TECHNOLOGY REPORT

Generation of Conditional Hoxc8 Loss-of-Function andHoxc8?Hoxc9 Replacement Alleles in MiceJessica Blackburn, Melissa Rich, Nima Ghitani, and Jeh-Ping Liu*Department of Neuroscience, University of Virginia School of Medicine, Charlottesville, Virginia

Received 23 April 2009; Revised 8 June 2009; Accepted 12 June 2009

Summary: The Hox family of transcription factors areexpressed at different domains along the rostrocaudal(R-C) body axis during development. To examine thefunction of Hoxc8 and Hoxc9 in specific cell types and atdifferent developmental times, we have generatedand characterized loxP flanked (floxed) Hoxc8 andHoxc8?Hoxc9 replacement alleles of mice, with eitherGFP or LacZ reporters. Although all four alleles of micebehave like wild-type controls in motor behavioral test-ing, slight differences in endogenous Hox gene expres-sion were observed among these alleles depending onthe type of reporters used and the presence of Hoxc9cDNA in the targeting constructs. The efficiency of Cre-mediated recombination was evaluated by crossingthese mice with the Nestin-cre and Isl1-cre mice, andthe loss of Hoxc8 expression with or without Hoxc9 mis-expression was confirmed in embryonic spinal cord. Inaddition, an upregulation of reporter gene expressionwas observed after Cre-mediated recombination. Thesemice will be useful tools to analyze Hox gene function ina cell type-specific manner. genesis 47:680–687, 2009.VVC 2009 Wiley-Liss, Inc.

Key words: Hoxc8; Hoxc9; gene targeting; Cre-loxP;mouse

Homeotic/Hox genes play important roles in definingcellular identity along the rostrocaudal (R-C) body axisduring development (Krumlauf, 1994). The function ofHox genes in determining neuronal identity in the hind-brain has been well studied (Keynes and Krumlauf,1994), while much less is known about their roles inspinal cord development. The expression domains ofvarious Hox-c genes have been shown to correlate withthe positions of motor neuron (MN) columns and pools(Dasen et al., 2003, 2005; Liu et al., 2001). In addition,both gain- and loss-of-function experiments performedin chick embryos demonstrate that Hox6 and Hox9paralog groups play instructive roles in defining MNcolumnar identity, while the Hox4, Hox5, Hox7, andHoxc8 groups of genes delineate different motor pools(Dasen et al., 2003, 2005). Mouse mutants that have lostHox9 and Hox10 function exhibit locomotion deficits inthe hindlimb region (Carpenter et al., 1997; de la Cruz

et al., 1999; Lin and Carpenter, 2003; Wu et al., 2008),while loss of Hoxc8 function results in a forelimbprehension-deficiency phenotype (Tiret et al., 1998).

Hox genes are expressed in multiple tissues duringdevelopment and their expression patterns change withtime, and therefore, the motor behavior deficitsobserved in Hox mutants could be a compound effect oflosing Hox function in both neural and mesodermal tis-sues. Moreover, neural expression of Hox genes is notlimited to MNs, as many spinal interneurons required forcoordinated locomotion also express various Hox genes.Thus, cell type-specific analyses will be required to fur-ther decipher the role of Hox genes in spinal cord devel-opment. To generate conditional loss-of-function andgain-of-function alleles of Hox genes in mouse, we firstfocused on the Hoxc8 locus, and used the forelimb grip-deficiency phenotype as a landmark to evaluate floxedHoxc8 and Hoxc9 (Hoxc8?c9) alleles of mice.

To generate the floxed Hoxc8 allele, a loxP site wasinserted in the 50noncoding region of the Hoxc8 gene,and a second loxP site in the same orientation wasinserted 30 to the three known Hoxc8 polyadenylation(pA) signals. The endogenous 50 splicing donor site(50SD), Intron 1, and the endogenous 30 splicingacceptor site (30SA) were also inserted downstream ofthe second loxP site. We preserved the endogenousintron not only because it contains essential regulatoryelements (Awgulewitsch et al., 1990), but also becauseit has the potential to stabilize the primary, and splicedtranscript (Huang and Gorman, 1990). A ribosome

Current address for Jessica Blackburn: Department of Biochemistry,

Dartmouth Medical School, Hanover, NH.

