Thomas Lufkin

Bapxl: an evolutionary conserved homologue of the Drosophila bagpipe homeobox gene is expressed in splanchnic mesoderm and the embryonic skeleton

In Drosophila, the visceral mesoderm giving rise to gut musculature is specified by the bagpipe homeobox gene. We have isolated, from both mouse and human, homologues of the bagpipe gene designated Bapx1 and BAPX1, respectively. Bapx1 encodes a predicted protein of 333 amino acids, and has significant regions of homology outside the homeodomain with members of the NK homeobox gene superfamily. Bapx1 maps to the proximal end of chromosome 5 in mouse, near the Msx1 gene. The syntenic region in human corresponds to a chromosomal region containing loci for several skeletal disorders. Bapx1 is first detectable in embryos just prior to axis rotation in lateral plate mesoderm (splanchnic mesoderm) adjacent to the endodermal lining of the prospective gut, and in the most newly formed somites in the region corresponding to the presclerotome, the precursor of the vertebrae. Thus, Bapx1 is one of the earliest developmental markers for the sclerotome portion of the somite and the gut mesentery. Bapx1 continues to be expressed well into organogenesis in lateral plate mesoderm surrounding the mid- and hindgut, and in essentially all cartilaginous condensations which will subsequently undergo endochondral bone formation. The expression pattern of Bapx1 in murine embryos suggests that there are evolutionary conserved mechanisms of visceral mesoderm development across the animal kingdom, and that the mammalian Bapx1 gene may have recently acquired an additional developmental role in skeletal patterning.

MSX3 : A NOVEL MURINE HOMOLOGUE OF THE DROSOPHILA MSH HOMEOBOX GENE RESTRICTED TO THE DORSAL EMBRYONIC CENTRAL NERVOUS SYSTEM

We have isolated Msx3, the third member of the murine Msx homeobox gene family which is homologous to the msh gene of Drosophila. The Msx3 cDNA encodes a protein of 204 amino acids which has striking regions of homology in addition to the homeodomain when compared to the other Msx family members. Msx3 maps to the distal end of mouse chromosome 7, thus it is unlinked to either Msx1 or Msx2. RNA in situ analysis of Msx3 gene expression during early development revealed that it is restricted to the dorsal portion of the neural tube with a rostral boundary in the rostral rhombencephalon. This sole expression domain for Msx3 overlaps with both Msx1 and Msx2 at early stages, but in older embryos, Msx3 expression becomes restricted to the ventricular zone of the dorsal neural tube, whereas Msx1 and Msx2 become localized to the non-neuronal roof plate region. The absence of detectable levels of Msx3 expression outside this restricted dorsal region of the developing central nervous system (CNS) is in marked contrast to Msx1 and Msx2, which have extensive expression domains in other embryonic tissues, particularly craniofacial structures and the limbs. By analogy with the expression of msh/Msx genes in other organisms, it is likely that one of the ancestral functions of the msh/Msx gene family is in patterning of the CNS.

Hmx: an evolutionary conserved homeobox gene family expressed in the developing nervous system in mice and Drosophila.

Three homeobox genes, one from Drosophila melanogaster (Drosophila Hmx gene) and two from mouse (murine Hmx2 and Hmx3) were isolated and the full-length cDNAs and corresponding genomic structures were characterized. The striking homeodomain similarity encoded by these three genes to previously identified genes in sea urchin, chick and human, as well as the recently cloned murine Hmx1 gene, and the low homology to other homeobox genes indicate that the Hmx genes comprise a novel gene family. The widespread existence of Hmx genes in the animal kingdom suggests that this gene family is of ancient origin. Drosophila Hmx was mapped to the 90B5 region of Chromosome 3 and at early embryonic stages is primarily expressed in distinct areas of the neuroectoderm and subsets of neuroblasts in the developing fly brain. Later its expression continues in rostral areas of the brain in a segmented pattern, suggesting a putative role in the development of the Drosophila central nervous system. During evolution, mouse Hmx2 and Hmx3 may have retained a primary function in central nervous system development as suggested by their expression in the postmitotic cells of the neural tube, as well as in the hypothalamus, the mesencephalon, metencephalon and discrete regions in the myelencephalon during embryogenesis. Hmx1 has diverged from other Hmx members by its expression in the dorsal root, sympathetic and vagal nerve (X) ganglia. Aside from their expression in the developing nervous system, all three Hmx genes display expression in sensory organ development, and in the adult uterus. Hmx2 and Hmx3 show identical expression in the otic vesicle, whereas Hmx1 is strongly expressed in the developing eye. Transgenic mouse lines were generated to examine the DNA regulatory elements controlling Hmx2 and Hmx3. Transgenic constructs spanning more than 31 kb of genomic DNA gave reproducible expression patterns in the developing central and peripheral nervous systems, eye, ear and other tissues, yet failed to fully recapitulate the endogenous expression pattern of either Hmx2 or Hmx3, suggesting both the presence and absence of certain critical enhancers in the transgenes, or the requirement of proximal enhancers to work synergistically.

