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Homeodomain-containing (HOX) factors such as the abdominal class homeodomain protein HOXA10 and the TALE-family protein PBX1 form coregulatory complexes and are potent transcriptional and epigenetic regulators of tissue morphogenesis. We have identified that HOXA10 and PBX1 are expressed in osteoprogenitors; however, their role in osteogenesis has not been established. To determine the mechanism of HOXA10-PBX-mediated regulation of osteoblast commitment and the related gene expression, PBX1 or HOX10 were depleted (shRNA or genetic deletion, respectively) or exogenously expressed in C3H10T1/2, bone marrow stromal progenitors, and MC3T3-E1 (preosteoblast) cells. Overexpression of HOXA10 increased the expression of osteoblast-related genes, osteoblast differentiation and mineralization; expression of PBX1 impaired osteogenic commitment of pluripotent cells and the differentiation of osteoblasts. In contrast, the targeted depletion of PBX1 by shRNA increased the expression of bone marker genes (osterix, alkaline phosphatase, BSP, and osteocalcin). Chromatin-associated PBX1 and HOXA10 were present at osteoblast-related gene promoters preceding gene expression, but PBX1 was absent from promoters during the transcription of bone-related genes, including osterix (Osx). Further, PBX1 complexes were associated with histone deacetylases normally linked with chromatin inactivation. Loss of PBX1 but not of HOXA10 from the Osx promoter was associated with increases in the recruitment of histone acetylases (p300), as well as decreased H3K9 methylation, reflecting transcriptional activation. We propose PBX1 plays a central role in attenuating the activity of HOXA10 as an activator of osteoblast-related genes and functions to establish the proper timing of gene expression during osteogenesis, resulting in proper matrix maturation and mineral deposition in differentiated osteoblasts.

Sex determination in the American alligator depends on the incubation temperature experienced during a thermo-sensitive period (TSP), although sex determination can be ‘reversed’ by embryonic exposure to an estrogenic compound. Thus, temperature and estrogenic signals play essential roles during temperature-dependent sex determination (TSD). The genetic basis for TSD is poorly understood, although previous studies observed that many of the genes associated with genetic sex determination (GSD) are expressed in species with TSD. Heat shock proteins (HSPs), good candidates because of their temperature-sensitive expression, have not been examined in regard to TSD but HSPs have the ability to modify steroid receptor function. A number of HSP cDNAs (HSP27, DNAJ, HSP40, HSP47, HSP60, HSP70A, HSP70B, HSP70C, HSP75, HSP90α, HSP90β, and HSP108) as well as cold-inducible RNA binding protein (CIRBP) and HSP-binding protein (HSPBP) were cloned, and expression of their mRNA in the gonadal-adrenal-mesonephros complex (GAM) was investigated. Embryonic and neonatal GAMs exhibited mRNA for all of the HSPs examined during and after the TSP. One-month-old GAMs were separated into 3 portions (gonad, adrenal gland, and mesonephros), and sexual dimorphism in the mRNA expression of gonadal HSP27 (male > female), gonadal HSP70A (male < female), and adrenal HSP90α (male > female) was observed. These findings provide new insights on TSD and suggest that further studies examining the role of HSPs during gonadal development are needed.

For many years the molecular mechanisms governing bone morphogenetic protein 4 (Bmp4) expression in tooth bud mesenchyme could be explained by an uncomplicated model involving the interaction of the homeobox gene Msx1 and the paired domain gene Pax9 and a limited proximal promoter segment of Bmp4. New insights have led to major revisions, but we are still far from understanding the role of Msx1 and Pax9 in the complex processes that result in the expression of Bmp4 in the mesenchymal layer of the developing tooth bud. The objective of these studies was to gain further insight into the molecular relationship between Pax9, Msx1, and Bmp4 in dental mesenchyme and explore its association with nonsyndromic tooth agenesis in humans.

The greatest burden of ill-health for adults in most societies relates to disorders of the cardiovascular system and to cancers [1]. These diseases are caused by lifestyle choices in which poor diet and relatively low levels of activity play a major role [1]. There is a large body of literature which shows that environmental exposures, including nutrition, play a significant role in the etiology of the disease, but there is variable susceptibility amongst individuals exposed to seemingly similar risk factors or environments. Cancer is a condition which results from a derangement in the cellular processes which regulate cell division, terminal differentiation and apoptosis, with damage to the genetic material of the cell being central. Exposures which lead to these derangements usually precede the appearance of the clinical disease by a prolonged period of time, often several decades [2]. While gene mutation has a role in the etiology of cancer, there is increasing evidence showing that epigenetic processes such as DNA methylation and covalent modification to histones are also involved [3]. Epigenetic changes of this kind represent potential for altered gene activity, and hence cellular dysregulation. However, the underlying propensity may only be manifest when the gene is exposed to an appropriate environmental signal with the direction of the response and nature of the cellular dysregulation a function of the specific epigenetic change [4, 5]. It has been known for many years that exposure of the individual to nutritional and other environmental challenges during critical periods of early development can markedly affect later size, shape, structure, function and behavior. However, Barker et al. [6] were the first to provide clear evidence that there might also be a direct link between early nutritional exposure and risk of chronic disease, building the evidence that is supportive of a causal association [6, 7]. In this they have refined a new conceptual approach to the way in which we consider the evolution of this group of diseases based upon a new developmental model, the so called ‘fetal origins hypothesis’. Based initially on ecological observations and later on retrospective cohort studies, there is now a considerable body of epidemiological and experimental data that supports the hypothesis. In the earlier observations, the size and shape of the baby at birth was shown to be related to the risk during adult life of coronary heart disease, hypertension, stroke, type 2 diabetes, obesity and some cancers [6]. These relations were shown to be graded across the usual range of birth weights seen in the population and not a special feature of very high or very low birth weight, indicating that they might be a consequence of the usual range of exposure found within a population [4, 7]. The results have been reproduced across a number of populations, and although there may be some debate around the details, the general principal appears to apply widely [4, 6, 7].