Current address for Melissa Rich: MD Program, Virginia Commonwealth

University School of Medicine, Richmond, VA.

Current address for Nima Ghitani: Neuroscience Training Program,

University of Wisconsin, Madison, WI.

* Correspondence to: Jeh-Ping Liu, Department of Neuroscience, Univer-

sity of Virginia, 409 Lane Road, MR4, Rm. 5032, Charlottesville, VA 22908.

E-mail: jl7nf@virginia.edu

Contract grant sponsor: NIH, Contract grant number: NS045933.Published online 20 July 2009 in

Wiley InterScience (www.interscience.wiley.com).

DOI: 10.1002/dvg.20547

' 2009 Wiley-Liss, Inc. genesis 47:680–687 (2009)

reentry site (IRES) followed by either an eGFP(Mombaerts et al., 1996, for tracing axons) or nlsLacZ(nuclear localized LacZ, for marking nuclei) reportercassette was inserted 30 to the 30SA to be used as tracersfor the Cre-mediated recombination event. The endoge-nous 30 untranslated region (30UTR) was added 30 to thereporter sequences. The 30UTR is included to conservepotential miRNA binding sites that regulate the exp-ression of Hoxc8 (Yekta et al., 2004). A neomycin phos-photransferase (Neo) resistance cassette was alsoincluded for positive selection of the transfected ES cells(Fig. 1a).

We also generated conditional Hoxc8?c9 replace-ment alleles to examine the long-term effects of Hoxc9misexpression in mouse. To generate the floxedHoxc8?c9 allele, a DNA fragment containing the Hoxc9coding region was inserted between the second loxPsite and the additional Hoxc8 intron (Fig. 1a). Twofloxed Hoxc8 alleles-one with a GFP reporter, the otherwith a LacZ reporter, and two floxed Hoxc8?c9 alleles,with either GFP or LacZ reporters were generated usingthis strategy.

Since the Hox locus is tightly regulated, any alterationat this locus could potentially affect the expression of sur-rounding genes. We therefore, characterized these floxedalleles prior to Cre-mediated recombination to ascertainthat they behave similar to the wild-type (WT) alleles. Wefirst examined Hoxc6-Hoxc9 mRNA expression in e10.5mouse embryos using whole-mount in situ hybridization.The expression domains of Hoxc6 and Hoxc9 are verysimilar among embryos carrying different floxed alleles

and their WT littermates at e10.5 (data not shown). How-ever, a �1-segment rostral extension in neural and meso-dermal Hoxc8 expression domain was observed in theGFP-tagged Hoxc8?c9F/F (Hoxc8?c9F/F-GFP) embryos(Fig. 2c), while a �2-segment rostral extension in the neu-ral and a �3-segment rostral extension in the mesodermalHoxc8 expression domains were observed in the LacZ-tagged Hoxc8F/F (Hoxc8F/F-LacZ) and Hoxc8?c9F/F

(Hoxc8?c9F/F-LacZ) embryos (Fig. 2d,e). The GFP-taggedHoxc8F/F (Hoxc8F/F-GFP) embryos exhibit no apparentchange in Hoxc8 expression domain as compared to theWT controls (Fig. 2a,b).

To examine the phenotypic consequences of thesechanges in mesodermal tissues, we performed skeletalstaining in e18.5 mouse embryos from different allelesprior to Cre-mediated recombination. WT and theHoxc8F/F-GFP embryos have seven cervical vertebrae(C1–C7) and their 6th and 7th ribs (R6s and R7s) areattached to the sterna (Fig. 2f,g,k,l). However, extra ribsextending from the C7 and elongated R8s attached tothe sterna were observed in Hoxc8F/F-LacZ andHoxc8?c9F/F-LacZ embryos (Fig. 2i,j,n,o). The F/1embryos derived from these two LacZ-tagged alleleshave a milder phenotype with either a partial rib extend-ing from the C7 or only one of the R8s attached to thesternum (data not shown). The majority of theHoxc8?c9F/F-GFP embryos have normal C7 vertebra,but their R8s are attached to the sterna (Fig. 2m). Noobvious homeotic transformation in skeletons wasobserved in the Hoxc8?c9F/1-GFP embryos (data notshown).

