Examining the role of Lunatic fringe dosage in somitogenesis. A Senior Honors Thesis. Jason D. Lather

Transcription

1 Examining the role of Lunatic fringe dosage in somitogenesis A Senior Honors Thesis Presented in partial fulfillment of the requirements for graduation with distinction in Molecular Genetics in the undergraduate colleges of The Ohio State University By Jason D. Lather The Ohio State University June 2008 Project Advisor: Doctor Susan Cole, Department of Molecular Genetics Thesis Committee: Dr. Susan Cole Dr. Harold Fisk Dr. Heithem El-Hodiri

2 Abstract The repeating structures of the vertebrate axial skeleton are produced by the process of somitogenesis. Somites bud from the presomitic mesoderm (PSM) in an anterior to posterior direction, and are the embryonic precursors of the vertebrae and ribs. This process is highly regulated by one signaling pathway in particular: Notch signaling. This study focuses on the functions of Lunatic fringe (Lfng) which encodes a glycosyltransferase that inhibits Notch signaling. The PSM is divided into two regions: one in the posterior region (region I), a clock that times somitogenesis. In the anterior PSM (region II), rostral-caudal (R/C) patterning of somites occurs. Lfng and Notch signaling play roles in both regions of the PSM. Our lab created a novel Lfng allele that perturbs Lfng function in the clock, but preserves its function in R/C somite patterning, and found that while cyclic Lfng actually is important in anterior skeletal development, the Lfng patterning function is specifically required for tail development. This project focuses on the roles played by Lfng and somite patterning in tail development. We examined skeletal development in embryos expressing different amounts of Lfng during R/C patterning. In the absence of anterior Lfng tail development is severely affected and tail truncation is observed. As the expression of Lfng in the anterior PSM increases, less severe tail phenotypes are observed. Data obtained from this experiment suggests that anterior expression of Lfng in the rostral-caudal patterning region is critical for development of the tail and that tail development is sensitive to Lfng dosage. In order to test this hypothesis, a novel transgene expressing exogenous Lfng only in the anterior PSM has been created. We hypothesize that this anterior-specific Lfng will rescue tail development in Lfng null mice. We are currently investigating the effects of this transgene by first analyzing Lfng expression by whole mount in situ hybridization. Currently, mice are being bred in order to provide us with data to support our hypothesis. i

3 Table of Contents Abstract... Table of Contents... List of Figures... List of Tables... i ii iii iv Chapters Chapter 1 Introduction... 1 Overview of Somitogenesis... 1 Primary vs. Secondary Body Formation... 1 Two regions in the PSM: Clock vs. Patterning... 3 Notch Pathway and somitogenesis... 3 Lfng plays many roles in somitogenesis... 6 Chapter 2 Results Examining the requirements for anterior development Quantifying the posterior defects in Lfng mutant mice Production of an anterior specific Lfng transgene Chapter 3 Discussion Clock function in somitogenesis is required for proper anterior skeletal development Patterning of presomites in region II is required for proper tail development The Mesp2 promoter linked Lfng transgene lacks expression in the anterior PSM Future Directions Conclusions Chapter 4 Materials and Methods Works Cited ii

4 List of Tables Table Medians and Means of quantification Table PCR Primers iii

5 List of Figures Figure 1.1 Schematic of Presomitic Mesoderm... 9 Figure 1.2 Notch signaling Figure 1.3 Interactions in the posterior and anterior PSM Figure 1.4 The FCE1 deletion and corresponding phenotypes Figure 2.1 Lfng genotyping Figure 2.2 Anterior body defects Figure 2.3 Posterior body defects Figure 2.4 Anterior Specific transgene Figure 3.1 Transgene knocking out region II iv

6 Chapter 1 Introduction Overview of Somitogenesis Pairs of somites bud from the presomitic mesoderm (PSM) during somitogenesis. These somites later differentiate into the repeating structures of the back and tail. In mouse, a pair of somites forms every two hours; however, the time required is species specific. In chick embryos, somites bud every ninety minutes. The consistent time required for a pair of somites to bud has led many to believe that a molecular clock underlies somite formation. Notch signaling is a prime candidate for acting in the clock in somitogenesis. In addition, Notch has been shown to play an important role in the patterning of somites into their anterior and posterior regions. Analysis of one particular Notch pathway member, Lunatic fringe (Lfng), has supported conclusions that Notch signaling is important in somitogenesis. Primary vs. secondary body formation The mechanism for body development has been highly debated since 1925, Some believe that the posterior body, the tail, forms as a continuation of primary body formation, or the formation of the head and trunk. However, some also believe that the anterior body and posterior body form by two different mechanisms. In primary neurulation, or primary body formation, the anterior regions of the body are formed. The head and trunk differentiate and develop from tissues formed in early development. Regions of the vertebral column formed at this stage in develop are the cervical, thoracic, and lumbar vertebrae (Reviewed in Handrigan, et al., 2003). It is believed that primary and secondary body formation occurs through different mechanisms because different 1

