate Medical Center, Brooklyn, NY 11203, USA�Author for correspondence (email: rrb@mdanderson.org)

SUMMARY Bat forelimbs are highly specialized forsustained flight, providing a unique model to explore thegenetic programs that regulate vertebrate limb diversity.Hoxd9–13 genes are important regulators of stylopodium,zeugopodium, and autopodium development and thusevolutionary changes in their expression profiles andbiochemical activities may contribute to divergent limbmorphologies in vertebrates. We have isolated the genomicregion that includes Hoxd12 and Hoxd13 from Carolliaperspicillata, the short-tailed fruit bat. The bat Hoxd13 geneencodes a protein that shares 95% identity with human and

mouse HOXD13. The expression pattern of bat Hoxd13mRNA during limb development was compared with that ofmouse. In bat and mouse hindlimbs, the expression patternsof Hoxd13 are relatively similar. However, although theforelimb Hoxd13 expression patterns in both organismsduring early limb bud stages are similar, at later stages theydiverge; the anterior expression boundary of bat Hoxd13 isposterior-shifted relative to the mouse. These findings,compared with the Hoxd13 expression profiles of othervertebrates, suggest that divergent Hoxd13 expressionpatterns may contribute to limb morphological variation.

INTRODUCTION

Vertebrate appendage (limb and fin) diversity includes vari-

ations in limb number, the position of limb formation, and

morphology (Gerhart and Kirschner 1997; Carroll et al.

2001). Vertebrate limb consists of the stylopodium (upper arm

and leg), zeugopodium (lower arm and leg), and autopodium

(hand and foot). The development of the vertebrate limb

serves as a paradigm of developmental biology and great

progress has been made in defining the genetic pathways

that regulate limb formation and patterning (Capdevila and

Izpisua-Belmonte 2001; Tickle 2003). HoxA and D 9–13

genes are essential for limb formation and patterning

(Favier and Dolle 1997; Zakany and Duboule 1999). The

various expression patterns of these Hox genes may be im-

portant factors in vertebrate limb evolution (Duboule 1994;

Hinchliffe 2002). Among these Hox genes, Hoxd13 has been

shown to be an important regulator of vertebrate autopod

development (Favier and Dolle 1997; Zakany and Duboule

1999).

In mouse, whole-mount RNA in situ hybridization studies

showed that Hoxd13 expression initiates in the fore- and

hindlimb buds at 9.5 and 10.0 days postcoitus (dpc), respec-

tively. Expression is initially detected in the posterior mesen-

chyme, and then progressively extends to a distal position. At

14.5–15.5 dpc, Hoxd13 transcripts are detected in the me-

senchymal cells surrounding the digit anlagen (Dolle et al.

1989, 1991). Hoxd13 expression has also been detected in the

limb and fin buds of chick and zebrafish, respectively (Sordino

et al. 1995; Nelson et al. 1996).

Interestingly, overexpression ofHoxd12 andHoxd13 in the

prospective posterior region of the zeugopod of mouse limb

buds downregulates Hoxd10, Hoxd11, and Hoxa11, which

are required for zeugopodal and distal stylopodal skeletal el-

ement morphogenesis. These decreases in Hox gene expres-

sion correlate with a reduction in the size of these elements

and malformations (Herault et al. 1997; Peichel et al. 1997).

Mice homozygous for mutations in Hoxd13 develop severe

defects in their autopodal elements, including reduction in

length or loss of phalanges, bone fusions, and the presence of

sixth digit rudiments (Dolle et al. 1993; Davis and Capecchi

1996; Fromental-Ramain et al. 1996). These data suggest that

Hoxd13 is required for autopod growth and patterning. To-

gether, these findings support the idea that the biochemical

activity and pattern of Hoxd13 expression may regulate the

expression of other 50 HoxA and HoxD genes in limb buds;

a Hox expression code could shape the morphology of the

limb.

EVOLUTION & DEVELOPMENT 7:2, 130–141 (2005)

& BLACKWELL PUBLISHING, INC.130

It has become increasingly clear that the limited numbers

of primary model organisms are not sufficient for under-

standing the diversity of developmental mechanisms that have

evolved (Eakin and Behringer 2004). The use of new and

divergent animal models will be useful for understanding the

molecular mechanisms that govern morphological diversity

among species. Bats are mammals that belong to the order

Chiroptera (Wilson 1997; Nowak 1999; Neuweiler 2000). Bats

are very unique among mammals because they are the only

mammals capable of sustained flight. Their ability to fly is

primarily because of their highly modified forelimbs and the

presence of wing membranes (patagia) (Fig. 1, A and B). The

most significant modification of the forelimbs of bats is the

extensive distal expansion of the autopodium and persistence

of interdigital tissue that contributes to the wing membrane.