FIG. 1. Generation of Hoxc8 conditional loss-of-function and Hoxc8 replaced by Hoxc9 mouse alleles (a) Hoxc8 genomic locus. E1 and E2represent Exon1 and 2, yellow boxes represent coding region, purple lines represent the intron, and pink boxes represent the 30 untranslatedregion (UTR). X 5 Xba1, H 5 HindIII, Sp 5 Sph1, Sc 5 SacII, N 5 Nco1, Sl 5 Sal1, Ea 5 Ear1. Targeting constructs contain �9.5-kbgenomic DNA from the 50 Sph1 site to the 30 Xba1 site. ‘‘IR’’ represents internal ribosome reentry site, ‘‘marker’’ represents either eGFP ornlsLacZ reporters used in various versions of the targeting constructs. A Black box indicates the HindII-Sph1 fragment used as a probe forthe Southern blot shown in (b). Black triangles indicate the positions of LoxP1 and LoxP3 primers used in (c). (b) Southern analyses.Genomic DNA digested with Xba1 and probed with a 500-bp HindIII-Sph1 probe identified a �10.4-kb wild-type (1) band. For the Hoxc8-LacZ allele, the size of the targeted band (F) is �19.2 kb (Group 1). For the Hoxc8-GFP, Hoxc8?c9-GFP, and Hoxc8?c9-LacZ alleles, thesize of the targeted band (F) is �7kb (Group 2).(c) PCR reactions using LoxP1 and LoxP3 primers. The wild-type allele (1) produces a �230bp band while the targeted allele (F) produces a �300-bp band.

681HOXC8 AND HOXC8?HOXC9 REPLACEMENT ALLELES

To ascertain that the minor changes observed in theHoxc8 expression domain did not impair motor func-tion, we examined 2-month-old male mice for their fore-

limb grip strength, a phenotype that was observed inthe Hoxc82/2 animals previously (Tiret et al., 1998). Wefound no deficit in the grip strength in all four floxed

FIG. 2. Phenotypic evaluation of floxed Hoxc8 and floxed Hoxc8?c9 alleles prior to Cre-mediated recombination (a–e) Hoxc8 mRNAexpression in e10.5 embryos revealed by whole mount in-situ hybridization, dashed lines mark the position of the forelimbs. In wild-type (a)and Hoxc8F/F- GFP (b) embryos, the rostral boundary of Hoxc8 expression in the ventral neural tube is located at �s8 (white arrows), whilethe rostral boundary of mesodermal expression is located �s13 (yellow arrows). In a Hoxc8?c9F/F- GFP embryo (c), the rostral boundary ofneural Hoxc8 expression is located at �s7 (white arrow), while the rostral boundary of mesodermal expression is located �s12 (yellowarrow). In Hoxc8F/F- LacZ (d) and Hoxc8?c9F/F- LacZ (e) embryos, the rostral boundary of neural Hoxc8 expression is located at �s6 (whitearrows), while the rostral boundary of mesodermal expression is located �s10 (yellow arrows). Scale bar 5 0.5 mm. (f–j) Lateral view of ver-tebral columns at cervical to rostral thoracic levels from e18.5 embryos. Cartilage is stained by Alcian Blue BGX and ossified bones arestained by Alizarin Red S. Wild-type (f), Hoxc8F/F- GFP (g), and Hoxc8?c9F/F- GFP (h) embryos have 7 cervical vertebrae with their first riboriginating from the first thoracic vertebra (open arrows), while Hoxc8F/F- LacZ (i), and Hoxc8?c9F/F- LacZ (j) embryos exhibit a posteriortransformation with an extra rib originating from C7 (green arrows). (k–o) Ventral view of the rib cages from e18.5 embryos. In wild-type (k)and Hxoc8F/F- GFP (I) embryos, the 6th and the 7th ribs are the last two ribs attached to the sterna (black brackets). In Hoxc8?c9F/F- GFP(m), as well as Hoxc8F/F- LacZ (n) and Hoxc8?c9F/F- LacZ (n) embryos, their 8th ribs are also attached to the sterna (black brackets). Scalebars 5 1 mm. (p) Forelimb grip strength of Hoxc8F/1, Hoxc8F/F, Hoxc8?c9F/1, and Hoxc8?c9F/F adult male mice with either GFP or LacZreporters, compared to their wild-type littermates. No significant difference was observed between different genotypes within each allele.

