Catherine E. Ovitt

The murine winged-helix transcription factor Foxl2 is required for granulosa cell differentiation and ovary maintenance

Human Blepharophimosis/ptosis/epicanthus inversus syndrome (BPES) type I is an autosomal dominant disorder associated with premature ovarian failure (POF) caused by mutations in FOXL2, a winged-helix/forkhead domain transcription factor. Although it has been shown that FOXL2 is expressed in adult ovaries, its function during folliculogenesis is not known. Here, we show that the murine Foxl2 gene is essential for granulosa cell differentiation and ovary maintenance. In Foxl2lacZ homozygous mutant ovaries granulosa cells do not complete the squamous to cuboidal transition leading to the absence of secondary follicles and oocyte atresia. We further demonstrate that activin-βA and anti-Mullerian inhibiting hormone expression is absent or strongly diminished in Foxl2lacZ homozygous mutant ovaries. Unexpectedly, two weeks after birth most if not all oocytes expressed Gdf9 in Foxl2lacZ homozygous mutant ovaries, indicating that nearly all primordial follicles have already initiated folliculogenesis at this stage. This activation, in the absence of functional granulosa cells, leads to oocyte atresia and progressive follicular depletion. In addition to providing a molecular mechanism for premature ovarian failure in BPES, these results suggest that granulosa cell function is not only crucial for oocyte growth but also to maintain follicular quiescence in vivo.

A mouse model for hereditary thyroid dysgenesis and cleft palate.

Alteration of thyroid gland morphogenesis (thyroid dysgenesis) is a frequent human malformation. Among the one in three to four thousand newborns in which congenital hypothyroidism is detected, 80% have either an ectopic, small and sublingual thyroid, or have no thyroid tissue. Most of these cases appear sporadically, although a few cases of recurring familial thyroid dysgenesis have been described. The lack of evidence for hereditary thyroid dysgenesis may be due to the severity of the hypothyroid phenotype. Neonatal screening and early thyroid hormone therapy have eliminated most of the clinical consequences of hypothyroidism such that the heritability of this condition may become apparent in the near future. We have recently cloned cDNA encoding a forkhead domain-containing transcription factor, TTF-2, and have located the position of the gene, designated Titf2, to mouse chromosome 4 (ref. 3). Titf2 is expressed in the developing thyroid, in most of the foregut endoderm and in craniopharyngeal ectoderm, including Rathkes pouch. Expression of Titf2 in thyroid cell precursors is down-regulated as they cease migration, suggesting that this factor is involved in the process of thyroid gland morphogenesis. Here we show that Titf2-null mutant mice exhibit cleft palate and either a sublingual or completely absent thyroid gland. Thus, mutation of Titf2 –/– results in neonatal hypothyroidism that shows similarity to thyroid dysgenesis in humans.

Differential sorting of lysosomal enzymes in mannose 6-phosphate receptor-deficient fibroblasts.

In higher eukaryotes, the transport of soluble lysosomal enzymes involves the recognition of their mannose 6‐phosphate signal by two receptors: the cation‐independent mannose 6‐phosphate/insulin‐like growth factor II receptor (CI‐MPR) and the cation‐dependent mannose 6‐phosphate receptor (CD‐MPR). It is not known why these two different proteins are present in most cell types. To investigate their relative function in lysosomal enzyme targeting, we created cell lines that lack either or both MPRs. This was accomplished by mating CD‐MPR‐deficient mice with Thp mice that carry a CI‐MPR deleted allele. Fibroblasts prepared from embryos that lack the two receptors exhibit a massive missorting of multiple lysosomal enzymes and accumulate undigested material in their endocytic compartments. Fibroblasts that lack the CI‐MPR, like those lacking the CD‐MPR, exhibit a milder phenotype and are only partially impaired in sorting. This demonstrates that both receptors are required for efficient intracellular targeting of lysosomal enzymes. More importantly, comparison of the phosphorylated proteins secreted by the different cell types indicates that the two receptors may interact in vivo with different subgroups of hydrolases. This observation may provide a rational explanation for the existence of two distinct mannose 6‐phosphate binding proteins in mammalian cells.