In contrast to humans and mouse, the second to fifth digits of

the forelimbs of bats are elongated 6- to 8-fold to form a

posteriorly expanded hand plate. In addition, the zeugopodi-

um is about twice as long as the stylopodium of the forelimb

of most bats, whereas these elements are about equal in length

in human and mouse. Furthermore, in most mammals the

ulna is the same length as the radius, whereas the distal ulna

of the bat forelimb is reduced and fused to the radius (Fig.

1B). The specialized forelimb structures of the wing facilitate

the movements required by bats to fly (Nowak 1999; Ri-

chardson 2002). In contrast to the forelimbs, the hindlimbs of

bats are generally quite reduced with essentially no difference

in digit length (Fig. 1C). The molecules that regulate limb

development in bats have not been examined.

We have isolated and characterized the Hoxd13–d12 gen-

omic region from the short-tailed fruit bat, Carollia per-

spicillata. Hoxd13 expression in the developing fore- and

hindlimbs of bat embryos was examined by whole-mount in

situ hybridization and compared with equivalent stages of

mouse limb development. Our analysis reveals initial similar-

ities but subsequent differences inHoxd13 expression patterns

in the developing forelimbs of these two mammals. In con-

trast, Hoxd13 expression patterns in the hindlimbs appear to

be more similar between bat and mouse. These results dem-

onstrate that there are divergent Hoxd13 expression patterns

in the forelimbs of bat embryos relative to the mouse, sug-

gesting that Hoxd13 expression may be a regulator of verte-

brate limb morphological diversity.

MATERIALS AND METHODS

Sources of bat and mouse embryosAll of the bat embryos examined in this study were removed from

pregnant Carollia females collected in the wild on the West Indian

island of Trinidad. The females were generally collected during the

morning with hand nets from diurnal roosts in northern or central

Trinidad. They were temporarily held and transported in specially

designed, darkened cages. These were constructed of wood and

wire mesh and were well ventilated to prevent overheating of the

animals. The bats were then held briefly (for up to 12 h) in an air-

conditioned laboratory in the Department of Life Sciences at the

University of the West Indies (St. Augustine, Trinidad) until proc-

essed. Mouse embryos were generated by timed matings of Swiss

outbred mice (Taconic, Germantown, NY, USA).

Collection and staging of specimensMost adult female C. perspicillata in the wild population on Trini-

dad carry two pregnancies per year and exhibit substantial repro-

ductive synchronization. For many of these females, the first

pregnancy appears to be established between September and early

November, includes a period of postimplantational developmental

delay at the primitive streak stage, and is completed in March or

April (Rasweiler and Badwaik 1997; Badwaik and Rasweiler 2001).

In a previous study, a peak in births was observed in the collection

area around April 1 (Rasweiler and Badwaik 1997; Badwaik and

Rasweiler 2001). Most parous females then apparently conceive

again at a postpartum estrus. In captive animals, this estrus usually

Fig. 1. Anatomy of bat limbs and wings. (A) Adult specimen ofCarollia perspicillata, dorsal view, head to the top. Digits are num-bered with roman numerals. Arrowhead, the distal tip of digit II.Yellow dots, joints. (B and C) Alizarin red (bone) and alcian blue(cartilage) stained juvenile skeleton. (B) Right forelimb, dorsalview, anterior to the top. Black dots, distal tips of phalangeal car-tilage. (C) Right hindlimb, dorsal view, anterior to the left. Thephalanges (P) are numbered with Arabic numerals. C, calcar; H,humerus; M, metacarpal; P1/2, the fused phalanges 1 and 2 of digitI; R, radius; U, ulna. Scale bars: B50.5 cm; C50.25 cm.

Hoxd13 in bat limb development 131Chen et al.

occurs between 3 and 6 days after parturition, but occasionally may

be up to several days later (Rasweiler and Badwaik 1996). The

second pregnancy in the wild population does not appear to in-

clude a significant period of delay. When parous females were

collected and examined during late May in 5 successive years

(2000–2004), most carried conceptuses that had progressed to the

somite stage or beyond (unpublished observations). Based on care-

fully controlled studies of pregnancies in captive-bred C. per-

spicillata (Rasweiler and Badwaik 1997; Badwaik and Rasweiler

2001), this is what would be expected in normal (nondelayed)

pregnancies if conception had occurred at a postpartum estrus

during March or April in the wild.