7 genes are expressed during primary and secondary body formation. In primary development, gastrulation continues until the primitive streak is at the end of the PSM, but secondary development begins when gastrulation ceases and the tailbud forms. Secondary body formation seems to occur by a differently regulated mechanism than primary body formation. Secondary body formation in mouse begins around embryonic day 10.0 and ceases when somitogenesis terminates at embryonic day Structures formed during secondary body formation later differentiate into parts of the adult tail the sacral region and vertebrae of the tail. An important tissue in forming somites is the Tail Bud Mesenchyme (TBM), which is proposed to be undifferentiated mesenchyme that gives rise to somites. Without the TBM, somitogenesis ceases; in wildtype mouse embryos, the TBM is lost at E The TBM s role in tail length has been shown by grafting the TBM (Goldman, et. al, 2000). The TBM receives important signals from the Ventral Ectodermal Ridge (VER) which allows for somitogenesis to occur. The VER develops during secondary body formation and acts to signal for cell proliferation, promoting tail outgrowth. The VER appears to be a posterior body analog of the Apical Ectodermal Ridge of limb development and appear to have functional homology in their respective regions of development. Tails where the VER is removed have a normal segment appearance, but have truncated tails as a result of the cessation of somite development (Goldman, et. al, 2000). Somites are all of equal size and are equally patterned showing that the VER s role is only in tail elongation and not in patterning of somites. Further experiments showed that a VER implant on tails lacking a VER allowed somitogenesis to occur. The 2

8 VER s obvious role in tail development suggests that the PSM and the VER likely have some interaction. Two regions in the PSM: Clock vs. Patterning The tail develops from the PSM, which is divided into two regions. The posterior is termed region I and functions using a molecular clock used for cell allocation (Reviewed in Andrade, et. al, 2005). The oscillation in this particular region was suggested by finding oscillation of a gene, hairy1, in the PSM. Comparing the oscillation with embryos of the same somite number, that is, chicks less than 90 minutes apart in development showed that different localizations of hairy1 were found (Palmeirim, et. al, 1997), meaning expression of hairy1 oscillates in the chick PSM. The second region of the PSM, region II, functions to pattern each somite pair into rostral/caudal halves. Genes related to both of these regions are also members of the Notch signaling pathway. Therefore, it is reasonable to believe that Notch signaling plays an important role both in region I and region II of the PSM (Figure 1.1). Notch Pathway and somitogenesis Notch signaling functions in many important biological processes in development and cell differentiation. Notch signaling is one way cells can interact with one another. A signaling cell in this pathway displays a membrane bound ligand, often Delta, Jagged/Serrate-like. This ligand then binds to the Notch receptor found on a receiving cell. Following contact between a Notch ligand and the Notch receptor, the receptor undergoes a cleavage by Presenilin 1(Psen1), allowing the Notch Intracellular Domain (NICD) to enter the nucleus of the receiving cell and behave as a transcription factor. Notch can be modified by other members of the pathway such as Lunatic fringe, which 3

9 acts to glycosylate the Notch receptor, modulating its interactions with its ligands when expressed on the extracellular surface (Figure 1.2). Notch signaling has roles in both regions of the PSM (Reviewed in Weinmaster and Kintner, 2003). Mutations in Notch pathway members appear to have similar phenotypes. By mutating Hes7 (Chen, et. al, 2005), Notch signaling in the posterior PSM, or region I, is altered. This affects the molecular clock. The region II specific Mesp2 mutants only affect the patterning function of Notch (Morimoto, et. al, 2005). And lastly, mutating Lfng influences Notch activity both in region I and region II of the PSM (Zhang and Gridley, 1998; Evrard, et. al, 1998). Each mutant, though affecting different aspects of Notch signaling in the PSM, has a similar phenotype: a short stature accompanied by rib and vertebral defects. The similarity in phenotype leads to difficulties in determining the specific roles of each Notch member in somitogenesis. Regulation of Notch varies between region I and region II of the PSM. In region I, Lunatic fringe (Lfng) plays a key role in directing oscillatory Notch signaling during clock function. The action of LFNG, the glycosylating protein encoded by Lfng, serves to prevent communication between cells by placing sugars on the EGF repeats of the Notch receptor (reviewed in Shifley and Cole, 2007). This periodic inhibition caused by the addition of sugars tends to support to the oscillation both of active Notch signaling as well as Lfng transcript. Oscillation of the Lfng mrna plays a role in the ability to produce protein (Chen et. al, 2005) In the posterior PSM, it has been shown that oscillatory Notch activity is regulated by the interplay between Lfng and Hes7, a bhlh repressor (Chen, et. al, 2005). It has been suggested that though Notch can activate Lfng expression by its ICD, Hes7 s ability to repress Lfng expression overrides this particular 4

10 form of activation. Hes proteins encode transcription factors which behave both as activators and repressors of different genes. Though Hes7 acts to repress both its own transcription (Chen, et. al, 2005) as well as the transcription of Lfng, Hes7 actually activates Notch signaling (Reviewed in Kageyama, et. al, 2007). Hes7 transcription, like Lfng transcription, oscillates in the posterior PSM. The time required for Hes7 oscillation in mouse equals the time required for Lfng two hours. Hes7 expression is limited to the posterior PSM, but Hes1 is expressed in both regions of the PSM. Oscillation of these two transcription factors is in phase. Experiments relating to Hes7 null animals have suggested that Hes7 plays a critical role in the cell allocation clock (Figure 1.3a). Notch in region II of the PSM is likely under control of a different mechanism, as suggested by the difference in gene expressions. Interactions between Mesp2 and Notch also appear to influence patterning in the anterior PSM. Lfng expression is freed from the Notch signaling pathway and becomes under control of Mesp2 in region II. The loss of control by Notch, along with the gain of control by Mesp2 leads to the different type of expression found in the anterior PSM compared to that of the posterior PSM (Shifley and Cole, 2007). Notch signaling is not the only factor in Lfng expression. Mesp2 also has been shown to play roles in the expression of Lfng in the anterior PSM. Experiments have shown that Lfng expression is enhanced when Mesp2 is expressed (Morimoto, et. al, 2005). Using previous data, it is likely that this upregulation of Lfng in the rostral compartment leads to the downregulation of Notch only in that compartment. Some variety of change in regulation seems to play a role in this alteration in Lfng expression (Figure 1.3b). Mesp2 can bind to the Lfng promoter and affect its transcription in the 5

