Proteins In Embryonic Development Research Articles

Novel patients with NHLRC2 variants expand the phenotypic spectrum of FINCA disease.

FINCA disease (Fibrosis, Neurodegeneration and Cerebral Angiomatosis, OMIM 618278) is an infantile-onset neurodevelopmental and multiorgan disease. Since our initial report in 2018, additional patients have been described. FINCA is the first human disease caused by recessive variants in the highly conserved NHLRC2 gene. Our previous studies have shown that Nhlrc2-null mouse embryos die during gastrulation, indicating the essential role of the protein in embryonic development. Defect in NHLRC2 leads to cerebral neurodegeneration and severe pulmonary, hepatic and cardiac fibrosis. Despite having a structure suggestive of an enzymatic role and the clinical importance of NHLRC2 in multiple organs, the specific physiological role of the protein is unknown. The clinical histories of five novel FINCA patients diagnosed with whole exome sequencing were reviewed. Segregation analysis of the biallelic, potentially pathogenic NHLRC2 variants was performed using Sanger sequencing. Studies on neuropathology and NHLRC2 expression in different brain regions were performed on autopsy samples of three previously described deceased FINCA patients. One patient was homozygous for the pathogenic variant c.442G > T, while the other four were compound heterozygous for this variant and two other pathogenic NHLRC2 gene variants. All five patients presented with multiorgan dysfunction with neurodevelopmental delay, recurrent infections and macrocytic anemia as key features. Interstitial lung disease was pronounced in infancy but often stabilized. Autopsy samples revealed widespread, albeit at a lower intensity than the control, NHLRC2 expression in the brain. This report expands on the characteristic clinical features of FINCA disease. Presentation is typically in infancy, and although patients can live to late adulthood, the key clinical and histopathological features are fibrosis, infection susceptibility/immunodeficiency/intellectual disability, neurodevelopmental disorder/neurodegeneration and chronic anemia/cerebral angiomatosis (hence the acronym FINCA) that enable an early diagnosis confirmed by genetic investigations.

Epidermal PAR-6 and PKC-3 are essential for larval development of C. elegans and organize non-centrosomal microtubules.

The cortical polarity regulators PAR-6, PKC-3, and PAR-3 are essential for the polarization of a broad variety of cell types in multicellular animals. In C. elegans, the roles of the PAR proteins in embryonic development have been extensively studied, yet little is known about their functions during larval development. Using inducible protein degradation, we show that PAR-6 and PKC-3, but not PAR-3, are essential for postembryonic development. PAR-6 and PKC-3 are required in the epidermal epithelium for animal growth, molting, and the proper pattern of seam-cell divisions. Finally, we uncovered a novel role for PAR-6 in organizing non-centrosomal microtubule arrays in the epidermis. PAR-6 was required for the localization of the microtubule organizer NOCA-1/Ninein, and defects in a noca-1 mutant are highly similar to those caused by epidermal PAR-6 depletion. As NOCA-1 physically interacts with PAR-6, we propose that PAR-6 promotes non-centrosomal microtubule organization through localization of NOCA-1/Ninein.

Differential proteomic analysis revealed crucial egg white proteins for hatchability of chickens

For healthy development, an avian embryo needs the nutritional and functional molecules maternally deposited in avian eggs. Egg white not only provides nutritional components but also exhibits functional properties, such as defenses against microbial invasion. However, the roles of the more detailed messages in embryo development remain unclear. In this study, a tandem mass tag labeling quantitation approach was used to innovatively identify the differential proteins in the egg whites of fresh eggs produced by hens with divergent high/low hatchability and in the egg whites of embryonated eggs with healthy and dead embryos. A total of 378 proteins were quantified in egg white, which is the most complete proteome identified for egg white to date, and up to 102 differential proteins were identified. GO enrichment, pathway, and hierarchical clustering analysis revealed some of the differential proteins that are the main participants in several biological processes, including blood coagulation, intermediate filament, antibacterial activity, and neurodevelopment. A list of 11 putative protein biomarkers, such as keratin (KRT19, KRT12, KRT15, and KRT6A), which is involved in cell architecture, and fibrinogen (fibrinogen alpha chain, fibrinogen beta chain, and fibrinogen gamma chain), which is related to blood coagulation, were ultimately screened. The current study screened egg white proteins that can predict low hatchability and embryonic death and deciphered the role of these proteins in embryonic development, which is meaningful for the comprehensive understanding of embryonic growth.