682 BLACKBURN ET AL.

alleles of mice when compared to their WT littermates(Fig. 2p), indicating that the minor change observed inthe Hoxc8 expression domain did not cause impairmentof motor function.

Because of our interest in Hox gene function duringspinal cord development, we further evaluated the en-dogenous Hox gene expression in open-book spinalcord preparations from e13.5 embryos by whole mountin situ hybridization prior to Cre-mediated recombina-tion. We focused our examination on the GFP-taggedfloxed alleles that exhibit little or no deviation in endog-enous Hox gene expression from the WT allele. The R-Clevels were estimated from the positions of the dorsalroot ganglia (DRG) that remain attached to the spinalcord during this procedure (see Methods). Slightchanges in the Hoxc8 mRNA rostral expression bound-ary were observed in Hoxc8F/F-GFP, Hoxc8?c9F/1-GFP,and Hoxc8?c9F/F-GFP embryos when compared to WT

controls (Fig. 3a,c–e), including a caudal shift of expres-sion at the intermediate region and a more uniformexpression in the ventral region of the spinal cord. Noobvious change in Hoxc8 mRNA expression wasdetected in the Hoxc8F/1-GFP embryos (Fig. 3b). No sig-nificant change in Hoxc6 mRNA expression wasobserved between various floxed alleles and WT con-trols (data not shown). In contrast, the rostral boundaryof Hoxc9 mRNA expression in the Hoxc8?c9F/F-GFPventral spinal cord is shifted �1 segment caudally whencompared to WT controls (Fig. 3f,j), while no significantchange in Hoxc9 mRNA expression was detected inHoxc8F/1-GFP, Hoxc8F/F-GFP, and Hoxc8?c9F/1-GFPembryos (Fig. 3g–i).

Hoxc6 protein induces lateral motor column (LMC)formation in the brachial level spinal cord while Hoxc9protein induces the formation of sympatheticpreganglionic neurons in the thoracic level spinal cord.

FIG. 3. Marker analyses in e13.5 embryonic spinal cords derived from GFP-tagged floxed Hoxc8 and floxed Hoxc8?c9 alleles prior toCre-mediated recombination (a–e) Hoxc8 mRNA expression in open-book preparations of e13.5 embryonic spinal cords. Slight changes inthe rostral boundary of Hoxc8 expression domain in the ventral (red boxed areas) and intermediate (open arrows) spinal cord are observedin Hoxc8F/F-GFP (c), Hoxc8?c9F/1-GFP (d), and Hoxc8?c9F/F -GFP (e) embryos as compared to the wild-type (a) and Hoxc8F/1 -GFP (b)embryos. (f–j) Hoxc9 mRNA expression in open-book preparations of e13.5 embryonic spinal cords. Hoxc9 rostral expression boundary(red boxed area) in the Hoxc8?c9F/F -GFP (j) embryos is placed �1 segment caudally when compared to the rest of embryos (f–i). (k–o)mRNA expression of an LMC marker, Raldh2, in open-book preparations of e13.5 embryonic spinal cords. �1 segment caudal extension ofRaldh2 expression is observed in the Hoxc8?c9F/F -GFP embryo (o) compared to the rest of embryos (k–n). (p–t) mRNA expression of amotor pool marker, Pea3, in open-book preparations of e13.5 embryonic spinal cords. A caudal expansion of Pea3 expression is observedin Hoxc8?c9F/F-GFP embryos (t). Scale Bars 5 0.5 mm.

683HOXC8 AND HOXC8?HOXC9 REPLACEMENT ALLELES

Cross-inhibitory actions between these two proteins atthe junction between brachial and thoracic levels delin-eate the brachial vs. thoracic identity of individual cells(Dasen et al., 2003). We therefore examined the mRNAexpression of an LMC marker, Raldh2, which is inducedby Hoxc6 and suppressed by Hoxc9 proteins. TheRaldh2 expression domain extends caudally by �1segment in the Hoxc8?c9F/F-GFP embryos compared tothe WT (Fig. 3k,o), and this could result from thecaudally displaced Hoxc9 rostral expression boundary.No significant change in Raldh2 mRNA expression wasobserved in Hoxc8F/1-GFP, Hoxc8F/F-GFP, andHoxc8?c9F/1-GFP spinal cords compared to the WTcontrols (Fig. 3l–n).