11 anterior PSM (Takahashi, et. al, 2007). Without Mesp2 expression in the anterior PSM, Lfng does not completely localize in the anterior (Takahashi, et. al, 2007). Lfng plays many roles in somitogenesis Lunatic fringe s (Lfng) roles in somitogenesis have been examined in great detail. When the Notch receptor is modified by the addition of the sugars added by Lfng, it is believed that the modified Notch receptor is rendered unable to interact with the signaling cell s ligands. The lack of communication between cells prevents the endocytosis of the Notch receptor and furthermore prevents cleavage by Psen1. A negative feedback loop between Notch and Lfng is one of the driving forces underlying somitogenesis (reviewed in Weinmaster and Kintner, 2003). Distinct patterns of Lfng expression are found in the two regions of the PSM. Expression oscillates every two hours in mouse embryos in the posterior PSM while it is ubiquitously expressed in region II of mouse embryos. The stable band of expression in region II is found in the rostral compartment of the presomite. It has been shown that the oscillation of Lfng in the posterior PSM is necessary for proper somitogenesis in mice (Serth, et. al, 2002). Mice with ubiquitously expressed Lfng were created and analyzed. It was found that the phenotypic defects are similar to those found in Lfng null animals. It has also been found that constitutive expression of Notch in chick embryos leads to an increase in Lfng expression (Dale, et. al, 2003). Animals defective in Lfng exhibit severe phenotypic consequences, showing the importance Lfng plays in the segmentation clock (Zhang and Gridley, 1998 and Evrard, et. al, 1998). Defects found in Lfng null animals include disordered vertebrae, resulting from the altered dynamic expression of Lfng, and truncated tails. It is unknown exactly 6

12 why Lfng null tails are severely truncated; and further study must be done. Not only do these animals exhibited truncated tails and disordered vertebrae, but they also show a variety of rib defects. Skeletal preps were done in order to visualize these defects. The loss of patterning in Lfng null animals leads to the hypothesis that patterning is required for secondary body formation. A novel allele that perturbs cyclic Lfng expression in the clock was created by removing a clock enhancer, Fringe Clock Element 1 (FCE1) (Cole, et al., 2002). In Lfng FCE mice, dynamic expression of Lfng is lost in the posterior PSM; however, stable, though slightly reduced, expression of Lfng is maintained in the anterior PSM where R/C somite patterning occurs (Figure 1.4). The difference in Lfng expression between the anterior and posterior PSM suggest different mechanisms control the anterior and posterior PSM. It has been shown that FCE1 mutants display phenotypes similar to those of Lfng null animals. Phenotypes in ΔFCE1/ΔFCE1 are similar to Lfng -/- in the anterior skeleton of the body. An interesting phenomenon occurs in the development of the spine in these mice; their anterior skeleton is very disordered, but upon reaching the posterior skeleton, the phenotype is rescued. The defects in segmentation of ΔFCE1 mutants are confined mostly to the anterior region of the body while those of the Lfng nulls range from the thoracic region through the tail (Shifley, et. al, 2008). Lfng expression in both region I and region II are required for proper development of both the anterior and posterior body. Oscillatory Lfng/Notch in the clock is important during primary somitogenesis while Lfng in region II during patterning is necessary during secondary somitogenesis. It is possible to begin analyzing what is happening with Notch1 in region II during secondary somitogenesis in order to produce these differences 7

13 in phenotype. To address this role, the dosage effects of the various transgenic mice will be examined. Next, the Lfng dosage will be manipulated by adding Lfng in an anteriorspecific manner. Lastly, potential mechanisms for regulation in region II will be analyzed. To analyze the effects of Lfng dosage on development, we first quantify the defects found amongst the various transgenic mice created, including a new genotype of FCE/-. This genotype appears to have its own unique phenotype associated with it. This quantification will be followed by the production of transgenic mice containing an anterior specific dose of Lfng driven by the Mesp2 promoter. It has been shown (Haraguchi, et. al, 2001) that the Mesp2 promoter can drive expression of LacZ in the anterior PSM. The anterior specific Lfng could serve to rescue the phenotype of some transgenic animals. If our hypothesis is correct, we would expect to see the rescue of tail development in Lfng -/- embryos. We believe rescue will occur because FCE/ FCE mice have some tail development. The sizes of somites will likely still vary since the clock will still be deleted. Creating a transgene containing a posterior-specific promoter and a Lfng coding sequence might lead to the restoration, to some extent, of somite size. This experiment would require a promoter that is dynamically expressed, as shown by the results of both Serth, et al. (2002), and Dale, et. al (2003). Oscillatory expression of Lfng is key in somite formation. 8

14 Figure 1.1 Schematic of Presomitic Mesoderm: The PSM is divided into two regions, cell allocation and rostral caudal (R/C) patterning. In region I cells are allocated while in region II, presomites are patterned into rostral and caudal halves. Somites bud in an anterior to posterior fashion, S0 through S-II represent presomites, while SI represents the most recently formed somite pair. 9

15 Figure 1.2 Notch signaling: This is a representation of the Notch Signaling pathway as it pertains to Lfng. The Notch receptor is expressed with the Notch Intracellular Domain (NICD) attached inside the cell. When a signal is received from a cell through a Delta, Serrate, or Jagged domain, the NICD is cleaved and enters the nucleus where transcription of target genes is affected. 10