Genetic monkeys reveal a new role for a longevity protein in embryonic development.

Genetic monkeys reveal a new role for a longevity protein in embryonic development.

The Unique Features of Proteins Depicting the Chicken Amniotic Fluid

In many amniotes, the amniotic fluid is depicted as a dynamic milieu that participates in the protection of the embryo (cushioning, hydration, and immunity). However, in birds, the protein profile of the amniotic fluid remains unexplored, even though its proteomic signature is predicted to differ compared with that of humans. In fact, unlike humans, chicken amniotic fluid does not collect excretory products and its protein composition strikingly changes at mid-development because of the massive inflow of egg white proteins, which are thereafter swallowed by the embryo to support its growth. Using GeLC-MS/MS and shotgun strategies, we identified 91 nonredundant proteins delineating the chicken amniotic fluid proteome at day 11 of development, before egg white transfer. These proteins were essentially associated with the metabolism of nutrients, immune response and developmental processes. Forty-eight proteins were common to both chicken and human amniotic fluids, including serum albumin, apolipoprotein A1 and alpha-fetoprotein. We further investigated the effective role of chicken amniotic fluid in innate defense and revealed that it exhibits significant antibacterial activity at day 11 of development. This antibacterial potential is drastically enhanced after egg white transfer, presumably due to lysozyme, avian beta-defensin 11, vitelline membrane outer layer protein 1, and beta-microseminoprotein-like as the most likely antibacterial candidates. Interestingly, several proteins recovered in the chicken amniotic fluid prior and after egg white transfer are uniquely found in birds (ovalbumin and related proteins X and Y, avian beta-defensin 11) or oviparous species (vitellogenins 1 and 2, riboflavin-binding protein). This study provides an integrative overview of the chicken amniotic fluid proteome and opens stimulating perspectives in deciphering the role of avian egg-specific proteins in embryonic development, including innate immunity. These proteins may constitute valuable biomarkers for poultry production to detect hazardous situations (stress, infection, etc.), that may negatively affect the development of the chicken embryo.

Genetic Suppression of Basement Membrane Defects in Caenorhabditis elegans by Gain of Function in Extracellular Matrix and Cell-Matrix Attachment Genes.

Basement membranes are extracellular matrices essential for embryonic development in animals. Peroxidasins are extracellular peroxidases implicated in the unique sulfilimine cross-links between type IV basement membrane collagens. Loss of function in the Caenorhabditis elegans peroxidasin PXN-2 results in fully penetrant embryonic or larval lethality. Using genetic suppressor screening, we find that the requirement for PXN-2 in development can be bypassed by gain of function in multiple genes encoding other basement membrane components, or proteins implicated in cell-matrix attachment. We identify multiple alleles of let-805, encoding the transmembrane protein myotactin, which suppress phenotypes of pxn-2 null mutants and of other basement membrane mutants such as F-spondin/spon-1. These let-805 suppressor alleles cause missense alterations in two pairs of FNIII repeats in the extracellular domain; they act dominantly and have no detectable phenotypes alone, suggesting they cause gain of function. We also identify suppressor missense mutations affecting basement membrane components type IV collagen (emb-9, let-2) and perlecan (unc-52), as well as a mutation affecting spectraplakin (vab-10), a component of the epidermal cytoskeleton. These suppressor alleles do not bypass the developmental requirement for core structural proteins of the basement membrane such as laminin or type IV collagen. In conclusion, putative gain-of-function alterations in matrix proteins or in cell-matrix receptors can overcome the requirement for certain basement membrane proteins in embryonic development, revealing previously unknown plasticity in the genetic requirements for the extracellular matrix.