Expression of Tfap2d, the gene encoding the transcription factor Ap-2δ, during mouse embryogenesis

Tfap2d encodes a novel member of the AP-2 family of transcription factors, called Ap-2 delta. Ap-2 delta has a unique DNA sequence binding specificity and lacks a PY motif as well as several highly conserved residues in the transactivation domain that are essential for transcriptional activation for other AP-2 proteins. In this study, we characterized the expression pattern of Tfap2d in mouse embryos from E7.5 to E16.5 using in situ hybridization as a first step towards understanding the role of Ap-2 delta during development. Overall, Tfap2d is expressed with a more restricted temporal and spatial pattern than the other Tfap2 genes, with signals observed in the central nervous system, retina, and, uniquely, in the developing heart. Signals were not detected in tissues such as the neural crest, facial mesenchyme, and limbs where the other Tfap2 genes are expressed. The expression of Tfap2d was first noted at E9.5, later than other Tfap2 genes, with signals from the heart and brain. The expression in the developing myocardium persisted through E10.5. Expression of Tfap2d was diffuse in the developing brain at E10.5 to E11.0, but became restricted to the roof of the third vesicle with two narrow columns extending forward along the wall of the third vesicle and the roof of the heopallial cortex in the forebrain from E12.5 onwards. Tfap2d expression was also observed in the spinal cord at E10.5. From E13.5 to E16.5, Tfap2d was expressed in the retinal epithelium. Based on Tfap2ds unique expression pattern and functional features, we believe that Ap-2 delta plays a role during mammalian development that is non-overlapping with those of the other Ap-2 transcription factors.

Mammalian Dlx homeobox gene control of craniofacial and inner ear morphogenesis

The Dlx homeobox gene family is of ancient origin, with apparent ancestral developmental functions in both nervous system regionalization and appendage (limb) outgrowth. Additional roles in inner ear and craniofacial development were likely acquired by the Dlx gene family during the course of animal evolution. Loss‐of‐function genetic mutations generated in the mouse have revealed a striking role for Dlx genes in patterning of the mammalian central nervous system, craniofacial structures and inner ear. Interestingly, none of the individual murine Dlx gene mutations to date have resulted in limb defects, suggesting a potentially significant developmental overlap of Dlx activity in this embryonic structure. J. Cell. Biochem. Suppls. 32/33:133–140, 1999.

MOLECULAR CLONING, CHROMOSOMAL MAPPING AND DEVELOPMENTAL EXPRESSION OF BAPX1, A NOVEL HUMAN HOMEOBOX-CONTAINING GENE HOMOLOGOUS TO DROSOPHILA BAGPIPE

We describe here the cloning of the human BAPX1 gene, a homologue of the Drosophila bagpipe gene which has 87% aa identity within the homeodomain relative to the fly gene. We recently have identified the murine bagpipe homolog. The predicted aa sequence of the human gene has 85% overall identity to the murine gene, with 100% identity in the homeodomain. In mouse, this gene maps to the proximal portion of chromosome 5. We show that the human gene maps to 4p16.1, the human region syntenic with mouse chromosome 5. Expression of BAPX1 was evaluated during human embryonic development by RT-PCR analysis and by RNA in situ hybridization. RT-PCR analysis showed that BAPX1 is expressed in embryo tissues, particularly the limb, and at a lower level in an embryonic lung cell line. RNA in situ hybridization revealed that BAPX1 is predominantly expressed in mesenchymal condensations of the fetal limb and axial skeleton, and in lateral plate mesoderm giving rise to visceral muscle. The expression pattern of BAPX1 combined with the chromosomal localization to 4p16.1, where several human genetic diseases involving dysmorphology of the skeleton have been assigned, raises the potential of it being a candidate gene for one of these disorders. O

