Jeffrey W. Innis, M.D., Ph.D.
Morton S. and Henrietta K. Sellner Professor in Human Genetics
Professor of Human Genetics and Pediatrics
Division Director, Pediatric Genetics
Early Post-Implantation Mammalian Body Patterning
Post-implantation development requires precisely coordinated cellular movements in preparation for gastrulation. We have characterized and genetically mapped a spontaneous X-linked mouse mutant, Polypodia (Ppd), which we discovered and established as a mutant line using in vitro fertilization and genetic crosses to explore early post-implantation body plan patterning. Ppd mice exhibit ectopic caudal limbs and other extraordinary malformations, which has been observed in humans and other animals. We hypothesize that Ppd alters vertebrate patterning during pregastrulation or early gastrulation stages by changing expression of patterning genes in the post-implantation embryo. Because of the strikingly similar malformations of Ppd mice by comparison with embryos treated with exogenous retinoic acid at E4.5-E5.5, we hypothesize that Ppd malformations are secondary to alterations of primitive streak formation or mesodermal cell allocation via premature activation of retinoic acid synthesis or alterations of Wnt/β-catenin signaling. We are determining the timing and nature of lethality and testing for alterations in post-implantation gene expression using RNA probes for genes expressed in the primitive streak and proximal epiblast and overlying visceral endoderm; distal visceral endoderm; extraembryonic ectoderm; and epiblast. To determine whether induced alterations in gene expression occur in common between Ppd and RA-treated mice, we are comparing RA-treated pregnant wild-type mouse embryos at E4.5 �?? E5.5 to Ppd mutant mice. We are studying the functional consequences of the mutation using transgenic mice and other tools.
Long-range Transcriptional Regulation of Hoxa13 in Developing Limbs and the Urogenital Tract
While we have learned a great deal about the underlying roles of many transcription factors in developmental fate and in human malformations, we know less about how such genes are targeted for expression in selected tissues at specific times and abundance. Proper regulation of such aspects occurs via cis elements, located within those genes or more often at great genomic distances away, as well as by trans-acting factors bound to those elements in multiprotein complexes. However, many questions remain as to how these elements are used mechanistically.
Hoxa13 is a critical Hox transcription factor in the development of distal limbs and the genitourinary tract. We recently published data showing that three genes, Hibadh, Tax1bp1, and Jazf1, upstream of Hoxa13 and the Hoxa cluster, together with Hoxa13 and Evx1 constitute a domain of 5 genes, which we call the HEHTJ domain, that appear to be coordinately expressed during murine limb and genital bud development. We also reported the enhancer capabilities of a highly conserved 2.25 kb sequence (mmA13CNS) 300 kb upstream of the HoxA cluster in transgenic mice. Our long-term goals are to determine the mechanisms that govern transcriptional regulation of the Hoxa13 gene in limbs and genitourinary structures in the larger context of the HEHTJ domain. The proximity of a highly conserved cluster of Hox genes and coordinately regulated upstream genes offers novel opportunities to study enhancer regulatory mechanisms.
Specifically, we are 1) using BAC transgenesis to capture enhancers for HEHTJ gene expression; and 2) working to identify cis-acting sequences necessary for Hoxa13 expression in mouse tissues. We created mice transgenic for a two BACs encompassing mmA13CNS and have shown the existence of at least two enhancers for proper expression in murine limbs and genital bud development. We are also making a Hoxa13 gene/β-globin lacZ reporter linked to candidate enhancers derived from our BAC transgene work. The plan is to introduce these constructs into the Hoxa13 null background to test for rescue of in utero lethality, limb and urogenital malformation in Hoxa13 -/- embryos.
Lehoczky J, Innis JW. BAC transgenic analysis reveals enhancers sufficient for Hoxa13 and neighborhood
gene expression in mouse embryonic distal limbs and genital bud. Evolution & Development, 2008,
Lehoczky J, Innis JW. Expanded Hoxa13 Polyalanine Tracts in the Monotreme. Evolution & Development,
2008, IN PRESS.
Williams ME, Lehoczky JA, Innis JW. A group 13 homeodomain is neither necessary nor sufficient for posterior prevalence in the mouse limb. Developmental Biology 2006, 297: 493-507.
Lahidji SF, Buchman SR, Muraszko K, Innis JW, Keegan CE. Craniofacial Dyssynostosis in Two Boys with Apparently Normal Cognitive Development. Amer J Med Gen 2006, 140(12):1333-6.
Lehoczky J, Cai W-W, Douglas J, Moran J, Beier D, Innis JW. Description and genetic mapping of Polypodia: an X-linked dominant mouse mutant with ectopic caudal limbs and other malformations. Mammalian Genome 2006, 17(9):903-13 (Cover).
DeScipio C, Kaur M, Yaeger D, Davis R, Innis JW, Spinner NB, Jackson LG, Krantz ID. Chromosome rearrangements in Cornelia de Lange syndrome (CdLS): Report of a Der(3)t(3;12)(p25.3;p13.3) in two half sibs with features of CdLS and review of reported CdLS cases with chromosome rearrangements. Am. J. Med. Gen., 2005, 137A: 276-282.