Hoxc8 protein defines motor pool identity in the cau-dal cervical level spinal cord, and we therefore examinedthe mRNA expression of a motor pool marker, Pea3, thatis a downstream target of Hoxc8 (Vermot et al., 2005).We did not observe significant differences in the Pea3expression domains in Hoxc8F/1-GFP, Hoxc8F/F-GFP, andHoxc8?c9F/1-GFP embryos compared to the controls(Fig. 3p–s). However, in the Hoxc8?c9F/F-GFP embryos,the Pea3 expression domain is extended caudally (�1segment, Fig. 3t), and this could be a secondary effect ofthe caudally expanded Raldh2 expression domainwhich also extended the caudal motor pools (Fig. 3o).These results further demonstrate the sensitivity of theHoxc locus to genomic manipulation, although thesefour targeting constructs share the same basic design,subtle changes in endogenous Hox gene expressioncould occur depending on the type of reporters used,and the presence of the Hoxc9 cDNA in individual tar-geting constructs. Although we are not certain whichcomponent of the exogenous DNA caused this differ-ence, the size difference between the GFP and LacZreporters (�0.8 kb vs. �3 kb) and the presence of anadditional �0.8 kb Hoxc9 cDNA sequence could be oneof the possibilities.

To determine if Hoxc8 expression can be eliminatedor replaced by Hoxc9 expression after Cre-mediatedrecombination, we crossed the floxed Hoxc8-GFP andHoxc8?c9-GFP mice with Nestin-cre (Nescre) (Troncheet al., 1999), and Isl1-cre (Isl1cre) (Srinivas et al., 2001)transgenic mice, and examined Hox protein expressionusing immunohistochemistry on cross-sectioned spinalcord tissue obtained from e12.5 mouse embryos. Bye12.5, the majority of cells in the spinal cord of theHoxc8F/F-GFP;Nescre and Hoxc8?c9F/F-GFP;Nescreembryos had already lost Hoxc8 protein expression (Fig.4d,f, and data not shown). In the Hoxc8?c9F/F-GFP;Nes-cre embryos, ectopic Hoxc9 protein expression at theexpense of endogenous Hoxc8 protein is clearly visibleusing antibodies against either Hoxc8 or Hoxc9 (Fig.4d,e). However, the Hoxc9 protein expression is not ashigh as that of the endogenous Hoxc8 protein butinstead, is more similar to the endogenous Hoxc9expression observed at the thoracic levels (Fig. 4a,d, anddata not shown), this could reflect a difference in theproperties between the anti-Hoxc8 and anti-Hoxc9 anti-

bodies used, or a brachial to thoracic fate changeinduced by Hoxc9 mis-expression. A reduction of Hoxc6protein expression in these cells is also apparent whencompared to the controls (Fig. 4b,c,e,f), demonstratingthat misexpression of Hoxc9 reduces the expression ofendogenous Hoxc6. Essentially none of the Isl11 neu-rons coexpress Hoxc8 in e12.5 Hoxc8F/F-GFP;Isl1creand Hoxc8?c9F/F-GFP;Isl1cre embryos, even in the lat-eral LMC (LMCl) MNs where Isl1 is only expressed transi-ently (Fig. 4g,h, and data not shown), suggesting thatIsl1-cre has a higher recombination efficiency than Nes-tin-cre. A higher level of GFP expression can be activatedin embryos that have undergone the Cre-mediatedrecombination and can be observed in freshly dissectedembryos under a fluorescent stereomicroscope in com-parison to the low levels of GFP expression observedprior to the recombination. Unfortunately, fixation withparaformaldehyde diminishes the GFP signal and an anti-GFP antibody is required to visualize GFP expression incross-sectioned spinal cord tissues (Fig. 4i, and data notshown). Similarly, high levels of b-galactosidase activitycan be observed in the spinal cord of e11.5 Hoxc8F/F-LacZ;Isl1cre and Hoxc8?c9F/F-LacZ;Isl1cre embryos(Fig. 4k and data not shown) in contrast to the low-levelb-galactosidase activity observed prior to Cre-mediatedrecombination (Fig. 4j). The presence of low-levelreporter gene expression prior to Cre-mediated recombi-nation indicates that a small percentage of Hoxc8 tran-scripts are not ployadenylated by the three known pAsignals.