Molecular identification of disk abalone (Haliotis discus discus) tetraspanin 33 and CD63: Insights into potent players in the disk abalone host defense system

Tetraspanins are a superfamily of transmembrane proteins involved in a diverse range of physiological processes including differentiation, adhesion, signal transduction, cell motility, and immune responses. In the present study, two tetraspanins, CD63 and tetraspanin 33 (TSPAN33) from disk abalone (AbCD63 and AbTSPAN33), were identified and characterized at the molecular level. The coding sequences for AbCD63 and AbTSPAN33 encoded polypeptides of 234 and 290 amino acids (aa) with predicted molecular mass of 25.3 and 32.5 kDa, respectively. The deduced AbCD63 and AbTSPAN33 protein sequences were also predicted to have a typical tetraspanin domain architecture, including four transmembrane domains (TM), short N- and C- terminal regions, a short intracellular loop, as well as a large and small extracellular loop. A characteristic CCG motif and cysteine residues, which are highly conserved across CD63 and TSPAN33 proteins of different species, were present in the large extracellular loop of both abalone tetraspanins. Phylogenetic analysis revealed that the AbCD63 and AbTSPAN33 clustered in the invertebrate subclade of tetraspanins, thus exhibiting a close relationship with tetraspanins of other mollusks. The AbCD63 and AbTSPAN33 mRNA transcripts were detected at early embryonic development stages of disk abalone with significantly higher amounts at the trochophore stage, suggesting the involvement of these proteins in embryonic development. Both AbCD63 and AbTSPAN33 were ubiquitously expressed in all the tissues of unchallenged abalones analyzed, with the highest expression levels found in hemocytes. Moreover, significant induction of AbCD63 and AbTSPAN33 mRNA expression was observed in immunologically important tissues, such as hemocytes and gills, upon stimulation with live bacteria (Vibrio parahaemolyticus and Listeria monocytogenes), virus (viral hemorrhagic septicemia virus), and two potent immune stimulators [polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS)]. Collectively, these findings suggest that AbCD63 and AbTSPAN33 are involved in innate immune responses in disk abalone during pathogenic stress.

Potential Involvement of Snail Members in Neuronal Survival and Astrocytic Migration during the Gecko Spinal Cord Regeneration.

Certain regenerative vertebrates such as fish, amphibians and reptiles are capable of regenerating spinal cord after injury. Most neurons of spinal cord will survive from the injury and regrow axons to repair circuits with an absence of glial scar formation. However, the underlying mechanisms of neuronal anti-apoptosis and glia-related responses have not been fully clarified during the regenerative process. Gecko has becoming an inspiring model to address spinal cord regeneration in amniotes. In the present study, we investigated the regulatory roles of Snail family members, the important transcriptional factors involved in both triggering of the cell migration and cell survival, during the spontaneous spinal cord regeneration. Both Snail1 and Snail3 have been shown to promote neuronal survival and astrocytic migration via anti-apoptotic and GTPases signaling following gecko tail amputation. Transforming growth factor-beta (TGFβ), together with other cytokines were involved in inducing expression of Snail protein. Our data indicate a conserved function of Snail proteins in embryonic development and tissue regeneration, which may provide clues for CNS repair in the mammals.

Structure and function of gap junction proteins: role of gap junction proteins in embryonic heart development.

Intercellular (cell-to-cell) communication is a crucial and complex mechanism during embryonic heart development. In the cardiovascular system, the beating of the heart is a dynamic and key regulatory process, which is functionally regulated by the coordinated spread of electrical activity through heart muscle cells. Heart tissues are composed of individual cells, each bearing specialized cell surface membrane structures called gap junctions that permit the intercellular exchange of ions and low molecular weight molecules. Gap junction channels are essential in normal heart function and they assist in the mediated spread of electrical impulses that stimulate synchronized contraction (via an electrical syncytium) of cardiac tissues. This present review describes the current knowledge of gap junction biology. In the first part, we summarise some relevant biochemical and physiological properties of gap junction proteins, including their structure and function. In the second part, we review the current evidence demonstrating the role of gap junction proteins in embryonic development with particular reference to those involved in embryonic heart development. Genetics and transgenic animal studies of gap junction protein function in embryonic heart development are considered and the alteration/disruption of gap junction intercellular communication which may lead to abnormal heart development is also discussed.

Review for the regulatory functions of KRAB zinc finger proteins in embryonic development and tumorgenesis of higher vertebrates

KRAB-containing zinc-finger proteins (KRAB-ZFPs) first arose in the tetrapod vertebrates, and evolved quickly. Till Homo sapiens, they have become the largest family of transcription factors. Despite the molecular mechanism of transcription regulation by KRAB-ZFPs has been clarified in some degree, the higher-vertebrate-specific biological functions of the KRAB-ZFP family are still largely unknown. This review focused on the important regulatory functions of the KRAB-ZFP in embryonic development and tumorgenesis, which will benefit to the comprehensive understanding of biological roles of KRAB-ZFP in different physiological and pathological states. All of the systematic information will facilitate the further theoretical and applied studies of KRAB-ZFPs.