Requirement for the ryanodine receptor type 3 for efficient contraction in neonatal skeletal muscles

The skeletal isoform of Ca2+ release channel, RyR1, plays a central role in activation of skeletal muscle contraction. Another isoform, RyR3, has been observed recently in some mammalian skeletal muscles, but whether it participates in regulating skeletal muscle contraction is not known. The expression of RyR3 in skeletal muscles was studied in mice from late fetal stages to adult life. RyR3 was found to be expressed widely in murine skeletal muscles during the post‐natal phase of muscle development, but was not detectable in muscles of adult mice, with the exception of the diaphragm and soleus muscles. RyR3 knockout mice were generated, and it was shown that skeletal muscle contraction in these mice was impaired during the first weeks after birth. In skeletal muscles isolated from newborn RyR3−/− mice, but not in those from adult mice, the twitch elicited by electrical stimulation and the contracture induced by caffeine were strongly depressed. These results provide the first evidence that RyR3 has a physiological role in excitation–contraction coupling of neonatal skeletal muscles. The disproportion between the low amount of RyR3 and the large impact of the RyR3 knockout suggests that this isoform contributes to the amplification of Ca2+ released by the existing population of ryanodine receptors (RyR1).

Odd-skipped related 2 (Osr2) encodes a key intrinsic regulator of secondary palate growth and morphogenesis

Development of the mammalian secondary palate involves multiple steps of highly regulated morphogenetic processes that are frequently disturbed during human development, resulting in the common birth defect of cleft palate. Neither the molecular processes governing normal palatogenesis nor the causes of cleft palate is well understood. In an expression screen to identify new transcription factors regulating palate development, we previously isolated the odd-skipped related 2 (Osr2) gene, encoding a zinc-finger protein homologous to the Drosophila odd-skipped gene product, and showed that Osr2 mRNA expression is specifically activated in the nascent palatal mesenchyme at the onset of palatal outgrowth. We report that a targeted null mutation in Osr2 impairs palatal shelf growth and causes delay in palatal shelf elevation, resulting in cleft palate. Whereas palatal outgrowth initiates normally in the Osr2 mutant embryos, a significant reduction in palatal mesenchyme proliferation occurs specifically in the medial halves of the downward growing palatal shelves at E13.5, which results in retarded, mediolaterally symmetric palatal shelves before palatal shelf elevation. The developmental timing of palatal growth retardation correlates exactly with the spatiotemporal pattern of Osr1 gene expression during palate development. Furthermore, we show that the Osr2 mutants exhibit altered gene expression patterns, including those of Osr1, Pax9 and Tgfb3, during palate development. These data identify Osr2 as a key intrinsic regulator of palatal growth and patterning.

The Atypical Homeodomain Transcription Factor Mohawk Controls Tendon Morphogenesis

ABSTRACT The Mohawk homeobox (Mkx) gene encodes a new atypical homeodomain-containing protein with transcriptional repressor activity. Mkx mRNA exhibited dynamic expression patterns during development of the palate, somite, kidney, and testis, suggesting that it may be an important regulator of multiple developmental processes. To investigate the roles of Mkx in organogenesis, we generated mice carrying a null mutation in this gene. Mkx−/− mice survive postnatally and exhibit a unique wavy-tail phenotype. Close examination revealed that the mutant mice had smaller tendons than wild-type littermates and that the rapid postnatal growth of collagen fibrils in tendons was disrupted in Mkx−/− mice. Defects in tendon development were detected in the mutant mouse embryos as early as embryonic day 16.5 (E16.5). Although collagen fibril assembly initially appeared normal, the tendons of Mkx−/− embryos expressed significantly reduced amounts of collagen I, fibromodulin, and tenomodulin in comparison with control littermates. We found that Mkx mRNA was strongly expressed in differentiating tendon cells during embryogenesis and in the tendon sheath cells in postnatal stages. In addition to defects in tendon collagen fibrillogenesis, Mkx−/− mutant mice exhibited abnormal tendon sheaths. These results identify Mkx as an important regulator of tendon development.