In summary, we have generated conditional Hoxc8loss-of-function and Hoxc8 ?Hoxc9 replacement allelesof mice with either GFP or LacZ reporters. Although allfour alleles are indistinguishable from WT controls inmotor behavioral testing, molecular analyses revealedthat the floxed Hoxc8-GFP allele behaved most like theWT in the endogenous Hox gene expression. The floxedHoxc8?Hoxc9-GFP allele displayed a �1 segment ros-tral expansion of Hoxc8 mRNA expression at e10.5, anda �1 segment caudal displacement of the Hoxc9 mRNArostral expression boundary in e13.5 spinal cord, whilethe floxed Hoxc8-LacZ and floxed Hoxc8?Hoxc9-LacZalleles exhibited a �2-segment rostral extension in theneural and a �3-segment rostral extension in the meso-dermal Hoxc8 mRNA expression domains at e10.5.Upon Cre-mediated recombination, deletion of Hoxc8and misexpression of Hoxc9 along with upregulatedGFP or LacZ expression were observed, demonstratingthat these alleles of mice will be useful tools to studyHox function in a cell type-specific manner.

METHODS

Targeting Constructs

To generate Hoxc8 conditional targeting constructs, a65-bp fragment containing the LoxP sequence wascloned into a SacII site within Exon 1, 50 to the transla-tion start site. The following sequences were inserted

684 BLACKBURN ET AL.

into the Ear1 site (30 to the three known polyadenylationsites) of the Hoxc8 locus in tandem: a second LoxP sitein the same orientation followed by a �1.4 kb Nco1-Sal1fragment containing the Hoxc8 intron, an IRES-eGFP oran IRES-nlsLacZ fragment followed by a �1.5-kb BseR1fragment containing the 30 UTR of Hoxc8 and the poly-adenylation sequence from bovine albumin gene, and apGKneo cassette for positive selection. The final target-ing constructs were assembled in the pMoluc vector(Feng et al., 2002) and contain a �9.5-kb Sph1-Xba1Hoxc8 genomic region with the above modifications.

The homologus sequence at the 50 and the 30 arms are�2.5 and �3.4 kb, respectively. To generate Hoxc8?c9conditional replacement targeting constructs, a �0.8-kbfragment containing Hoxc9 coding region was insertedbetween the 30LoxP site and the 1.4-kb Hoxc8 intronsequence in the Hoxc8 conditional knock-out con-structs. Targeting constructs were digrested with Not1and electroporated into W9.5 ES cells as described (Dra-gatsis et al., 2000). The targeting frequency is �3–4%(three or four targeted clones from 96 G418-resistentcolonies picked for each construct).

FIG. 4. Changes in Hox and reporter protein expression after Cre-mediated recombination (a–f) Immunohistochemistry using anti-Hoxc6,anti-Hoxc8, and anti-Hoxc9 antibodies in cross-sectioned e12.5 caudal cervical level spinal cord. Many Hoxc61 and Hoxc81, but fewHoxc91 cells are present in Hoxc8?c9F/1-GFP embryos (a–c). Several remaining Hoxc81 cells along with increased Hoxc9 and decreasedHoxc6 expression are visible in the Hoxc8?c9F/F-GFP;Nescre spinal cord (d–f). (g–h) Immunohistochemistry using anti-Hoxc8 and anti-Isl1antibodies in cross-sectioned caudal cervical level e12.5 spinal cord. There is no overlap between Isl1 and Hoxc8 expression in a Hoxc8F/F-GFP;Islcre (h) embryo compared to the control littermate (g). Hoxc8 expression is also absent in LMCl MNs (marked by white dashed lines)where Isl1 is only expressed transiently (h). (i) Cross-sectioned spinal cord from an e12.5 Hoxc8F/F-GFP;Islcre embryo showing high levelsof GFP expression revealed by an anti-GFP antibody in dI3 interneurons (white arrow) and in MNs lacking Hoxc8 expression, while low lev-els of GFP expression are observed in Hoxc81 neurons. GFP expression is also visible in the axons. Scale bars 5 100 lm. (j, k) High levelsof b-galactosidase activity are observed in the spinal cord of an e11.5 Hoxc8?c9F/F-LacZ;Islcre embryo (k) compared to the low-level b-ga-lactosidase activity present in the spinal cord and mesoderm in their Hoxc8?c9F/1 littermates (i). Red dotted lines mark the position of theforelimbs. Scale bar5 1 mm.