16 Figure 1.3 Interactions in the posterior and anterior PSM these schematics show the gene interactions in the posterior (A) and anterior (B) PSM. Notch is capable of activating expression of Lfng and Hes7. Hes7 inhibits Lfng, and itself. Lastly, it is believed that Lfng inhibits Notch signaling, thus leading to the oscillatory expression of Lfng. (B) describes expression in the anterior PSM. Here, Mesp2 expression overrides Notch signaling and activates Lfng in the rostral compartment of the somite. 11

17 Figure 1.4 The FCE1 deletion and corresponding phenotypes (a) shows the targeted deletion of Fringe Clock Element 1 and the corresponding change in expression. (b) shows the dosage gradient produced by our various mice. Notice the decrease in loss of r/c patterning as we move from left to right. (c) shows corresponding phenotypes of adult mice. The body progressively shortens as the dosage of Lfng is lowered. The tail especially shortens. Photo s courtesy of Susan Cole and Emily Shifley 12

18 Chapter 2 Results Examining the requirements for anterior development We want to examine the role Lfng dosage plays in development of the skeleton. Since Lfng is expressed in different manners, we decided to see which region of expression is absolutely required for development of the axial skeleton. If development was solely dependent upon the clock, we would expect all mutant mice to have the same phenotype. However, if patterning is important, we would expect the FCE/ FCE and FCE/- embryos to have a different phenotype from the null and wild type embryos. To obtain our various genotypes, we cross Lfng +/- mice with other strains, such as Lfng FCE/-, or some other variety. The null allele must be kept in the heterozygous form, however, because null males tend to be sterile. We collected embryos 18.5 d.p.c from adult mice by non-survival Cesarean section, and then prepared them for examination as described in the materials and methods. After approximately one month, the end result was available for examination. Various axial skeleton defects were quantified starting with the anterior regions of the mice first. A comparison of the length of the anterior body was done by measuring the number of vertebrae present from the base of the skull to the pelvic girdle. In wild type embryos (n=25), we counted 26 ossification sites, or vertebrae, from the skull to the top of the pelvic girdle (Figure 2.2(e-h)). By removing the clock, or deleting FCE1, anterior vertebrae lost their well-maintained shape. Additionally, this removal of the clock found in our FCE/ FCE appears to impact the number of vertebrae contained in the anterior body. We found by direct counting that the total number of vertebrae in this region ranges from 16 to 21 (n=25), with an average of 18 13

19 vertebrae. This is a loss compared to the anterior body s normal length; loss of the clock leads to a loss of approximately 23% of the body length (6 missing/26 in wild type). Not only did these animals display a decrease in the anterior body length, but also the vertebrae lack consistent structure. Continuing our analysis of Lfng s role in axial skeleton development, we examined our FCE/- mice. The results were not quite as expected, with these mutant embryos showing a similar range of cervical to pelvic girdle vertebrae (17 to 20), and also sharing the average of 18 vertebrae per embryo (n=23). The expected result was to have further loss of anterior structures for a shorter anterior body. The expected results for these embryos would have been that they would have looked like the FCE/ FCE in their entire body shape. However, this finding suggests that dosage of Lfng in the patterning region influences development of the secondary body structures, as well as the primary body structures. Like the FCE/ FCE mice, anterior vertebrae of the FCE/- mice were always deformed. Our last genotype examined were Lfng -/-, those embryos lacking all Lfng. As expected, the complete loss of all Lfng played a clear role in the shortening of the anterior body; vertebral counts ranged from 13 to 17 vertebrae, with a median of 16 vertebrae (n=15). In addition to the much shorter stature of the Lfng -/- embryos, all vertebrae for these were poorly formed. These data suggest to us that the formation of vertebrae is dependent upon only the clock, and region II of PSM is insufficient to rescue anterior skeleton development. The vertebral column is not the only region of the anterior body affected by the varying levels of Lfng. We noticed that the ribcage is affected and thus determined three ways to quantify these defects. First, we counted the number of rib attachments on the sternum. Next, we examined the sternum of each ribcage for the number of misaligned 14

20 rib attachments (Figure 2.2(a-d; i)). The last area of rib quantification was simply to count the rib defects; the main classes of rib defects quantified were bifurcations, trifurcations, and rib fusions (Figure 2.2(a-h; j). Our wild type embryos were simply that: they had 7 attached ribs on each side of the sternum; they lacked misalignments on the sternum; they lacked any of the rib defects expected in our other mutant mice. Our next set of mice we examined were the FCE/ FCE mutants: their defects were quite evident. We found that some ribs never formed, but this lack of formation was not necessarily symmetric. For example, we counted 3 embryos that had fewer ribs on the right side of the sternum than the left. The range for right ribs was 5 to 7 and left ribs were 6 to 7. This rib asymmetry likely lead to the number of misalignments found in these embryos (median of 1, range= 0-4). Lastly, we examined the number of actual defects of ribs; these were most prominent in the dorsal ribcage. We found that all FCE/ FCE embryos exhibit at least some sort of rib abnormality, with the most common form of defect being a bifurcation. FCE / FCE embryos displayed about 3 rib defects per animal, but ranged from 2 to 5 defects. We next analyzed FCE/- embryos for defects in the ribcage region and found similar results to those obtained from analysis of the FCE/ FCE embryos. These embryos also showed asymmetry in number of ribs on each side of the sternum with the lower number most frequently found on the right side. Thus we found the median number of ribs on the left and right to both be 7, but the difference between FCE/ FCE and FCE/- was, however, the frequency of fewer than 7 ribs was higher. The range was the same as our FCE/ FCE embryos. The number of misalignments in our sample ranged from 1 to 3 with a median of 2 misalignments per embryo. We counted the number of rib abnormalities in the FCE/- animals, with a median of 4 rib 15