Targeted disruption of the mouse cation-dependent mannose 6-phosphate receptor results in partial missorting of multiple lysosomal enzymes.

In mammalian cells two mannose 6‐phosphate receptors (MPRs) are involved in lysosomal enzyme transport. To understand the precise function of the cation‐dependent mannose 6‐phosphate receptor (CD‐MPR), one allele of the corresponding gene has been disrupted in mouse embryonic stem cells and homozygous mice lacking this receptor have been generated. The homozygous mice appear normal, suggesting that other targeting mechanisms can partially compensate for the loss of the CD‐MPR in vivo. However, homozygous receptor‐deficient cells and animals clearly exhibit defects in targeting of multiple lysosomal enzymes when compared with wild‐types. Increased levels of phosphorylated lysosomal enzymes were present in body fluids of homozygous animals. In thymocytes from homozygous mice or in primary cultures of fibroblasts from homozygous embryos, there is a marked increase in the amount of phosphorylated lysosomal enzymes that are secreted into the extracellular medium. The cultured fibroblasts have decreased intracellular levels of multiple lysosomal enzymes and accumulate macromolecules within their endosomal/lysosomal system. Taken together, these results clearly indicate that the CD‐MPR is required for efficient intracellular targeting of multiple lysosomal enzymes.

Salivary Gland Homeostasis Is Maintained through Acinar Cell Self-Duplication

Current dogma suggests that salivary gland homeostasis is stem cell dependent. However, the extent of stem cell contribution to salivary gland maintenance has not been determined. We investigated acinar cell replacement during homeostasis, growth, and regeneration, using an inducible CreER(T2) expressed under the control of the Mist1 gene locus. Genetic labeling, followed by a chase period, showed that acinar cell replacement is not driven by the differentiation of unlabeled stem cells. Analysis using R26(Brainbow2.1) reporter revealed continued proliferation and clonal expansion of terminally differentiated acinar cells in all major salivary glands. Induced injury also demonstrated the regenerative potential of pre-labeled acinar cells. Our results support a revised model for salivary gland homeostasis based predominantly on self-duplication of acinar cells, rather than on differentiation of stem cells. The proliferative capacity of differentiated acinar cells may prove critical in the implementation of cell-based strategies to restore the salivary glands.

Oct‐4: more than just a POUerful marker of the mammalian germline?

Mammals lack visible cytoplasmic components in the oocyte that could account for ‘germline determinants’ as identified in various non‐mammalian species. Actually, mammals might not define the germline autonomously by localized ‘germline determinants’ but conditionally depending on the position of cells within the embryo. The Oct‐4 gene encodes a transcription factor that is specifically expressed in the toti‐ and pluripotential stem cells of the mouse embryo and so far has only been found in mammalian species. Oct‐4‐expressing embryonal cells retain the capacity to differentiate along multiple lineages and they have been suggested to be part of a ‘totipotent germline cycle’ that links one generation to the next.

Ascl3 expression marks a progenitor population of both acinar and ductal cells in mouse salivary glands

Ascl3, also know as Sgn1, is a member of the mammalian achaete scute (Mash) gene family of transcription factors, which have been implicated in cell fate specification and differentiation. In the mouse salivary gland, expression of Ascl3 is restricted to a subset of duct cells. Salivary gland function depends on the secretory acinar cells, which are responsible for saliva formation, and duct cells, which modify the saliva and conduct it to the oral cavity. The salivary gland ducts are also the putative site of progenitor cells in the adult gland. Using a Cre recombinase-mediated reporter system, we followed the fate of Ascl3-expressing cells after the introduction of an EGFP-Cre expression cassette into the Ascl3 locus by homologous recombination. Lineage tracing shows that these cells are progenitors of both acinar and ductal cell types in all three major salivary glands. In the differentiated progeny, expression of Ascl3 is down-regulated. These data directly demonstrate a progenitor-progeny relationship between duct cells and the acinar cell compartment, and identify a population of multipotent progenitor cells, marked by expression of Ascl3, which is capable of generating both gland cell types. We conclude that Ascl3-expressing cells contribute to the maintenance of the adult salivary glands.