Leon W. Browder

Acquisition of the heat-shock response and thermotolerance during early development of Xenopus laevis☆

The ability to synthesize a 68,000- to 70,000-Da protein (hsp) in heat-shocked early Xenopus laevis embryos is dependent on the stage of development. Whereas late blastula and later stage embryos synthesize hsp68-70 after heat shock, cleavage stages are incompetent with respect to hsp synthesis. In vitro translation experiments and RNA blot analyses demonstrate that enhanced synthesis of hsp68-70 is associated with an accumulation of hsp68-70 mRNA. Examination of the effect of heat shock on preexisting actin mRNA reveals that heat shock promotes a reduction in the levels of actin mRNA in cleavage embryos but has no discernible effect on actin mRNA levels in neurula embryos. Finally, the acquisition of the heat-shock response (i.e., synthesis of hsp68-70 and accumulation of hsp70 mRNA) during early Xenopus development is correlated with the acquisition of thermotolerance.

Short‐faced mice and developmental interactions between the brain and the face

The length of the face represents an important axis of variation in mammals and especially in primates. Mice with mutations that produce variation along this axis present an opportunity to study the developmental factors that may underlie evolutionary change in facial length. The Crf4 mutant, obtained from the C57BL/6J (wt/wt) background by chemical mutagenesis by the Baylor Mouse Mutagenesis Resource, is reported to have a short‐faced phenotype. As an initial step towards developing this model, we performed 3D geometric morphometric comparisons of Crf4 mice to C57BL/6J wild‐type mice focusing on three stages of face development and morphology – embryonic (GD 9.5–12), neonatal, and adult. Morphometric analysis of adult Crf4 mutants revealed that in addition to a shortened face, these mice exhibit a significant reduction in brain size and basicranial length. These same features also differ at the neonatal stage. During embryonic face formation, only dimensions related to brain growth were smaller, whereas the Crf4 face actually appeared advanced relative to the wild‐type at the same somite stage. These results show that aspects of the Crf4 phenotype are evident as early as embryonic face formation. Based on our anatomical findings we hypothesize that the reduction in facial growth in Crf4 mice is a secondary consequence of reduction in the growth of the brain. If correct, the Crf4 mutant will be a useful model for studying the role of epigenetic interactions between the brain and face in the evolutionary developmental biology of the mammalian craniofacial complex as well as human craniofacial dysmorphology.

The effects of cinnamon on xanthine dehydrogenase, aldehyde oxidase, and pyridoxal oxidase activity during development in Drosophila melanogaster

Abstract The cinnamon (cin) eye color mutant of Drosophila melanogaster was characterized to determine biochemical correlations with another mutant, maroon-like . As with maroon-like, cinnamon flies lack three enzymatic activities: xanthine dehydrogenase, aldehyde oxidase, and pyridoxal oxidase. Xanthine dehydrogenase (XDH) is subject to a maternal effect in both mutants; i.e., mutant progeny of heterozygous mothers have XDH activity, resulting in wildtype eye color. However, the maternal effect is stronger in cinnamon than in maroon-like . Whereas maternally affected cinnamon show a large increase in XDH activity during larval stages, and XDH activity is still detectable after eclosion, the magnitude of increase in XDH activity is less in maroon-like , and activity is no longer detectable in second-day pupae and all later stages. The large increase in XDH activity in maternally affected cinnamon suggests that there is de novo synthesis of enzymatically active XDH during development in the absence of the cin + gene. Cinnamon is also unique in that maternally affected flies retain isoxanthopterin (IXP), the product of XDH activity. These flies appear to be deficient in some aspect of either pteridine metabolism or excretion.

Comparative morphometrics of embryonic facial morphogenesis: Implications for cleft‐lip etiology

Cleft lip (CL) with or without cleft palate (CL[P]) has a complex etiology but is thought to be due to either genetic or environmentally induced disruptions of developmental processes affecting the shape and size of the facial prominences (medial nasal, lateral nasal, and maxilla). Recent advances in landmark‐based morphometrics enable a rigorous reanalysis of phenotypic shape variation associated with facial clefting. Here we use geometric morphometric (GM) tools to characterize embryonic shape variation in the midface and head of six strains of mice that are both cleft‐liable (A, A/WySn, CL/Fr) and normal (BALB/cBy, C57BL, CD1). Data were comprised of two‐dimensional landmarks taken from frontal and lateral photographs of embryos spanning the time period in which the facial prominences fuse (GD10‐12). Results indicate that A/‐ strain mice, and particularly A/WySn, have overall smaller midfaces compared to other strains. The A/WySn strain also has significant differences in facial shape related to retarded development. Overall, CL/Fr strain mice are normal‐sized, but tend to have undersized maxillary prominences that do not project anteriorly and have a small nasal contact area. These results suggest that the etiology of clefting differs in A/WySn and CL/Fr strains, with the former strain suffering disruptions to developmental processes affecting overall size (e.g., neural crest migration deficiencies and lower mitotic activity), while the latter strain has defects restricted to the shape and size of the maxilla. A combination of molecular experimentation and phenotypic analysis of shape is required to test these hypotheses further. Anat Rec, 290:123–139, 2007.

Region-specific heat-shock protein synthesis correlates with a biphasic acquisition of thermotolerance in Xenopus laevis embryos

Abstract Embryos of Xenopus laevis , heat shocked before the midcell blastula stage, do not survive heat shock. Heat-shocked midcell blastulae only partially survive heat shock, exhibiting normal development of animal hemisphere derivatives (ectoderm and mesoderm) but exogastrulation of the vegetally derived endoderm. Fine-cell blastulae and early gastrulae are completely thermotolerant. We examined the synthesis of heat-shock proteins (hsps) in whole embryos and in their animal and vegetal hemispheres during heat shock. Hsp synthesis was first observed in embryos that are partially thermotolerant (midcell blastulae). Cells from both the animal and vegetal hemispheres synthesize 87- and 68- to 70-kDa hsps at this time. Completely thermotolerant fine-cell blastulae and early gastrulae synthesize additional hsps upon heat shock. Animal hemisphere cells synthesize 87- and 68- to 70-kDa hsps and small amounts of 57- and 43-kDa hsps. The vegetal cells also synthesize the 57- and 43-kDa hsps, but in large amounts. In addition, they synthesize a 35-kDa hsp not observed in the animal cells. The latter is the earliest observed example of region-specific protein synthesis in amphibians and may account for the delayed acquisition of thermotolerance of the vegetal hemisphere cells.

Decay of the oocyte-type heat shock response of Xenopus laevis

Xenopus oocytes have a complex heat shock response. During transition of the oocyte into fertilized egg, the heat shock response undergoes several qualitative and quantitative changes culminating in its complete extinction. Heat shock induces oocytes to synthesize four heat shock proteins (hsps): 83, 76, 70, and 57. After ovulation, two additional proteins (hsps 22 and 16) are inducible. The heat shock response of spawned eggs can be modified by changing the ionic configuration of the external medium and by adding pyruvate and oxaloacetate to the media. Since Xenopus eggs do not synthesize mRNA, these modifications to the external medium apparently alter the utilization of preexisting messenger RNAs in protein synthesis. Artificial activation terminates inducibility of hsps 76, 57, and 16 and diminishes the hsp 70 response. Two new heat shock proteins-66 and 48-are also inducible in artificially activated eggs. Fertilization, on the other hand, terminates the heat shock response; no hsps can be induced. However, hsp 70 appears to be made constitutively in fertilized eggs. RNA blot analyses reveal that oogenic hsp 70 messenger RNA is retained in eggs and early embryos. This messenger is apparently used for heat-induced synthesis of hsp 70 before fertilization and for constitutive synthesis of hsp 70 in zygotes.

Developmental Regulation of the Heat-Shock Response

Cell differentiation in the development of multicellular organisms occurs as a consequence of the generation of chronologically and spatially distinct patterns of protein synthesis. These unique constellations of proteins confer on cells the functional and structural characteristics that enable them to perform their specialized roles in the organism. However, in addition to the overt protein synthetic profile, cells may retain the potential to produce proteins that they would not normally produce in significant amounts. During periods of stress, this potential is realized and is evidenced by the synthesis of a set of stress proteins. As discussed in this chapter, the pattern of stress protein synthesis is also subject to developmental regulation. Thus, cells are simultaneously engaged in two parallel developmental processes: regulation of the patterns of overt and stress-inducible protein synthesis. Stress-inducible protein synthesis can be evoked by regulation at either the transcriptional or translational levels, or both. Thus, regulation at both levels must be subject to developmental modulation.

Expression of xanthine dehydrogenase activity during embryonic development of Drosophila melanogaster

Abstract The contributions of oogenesis and zygotic genome expression to xanthine dehydrogenase activity during embryogenesis were investigated utilizing the mal and ry 2 mutants. In vitro complementation experiments demonstrated the presence of the mal + complementation factor in the oocyte, suggesting an explanation for the mal maternal effect. The ry + complementation factor synthesized from paternal template was detected at gastrulation. This is the earliest detection of a paternal enzyme during nonmammalian embryonic development.

Preparative labeling of proteins with [35S] methionine

The most common technique for preparative labeling of proteins with radioisotopes for experimental purposes utilizes 125I. This isotope has certain limitations, including the emission of gamma- and X-irradiation, the release of gaseous 125I2 from solutions of Na 125I, and the potential for concentration of 125I in thyroid glands. We have discovered a means for labeling proteins rapidly and simply with [35S]methionine. The technique is applicable to a wide variety of proteins. Antibodies labeled by our technique remain functional.

Compact intergenic regions of the pufferfish genome facilitate isolation of gene promoters: characterization of Fugu 3′-phosphoadenosine 5′-phosphosulfate synthase 2 (fPapss2) gene promoter function in transgenic Xenopus

The highly compact nature of the pufferfish (Fugu rubripes) genome renders it a useful tool not only for annotating coding regions within vertebrate genomes, but also for the identification of sequences important to gene regulation. Indeed, owing to this compaction it will be feasible in many instances to initiate analyses using entire intergenic regions when mapping gene promoters; a strategy that is very rarely feasible with the expanded genomes of other species. Stemming from our interest in studying promoters expressed in chondrocytes, we selected for study the intergenic region upstream of Fugu 3′‐phosphoadenosine 5′‐phosphosulfate synthase 2, fPapss2, a gene required for the normal development of cartilage extracellular matrix. Functional characterization of the entire fPapss2 5′ intergenic region was carried out by monitoring expression of the enhanced green fluorescent protein (EGFP) gene reporter in the developing cartilage of transgenic Xenopus laevis. By evaluating a series of 5′ intergenic region deletions we defined a minimal fPapss2 sequence of ∼300 bp that was essential for EGFP expression in tadpole cartilage. This functional analysis of an entire Fugu intergenic region, combined with the efficiency of Xenopus transgenesis, serves as a model for the rapid characterization of evolutionarily‐conserved regulatory regions of other pufferfish genes.