21 defects per embryo, and a range of 3 to 5. Our median suggests a slight increase in the number of rib defects between the FCE/ FCE and the FCE/- embryos, but this is rather insignificant as shown by the error. The Lfng -/- embryos were expected to have the most severe phenotype because they completely lack all Lfng in both regions of the PSM. We counted ribs on each side of the sternum and found that the left and right had medians of 7 and 6, respectively. The right had a low of 4 ribs while the left had a minimum of 5 attached ribs. Additionally, we noticed more rib misalignments, an average of 3.6, along with a low of at least two misalignments. We counted defects in these embryos and found that of our sample size, we had a median of 5 rib defects per animal; coupled with this, we had a higher range than in other genotypes (4 to 6). Quantifying the posterior defects in Lfng mutant mice We next turned our attention to the posterior skeleton, where development appears to occur by a different mechanism. We want to look in these structures to compare which level of Lfng expression leads to a loss of structures and which expression changes the organization of the posterior skeleton. To begin analysis of the posterior body, we counted the number of vertebrae from the pelvic girdle to the tip of the tail. In our wild type sample, this was the only real source of variation; posterior vertebrae totaled between 25 and 28 vertebrae (Figure 2.3(a-e)), which could be explained by the idea that tail length might be a polymorphic trait, or that some of the vertebrae at the tip of the tail were unable to be seen. The tails of these animals are best described as smooth; they lack rough bends, or kinks. We then moved on to quantify the FCE/ FCE posterior body. Our finding in these embryos was quite interesting: vertebral phenotype was rescued and tail length became mostly normal, with a median length of about 26 16

22 vertebrae and a range from 18 to 30. The number of deformed vertebrae in embryos with the homozygous deletion of FCE1 was surprisingly small: with between 0 and 2 vertebral defects in the posterior vertebral column. We found that some FCE/ FCE embryos had a tail kink. We counted a tail kink as a bend in the tail where the vertebra leads to a sharp change in direction. Our examination of Lfng FCE/- embryos showed that one less copy of Lfng in the patterning region lead to the loss of tail length (median length = 22 vertebrae), as well as the increase in tail kinks (median number of kinks = 2), compared to that of FCE/ FCE embryos. We also found that the number of vertebral defects extended further down the spine into the pelvic girdle, but subsequent vertebrae were nearly wild type. Our last sample, the Lfng -/- embryos, was examined for the same parameters (posterior body length and tail kinks). We found, however, that the body was so short, that the tail lacked length, and we were unable to count tail kinks. Thus, we classified their tails as truncated (Figure 2.3(a-d;f). The tail length for these embryos was shorter than those of the other genotypes, with a median length of 14 vertebrae. Our results, at this time, appear to support our hypothesis that Lfng dosage is important in the formation of the tail. Production of an anterior specific Lfng transgene It is known that Mesp2 is expressed only in the anterior region of a somite pair, along with Lfng. Because Mesp2 is expressed solely in the anterior PSM, we believed that when the transgene is activated for transcription, we will obtain Lfng in the anterior PSM only (Figure 2.3b) (Haraguchi, 2001). We would like to examine the effect this has on development, especially in Lfng -/- embryos. We constructed a transgene containing the Mesp2 promoter followed by the Lfng coding sequence, along with IRES and Bgeo in 17

23 order to test for expression (Figure 2.3a). The pieces of our transgene were assembled by a four-piece ligation; following assembly, we had mice injected with the transgene. The most evident way to address the issue of the importance of Lfng in secondary body formation is to breed our transgenic mice with Lfng -/- animals. These would give the most evident change in phenotype, as the tail would no longer be truncated. Tails obtained from offspring from the chimeric mouse were prepared as described in materials and methods. We analyzed the DNA with PCR to identity mice that were positive for our transgene. Our PCR showed that two transgenic mice were found (Figure 2.3c). We then used these founder mice to breed and produce more transgenic positive mice. We examined the expression of the transgene by RNA whole mount in situ hybridization on embryos (n=12) 10.5 d.p.c. and expected to find localized expression in the anterior region of the somites; our results, however, do not show expression of the transgene (Figure 2.3d) 18

24 Figure 2.1 Lfng genotyping Figures A-C show the chromosomes present in our various transgenic mice. 19

25 Figure 2.2 Anterior body defects (a-d) show the defects found on the ventral ribcage of these mice. The development of misalignments in the rib attachments is evident in those mice lacking oscillatory Lfng. (e-h) show dorsal rib defects. Notice the condensed cervical vertebrae in (f) compared to (e). Also, bifurcations and other rib defects are marked by arrows. Additionally, the vertebral disorder is marked in a red box, as an example. The numbers of attached ribs are shown in (i), while the total number of rib defects are shown in (j). (k) shows a comparison of the number of vertebrae to the number of defective vertebrae by genotype. Notice the progressive shortening of the anterior body. 20

26 Figure 2.3 Posterior body defects (a-d) are posterior skeletons of 18.5 d.p.c embryos. Notice the change in phenotype at the pelvic girdle. Also, (b) shows kinks in the tail of the FCE/ FCE mouse, which continue into the FCE/-. (d) shows the shorter, truncated tail evident in Lfng -/-, as well as the lack of change in disorder throughout the posterior body. A comparison of posterior body length and posterior body defects is shown in figure (e), with the FCE/ FCE being almost completely wild type. In (f), we counted the tail kinks and displayed them as a percent based on genotype. 21

27 Figure 2.4 Anterior Specific transgene The structure of our transgene is shown in (a), with the Mesp2 promoter linked to the Lfng gene. We included an IRES and Bgeo (LacZ + Neo) for verifying expression. (b) is the proposed effect of adding the transgene to a Lfng -/- mouse. Our initial founders yielded two positive transgenic mice (labeled). Embryos aged to 10.5 d.p.c. were examined by whole-mount in situ hybridization but we found not detectable expression in the anterior region of the somites. 22