McDermott DA, Bressan MA, He J, Lee JS, Aftimos S, Brueckner M, Gilbert F, Graham GE, Innis JW, Pierpont MEM, Raas-Rothschild A, Shanske AL, Smith WE, Spencer RH, St. John-Sutton MG, van Maldergem L, Waggoner DJ, Basson CT. TBX5 genetic testing validates strict clinical criteria for Holt-Oram syndrome. Pediatric Research, in press; PMID: 16183809.
Misra VK, Struys EA, O'Brien W, Salomons GS, Glover T, Jakobs C, Innis JW. Phenotypic heterogeneity in the presentation of D-2-hydroxyglutaric aciduria in monozygotic twins. Molecular Genetics and Metabolism, 2005, 86: 200-205.
Williams TM, Williams ME, Heaton JH, Gelehrter TD, Innis JW. Group 13 HOX proteins interact with the MH2 domain of R-Smads and modulate Smad transcriptional activation functions independent of HOX DNA binding capability. Nucleic Acids Research, 2005, 33: 4475-84.
McCabe C, Innis JW. A genomic approach to identification and characterization of
HOXA13 functional binding elements. Nucleic Acids Research, 2005, 33:6782-94.
Williams T, Williams ME, Innis JW. Range of HOX/TALE Superclass Associations and Protein
Domain Requirements for HOXA13/MEIS Interaction. Developmental Biology, 2005, 277:457-471.
Innis JW, Mortlock DP, Chen Z, Ludwig M, Williams ME, Williams TM, Doyle CD, Shao Z, Glynn
M, Mikulic D, Lehmann K, Mundlos S, Utsch B. Polyalanine expansion in HOXA13: Three new affected families and the molecular consequences in a mouse model. Human Molecular Genetics, 2004, 13:2841-2851.
Williams TM, Williams ME, Kuick R, Misek D, McDonagh K, Hanash S, Innis JW. Candidate downstream-regulated genes of HOX group 13 transcription factors with and without monomeric DNA binding capability. Developmental Biology, 2005, 279: 462-480.
Lehoczky JA, Williams ME, Innis JW. Conserved Expression Domains For Genes Upstream and
Within of HoxA and HoxD Clusters Suggests a Long-range Enhancer Existed Prior to Cluster Duplication. 2004, Evolution and Development, 2004, 6:423-430.
Keegan CE, Vilain E, Mohammed M, Lehoczky J, Dobyns WB, Archer SM, Innis JW.
Microcephaly, jejunal atresia, aberrant right bronchus, ocular anomalies, and XY sex reversal.. Am. J. Med. Gen. 2004, 125: 293-298.
Wechsler SB, Lehoczky JA, Hall JG, Innis JW. Tibial aplasia and preaxial polydactyly,
brachyphalangy, craniofacial dysmorphisms and genital hypoplasia: Further delineation and mutational analysis. Clinical Dysmorphology, 2004, 13: 63-69.
Innis JW, Goodman F, Williams T, Mortlock D, Sateesh P, McKinnon W, Guttmacher A.
A HOXA13 allele with a missense mutation in the homeobox and a dinucleotide deletion in the promoter underlies Guttmacher syndrome. Human Mutation, 2002, 19: 573-574.
Margulies EH, Kardia SLR, Innis JW. Identification and prevention of a GC content bias in SAGE
libraries. Nucleic Acids Res. 2001, 29 (12): e60.
Margulies EH, Kardia SLR, Innis JW. A comparative molecular analysis of developing
mouse forelimbs and hindlimbs using serial analysis of gene expression (SAGE). Genome Research 2001, 11: 1686-1698.
Post LC, Margulies EH, Kuo A, Innis JW. Severe limb defects in Hypodactyly mice result from
expression of a novel, mutant HOXA13 protein. Developmental Biology 2000, 217: 290-300.
Mortlock DP, Sateesh P, Innis JW. Evolution of N-terminal sequences of the vertebrate HOXA13
protein. Mammalian Genome 2000, 11: 151-158.
Goodman FR, Fryns J-P, Mortlock DP, Innis JW, Holmes LB, Donnenfeld AE, Feingold M, Beemer
FA, Hennekam RCM, Scambler PJ. Novel HOXA13 mutations and the phenotypic spectrum of Hand-Foot-Genital Syndrome. Amer. J. Hum. Gen. 2000, 67: 197-202.
Margulies EH, Innis JW. eSAGE: Managing and analyzing data generated with Serial Analysis of
Gene Expression (SAGE). Bioinformatics 2000, 16: 649-650.
Post LC and Innis JW. Altered Hox expression and increased cell death distinguish Hypodactyly
from Hoxa13 null mice. Int. J. Dev. Biol. 1999, 43: 287-294.
Post LC and Innis JW. Infertility in Hypodactyly mice is associated with hypoplasia of distal reproductive structures. Biology of Reproduction 1999, 61: 1402-1408.
Mortlock DP and Innis JW. Mutation of HOXA13 in Hand-foot-genital syndrome. Nature Genetics
1997, 15: 179-180.
Kondo T, Zakany J, Innis JW, and Duboule D. Of fingers, toes and penises. Nature 1997, 390: 29.
Mortlock DM, Nelson M, and Innis JW. An efficient method for isolating putative promoters and 5'
transcribed sequences from large genomic clones. Genome Research, 1996, 6: 327-335.
Mortlock DP, Post LC, and Innis JW. The Molecular Basis of Hypodactyly (Hd): A Deletion in
Hoxa13 Leads to Arrest of Digital Arch Formation. Nature Genetics 1996, 13: 284-289.