Abstract 1069: The role of mammalian Ecdysoneless in cell cycle regulation and cancer

Abstract Our long term research goal is to understand biochemical mechanisms of immortalization of human mammary epithelial cells (hMECs) that represents an early step in breast cancer progression. We have generated several hMEC immortalization models using viral oncogenes, mutated cancer genes as well as radiation treatment of cells. In one of such immortalization model, we identified Ecdysoneless (Ecd) protein as the human papilloma virus (HPV) E6 interacting protein. We have reported earlier that HPV16 E6 protein is highly efficient for hMEC immortalization. Here, we present evidence that Ecd, an evolutionarily conserved protein, is a novel regulator of the Rb/E2F pathway. We generated a conditional Ecd knockout mouse model and observed that complete deletion of Ecd leads to early embryonic lethality in mice. The experiments using conditional Ecd-null MEFs created from Ecdlox/lox revealed that lack of Ecd inhibits cellular proliferation by delaying G1-S cell cycle progression and down-regulates E2F target gene expression. Furthermore, Ecd directly binds to Rb at the pocket domain and disrupts the interaction of Rb with E2F, leading to impairment of repressive effect of Rb on E2F target promoters. Thus, we demonstrate that Ecd is an essential protein in embryonic development and a novel regulator of G1-S cell cycle transition by modulating Rb/E2F pathway. Consistent with these observations, overexpression of Ecd in hMECs hastens the exit of cells from G1 phase of the cell cycle. Significantly, immunohistochemical analyses showed Ecd is overexessed in ductal carcinoma in situ as well as in infiltrating ductal carcinoma of breast. In conclusion, Ecd plays an important role in cell cycle progression and its deregulation can lead to abnormal cell proliferation leading to breast cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1069.

Development of Transgenic Chickens Expressing Human Parathormone Under the Control of a Ubiquitous Promoter by Using a Retrovirus Vector System

Transgenic chickens, ubiquitously expressing a human protein, could be a very useful model system for studying the role of human proteins in embryonic development as well as for efficiently producing pharmaceutical drugs as bioreactors. Human parathormone (hPTH) secreted from parathyroid glands plays a significant role in calcium homeostasis and is an important therapeutic agent for the treatment of osteoporosis in humans. Here, by using a robust replication-defective Moloney murine leukemia virus-based retrovirus vector encapsidated with vesicular stomatitis virus G glycoprotein, we generated transgenic chickens expressing hPTH under the control of a ubiquitous Rous sarcoma virus promoter. The recombinant retrovirus was injected into the subgerminal cavity of freshly laid eggs at the blastodermal stage. After 21 d of incubation, 42 chicks hatched from 473 retrovirus-injected eggs. All 42 living chicks were found to express the vector-encoded hPTH gene in diverse organs, as revealed by PCR and reverse transcription-PCR analysis by using primer pairs specific for hPTH. Four days after hatching, 6 chicks died and 14 chicks showed phenotypic deformities. At 18 wk of age, only 3 G0 chickens survived. They also released the hPTH hormone in their blood and transmitted the hPTH gene to G1 embryos. However, although the embryos were alive at d 18 of incubation, none hatched. An electrochemiluminescence immunoassay further showed that the hPTH expression level was markedly elevated in mammalian cells infected by the retrovirus vector. Thus, we demonstrated that transgenic chickens, expressing a human protein under the control of a ubiquitous promoter, not only could be an efficient bioreactor for the production of pharmaceutical drugs, but also could be useful for studies on the role of human proteins in embryonic development. To our knowledge, this is the first report on the production of a human protein (hPTH) in transgenic chickens under the control of a ubiquitous promoter by using a replication-defective Moloney murine leukemia virus-based retrovirus vector system.