NKX2.6 EXPRESSION IS TRANSIENTLY AND SPECIFICALLY RESTRICTED TO THE BRANCHIAL REGION OF PHARYNGEAL-STAGE MOUSE EMBRYOS

The Nkx2.6 gene belongs to the NK superfamily of homeobox genes (Harvey, 1996). We report here the expression pattern of the murine Nkx2.6 gene during early mouse development, which is unique among the NK family of homeobox genes in that its expression is restricted to the very narrow development period between stages E8.5 and E10.5 of embryogenesis. The distribution of Nkx2.6 transcripts is also quite restricted spatially, with expression detected uniquely within the caudal branchial arches. Nkx2.6 is expressed in all three layers comprising the caudal branchial arches (ectoderm, mesectoderm and endoderm) with the strongest expression being detected in the surface ectoderm.

The murine fork head gene Foxn2 is expressed in craniofacial, limb, CNS and somitic tissues during embryogenesis.

The fork head domain-containing gene family (Fox) comprises over 20 members in mammals and is defined by a conserved 110 amino-acid motif containing a winged helix structure DNA-binding domain. The members of this gene family have been implicated as key regulators of embryogenesis, cell cycling, cell lineage restriction and cancer. The Foxn2 gene (Ches1) is expressed in postgastrulation embryos in multiple tissues that serve as important signaling centers as well as end-stage-differentiated cell types that arise from different germ layers of the developing embryo. The dynamic and specific expression of Foxn2 during embryonic development suggest multiple independent roles for Foxn2 function during gestation.

Linkage mapping of Sax2 to mouse Chromosome 5

Species: Mouse Locus name: Sax homeobox gene 2 Locus symbol: Sax2 Map position: Sax2 is located on mouse Chromosome (Chr) 5: proximal-D5Mit1-4.25 +_ 2.08 (cM _+ SE)-Sax2, Dpp6, Znt3, D5Mit149, D5Bir6-1.06 +_ 1.06-D5Mit351-1.06 +_ 1.06--D5Bir7distal. Method of mapping: 94 DNAs from N 2 animals of a [(C57BL/6J x SPRET/Ei) F 1 females x SPRET/Ei males] backcross (obtained from The Jackson Laboratory Backcross DNA Panel Map Service, Bar Harbor, Maine [1]. Database deposit information: Typing data may be obtained through the World Wide Web at the following location: http:// www.jax.org/resources/documents/cmdata/. Molecular reagents: A 1.0-kb genomic XbaI fragment isolated from a 129/sv lambda genomic phage containing the Sax2 gene was used as the probe on Southern blots of the backcross panel (BSS2). A genomic clone containing the Sax2 locus was isolated as follows. A PCR-generated fragment corresponding to the Drosophila bagpipe homeobox [2] was labeled with [32p]-dCTP and hybridized to filters containing a mouse 129/Sv genomic library constructed in lambda DashlI at 35~ in a hybridization solution containing: 50% formamide, 0.9 M NaC1, 50 rnM NaH2PO 4 pH 6.5, 2 rnN EDTA, 2x Denhardts solution, 5% Dextran sulfate, 1% SDS, 50 ixg/ml denatured salmon sperm DNA (normal stringency hybridization is at 42~ Blots were washed in 2x SSC, 0.1% SDS at 55~ for 4 x 30 min and exposed to film. Sax2 and Bapxl containing genomic phage were isolated, and the genomic inserts containing the homeobox were subcloned into plasmid (pBSKS+) and sequenced by the dideoxynucleotide chain termination method and Sequenase enzyme (U.S.Biochemicals). DNA sequence analysis of both strands confirmed that one of the genomic phage contained Sax2. Allele detection: By Southern blot analysis, the genomic probe fragment hybridized to HindlII fragments of approximately 3.2 kb in C57BL/6J DNA and 7.5 kb in SPRET/Ei DNA. Previously identified homologs: Murine Sax2 was previously called Nkxl.1 [3]. Homologs to Sax2 are Drosophila

Otp maps to mouse Chromosome 13

59/NK1 [4,5] and mouse Saxl [6]. Discussion: The mouse Sax2 gene (previously called Nkxl.1 [3]) belongs to a small group of homeobox genes (Fig. 1A) that have strong amino acid homology to the Drosophila NK1/