To obtain postdelay conceptuses at the desired stages from

wild-caught females during their first pregnancy of the reproductive

year, an appropriate collecting period was scheduled by counting

backward from an expected April 1 birth peak. To obtain con-

ceptuses at the desired stages in the second pregnancy, a collecting

period was scheduled by counting forward from the estimated time

of a peak of conceptions in early April. Both techniques worked

well to provide a preponderance of embryos at the required stages.

The harvesting of bat embryos at such stages during May was

further enhanced, however, by also examining the extent of hair

regrowth around the teats of recently parous females. Although

there were exceptions, females were generally found to be more

likely to carry embryos at the stages focused upon in this study if

lactation had ceased and very early hair regrowth was evident

around the teats.

Bat embryos were staged according to the system developed by

Cretekos et al. (2005). The stages and inferred postcoital ages rel-

ative to nondelayed bat pregnancies were: St. 12, approximately 42

dpc; St. 13, approximately 43 dpc; St. 14, approximately 44 dpc; St.

15, approximately 46 dpc; St. 16, approximately 50 dpc. Mouse

embryos were staged according to the number of tail somites pos-

terior to the hindlimb bud (Hacker et al. 1995). Mouse embryos

with eight tail somites are approximately 10.5 dpc, 18 somites are

11.5 dpc, and 30 somites are approximately 12.5 dpc.

Skeletal analysisThe juvenile bat skeleton was stained with alizarin red for bone and

alcian blue for cartilage as described for neonatal mice (Nagy et al.,

2003).

Isolation of C. perspicillata Hoxd13Primers for polymerase chain reaction (PCR) amplification were

designed based on the conserved protein sequences LGYGYH

and YISMEGYQ in human, mouse, and chick HOXD13 proteins.

The forward and reverse primers were 50-CT(G/C)GGCTA(C/T)

GGCTACCAC-30 and 50-TG(G/A)TA(C/G)CCCTCCATGGA-

GATG-30, respectively. Using these primers, a C. perspicillata

(Cpe) Hoxd13 fragment was amplified, cloned into pBluescipt II

KS (� ) (Stratagene, La Jolla, CA, USA), and sequenced (March-

uk et al. 1991). A C. perspicillata genomic library composed of 94

sub-libraries (Cretekos et al. 2001) was screened by PCR using the

primer set 50-CTGGGCTACGGCTACCAC-30 and 50-TGGTA-

GCCCTCCATGGAGATG-30. PCR-positive sub-libraries were

further confirmed using two nested bat-specific primers, 50-ACGG-

TGTGGGCTTACAG-30 and 50-CGGAGCCGAAGGTAGAC-

ACC-30. The CpeHoxd13-specific probe was subsequently used to

screen the PCR-positive sub-libraries to purify CpeHoxd13 gen-

omic clones. Four genomic clones were isolated, spanning approx-

imately 21kb. The 21kb genomic DNA was digested with PstI or

SacI and individual fragments were subcloned and sequenced.

DNA sequences were aligned using the SeqMan program (DNA-

STAR, Madison, WI, USA).

Comparative analysis of genomic DNA and protein

sequencesVISTA (http://www.gsd.lbl.gov/vista/index.shtml) is a comprehen-

sive suite of programs and databases for comparative analysis of

genomic sequences. VISTA analysis was performed using a win-

dow size of 100bp. The Clustal method was used to compare

HOXD13 proteins (Higgins and Sharp 1988). Accession numbers

for the protein sequences are human HOXD13 (HOX-4I),

NP_000514 (Muragaki et al. 1996); mouse HOXD13 (HOX-4.8),

P70217 (Herault et al. 1996); chick HOXD13 (CHOX-4.8/4G),

P24344 (Rogina and Upholt 1993); horn shark HOXD13,

AAF44637 (Kim et al. 2000); and zebrafish HOXD13,

CAA61031 (Herault et al. 1996).

Whole-mount in situ hybridizationWhole-mount in situ hybridization was performed according to Xu

and Wilkinson (1999). Proteinase K digestion of bat embryos was

6min for St. 12, 8–10min for St. 13, 12–15min for St. 14, 18–

20min for St. 15, and 25–30min for St. 16. A ApaI–BamHI

CpeHoxd13 genomic fragment containing exon 1 was used as a

template to synthesize riboprobes. A mouse Hoxd13 cDNA that

includes sequence from within the homeobox to the 30 untranslated

region was used as a template to synthesize riboprobes (Dolle et al.

1991).