Comparative analysis of serine protease‐related genes in the honey bee genome: possible involvement in embryonic development and innate immunity

We have identified 44 serine protease (SP) and 13 serine protease homolog (SPH) genes in the genome of Apis mellifera. Most of these genes encode putative secreted proteins, but four SPs and three SPHs may associate with the plasma membrane via a transmembrane region. Clip domains represent the most abundant non-catalytic structural units in these SP-like proteins −12 SPs and six SPHs contain at least one clip domain. Some of the family members contain other modules for protein–protein interactions, including disulphide-stabilized structures (LDLrA, SRCR, frizzled, kringle, Sushi, Wonton and Pan/apple), carbohydrate-recognition domains (C-type lectin and chitin-binding), and other modules (such as zinc finger, CUB, coiled coil and Sina). Comparison of the sequences with those from Drosophila led to a proposed SP pathway for establishing the dorsoventral axis of honey bee embryos. Multiple sequence alignments revealed evolutionary relationships of honey bee SPs and SPHs with those in Drosophila melanogaster, Anopheles gambiae, and Manduca sexta. We identified homologs of D. melanogaster persephone, M. sexta HP14, PAP-1 and SPH-1. A. mellifera genome includes at least five genes for potential SP inhibitors (serpin-1 through -5) and three genes of SP putative substrates (prophenoloxidase, spätzle-1 and spätzle-2). Quantitative RT-PCR analyses showed an elevation in the mRNA levels of SP2, SP3, SP9, SP10, SPH41, SPH42, SP49, serpin-2, serpin-4, serpin-5, and spätzle-2 in adults after a microbial challenge. The SP41 and SP6 transcripts significantly increased after an injection of Paenibacillus larva, but there was no such increase after injection of saline or Escherichia coli. mRNA levels of most SPs and serpins significantly increased by 48 h after the pathogen infection in 1st instar larvae. On the contrary, SP1, SP3, SP19 and serpin-5 transcript levels reduced. These results, taken together, provide a framework for designing experimental studies of the roles of SPs and related proteins in embryonic development and immune responses of A. mellifera.

Function of PEA3 Ets transcription factors in mammary gland development and oncogenesis.

The Ets gene families of mice and man currently comprise 27 genes that encode sequence-specific transcription factors. Ets proteins share an approximately 85 amino acid structurally conserved ETS DNA binding domain. Genetic analyses in model organisms suggest roles for Ets proteins in embryonic development and various adult physiological processes. Chromosomal translocations involving several ETS genes are associated with Ewing's sarcomas and leukemias, whereas the overexpression of some ETS genes is linked with numerous malignancies, including breast cancer. Indeed PEA3, ETS-1, PDEF, and ELF-3 transcripts have all been reported to be elevated in human breast tumors. Some of the ETS genes that are overexpressed in human breast tumors are also overexpressed in mouse models of this disease. Notably, pea3, as well as its close paralogs er81 and erm, which comprise the pea3 subfamily of ets genes, are coordinately overexpressed in mouse mammary tumors. Genetic analyses in mice reveal required roles for one or more of the PEA3 subfamily Ets proteins in the initiation and progression of mouse mammary tumors. The pea3 subfamily genes are normally expressed in the primitive epithelium of mouse mammary buds during embryogenesis, and these three genes are expressed in epithelial progenitor cells during postnatal mammary gland development. Loss-of-function mutations in the mouse pea3 gene results in increased numbers of terminal end buds and an increased fraction of proliferating cells in these structures, suggesting a role for PEA3 in progenitor cell renewal or terminal differentiation. Taken together these observations suggest that the PEA3 subfamily proteins play key regulatory roles in both mammary gland development and oncogenesis.

Dual roles for the Dab2 adaptor protein in embryonic development and kidney transport.

The Disabled-2 (Dab2) gene has been proposed to act as a tumor suppressor. Cell culture studies have implicated Dab2 in signal transduction by mitogens, TGFbeta and endocytosis of lipoprotein receptors. To identify in vivo functions of Dab2, targeted mutations were made in the mouse. In the absence of Dab2, embryos arrest prior to gastrulation with a phenotype reminiscent of those caused by deletion of some TGFbeta signal transduction molecules involved in Nodal signaling. Dab2 is expressed in the extra-embryonic visceral endoderm but not in the epiblast. Dab2 could be conditionally deleted from the embryo without affecting normal development, showing that Dab2 is required in the visceral endoderm but dispensable in the embryo proper. Conditionally mutant Dab2(-/-) mice are overtly normal, but have reduced clathrin-coated pits in kidney proximal tubule cells and excrete specific plasma proteins in the urine, consistent with reduced transport by a lipoprotein receptor, megalin/gp330, in the proximal tubule. This evidence indicates that Dab2 is pleiotropic and regulates both visceral endoderm function and lipoprotein receptor trafficking in vivo.