685HOXC8 AND HOXC8?HOXC9 REPLACEMENT ALLELES

ES Cell Injection and Mouse Genotyping

ES cells carrying the targeted alleles were verified bySouthern analyses (see below) and were injected into129/Sv blastocysts using standard procedures (Bradley,1987). Male chimeras were crossed with C57BL6 femalesto generate heterozygous offspring. Mice carrying floxedalleles are maintained in a mixed 129/Sv and C57BL6background and are housed in a barrier facility under 12-h light/dark cycle. All protocols for animal use wereapproved by the Institutional Animal Care and Use Com-mittee of the University of Virginia and were inaccordance with NIH guidelines.

Genotyping was first performed by Southern blotanalysis using Xba1-digested genomic DNA probedwith a 500 bp 50-flanking HindIII-Sph1 fragment. PCRgenotyping was performed with following primerpairs: LoxP1: 50-CGCTCTACCTACCGACAGTGAG-30,and LoxP3: 50-GGAAAACAGGGGGTTGACGAAG-30 (forfloxed Hoxc8, and floxed Hoxc8?c9 alleles), LoxP1and mHoxc8I: 50-GGAGCGCGGGAGACCAAG-30 (forDfloxed Hoxc8 alleles), LoxP1 and Hoxc95R: 50-CCTGGACGCTAGGAGGTC-30 (for Dfloxed Hoxc8?c9alleles).

In Situ Hybridization, b-Galactosidase Activity,Immunohistochemistry, and Skeletal Staining

For whole mount in-situ hybridization, mouseembryos were harvested at e10.5 (noon of the day thatthe vaginal plug was present is considered e0.5), and spi-nal cords were isolated from e13.5 embryos. Dorsal rootganglia (DRG) were left on the dissected spinal cordthrough the in-situ hybridization procedure, and imageswere taken with a Nikon SMZ1500 stereoscope beforeand after DRG removal to estimate the R-C extent ofgene expression. Raldh2, Pea3, and Hoxc6 probes wereprovided by Drs. T. Jessell and J. Dasen. Hoxc8 andHoxc9 probes were provided by Dr. P. Gruss.

For b-galactosidase staining, e11.5 mouse embryoswere fixed for 200 in 4% paraformaldehye in 0.1 M PB(phosphate buffer, pH 7.4), then washed 2 3 150 withBuffer A (5 mM EGTA, 2 mM MgCl2 in 0.1M PB), and 2 3150 with Buffer B (0.2% NP40, 0.1% Sodium Desoxycho-late, 2 mM MgCl2 in 0.1M PB). Color reaction was per-formed in Buffer C (0.6 mg ml21 X-Gal, 5 mMK3Fe(CN6), 5 mM K4Fe(CN6), in Buffer B) at 378C indark and images were taken using a Nikon SMZ1500stereoscope.

Immunohistochemistry was performed on 10-lm sec-tions from e12.5 embryos using the following antibodiesas described (Liu, 2006): rabbit anti-GFP (Invitrogen),mouse anti-Hoxc8 (Covance), rabbit and guinea pig anti-Isl1 (provided by Dr. T. Jessell), guinea pig anti-Hoxc6,and rabbit anti-Hoxc9 (Liu et al., 2001). Images weretaken with a Nikon C1 confocal microscope.

Mouse embryos were harvested at e18.5 andprocessed for skeleton staining as described (McLeod,1980).

Grip Strength Testing

Forelimb grip strength testing was performed withthree trials using a grip strength apparatus (San DiegoInstruments). Graphs and statistical analyses were gener-ated using SigmaPlot.

ACKNOWLEDGMENTS

The authors thank S. Zeitlin for discussion and com-ments on this manuscript. They also thank T.-C. He forproviding the pMOLUC vector.

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687HOXC8 AND HOXC8?HOXC9 REPLACEMENT ALLELES