RESULTS

Isolation and structure of the bat Hoxd13 locus

PCR was used to amplify a Carollia Hoxd13 genomic frag-

ment and to isolate phage clones from the Carollia genomic

library (Cretekos et al. 2001). Two Hoxd13-containing

clones that spanned 20,332bp (GenBank accession number

AY744676) were isolated and completely sequenced. Se-

quence analysis comparisons with human and mouse gen-

omes revealed that in addition to Hoxd13, the bat clones also

includedHoxd12 (Fig. 2A). These comparisons were also used

to determine the structure of the bat Hoxd13 coding region

and exon–intron junctions (Fig. 2A). The deduced amino acid

sequence of bat HOXD13 had mammalian HOXD13 char-

acteristics, including a HOXD13-specific homeodomain, and

polyserine and polyalanine stretches (Figs. 3 and 4). Align-

ment of the bat HOXD13 amino acid sequence with other

vertebrate HOXD13 orthologs showed 95.20% identity with

human HOXD13, 94.59% identity with mouse HOXD13,

76.88% identity with chick HOXD13, 32.43% identity with

zebrafish HOXD13, and 53.45% identity with horn shark

132 EVOLUTION & DEVELOPMENT Vol. 7, No. 2, March^April 2005

HOXD13. The HOXD13 polyserine stretch was present only

in the three mammals although the number of serine residues

varied among the species. In addition, the three mammals also

have the same number of alanine residues in the HOXD13

polyalanine stretch. The four tetrapods share identical homeo-

domains.

Previous studies have identified cis-elements that regulate

mouse Hoxd13 and Hoxd12 transcription. In mouse, RX de-

fines the anterior boundary of Hoxd12 expression in sclero-

tome (Beckers and Duboule 1998); RXI directs Hoxd12

expression in the posterior margin of the limb buds (Herault

et al. 1998); and RXII is a transcriptional insulator that pre-

vents 50 HoxD gene expression in the central nervous system

(Kmita et al. 2002b). RXII is composed of two elements

(RXIIA and RXIIB) that function redundantly (Kmita et al.

2002b). Sequence analysis revealed that three of these ele-

ments (RX, RXI, and RXIIB) also exist in the Hoxd13–d12

locus of bat and humans (Fig. 2, A and B). Each element

shows approximately 90% identity among the three mam-

mals. The bat genomic DNA clones did not contain RXIIA

presumably because it resides outside of the genomic interval

covered by our clones.

VISTA is a software program that permits the visualiza-

tion of DNA sequence alignments (Mayor et al. 2000). VIS-

TA analysis of bat, mouse, and humans also revealed

homologies between the loci of the three mammals (Fig.

2B). There are 43 regions with475% homology between bat

and humans, 23 regions between bat and mouse, and 29 re-

gions between humans and mouse. These findings suggest that

bat and human share more similarity within the Hoxd13–d12

locus compared with the homologous region in mouse.

Expression of Hoxd13 during bat limbdevelopment

Hoxd13 expression was examined in St. 12–16 (approximately

42–50dpc) bat embryos during limb formation (Figs. 5, 6, A–

J, and 7, A–G). Hoxd13 expression is first detected in the

posterior region of the forelimb buds at St. 13 (Figs. 5A and

6A). This expression domain expands anteriorly until late St.

14 (Figs. 5, B–D and 6, B–D). The anterior limit of the ex-

pression domain is unchanged from late St. 14 to early St. 15

(Figs. 5, D–F and 6, D–F). From early St. 15 to early St. 16,

the expression domain expands distally with limb outgrowth

(Figs. 5, E–I and 6, E–I). At St. 15 and late St. 15, me-

senchymal condensations are easily observed in the posterior

limb region (Figs. 5, G and H and 6, G and H), which will

subsequently generate the fourth and fifth digits. At early

Hoxd13 Hoxd12

RXIIB RXI RX

Bat

Human

Mouse

1kb

A

B

RXIIA

Bat/Human

Bat/Mouse

Human/Mouse

RXIIB

Hoxd13CDS1 CDS2

Hoxd12CDS1RX1 RXCDS2

0 2500 5000 7500 10000 12500 15000 17500 20000 bp

Fig. 2. Structure of bat Hoxd13 and Hoxd12 genes and comparison with humans and mouse. (A) Bat, human, and mouse Hoxd13 andHoxd12 coding and flanking regions. The dashed lines define the synteny. Solid boxes, Hoxd13 and Hoxd12 coding regions. Threeconserved intergenic regions (RX, RXI, RXII) are cis-elements for Hoxd gene transcription. In mouse, RXII has two conserved regions:RXIIA and RXIIB. Shaded bar, ApaI–BamHI bat genomic DNA fragment used for whole-mount in situ hybridization. (B) VISTA analysisof bat, human, and mouse Hoxd13/d12 genomic region. The Hoxd13 and Hoxd12 coding sequences (CDS) and three conserved intergenicregions (RX, RXI, and RXIIB) are indicated by solid bars. Horizontal axis, base pairs; vertical axis, % sequence homology.