The involvement of a conserved family of RNA binding proteins in embryonic development and carcinogenesis

Vg1 RBP is a member of a family of highly conserved proteins that appear to be involved in RNA localization, stability, and/or translational control in a wide variety of cell types and organisms. Over the last few years, the human homologs of these proteins have been found to be overexpressed in an increasing number of different kinds of cancers. Although the role of these proteins in neoplasia is not understood, results from several labs, including our own, are beginning to suggest that many of these proteins may be important in cell motility, a necessary requirement for metastasis. This paper will review these data and suggest a model for the role of Vg1 RBP and its homologs in embryonic development and carcinogenesis.

The mouse fidgetin gene defines a new role for AAA family proteins in mammalian development.

The mouse mutation fidget arose spontaneously in a heterogeneous albino stock. This mutant mouse is characterized by a side-to-side head-shaking and circling behaviour, due to reduced or absent semicircular canals. Fidget mice also have small eyes, associated with cell-cycle delay and insufficient growth of the retinal neural epithelium, and lower penetrance skeletal abnormalities, including pelvic girdle dysgenesis, skull bone fusions and polydactyly. By positional cloning, we found the gene mutated in fidget mice, fidgetin (Fign), which encodes a new member of the 'meiotic' or subfamily-7 (SF7; ref. 7) group of ATPases associated with diverse cellular activities (AAA proteins). We also discovered two closely related mammalian genes. AAA proteins are molecular chaperones that facilitate a variety of functions, including membrane fusion, proteolysis, peroxisome biogenesis, endosome sorting and meiotic spindle formation, but functions for the SF7 AAA proteins are largely unknown. Fidgetin is the first mutant AAA protein found in a mammalian developmental mutant, thus defining a new role for these proteins in embryonic development.

Expression of the highly conserved RNA binding protein KOC in embryogenesis

The human KOC gene which is highly expressed in cancer shows typical structural features of an RNA binding protein. We analyzed the temporal and spatial expression pattern of KOC in mouse embryos at different gestational ages. The expression of KOC seems to be ubiquitous at early stages. During advanced gestation highest KOC expression occurs in the gut, pancreas, kidney, and in the developing brain. The expression pattern of KOC was compared to its Xenopus homologue Vg1-RBP during frog development. Similar expression was found in these organs suggesting an important functional role of the homologous proteins in embryonic development.

P53 in embryonic development: maintaining a fine balance.

In addition to its role as a tumour suppressor and cell-cycle checkpoint control protein, p53 has been implicated as an important protein in embryonic development. Despite the viability of most p53 null mice, evidence has accumulated that p53 may regulate differentiation and the response of embryonic cells to diverse environmental stresses. Moreover, it appears that maintenance of a fine balance of p53 protein levels within embryonic cells is important for optimal development. Inappropriate overexpression or underexpression of p53 can lead to embryonic lethality or increased risk of malformations. The p53 protein may utilize multiple functional activities in its regulation of developmental processes.

Differences in water release with DNA binding by ultrabithorax and deformed homeodomains.

The amino acid sequences of the homeodomains (HD) within the Ultrabithorax (Ubx) and Deformed (Dfd) proteins from Drosophila melanogaster are highly conserved despite distinct genetic regulatory functions for these proteins in embryonic development. We reported recently that Ubx-HD binding to a single target site displayed significantly increased affinity and greater salt concentration dependence at lower pH; in contrast, Dfd-HD did not show pH dependence in its DNA binding properties [Li, L., et al. (1996) Biochemistry 35, 9832-9839]. We demonstrate in this study that water activity differentially affects Ubx-HD and Dfd-HD DNA binding affinity. The sensitivity of the protein-DNA binding constant to osmotic pressures generated by neutral solutes was measured, and the formation of the Ubx-HD-DNA complex is associated with significantly greater water release than that of the Dfd-HD-DNA complex. No influence of pH on water release was detected for either HD. Experiments with chimeric Ubx-Dfd homeodomains demonstrated that the C-terminal region of the Ubx-HD is the primary determinant for the greater water release associated with DNA binding for this protein. DNA sequences do not exert a significant effect on the magnitude of water release associated with protein-DNA binding for Ubx-HD and the chimeric HD, UDU.