28 Table 2.1 Medians and Means of quantification The above tables show the medians and means for the quantification of skeletal defects among the various genotypes. 23

29 Chapter 3 Discussion Clock function in somitogenesis is required for proper anterior skeletal development Our results indicate that a progressive decrease in Lfng leads to a shorter upper body. Removal of FCE1 leads to loss of oscillatory Lfng expression and produces defects in the anterior body. The anterior skeleton of the FCE/ FCE mice shows great disorder; FCE/ FCE mice lack all oscillatory Lfng. This suggests that the oscillatory expression of Lfng is required for proper body formation. However, this expression may not be sufficient to produce wild type appearance. This is because an examination of FCE/- animals displays a slightly more severe phenotypes in the anterior body (more rib defects and misalignments). Some other factor likely plays a role in this process though, because Lfng -/- produce somitic derivatives. The various phenotypes associated with our Lfng mutant mice suggest to us that the clock function, or region I expression of Lfng, is required for proper development of the anterior skeleton. Patterning of presomites in region II is required for proper tail development Upon examination of FCE/ FCE mice, we found an interesting phenomenon; formation of vertebrae at the lumbo-sacral junction is rescued and almost all subsequent vertebrae are wild type in appearance. Looking at the tail length, we found that it was essentially wild-type. Although we occasionally found tail kinks in these embryos, the observation of essentially wild-type embryos in the posterior body in mice with only patterning Lfng suggests that proper patterning is a requirement for formation of the posterior body. To examine this further, we examined the FCE/- mice, those that further reduced dosage of Lfng in the anterior PSM. We found that the lower dose has a slight, 24

30 but noticeable effect on development of the posterior skeleton. Most notably, the rescue found in the FCE/ FCE is essentially lost. Vertebrae become much more disordered and the tail becomes kinked in FCE/- embryos. The increase in tail kinks between FCE/ FCE and FCE/- suggests that tail kinks form as a result as a loss of Lfng in the anterior PSM. This is not as common in FCE/ FCE because they have two copies of Lfng, but because the FCE/- embryos lack an entire copy of Lfng, tail kinks are more common. Along with the noticeably shorter posterior region, we also noticed an increase in tail kinks and vertebral deformities in these mutant mice. A complete lack of both Lfng elements (clock and patterning) found in Lfng -/- embryos leads to poorly formed vertebrae along the entire axial skeleton of these mutant mice. Additionally, the truncation of the tail is a finding. The fact that the tail is truncated in these animals suggests that Lfng, in one way or another, is required for development of the tail. We therefore propose that exogenous expression of Lfng in the anterior PSM would lead to a rescued phenotype in null animals so they share a phenotype with the FCE/- embryos. The Mesp2 promoter linked Lfng transgene lacks expression in the anterior PSM The transgene we developed to directly assess the role of the patterning lacked expression in the PSM. One hypothesis to explain our result is that when injected into the mice, the transgene inserted in a region of a chromosome that is epigenetically repressed; an example of this could be insertion into the heterochromatin. This theory is supported by the fact that our transgene is transmitted down generations, as verified by PCR analysis of the offspring of our founders, but lacks RNA transcripts, determined through use of RNA whole mount in situ hybridization. 25

31 Another hypothesis to explain the lack of detectable expression is that the Mesp2 promoter is simply not robust enough to produce any product, although this promoter has been used in other transgenes (Haraguchi, 2001). In addition to this possibility, the Mesp2 promoter might require some additional factors not present in the promoter sequence we used. We could test this idea by transfecting cells with a modified transgene, one that has a reporter gene following the Mesp2 promoter, and looking for expression. If we found expression under wild type conditions, the previously mentioned hypothesis would be supported; however, if no expression occurs, additional factors would likely be needed. This experiment, however, would not directly reflect the way cells work in vivo. We could take a different approach to test the hypothesis that Lfng in the anterior PSM is required for proper development of the tail. We could look for genes that are expressed only in the anterior region of a presomite. To do this, we could do a microarray analysis on cells from the anterior PSM and cells from the posterior PSM. By comparing the results, we could determine which genes are expressed only in the anterior region of a somite. We could then produce a new transgene with the new promoter, followed by the Lfng coding sequence. Our hope would be to produce the anterior specific Lfng and rescue the skeletal phenotype. Additionally, we could use other regulatory sequences, such as the Lfng enhancer (Cole, 2002) or the EphA4 enhancer (Nakajima, 2006) to examine expression only at the anterior region of a presomite. Future Directions Because Lfng mutant animals show truncated tails, it is possible that some interaction between Lfng and the VER is present. The VER is a necessary component to proper tail formation and it is therefore reasonable to believe some interaction between 26