Hoxd13 in bat limb development 133Chen et al.

St. 16, the mesenchymal condensations for the anteriormost

three digits are apparent (Figs. 5I and 6I). Hoxd13 expression

is less strong in the mesenchymal condensation regions in

comparison with interdigital tissue. The anterior and posterior

limits of expression extend bi-directionally to the junction of

the zeugopod and autopod (Figs. 5, G–I and 6, G–I). At St.

16, expression appears reduced in the interdigital regions and

expression is predominantly adjacent to the condensed me-

senchymal regions and in the interdigital regions between

digits three and four (Figs. 5J and 6J).

Hoxd13 expression was also examined in the forming

hindlimbs (Figs. 5 and 7, A–G). Hoxd13 expression in hind-

limb buds was not detected until late stage 14 (Figs. 5D and

7A). The initial expression of Hoxd13 in the hindlimb buds

extended more anterior relative to the initial expression found

in the forelimb buds (Figs. 6A and 7A). Unlike Hoxd13 ex-

pression in forelimb buds, hindlimb expression continues to

expand anteriorly until early St. 15, resulting in a symmetrical

anterior–posterior expression domain (Fig. 7, B and C). The

reduced expression in mesenchymal condensation regions be-

gins at late St. 15 (Fig. 7E). At St. 16, Hoxd13 expression is

predominantly adjacent to the digit condensations (Fig. 7G).

Hoxd13 expression was also detected in the genital tubercle

region from early St. 14 but not in the tail bud (data not

shown).

Comparison of Hoxd13 limb expression betweenbat and mouse

For this study, we used forelimb morphological features, in-

cluding limb bud shape and mesenchyme condensations, to

suggest developmental equivalent stages for bat and mouse.

We compared the Hoxd13 expression profile in the forelimb

and hindlimb, in bat and mouse, respectively (Figs. 6 and 7).

In the forelimb, Hoxd13 expression is initially detected in bat

limb buds at St. 13, whereas Hoxd13 expression is initially

detected in mouse forelimb buds at 10.0dpc (Fig. 6, A and K).

Bat forelimb buds at St. 13 have an anterior–posterior length

to proximal–distal length ratio of about 5:2; 10.0dpc mouse

forelimb buds have a similar ratio. Bat and mouse forelimb

Fig. 3. Bat Hoxd13 coding region. Arrows, primer region used for isolating a Carollia Hoxd13 genomic fragment; bold and shaded aminoacids, polyserine and polyalanine stretches; arrowhead, predicted exon junction; boxed amino acids, homeodomain; asterisk, stop codon.

134 EVOLUTION & DEVELOPMENT Vol. 7, No. 2, March^April 2005

buds continue to grow distally, and their gene expression

domains expand anteriorly at late St. 13 and 10.5dpc,

respectively (Fig. 6, B and L). The limb buds of the two

species continue growing to a proximal–distal length that is

longer than their anterior–posterior length at early St. 14 for

bat and 11.0dpc for mouse (Fig. 6, C andM). At these stages,

both of their Hoxd13 expression domains spread anteriorly.

However, the anterior expression limit of the bat is located

near the distal tip of the forelimb bud, whereas the mouse

Hoxd13 anterior limit is located on the anterior side of the

limb bud (Fig. 6, C and M). Subsequently, the distal region of

the bat limb expands from late St. 14 to early St. 15. During

these stages there is an expansion of tissue adjacent to the

posterior forelimb that will form the plagiopatagium (Fig. 6,

D–F). Similarly, the distal region of the mouse limb bud ex-

pands at 11.5dpc (Fig. 6N). From late St. 14 to early St. 15,

the anterior limit of bat Hoxd13 expression in the limb is

located at the one o’clock position (Fig. 6, D–F), whereas

the anterior limit of mouse Hoxd13 expression at 11.5dpc

is located at the twelve o’clock position (Fig. 6N). At early

St. 15, the bat limb posterior autopod expands to a greater

extent than the anterior and the expression pattern of

Hoxd13 correlates with this posterior growth (Fig. 6, E and

F). The margin of the limb is quite smooth except the most

posterior region that becomes scalloped between digits four

and five from early St. 16 (Fig. 6, I and J). From 12.0 to

12.5dpc, the mouse limb continues to grow and the

margin becomes scalloped (Fig. 6, O and P). Hoxd13 expres-

sion is maintained and the anterior and posterior expression

limits are at the arm–hand junction in both species (Fig. 6, J

and P).

In bat, forelimb and hindlimb development looks very

different. Unlike the forelimb, the hindlimb always exhibits

symmetrical growth along the anterior–posterior axis (Figs. 6,

A–J and 7, A–G) and the resulting digits of the foot show no

overt anterior–posterior patterning before St. 18, that is each

digit looks identical to the others (unpublished results). After

St. 18, phalanges 1 and 2 of digit one fuse together; however,

all five digits are still identical in length (Fig. 1C). In mouse,

there is anterior–posterior patterning of the hindlimb digits,

with notable differences between digit one and the other dig-

its. Interestingly, after an initial posterior–distal expression

pattern (Fig. 7, A and B), Hoxd13 expression in the forming

bat hindlimb shows an anterior–posterior symmetry (Fig. 7,

C–G). This is in contrast to the mouse Hoxd13 expression

pattern in the hindlimb that shows an asymmetric pattern,

biased posteriorly with a relatively weak expression in the

anteriormost region (Fig. 7, K and L).

Fig. 4. HOXD13 alignment in vertebrates. Identical amino acid residues are shaded. Four domains are marked: I and III, polyserinestretch; II, polyalanine stretch; IV, homeodomain.

Hoxd13 in bat limb development 135Chen et al.

DISCUSSION

Here we describe the isolation of the batHoxd13 andHoxd12

genomic region from C. perspicillata and establish the first

temporal and spatial expression profiles for a Hox gene

during bat embryogenesis. Our expression studies reveal an-

terior differences in the limits of Hoxd13 forelimb expression

between bat and mouse embryos but more similar patterns

during hindlimb development.

The deduced overall amino acid sequence of bat HOXD13

is highly identical (approximately 95%) to the reported mam-

malian HOXD13 proteins but much less conserved among

nonmammalian chordates (Herault et al. 1996; Muragaki et

al. 1996). Whereas the HOXD13 homeodomains of the teleost

zebrafish and the chondrichthyes horn shark (Heterodontus

francisci) have diverged somewhat from the mammals, the

HOXD13 homeodomains of mammal and the chick are

identical (Rogina and Upholt 1993; Herault et al. 1996; Kim

et al. 2000). In addition, the three mammalian orthologs

studied here have the same number of alanines in the con-

served polyalanine stretch. Previous studies have shown that

mutations in either the homeodomain or polyalanine stretch

can cause synpolydactyly, a dominantly inherited malforma-

tion of the distal limbs, in humans and mouse (Ashley and

Warren 1995; Warren 1997; Bruneau et al. 2001; Goodman

et al. 2001; Debeer et al. 2002). The mutations in the homeo-

domain probably reduce the binding activity of HOXD13 for

DNA. The expansion of the polyalanine domain may function

to provide space between other domains, or protein interac-

tion sites for other transcription factors (Goodman 2002).

Because bat, mouse, and human HOXD13 are nearly identical

overall and have identical known functional domains, it seems

likely that they would have identical biochemical activity. In

contrast, chick HOXD13 has six fewer alanines within the

polyalanine domain and no polyserine domain. Moreover, the

two fish HOXD13 proteins do not have polyalanine or po-

lyserine domains, and have several amino acid differences in

the homeodomain. Thus, there are likely to be biochemical

differences between mammalian and nonmammalian HO-

XD13 proteins as well as between tetrapods and fish.

Bat and humans were found to share higher overall

sequence identity within the Hoxd13–Hoxd12 locus in

Fig. 5. Whole-mount in situ hy-bridization analysis of Hoxd13 ex-pression in bat embryos. Dashedline, distal boundary of the fore-limb buds; arrowhead indicates in-itial Hoxd13 expression in (A) and(B). St. 13, approximately 43dpc;St. 14, approximately 44dpc; St.15, approximately 46dpc; St. 16,approximately 50dpc. (E) and (F)are both early St. 15 but (F) ismore advanced than (E).

136 EVOLUTION & DEVELOPMENT Vol. 7, No. 2, March^April 2005

comparison with mouse. VISTA analysis of the bat Hoxd13–

Hoxd12 locus sequence led to the identification of conserved

intergenic regions that in mouse function as cis-acting

transcriptional elements that regulate Hoxd13 and Hoxd12

expression (Beckers and Duboule 1998; Herault et al. 1998;

Kmita et al. 2002b). Thus, these sequences may have similar

functions in bats.

The expression patterns of Hoxd13 in the developing fore-

and hindlimbs of bat and mouse embryos are summarized in

Fig. 8. The initiation of Hoxd13 expression appears to be

similar in both mammals (i.e., expression initiates in a discrete

posterior domain), although we were unable to detect this in

the bat hindlimb buds. The most significant difference in the

expression patterns of Hoxd13 between bat and mouse was

that Hoxd13 expression in the bat forelimb bud is posteriorly

shifted relative to mouse. Interestingly, this posterior restric-

tion relative to mouse corresponds with the posterior–distal

expansion of the bat forelimb autopod. Another significant

difference is that in the hindlimbs, batHoxd13 expression was

not detected in early limb bud stages but was detected at late

limb bud stages, corresponding to the phase when 50 Hox

gene expression changes from a more posterior localization to

a more distal and anteriorized expression pattern (Nelson

et al. 1996). At this later stage, Hoxd13 expression in the bat

hindlimb resembles that of the mouse forelimb and hindlimb,

that is all three exhibit an anterior–posterior symmetric ex-

pression domain. However, Hoxd13 expression in the mouse

hindlimb appears to have an anterior–posterior asymmetry

(anterior expression relatively weak in comparison with pos-

terior), whereas the bat is much more symmetric. These dif-

ferences correlate with the anterior–posterior asymmetry and

the symmetry found in the length of digits of mouse and bat

feet, respectively.

Hoxd13 expression in mouse, chick, and zebrafish, at early

limb/pectoral fin bud stages (mouse: 10.0/10.5dpc; chick: St.

19/20; and zebrafish: late prim-15/high-pec stages), is similar

Fig. 6. Comparison ofHoxd13 expression in forelimbs of bat and mouse embryos. (A–J)Hoxd13 expression in bat forelimbs between stage13 and 16. (E) and (F) are early St. 15 but (F) is more advanced than (E). (K–P) Hoxd13 expression in mouse forelimbs between 10.0 and12.5dpc. Arrowheads indicate initial Hoxd13 expression in (A) and (K). Open arrows in (D–J) indicate developing plagioplatagium. Ant,anterior; Dist, distal; Post, posterior; Prox, proximal.

Hoxd13 in bat limb development 137Chen et al.

(i.e., it is restricted to the posterior mesenchyme of the limb/

pectoral fin buds) among these three vertebrates (Kimmel et

al. 1995; Sordino et al. 1995; Nelson et al. 1996) and the initial

spatial pattern of expression ofHoxd13 in the forelimbs of the

bat embryo (St. 12) is the same. However, chick and zebrafish

Hoxd13 expression show differences in comparison with the

two mammals. In chick,Hoxd13 expression is first detected in

the hindlimb buds at St. 18 and subsequently in the forelimb

buds at St. 19. This is the opposite of what is found in bat and

mouse, in which Hoxd13 expression is first detected in fore-

limb buds and then later in hindlimb buds (Nelson et al.

1996). In addition to this temporal difference there is also a

spatial difference inHoxd13 expression between chick and the

two mammals. In chick at St. 27 (early limb plate stage),

Hoxd13 is expressed in the distal forelimb but this expression

does not extend to the anteriormost and posteriormost mar-

gins of the limb plate. However, expression in the hindlimb is

posteriorly shifted like the bat forelimb (Nelson et al. 1996;

Wada and Nohno 2001). Chick forelimbs and hindlimbs have

three and four digits, respectively, perhaps a consequence of

these distinct patterns of Hoxd13 expression. In zebrafish,

there is some Hoxd13 expression in older fins that is found in

the posterior margin and distally but apparently absent from

the fin fold (Sordino et al. 1995; Neumann et al. 1999). The

pattern of Hoxd13 expression and its levels are important

for regulating limb skeletal pattern and morphology (Dolle

et al. 1993; Davis and Capecchi 1996; Fromental-Ramain

et al. 1996; Herault et al. 1997; Peichel et al. 1997). These

divergent patterns of Hoxd13 expression in bat, mouse, chick,

and zebrafish suggest that Hoxd13 may contribute to the

divergent autopodal morphologies of these diverse vertebrate

species.

Currently, the mechanisms that regulate Hoxd13 expres-

sion during limb development are starting to become known

(Kmita et al., 2002a; Spitz et al., 2003; Zakany et al., 2004). In

mouse, at early limb bud stages, Hoxd13 expression initiates

in a posterior–distal domain, perhaps by Hand2 (Fernandez-

Teran et al. 2000; te Welscher et al. 2002a). Expression ap-

pears to be restricted to this posterior domain by Gli3, which

is present in the anterior region of the limb bud (Zuniga and

Zeller 1999; te Welscher et al. 2002a,b). At later stages, the

maintenance of Hoxd13 expression is dependent upon Shh,

whereas Gli3 controls the anterior extent of expression

(Zuniga and Zeller 1999; Litingtung et al. 2002; te Welscher

et al. 2002b).