Paracrine factor signaling regulates many aspects of vertebrate gonadal development. We investigated key ovarian and testicular morphological markers of the American alligator (Alligator mississippiensis) during the first 5 months post-hatching and correlated gonadal development with mRNA expression levels of a suite of regulatory factors. In both sexes, we observed significant morphology changes, including ovarian follicle assembly and meiotic progression of testicular germ cells. Concomitant with these changes were sexually dimorphic and ontogenetically variable mRNA expressions. In ovaries, FOXL2, aromatase, and follistatin mRNA expression was greater than in testes at all ages. At one week after hatching, we observed ovarian medullary remodeling in association with elevated activin/inhibin βA subunit, follistatin, and aromatase mRNA expressions. Three and 5 months following hatching and concomitant with follicle assembly, ovaries showed increased mRNA expression levels of GDF9 and the mitotic factor PCNA. In testes, the activin/inhibin α and βB subunit transcript levels were greater than in ovaries at all ages. Elevated testicular expression of GDF9 mRNA levels at 5 months after hatching aligned with increased spermatogenic activity. We propose that the mRNA expression levels and concomitant morphological changes observed here affect the establishment of alligator reproductive health and later fertility.

The osteoinductive BMP2 signal facilitates commitment to the osteoblast phenotype by inducing several classes of early response genes. Among these are bone-related HOX factors, homeodomain, RUNX and OSTERIX proteins. Here we demonstrate molecular events among BMP2-induced transcription factors that constitute a network of molecular switches on promoters of bone-related genes to coordinate their temporal expression during cellular differentiation. Our studies provide evidence for (1) selective association of HOXA10, MSX2, DLX3 and DLX5 homeodomain transcription factors on Runx2 and OC genes at stages of osteoblast maturation as well as (2) participation of these factors with RUNX2 in chromatin remodeling of bone-specific genes for repression, activation and attenuation of transcription. These findings reveal the requirement for multiple levels of control for the appropriate timing of osteoblast-related gene expression.

Aims—To establish the expression pattern of ccn3 (nov) in the central nervous system (CNS) of adult rats and to determine whether spatiotemporal variations in the expression of ccn3 (nov) are related to specific developmental stages and/or specific CNS functions. Methods—The sites of ccn3 (nov) expression have been identified by in situ hybridisation using didoxigenin labelled cRNA and by the reverse transcription-polymerase chain reaction (RT-PCR). The rat CCN3 (NOV) protein was characterised by western blotting performed on brain extracts. The localisation of the CCN3 (NOV) protein in the brain was established by immunocytochemistry. Results—Increased expression of ccn3 (nov) was detected in the developing brain of rats after birth, as shown by RT-PCR and immunocytochemistry analysis performed on a series of samples taken between day 5 (P5) and day 300 (P300), with a pronounced peak between P15 and P150, suggesting that CCN3 (NOV) might play a role in the maintenance or establishment of specific brain functions. The relatively high amounts of an N-terminal truncated CCN3 (NOV) related protein detected both in the brain tissues and cerebrospinal fluid suggested that post translational processing of CCN3 (NOV) might be particularly prevalent in the brain. Such processing might be of biological importance in the light of the previously reported growth stimulatory effects of N-terminal truncated CCN3 (NOV) isoforms. Conclusions—The postnatal differential expression of ccn3 (nov) in the brain of developing rats suggests that CCN3 (NOV) might be involved in the acquisition of specific functions. The rat species provides an as yet unequalled system for these studies. Because the CCN3 (NOV) protein is detected in restricted areas of the brain, it will be interesting to establish whether variations of ccn3 (nov) expression are associated with normal cognitive processes and whether ccn3 (nov) expression is affected by aging. In addition, because CCN3 (NOV) is found in the spinal cord and along the axonal processes, it will be of interest to determine the expression of the normal and truncated isoforms of CCN3 (NOV) in various pathological conditions, such as neurodegenerative diseases.

IN 1972, a paper on the developmental genetics of the mutation engrailed appeared in this journal. PEDRO SANTAMARIA, then a Ph.D. student in my laboratory, and I co-authored it. Being involved, I find it hard to comment on the paper itself, on its impact when it appeared and on its later relevance. I will, therefore, try to navigate along the strait between Scylla and Charybdis, avoiding my own views and considering what others may have thought of the findings.