32 Lfng and the VER exists. Since Lfng null animals show truncated tails, and the VER signals for tail elongation, it is likely that Lfng interacts with the VER by promoting signaling. In the absence of Lfng, the VER would be unable to signal for tail elongation, thus resulting in the characteristic truncated tails of Lfng null mice. Since the anterior VER seems to be unable to influence tail outgrowth, it is expected that Lfng has no impact on this region, yet some interaction with either the middle or the posterior VER. While Hes7 s most important activity is located in the posterior PSM, the Mesp family of genes exclusively functions in the anterior PSM. Genes in this particular family serve as transcription factors; the two genes of interest in the anterior PSM are Mesp1 and Mesp2. Expression of Mesp2 is limited only to the rostral region of the maturing somite. Studies on the effect of Mesp2 on somitogenesis have been conducted by analyzing null mice. Mice lacking Mesp2 are unable to properly pattern somites; they lack rostralization. Its counterpart, Mesp1, seems to lack comparable importance in somitogenesis. Expression of Mesp1 and Mesp2 overlaps in the PSM; this suggests that redundancy in function might be present (Takahashi, 2005). Chimeric analysis of Mesp1 and Mesp2 double null animals is necessary because Mesp1/Mesp2 do not develop to term. The chimeric analysis of the double knockouts showed the inability of cells lacking both Mesp1 and Mesp2 to epithelialize. Mesp2 null animals appear to show at least some epithelialization which therefore suggests that Mesp1 is able to compensate for the lack of Mesp2 (Takahashi, 2005). It has also been shown that varying levels of Mesp1 and Mesp2 seems to impact the ability of the axial skeleton to develop (Morimoto, 2006). Mice lacking Mesp2 at a later developmental stage display fused pedicles, disordered vertebrae, and appear to lack rostral properties. Mesp2 plays roles in establishing the 27

33 rostral properties of each somite pair formed (Takahashi, 2003; Takahashi, 2007). Mice lacking Mesp2 only lack an aspect of patterning in somitogenesis, yet share a comparable phenotype to those lacking Hes7, a cell-allocation gene. To examine the role played by region II in anterior body development, a transgene expressing the anterior-specific, complementary Lfng coding region would be produced (Figure 3.1). The formation of the anterior-specific, antisense transcript would interfere with the wild-type expression of Lfng by producing a double stranded RNA molecule that would be destroyed by cellular mechanisms. This transgene would therefore remove only Lfng in the anterior PSM, but would allow for the expression of the oscillatory Lfng found in the posterior PSM. If our hypothesis is correct that only the oscillatory Lfng expression is required for proper development of the anterior body, it would be plausible to see a nearly wild-type phenotype. However, our data suggests that the loss of region II Lfng could lead to rostral-caudal patterning issues, but examination of these mice would answer the question of the requirements for somitogenesis in the anterior and the posterior PSM. We would expect to see tail truncations in transgenic mice due to the lack of Lfng in the patterning region. Additionally, defects would be present in the anterior skeleton due to the loss of patterning. We would find tail truncations because the mrna formed by our transgene would be able to bind, and thus sequester, any Lfng produced; the anti-sense Lfng would behave as a trans acting regulatory sequence. Tail truncations would be present because our transgene would make region II behave as if it is Lfng -/-, thus leading to a truncated tail. 28

34 Conclusions Through careful quantification of skeletons, we have determined that the dose of Lfng present in the PSM impacts the development of the axial skeleton. We also have found that the region I and region II impact development of different structures: region I expression promotes development of the anterior skeleton, while region II expression promotes development of the posterior skeleton. Additionally, we found that the amount of Lfng in the anterior PSM impacts the quality of the tail by comparing the phenotypes of FCE / FCE embryos to that of FCE /- embryos. We hope to gain further knowledge of the process of somitogenesis by knocking out only the R/C patterning region while maintaining oscillatory expression of Lfng. This experiment would show the true role of the clock. 29

35 Figure 3.1 Transgene knocking out region II The structure of the transgene mimics that of our earlier transgenic experiments (a). The likely effect of this transgene is shown in (b). We believe if would result in the loss of region II Lfng, but not oscillatory. 30

36 Chapter 4 Materials and Methods Genotyping of mice: Agarose gel electrophoresis was used to analyze all genotyping reactions. Tail DNA A salt-out technique was used to isolate tail DNA for all PCR reactions. Approximately 1 cm of tissue is removed from the specimen and placed in a 6 μl Proteinase K: 600 μl TENS overnight at 55 C overnight. The next day, saturated NaCl( μl) is added and mixed for 15 seconds. The mixture is then placed in a room temperature centrifuge at maximum speed for ten minutes. The resulting supernatant is then decanted into a new, sterile, 1.5-mL test tube. 600 μl of 95% ethanol is added and centrifuged as before. The supernatant is then removed and 70% ethanol is added in its place. The resulting solution is rocked at room temperature for no less than one hour. To concentrate the DNA, the tube is centrifuged for five minutes at maximum speed. The supernatant is removed and the sample is left to dry. Once the remaining ethanol has evaporated, 100 μl Tris (10 mmol, ph 7.5) is added. Samples are then stored at -20 C until needed for PCR. Yolk Sac DNA Yolk sac DNA comes from the membrane surrounding an embryo ranging from d.p.c. in age. A small amount is removed in order to determine the genotype of the embryo. It is placed in a 1.5-mL eppendorph test tube and spun down to concentrate the tissue. Next, a fresh 0.05M NaOH solution (100μl), prepared from 10N NaOH, is added and the test tube is placed on a heating block set to 95 C for thirty minutes. Next, 1/3 M Tris (ph 7.5) is added and the solution is mixed and 31

37 centrifuged. Finally, 90 μl of the upper region of the solution is placed in a separate, clean test tube and kept at -20 C until needed for further usage. PCR Genotyping reactions were done as follows: Lfng was genotyped using primers FNG322, FNG325, and PGK3 (Table 4.1). The FCE deletion requires SC284 and SC285 (Table 4.1). Reactions are run with 35 cycles with an annealing temperature of 55 C. Lfng null animals show a band at 200 bp while wild types have a band at 170 bp. FCE mutants displayed a negative band at 182 bp and a wild type band at 340 bps. For examination of transgenic mice, we used SC340 and SC 341, along with FNG 237 and FNG 239 as positive controls; transgenic animals display bands at about 250 bps. Skeleton Preparations: Mice aged at approximately 18.5 DPC are acquired from the mother by Cesarean section. After obtaining genotype information as above for Lfng as well as ΔFCE deletions, embryos are skinned and eviscerated and placed in 100% ethanol for four days. The embryos are removed from the 100% ethanol and then placed in acetone. The acetone is used to remove any remaining fat. After three days in acetone, embryos are rinsed with water then placed in the staining solution containing 2 volumes Alcian blue, 1 volume Alizarin red, 8 volumes Glacial acetic acid and 50 volumes 70% ethanol. Following staining, the embryos are transferred to a 1% KOH in 20% glycerol solution and placed in a 37 C water bath overnight. To prevent destruction of the specimens, they are then removed and remain in the 1% KOH/20% glycerol until the specimens are completely cleared. Following the clearing step, they are placed in a 2:2:1 Ethanol:Glycerol:Benzyl alcohol solution for analysis. Whole Mount RNA in situ hybridization 32

