Daphne G. Trasler

Influence of Uterine Site on Occurrence of Spontaneous Cleft Lip in Mice

It has been found that within the A/Jax strain, embryos in the uterine site nearest the ovary develop cleft lip (with or without cleft palate) significantly more often than embryos in other positions in the uterus.

Time-Position Relationships

Most anatomical birth defects can be classified either as deformations, alterations in shape or structure of a normally formed part, such as congenital torticollis and some types of clubfoot; or malformations, primary structural defects that result from an error in morphogenesis. (Morphogenesis refers, in this context, to the progressive emergence of organized structures in the embryo.) Normal morphogenesis depends on a system of highly integrated and interacting sets of reactions. The embryonic cells are programmed in such a way that certain genes are turned on in the cells of certain tissues and others remain inactive, according to a progressive set of nuclear-cytoplasmic interactions. At this level of interaction, the production of abnormal molecules (enzymes, organelle components, membrane constituents, intercellular matrix) or the production of normal molecules at altered rates or at the wrong time may change the properties of the cells or their products, leading to errors in development. At another level of interaction, normal development depends on the properties of groups of cells, tissues, or organs and their relations to one another. There may be interaction of cells within a tissue, for example, when a group of cells has to reach a certain critical mass for its cellular components to proceed with differentiation and organ formation. Or there may be interaction between tissues. One tissue may require an inductive stimulus from another before it will proceed further along its developmental pathway. Or two or more tissues may need to meet one another by merging or fusion to form a structure. These kinds of interaction often involve a developmental threshold, in which the structure is morphologically normal if the threshold is reached and abnormal if it is not; this threshold concept has been dealt with in Chapter 3, Vol. 1.

High-resolution linkage map in the vicinity of the Lp locus

Looptail (Lp) is a mutation that profoundly affects neurulation in mouse and is characterized by craniorachischisis, an open neural tube extending from the midbrain to the tail in embryos homozygous for the mutation. Lp maps to the distal portion of mouse chromosome 1, and as part of a positional cloning approach, we have generated a high-resolution linkage map of the Lp chromosomal region. For this, we have carried out extensive segregation analysis in a total of 706 backcross mice informative for Lp and derived from two crosses, (Lp/+ x SJL/J)F1 x SJL/J and (Lp/+ x SWR/J) F1 x SWR/J. In addition, 269 mice from a (Mus spretus x C57BL/6J)F1 x C57BL/6J interspecific backcross were also used to order marker loci and calculate intergene distances for this region. With these mice, a total of 28 DNA markers corresponding to either cloned genes or anonymous markers of the SSLP or SSCP-types were mapped within a 5-cM interval overlapping the Lp region, with the following locus order and interlocus distances (in cM): centromere--D1Mit110/Atp1 beta 1/Cd3 zeta/Cd3 eta/D1Mit145-D1Hun14/D1Mit15- D1Mit111/D1Mit112-D1Mit114-D1Mit148/D1Mit205+ ++/D1Mit36/D1Mit146/D1Mit147/D1Mit270 / D1Hun13-Fcgr2-Mpp-Apoa2/Fcer1 gamma-Lp-D1Mit149/Spna1/Fcer1 alpha-Eph1-Hlix1/D1Mit62. These studies have allowed the delineation of a maximum genetic interval for Lp of 0.5 cM, a size amenable to physical mapping techniques.

Nhlh1, a basic helix-loop-helix transcription factor, is very tightly linked to the mouse looptail (Lp) mutation

Looptail (Lp) is a mutation on the distal portion of mouse Chromosome (Chr) 1 that affects neurulation in mouse and is phenotypically expressed by appearance of an open neural tube along the entire antero-posterior axis of the embryo (craniorachischisis). Nhlh1, a member of the basic helix-loop-helix family of transcription factors, is expressed in the developing neural tube in structures affected by the Lp mutation and has been regionally assigned to the distal part of mouse Chr 1. Using a large panel of looptail animals from an (Lp/+ x SWR/J)F1 x SWR/J segregating backcross progeny, we have determined that Nhlh1 maps very close to Lp, with no recombinant detected in 500 informative animals tested; both map within a 0.6-cM segment defined as D1Mit113/apoa2/Fcer1γ-(0.4 cM)-Nhlh1/Lp-(0.2 cM)-Fcer1α/D1Mit149/Spna1. Nucleotide sequencing of Nhlh1 cDNA clones from wild type (WT) and Lp/Lp embryos failed to identify sequence alterations associated with the mutant phenotype. Southern hybridization of genomic DNA from WT and Lp/Lp embryos failed to identify specific rearrangements at or near the Nhlh1 locus, and Northern RNA blotting and RT-PCR evaluation of Nhlh1 mRNA expression indicated that both the levels and types of Nhlh1 mRNAs produced in WT and Lp/Lp embryos were indistinguishable. These studies suggest that Nhlh1 and Lp are not allelic. Nevertheless, Nhlh1 is the Chr 1 marker most tightly linked to Lp identified to date and can, therefore, be used as an excellent entry probe to clone the Lp region.