Here we suggest a model to explain the differences between

bat and mouse Hoxd13 expression and the respective limb

morphologies of these two mammals. At early limb bud stag-

es, the initiation of Hoxd13 expression in the forelimbs of

both mammals, as well as the mouse hindlimb, is generated by

Fig. 7. Comparison of Hoxd13 ex-pression in hindlimbs of bat andmouse embryos. (A–G) Hoxd13expression in bat hindlimbs be-tween late stage 14 and 16. (B)and (C) are early St. 15 but (C) ismore advanced than (B). (H–L)Hoxd13 expression in mouse hind-limbs between 10.5 and 12.5 dpc.Ant, anterior; Dist, distal; Post,posterior; Prox, proximal.

138 EVOLUTION & DEVELOPMENT Vol. 7, No. 2, March^April 2005

similar signaling centers located in the posterior limb buds.

Gli3 expression in the anterior limb bud would restrict

Hoxd13 expression in the posterior position. However,

Hoxd13 expression in bat hindlimbs is undetectable by whole-

mount in situ hybridization until late limb bud stages. This

may be caused by a delay in the establishment of the Hand2-

regulated signaling center, and/or the existence of other in-

hibitory factors. At late limb bud stages, Shh expression in the

zone of polarizing activity (ZPA) could initiate Hoxd13 ex-

pression in the hindlimb. In addition, Shh could maintain

Hoxd13 expression and expand its domain anteriorly in both

the forelimb and hindlimb. Gli3 would also determine the

position of the anterior expression margin. In the bat fore-

limb, the anterior expression margin is more posterior in

comparison with mouse possibly because of a different Gli3

expression profile in bat forelimb buds relative to mouse. Al-

ternatively, any variation in the signaling configuration of

SHH, as well as BMP and FGF4, which are important for the

establishment of the SHH signaling center, could cause the

Hoxd13 expression divergence (Zuniga et al. 1999; Niswander

2003). Unlike mouse, the bat forelimb could have a unique

SHH/FGF4/BMP and GLI3 antagonism system, to cause the

posterior tilt in the Hoxd13 expression domain and also the

posterior expansion of the limb plate. Hoxd13 misexpression

has been shown to reduce 50 HoxA and 50 HoxD transcription

in the prospective zeugopod (Herault et al. 1997; Peichel et al.

1997). As the bat forelimb Hoxd13 expression domain is pos-

terior-shifted relative to mouse from late limb bud stages, the

posterior region of Hoxd13 expression may interact with the

posterio-distal-most part of the zeugopodal Hox expression

codes, and thus downregulate their expression at the

zeugopodal–autopod boundary. This could diminish zeugo-

podal skeleton morphogenesis (i.e., distal ulna) and result in a

distal regressive ulna. In contrast, bat hindlimb Hoxd13 ex-

pression is comparable to the mouse from late limb bud

stages; both mammals have well-developed fibulae. Exami-

nation of candidate signaling molecules and other 50 HoxA

and 50 HoxD gene expression profiles in developing bat and

mouse limbs should help to clarify this model.

AcknowledgmentsWe thank the Department of Life Sciences, University of the WestIndies, St. Augustine, Trinidad, and particularly Dr. Indira Omah-Maharaj, for generous assistance and the use of departmental facil-ities during the course of the field work. We are also grateful toSimeon Williams for field assistance, Peter Akinwunmi for skeletonpreparation, and Drs. Scott Weatherbee and Lee Niswander forhelpful comments on the manuscript. Finally, we appreciate the

Forelimb

Hindlimb

Mouse

10.5 dpc 11.0 dpc 11.5 dpc 12.5 dpc

Mouse

12.5 dpc11.5 dpc11.0 dpc10.5 dpc

Bat

Late St. 13 Early St. 14 Early St. 15 Early St. 16

Bat

Late St. 13 Early St. 14 Early St. 15 Early St. 16

*

*

*

*

Ant

Post

Prox Dist

Ant

Post

Prox Dist

Fig. 8. Summary of Hoxd13 expres-sion comparison between bat andmouse during limb development.Filled regions, Hoxd13 expressiondomains; asterisks, divergent anteri-or limits of Hoxd13 expression; grayto black gradient shown in 12.5dpcmouse hindlimb indicates anterior–posterior expression level (lower tohigher) asymmetry. Limbs are notdrawn to scale between species andstages. Ant, anterior; Dist, distal;Post, posterior; Prox, proximal.

Hoxd13 in bat limb development 139Chen et al.

assistance of the Wildlife Section, Forestry Division, Ministry ofPublic Utilities and the Environment in providing required collectingand export licenses. C. J. C. was supported by National Institutes ofHealth postdoctoral fellowships CA09299 and HD07325. This workwas supported by a grant from the National Science FoundationIBN 0220458 to R. R. B. DNA sequencing resources were supportedby the National Institutes of Health Cancer Center support grantCA16672.

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