38 Embryos aged from 8.5 d.p.c. to 12.5 d.p.c were obtained from the mother via Caesarian section. Embryos were stored at -20 C in 100% methanol until needed; they are then rehydrated to 100% PBT. Bleaching of embryos using 6% hydrogen peroxide in PBT was the first step, followed by treatment with Proteinase K for fifteen minutes. Following Proteinase K treatment, 2μg/mL glycine in PBT was added. Next, embryos were fixed using 0.2% gluteraldehyde in 4% PFA in PBT for thirty minutes. Embryos were then incubated in Prehyb for one hour at 70 C with rocking. A combination of Prehyb and Dig-labeled probe (1μg/mL) was mixed and heated to 70 C. Following the Prehyb wash, the Prehyb and Dig-labeled probe were added to the embryos and incubated overnight at 70 C. Washes consisting of formamide and SCC (ph 4.5) were made and were used to wash the embryos for several hours. A series of washes using MABT were then prepared and the embryos were washed with these for several hours more. The solutions, in order, were MABT, MABT + Boehringer Blocking Reagent (BBR), MABT + BBR + 20% heat inactivated sheep serum, and MABT + BBR+ 20% heat inactivated sheep serum + anti- DIG antibody. The embryos remained in the final solution overnight at 4 C. The following day involved washing the embryos with a solution of MABT + 2 mm Levamoisole, first changing the wash every hour for five hours, and then switching the wash every twelve hours. The last day consisted of washing embryos in a solution of freshly prepared NTMT + 2mM Levamisole, then exposed to the same solution only with the addition of NBT and BCIP in the absence of light until staining became apparent. Once the desired staining became apparent, embryos were washed in PBT ph 5.5 and fixed in 4% 33

39 PFA/0.1% gluteraldehyde. Clearing of embryos was done by washing first with 1:1 Glycerol:PBT then followed by 4:1 Glycerol:PBT. Stained embryos were stored in this last solution at 4 C. 34

40 FNG GAGCACCAGGAGACAAGCC-3 FNG AGAGTTCCTGAAGCGAGAG-3 PGK3 5 -CTTGTGTAGCGCCAAGTGC-3 SC TTTGGTGGGAATGGATTAGC-3 SC CTGGTCCATTTGCTCTGAGG-3 SC CAGAATCCACACCTCTGCAA-3 SC ACCAGGAGACAAGCCAACAG-3 FNG CGACATTTTGCAGCACAG-3 FNG TTCACCGATGGAGACGAC-3 SC GCGACATGCTGGCTCTTCTA-3 SC CCAGGTTTTGACACTAGCACA-3 Table PCR Primers - List of PCR Primers and corresponding sequences used for genotyping and isolation of coding regions 35

41 Works Cited Andrade, R., Pascoal, S., and Palmeirim, I Thinking clockwise. Brain Research Reviews 49: Chen, J., Kang, L., and Zhang, N Negative Feedback Loop Formed by Lunatic Fringe and Hes7 Controls their Oscillatory Expression During Somitogenesis. Genesis 43, Cole, Susan E., John M. Levorse, Shirley M. Tilghman, and Thomas F. Vogt Clock Regulatory Elements Control Cyclic Expression of Lunatic fringe during Somitogenesis. Developmental Cell 3: Dale, J., Maroto, M., Dequeant, M., Malapert, P., McGrew, M., and Pourquie, O Periodic Notch inhibition by Lunatic Fringe underlies the chick segmentation clock. Nature 421, Evrard, Y.A., et al (1998). Lunatic fringe is an essential mediator of somite segmentation and patterning. Nature 394: Goldman, D., Martin, G., Tam, P Fate and function of the ventral ectodermal ridge during mouse tail development. Development 127, Haraguchi, S., Kitajima, S., Takagi, A., Takeda, H., Inoue, T., Saga, Y Transcriptional regulation of Mesp1 and Mesp2 genes: differential usage of enhancers during development. Mechanisms of Development 108: Kageyama, R., Masamizu, Y., and Niwa, Y Oscillator Mechanism of Notch Pathway in the Segmentation Clock. Developmental Dynamics 236, Nakajima, Y., Morimoto, M., Takahashi, Y., Koseki, H., Saga, Y Identification of Epha4 enhancer required for segmental expression and the regulation by Mesp2. Development 133(13), Morimoto, M., Takahashi, Y., Saga, Y The Mesp2 transcription factor establishes segmental borders by suppressing Notch activity. Nature 435, Morimoto, M., Kiso, M., Sasaki, N., Saga, Y Cooperative Mesp activity is required for normal somitogenesis along the anterior-posterior axis. Developmental Biology 300, Palmeirim, I., Henrique, D., Ish-Horowicz, D., and Pourquie, O Avian hairy gene expression identifies a molecular clock linked to vertebrate segmentation and somitogenesis. Cell 91: